article stringlengths 0 826k | abstract stringlengths 174 4.34k |
|---|---|
the world health organization ( who ) co - sponsored an international conference on primary health care in alma - ata , ussr ( now in kazakhstan ) in september 1978 .
the declaration of alma - ata stated the need for urgent action by all governments , all health and development workers , and the world community to protect and promote the health of all the people of the world. unfortunately , at least in india , although progress has been achieved in different sectors , much needs to be done to achieve good health for its citizens .
chronic kidney disease ( ckd ) belongs to the category of non - communicable diseases ( ncds ) . according to a report ,
ncds were responsible for 63% of deaths worldwide in 2008 , and in india alone they were responsible for 53% of deaths . according to this report , causes of mortality were cardiovascular diseases in 24% , chronic respiratory diseases in 11% , cancer in 6% and diabetes in 2% population .
there is limited information available about the state of kidney health in india , and especially the rural and semi - urban population , who have limited access to health services for various reasons
. the age - adjusted incidence rate of end - stage renal disease ( esrd ) in india was reported to be 229 per million population ( pmp ) and > 100 000 new patients enter renal replacement programs annually in india [ 2 , 3 ] . in a recent multicenter study in india ,
the prevalence of ckd was observed to be 17.2% , with about 6% having ckd stage 3 or worse .
these reports suggest that much needs to be done to focus on kidney diseases in india .
kidney diseases may become a major threat to the health of the population of developing nations and particularly india in the near future .
hypertension , diabetes mellitus ( dm ) , obesity and stone diseases are the common risk factors for development of ckd , as well as less common causes such as infections and congenital anomalies of the urogenital tract .
world kidney day ( wkd ) is has been celebrated every year in many parts of the world since 2006 .
our aim in celebrating this day is to increase awareness of kidney diseases in the general population so that we can prevent the progression of kidney diseases and help suffering patients with therapeutic strategies .
however , there is limited benefit of such celebrations to the people in less developed areas of the world due to the lack of awareness about kidney diseases , a lack of commitment and will of the personnel engaged in health practices and the paucity of resources to address this issue .
kidney health of the semi - urban population of gujarat on the occasion of wkd in march 2014 .
we screened the population of six towns where we had established dialysis centers to serve the local population by organizing a kidney disease screening program as a pilot project .
all willing participants were included in the study except pregnant women , persons < 18 years of age and those with hypertension , dm , ckd , hepatitis b or c , hiv , cardiac / liver pathology or if they had undergone kidney transplantation .
this study was conducted in 2014 on sundays from the end of february to june to ensure that maximum participants could take advantage of these free services .
participants were informed about these camps by advertising in local newspapers and on television and by placing billboards in the villages for 1 week before the scheduled date of the camp .
every camp was open from 8 a.m. to 8 p.m. a team of nephrologists , pathologists , urologists , radiologists , medical officers , nurses , technicians and clerical staff trained to conduct interviews participated in the camps .
a structured questionnaire in the local language was developed and volunteer participants were requested to complete it on arrival and after enrollment ( appendix ) with the help of our staff .
it included history of illness , diabetes , hypertension , swelling of the face / eyelids in the morning , urinary complaints , medications and any significant family history related to kidney diseases .
demographic studies included height , weight , temperature , pulse rate , respiratory rate , blood pressure ( bp ) and body mass index .
lab investigations included complete blood counts , random blood sugar , serum creatinine ( scr ) and urinalysis .
ultrasonography of the abdomen was carried out and any anomaly related to the urogenital tract was noted .
after the reports were printed , each participant was examined by a nephrologist and urologist and advised for further management according to examination and reports .
overweight was defined as a bmi between 25 and 30 kg / m and bmi > 30 kg / m was considered obese .
blood samples were collected and sent to nearby labs accredited under the national accreditation board for testing and calibration laboratories ( nabl ) or to our own lab .
complete blood counts were performed by an automated hematology analyzer ( sysmex xn series , sysmex , kobe , japan ) .
reference ranges were hemoglobin 1214 g / dl for women and 1317 g / dl for men , and total leukocyte count 411 10/l .
scr was measured using a modified jaffe colorimetric method , with a reference range was 0.51.4 mg / dl .
plasma glucose was measured by the glucose oxidase peroxidase method , with a reference range of 70140 mg / dl .
standardization was carried out using a chemistry calibrator traceable to an isotope dilution mass spectrometry ( idms ) reference method using the national institute of standards and technology ( nist ) standard reference material 967 .
urinalysis was performed using a fully automated urinalysis system ( urisys 2400 , roche diagnostics india ) from midstream urine samples from all participants in the same season and similar weather conditions for all the centers .
estimated glomerular filtration rate ( egfr ) was calculated using the modified modification of diet in renal disease 3 equation using standardized scr , age , race and gender .
dm was defined as random blood sugar 200 mg / dl as per the american diabetes association guidelines .
proteinuria was considered for urine albumin 1 + , which is equivalent to 25 mg / dl .
all participants received their reports along with instructions from a nephrologist and urologist before they left the camp site .
they were advised to come for follow - up to our center for further care or if they needed any additional consultation .
further comparisons of towns from north gujarat were made with those of the coastal region of saurashtra to determine the prevalence of hypertension , diabetes and stone burden in these areas .
this comparison was made because north gujarat is a distance from coast and has hard water compared with other parts of the state .
continuous variables were expressed as mean sd and categorical variables were expressed as number ( % ) .
data followed normal and non - normal distributions ; continuous variables were tested for normally distributed variables using independent unpaired student 's t - test , and mann whitney u - test was used for non - normally distributed continuous variables . for categorical variables , pearson test was used .
continuous variables were expressed as mean sd and categorical variables were expressed as number ( % ) .
data followed normal and non - normal distributions ; continuous variables were tested for normally distributed variables using independent unpaired student 's t - test , and mann whitney u - test was used for non - normally distributed continuous variables . for categorical variables , pearson test was used .
of the 2800 participants screened from six towns in western india , 2350 fulfilling the selection criteria were included in the study .
the mean distance of camp venues from our premises was 132.33 82.48 km ( range 61311 ) .
none of the participants had a history of illness , diabetes , hypertension , swelling of the face / eyelids or urinary complaints .
this included a history of diagnosed kidney failure or any visit to the doctor for urinary problems , swelling of the face / eyelids , weakness , hypertension , diabetes or hereditary diseases related to kidney disease or birth defects .
of the studied participants , 16.1% had a positive history and hence were removed from the study .
bmi < 25 kg / m was observed in 1141 ( 48.55% ) participants and bmi 25 kg / m was observed in 1209 ( 51.45% ) participants .
dm was observed in 82 ( 7.19% ) participants of the lower bmi group versus 146 ( 12.08% ) in the higher bmi group ( p < 0.01 ) .
males had a higher incidence of dm than females in both groups , constituting 74.39% in the former and 65.07% in the latter .
hypertension was observed in 207 ( 18.14% ) participants belonging to lower bmi group versus 427 ( 35.32% ) in the higher bmi group ( p < 0.01 ) .
males had a higher incidence than females in both groups , constituting 73.43% in the former and 65.07% in the latter .
thus dm and hypertension were more common with higher bmi ( p < 0.01 ) .
all the participants with stone burden had multiple calculi and concretions in the kidneys with / without calculi in the ureter / bladder .
the size of the smallest calculus was 3 mm and the largest was 45 mm .
table 1.results of screening of participants for kidney diseasesparameters evaluatedtotalmalesfemalesp - valuesubjects , n23501438912<0.01age ( years ) , mean sd48.16 1449.77 14.4746.77 13<0.01bmi (
kg / m ) , mean sd25.61 5.2525.11 526.41 5.53<0.01overweight772 ( 32.85%)469 ( 32.61%)303 ( 33.22%)0.79obesity423 ( 18%)218 ( 15.16%)205 ( 22.48%)<0.01hypertension631 ( 26.85%)385 ( 26.78%)246 ( 26.97%)0.91diabetes mellitus230 ( 9.79%)151 ( 10.50%)79 ( 8.66%)0.16serum creatinine ( mg / dl ) , mean sd1.02 0.421.11 0.450.87 0.3<0.01egfr ( ml / min/1.73 m ) ,
mean sd87.84 2188.19 20.9187.30 21.240.32urine albumin 1 + 324 ( 13.79%)208 ( 14.46%)116 ( 12.72%)0.23stone burden404 ( 17.19%)277 ( 19.26%)127 ( 13.92%)<0.01hemoglobin < 12 g / dl638 ( 27.15%)207 ( 14.4%)431 ( 47.26%)<0.01egfr < 60 ml / min/1.73 m195 ( 8.29%)108 ( 7.5%)87 ( 9.5%)0.38ckd egfr < 60 ml / min/1.73 m urine albumin 1 + 492 ( 20.93%)302 ( 21.0%)190 ( 20.8%)0.96 results of screening of participants for kidney diseases on comparing the towns from north gujarat with those of saurashtra , the prevalence of hypertension , diabetes and stone burden was different in the two areas . their demographics , clinical , radiological and laboratory data along with gender distribution are summarized in table 2 .
the incidence of obesity , overweight , hypertension and dm was greater in saurashtra whereas the incidence of stone burden , elevated scr and proteinuria was higher in north gujarat ( p < 0.01 ) .
hemoglobin < 12 g / dl was observed in both regions without any significant difference between them ( p = 0.30 ) .
table 2.comparison of demographics and results of screening participants for kidney diseases from two regions of western indiaparametertotalmalesfemalesregion 1region 2p - valueregion 1region 2p - valueregion 1region 2p - valuetotal , n121311370.987436950.984704420.98age ( years ) , mean sd49.38 13.3447.8 14.680.0149.25 15.4250.24 13.530.2045.37 12.9848.04 12.9<0.01bmi ( kg / m ) , mean sd26.53 5.2424.59 5.03<0.0124.08 4.6526.02 5.12<0.0125.39 5.5227.32 5.38<0.01overweight445 ( 36.60%)327 ( 28.75%)<0.01269 ( 36.2%)200 ( 28.77%)<0.01130 ( 27.56%)173 ( 39.14%)<0.01obesity278 ( 22.91%)145 ( 12.75%)<0.01140 ( 18.84%)78 ( 11.22%)<0.0179 ( 16.74%)126 ( 28.51%)<0.01hypertension372 ( 30.66%)259 ( 22.77%)<0.01206 ( 27.72%)166 ( 23.88%)0.1187 ( 18.44%)159 ( 35.97%)<0.01diabetes mellitus129 ( 10.63%)101 ( 8.88%)0.7078 ( 10.49%)73 ( 10.50%)0.9329 ( 6.14%)50 ( 11.31%)0.01serum creatinine ( mg / dl ) , mean sd1.04 0.430.99 0.4<0.011.15 0.471.08 0.430.010.87 0.280.87 0.321urinary albumin 1 + 106 ( 8.73%)218 ( 19.17%)<0.0164 ( 8.61%)144 ( 20.71%)<0.0142 ( 8.93%)74 ( 16.74%)<0.01ckd190 ( 15.66%)302 ( 26.0%)<0.01116 ( 15.61%)186 ( 26.76%)<0.0173 ( 15.53%)117 ( 26.47%)<0.01egfr ( ml / min/1.73 m ) , mean sd78.84 21.9589.34 20.76<0.0187.22 21.2289.09 20.610.1084.64 21.2189.73 21.0<0.01stone burden118 ( 9.72%)286 ( 25.15%)<0.01114 ( 15.34%)163 ( 23.45%)<0.0157 ( 12.12%)70 ( 15.84%)0.13hemoglobin 12 g / dl341 ( 27.96%)297 ( 26.12%)0.30116 ( 15.61%)91 ( 13.09%)0.20225 ( 47.7%)206 ( 46.61%)0.75egfr < 60 ml / min/1.73 m91 ( 7.46%)104 ( 9.14%)0.1754 ( 7.26%)54 ( 7.76%)0.7937 ( 7.84%)50 ( 11.31%)0.10adpkd5 ( 0.41%)3 ( 0.26%)0.795 ( 0.67%)3 ( 0.43%)0.790 ( 0%)0 ( 0%)region 1 : coastal region ( saurashtra ) , region 2 : non - coastal region ( north gujarat ) .
comparison of demographics and results of screening participants for kidney diseases from two regions of western india region 1 : coastal region ( saurashtra ) , region 2 : non - coastal region ( north gujarat ) .
table 3 describes renal impairment observed in different age groups from 18 to > 70 years .
this includes egfr < 60 ml / min/1.73 m , albuminuria 25 mg / dl , with ckd stages 3 , 4 and 5 and stone burden .
it was observed that overall ckd peaked in the seventh decade of life in both genders .
significant albuminuria was observed in 291 ( 16.77% ) participants with an egfr > 60 ml / min/1.73 m. of 492 participants with ckd greater than stage 2 , 32.52% were found to have ckd stage 3a , 4.87% were found to have ckd stage 3b , 2.03% were found to have ckd stage 4 and 0.20% were found to have ckd stage 5 .
figure 1 depicts the findings of our study regarding kidney diseases in this population .
table 3.renal impairment in different age groupsage group ( years)egfr < 60 ml / min/1.73 m ( n = 195 ) , /totalalbuminuria ( n = 324 ) , /totalckd ( n = 492 ) , /totalstone burden ( n = 404 ) , /totalcreatinine clearance , mean sdtotal 183017 ( 5.94%)/28636 ( 12.59%)/28653 ( 18.53%)/28639 ( 13.64%)/28689.90 32.30 314019 ( 4.58%)/41541 ( 9.88%)/41557 ( 13.74%)/41578 ( 18.8%)/41591.17 33.12 415052 ( 8.47%)/61486 ( 14.01%)/614126 ( 20.52%)/614122 ( 19.87%)/61486.48 30.33 516046 ( 7.96% ) /57878 ( 13.49%)/578121 ( 20.93%)/57894 ( 16.26%)/57880.78 28.57
617034 ( 10.46%)/32558 ( 17.85%)/32587 ( 26.77%)/32551 ( 15.69%)/32575.56 27.29 > 7027 ( 20.45%)/13225 ( 18.93%)/13248 ( 36.36%)/13220 ( 15.15%)/13266.23 25.99males 183010 ( 5.78%)/17322 ( 12.72%)/17332 ( 18.5)%/17323 ( 13.29%)/17389.96 33.59 31408 ( 3.72%)/21518 ( 8.37%)/21525 ( 11.63%)/21547 ( 21.86%)/21593.92 32.15 415024 ( 6.88%)/34953 ( 15.19%)/34970 ( 20.06%)/34987 ( 24.93%)/34984.50 29.57 516028 ( 7.61%)/36858 ( 15.76%)/36884 ( 22.83%)/36871 ( 19.3%)/36881.96 29.12 617021 ( 9.33%)/22539 ( 17.33%)/22557 ( 25.33%)/22536 ( 16%)/22576.05 27.84 > 7017 ( 15.74%)/10819 ( 17.59%)/10834 ( 31.48%)/10814 ( 12.96%)/10868.94 26.69females 18307 ( 6.19%)/11314 ( 12.39%)/11321 ( 18.58%)/11316 ( 14.16%)/11389.80 30.33 314011 ( 5.5%)/20023 ( 11.5%)/20032 ( 16%)/20031 ( 15.5%)/20088.64 34 415028 ( 10.57%)/26533 ( 12.45%)/26556 ( 21.13%)/26535 ( 13.2%)/26589.12 31.17 516018 ( 8.57%)/21020 ( 9.52%)/21037 ( 17.62%)/21023 ( 10.96%)/21078.68 27.52 617013 ( 13%)/10019 ( 19%)/10030 ( 30%)/10015 ( 15%)/10074.44 26.12 > 7010 ( 41.67%)/246 ( 25%)/2414 ( 58.33%)/246 ( 25%)/2459.55 21.37
fig . 1.bar diagram depicting the parameters of renal impairment in the form of albuminuria > 1 + , egfr < 60 ml / min/1.73 m , ckd and stone burden . renal impairment in different age groups bar diagram depicting the parameters of renal impairment in the form of albuminuria > 1 + , egfr < 60 ml / min/1.73 m , ckd and stone burden .
its complications include cardiovascular mortality , kidney disease progression , cognitive decline , anemia , mineral and bone disorders and fractures .
screening and timely intervention can prevent ckd and its complications . in our study of a semi - urban population
interestingly , we also found a high stone burden in the non - coastal area as compared with the coastal area .
thus both regions showed a high risk of their populations progressing to ckd , but for different reasons .
they observed that the median prevalence of ckd was 7.2% in persons 30 years old , whereas in persons 64 years , the prevalence varied from 23.4 to 35.8% . according to the kidney dialysis outcomes quality initiative ( kdoqi ) practice guidelines published in 2002 by the national kidney foundation ( nkf ) , ckd is defined as creatinine clearance or gfr < 60 ml / min/1.73 m [ 20 , 21 ] .
these guidelines were adopted by all studies to arrive at the prevalence of ckd . in the cross - sectional multicenter screening and early evaluation of kidney disease ( seek ) indian study by singh et al . on 6120 subjects ,
in this study , ckd stages were defined as per the kdoqi guidelines ( egfr < 60 ml / min/1.73 m or proteinuria > 1 + on dipstick ) .
seek studies of thailand and saudi arabia reported ckd prevalences of 17.5 and 5.7% , respectively [ 23 , 24 ] .
the present study shows that the prevalence of ckd is 18.60% in our semi - urban population who are unaware of their present kidney health status .
about 400 participants from the total population studied were removed from the analysis since they were already suffering from dm / hypertension / stone disease / other sickness and are under treatment from local medical practitioners . these figures indicate the significance of wkd to focus attention on prevention and cure of kidney diseases globally .
the theme of wkd-2014 was kidney health for all. urban populations have easy access to medical care compared with rural and semi - urban populations of developing countries .
we decided to organize a screening program in a semi - urban population where we could easily set up the required basic facilities for a screening camp . in india , as in other developing nations that have adopted the lifestyle of developed nations , the population has started becoming more sedentary and eating a high fat and low fiber diet , giving rise to obesity and increases in bmi , which is associated with hypertension and diabetes [ 9 , 25 ] . in india itself , which has geographically varied areas , the prevalence of hypertension and diabetes are different in different areas , varying from 13 to 58% and 6 to 20% [ 26 , 27 ] . in the seek - india study , hypertension was noted in 64.5% and dm was noted in 31.6% of the ckd group . in that study ,
self - reported kidney stone disease was observed in 4.5% . in our study , the mean egfr of our population was 87.84 21
ml / min/1.73 m. finding hypertension in 26.85% and diabetes in 9.79% of a population that is otherwise
healthy according to their own belief suggests that this population is living on the brink of ckd , warranting immediate measures to improve their health status .
we compared two regions of this state that occupies the western part of india : north gujarat , which is inland , and saurashtra , which is on the coast .
interestingly , although the populations in both areas belong to the middle class income group as per the prevailing social norms , there was substantial difference in the patterns of affection .
north gujarat emerges as the stone belt , with most of the population harboring multiple calculi .
this population also had associated proteinuria and elevated scr , whereas hypertension and diabetes , which are believed to be afflictions of affluence , were observed in saurashtra .
the answer to this mammoth problem is timely intervention . in 2003 , mani reported that diabetes accounts for ~30% of chronic renal failure and hypertension for 10% of the patient load on waiting lists for kidney transplantation . in a house - to - house survey of 25 000 in south india
he used only reserpine , hydralazine and hydrochlorothiazide for hypertension , and glibenclamide and metformin for diabetes , the cheapest agents available , and was able to control blood pressure to 140/90 mmhg in 96% of cases and reduce glycosylated hemoglobin ( hba1c ) by 10% of the original reading in 77% and hba1c of 7% in 50% of the diabetic subjects .
this study suggests that early screening and intervention can control the progress of ckd in our semi - urban population .
this was a single - point cross - sectional study carried out to screen a population who was willing to participate .
the persons suffering from diabetes , hypertension or stone burden were excluded from the study . in
they shy away even for a routine health check - up , unless forced to by the men of the house or that area , which rarely happens .
thus more males participated in this study and the actual prevalence of ckd may be higher .
the equation for ckd employed here followed the kdoqi guidelines , which may not be ideal for our population .
this study does not elaborate on the observation of hemoglobin < 12 g / dl in ~28% of the population , which also could be a contributory factor of ill health .
we did not adhere to the criterion of persistence for 3 months to label this population as ckd .
hence , larger studies involving more participants from other parts of the state as well as the country need to be carried out . in conclusion , in a semi - urban population of india that does not have the facilities for screening and early intervention for kidney diseases , the prevalence of ckd was higher than that reported in developed nations .
this study may represent the tip of the iceberg since participants considered themselves healthy and individuals with risk factors for ckd were excluded .
a relatively high prevalence of undiagnosed hypertension , diabetes and stone burden indicates that a larger population is likely to develop ckd if timely intervention is not provided .
in addition , there are geographical differences , as stone burden associated with increased scr and proteinuria was more prevalent in the non - coastal region , whereas diabetes and hypertension were more prevalent in the coastal region .
there is an urgent need to draft preventive health policies and plan for the allocation of more resources to improve kidney health in india .
this study also suggests that the populations of other developing nations , especially in asia , are also likely to be progressing toward ckd due to hypertension , dm and obesity .
| backgroundglobally there is an increase in incidence of chronic kidney diseases ( ckds ) .
diabetes mellitus ( dm ) , hypertension and stone diseases are the major risk factors for ckd .
we organized kidney disease screening camps in a semi - urban population of gujarat , india on the occasion of world kidney day ( wkd).methodsvoluntary participants from six towns were screened .
estimated glomerular filtration rate ( egfr ) was calculated by the modification of diet in renal disease formula and ckd was defined as an egfr < 60 ml / min/1.73 m2 or albuminuria 1 + .
urogenital ultrasonography was performed with emphasis on stone burden .
participants with known diabetes , stone diseases , hypertension , kidney / liver / cardiac disease , hepatitis , hiv , transplant recipients , pregnant women and those < 18 years were excluded from the study.resultsof the 2350 participants ( 1438 men ) , ckd was found in 20.93% and egfr < 60 ml / min/1.73 m2 was noted in 8.29% of participants .
the prevalence of ckd peaked after the seventh decade of life in both genders .
there was no significant difference in the prevalence of ckd between coastal and non - coastal regions , however , obesity , hypertension and diabetes were more common in the coastal belt , whereas stone burden was greater in the non - coastal region.conclusionsthe prevalence of ckd in a semi - urban apparently healthy indian population was higher than the reported prevalence in developed countries .
significant differences between regions point to the need to evaluate and correctregion - specific risk factors . |
mycobacterium malmoense is an acid - fast environmental non - tuberculous mycobacterium ( ntm ) that rarely causes disease in humans but is becoming increasingly clinically significant , especially within europe .
it was first described in 1977 by schroder and juhlin , following isolation in respiratory tract culture samples .
however , extrapulmonary infection within lymph nodes , joints , and tendon sheaths has been reported.[12410 ] ntm may develop within the joint as a result of injection or aspiration .
the latter can be as a result of long - term corticosteroid therapy.[45710 ] diagnosis has improved over the years and this may account for an increase in incidence of this pathogen .
ziehl - neelsen ( zn ) staining has been used to detect ntm . however , a negative zn stain does not argue against a ntm infection and extended microbiological cultures are essential for diagnosis .
treatment of extrapulmonary m. malmoense varies , with some authors using stand - alone antibiotic therapy whereas others using surgical intervention , such as debridement , as well as antibiotic therapy .
we report the first case of m. malmoense septic arthritis of the shoulder and conclude that m. malmoense infection should be considered in the differential diagnosis of patients rendered immunocompromised , especially following shoulder procedures .
in may 2009 , a 60 year - old female on regular prednisolone and methotrexate for focal myositis and vasculitis presented to her rheumatologist with a 1-year history of right shoulder pain and swelling over the right acromioclavicular joint ( acj ) .
four weeks later , the swelling increased in size and she was again aspirated in clinic . following this , it began discharging and swabs were taken .
her symptoms resolved . in june 2010 , she was admitted after developing a discharging abscess anterior to the right acj .
investigations revealed that both her c - reactive protein ( crp ) and estimated sedimentation rate ( esr ) were raised at 82.7 mg / l and 89 mm / h , respectively .
a microbiology opinion was sought and she was commenced on oral rifampicin 300 ( mg ) twice daily ( bd ) and intravenous flucloxacillin 1 g four times a day ( qds ) .
she failed to improve and was commenced on oral sodium fusidate 500 mg three times a day ( tds ) and oral ciprofloxacin 500 mg bd . following a rheumatologic consultation ,
a magnetic resonance imaging ( mri ) scan of the shoulder was performed [ figure 2 ] . following referral to a shoulder surgeon
, she underwent an arthroscopic washout of the shoulder joint , open lateral clavicle excision , and closure of wound . in view of no frank pus
initial microbiology and histopathology results were negative for gram stain , zn stain , wade - fite stain , and periodic acid - schiff ( pas ) stain .
following surgery , there was a clinical and biochemical improvement . in view of the negative results from microbiology and histology ,
eight weeks later , m. malmoense was isolated which was sensitive to ethambutol , rifampicin , and clarithromycin .
ethambutol 1 g once a day ( od ) and oral clarithromycin 500 mg bd were commenced .
twelve months following surgical treatment , the patient had clinically , functionally , and biochemically improved . in july 2011 ,
ap radiograph of the shoulder showing a complete loss in the articular surface of the lateral end of the clavicle and resorption of the bone , with prominent swelling above the acj mri scan showing a large glenohumeral joint effusion with communication with the acj via a large rotator cuff defect
m. malmoense is a relatively uncommon slow - growing ( ntm ) , first described in 1977 by schroder and juhlin .
it is an opportunistic pathogen with an increasingly recognized clinical importance worldwide , especially in europe , and is second only to the mycobacterium avium complex as a cause of atypical mycobacterial infection .
in addition to this , extrapulmonary cases have been reported in both immunocompromised and immunocompetent patients.[12410 ] however , no case of isolated shoulder joint involvement with m. malmoense has been reported .
granulomatous infection caused by ntm may develop in tendon sheaths and joints after direct inoculation of the organisms through puncture wounds or injections including intraarticular or bursal steroids .
disseminated ntm disease is very rare with any form of immunosuppression other than advanced hiv disease .
however , dissemination of ntm in adult patients without aids has been reported in immunosuppressed patients with renal or cardiac transplantation , chronic corticosteroid use , and leukaemia .
early diagnosis of ntm can be difficult and may require recurrent aspiration and referral to a highly specialized laboratory for diagnosis .
m. malmoense is an acid - fast organism and zn staining is positive in the majority of cases .
issues around specific diagnosis can lead to a delay in correct treatment regimes and can lead to pain and disability .
furthermore , with the advent of molecular tools , especially 16s rdna gene sequence analysis , the identification process has sped up and improved . however , it is still recommended that mycobacterial culture for extended periods is the gold standard for diagnosis .
first - line treatment for the disease is 18 months treatment with rifampicin and ethambutol .
however , this is in relation to pulmonary m. malmoense infection and not joint involvement .
treatment of extrapulmonary m. malmoense infection has been described.[410 ] different treatment regimes have been reported including ; oral antibiotic treatment alone and surgical intervention ( debridement and washout ) coupled with antibiotic therapy .
however , despite prolonged treatment with antibiotics within the immunocompromised , symptoms may persist and may require surgical exploration and debridement .
either way , there is no randomized control trial found in the current literature , recommending a particular management option for the treatment of m. malmoense infection of the joint .
it is also important to note that both ethambutol and rifampicin have significant side effect profiles , including ocular neuritis and gastrointestinal disturbance , respectively .
these were both seen in our case , which may lead to suboptimal antimicrobial therapy . in conclusion ,
our follow - up continues due to incomplete resolution of symptoms and side effects , which is a limitation of the report .
however , we believe our case highlights several important issues . to our knowledge , this is the first reported case of m. malmoense infection within the shoulder , and we believe that infection with m. malmoense should be considered as a differential diagnosis in patients rendered immunocompromised , especially following previous injections to , or aspiration of the shoulder . moreover , we recommend that aspiration of the joint in the immunocompromised be performed in a clean environment other than clinic . additionally , in view of the difficulties encountered with diagnosis , an early referral to a specialist competent to perform surgical debridement , biopsies , and washout should be considered to ensure adequate tissue samples are obtained .
finally , this case highlights the side effect profile of long - term antibiotic therapy leading to significant morbidity .
we suggest regular multidisciplinary follow - up to ensure this is recognized and managed in what is an increasingly common entity . | mycobacterium malmoense is an acid - fast non - tuberculous organism that most commonly causes pulmonary infection .
extrapulmonary infection has also been reported . with an increased emphasis being placed on the clinical importance of this organism , especially within europe , we report the first case of septic arthritis of the shoulder caused by this organism .
we also highlight the importance of considering atypical mycobacterium infection in the differential diagnosis of shoulder infection and issues surrounding the management of this entity . |
creating peptide - based drugs includes the search of active sites or functionally important sites in the target protein and the selection of peptides capable of specific interaction with these sites .
such a selection usually can be accomplished using peptide libraries containing random fragments of amino acid sequences ( irrational design ) or fragments of natural proteins capable of reacting with target structural determinants .
developing approaches to rational design of potential peptide inhibitors of structural functional determinants of proteins can potentially expand the range of therapeutic medications based on peptides .
the key role in the process of development of an infection caused by the influenza virus is played by rna polymerase .
rna polymerase of the influenza virus consists of three subunits pa , pb1 , and pb2 .
assembling of pa and pb1 subunits of a polymerase complex occurs in cytoplasm of infected cells .
heterodimer is transported into the nucleus , where , upon accession of the third subunit
according to the data of several authors , the critical region of the interaction of proteins pa and pb1 is the n - terminal region of protein pb1 [ 2 , 3 ] .
a number of studies [ 4 , 5 ] showed that the structural determinant of the pa - pb1 interaction during formation of a polymerase complex of the influenza virus is fragment 1 - 15 of pb1 protein .
the x - ray structural analysis of the crystals of the complex of the fragment of pa protein and the peptides corresponding to n - terminus of pb1 was carried out , and it showed that this fragment forms a system of hydrogen bonds with the c - terminal domain of pa .
it was also shown that the presence of the polypeptide , containing the fragment of the amino acid sequence corresponding to amino acid residues 1 - 25 and 1 - 15 of pb1 , in a cell , prevents the infection of cells with the influenza virus .
for example , in , genetic construct that expresses protein which contains the given fragment was used to suppress the replication of the influenza virus ; in , influenza virus replication was blocked by the peptide functionalized with the fragment of human immunodeficiency virus tat protein and such a way transported from the medium to the cell .
it was assumed that peptide pb1 ( 1 - 25 ) inside the cell forms a structure close to the structure of the n - terminal region of pb1 in the structure of pa - pb1 complex , interacts with protein pa , and thus prevents the formation of a polymerase complex required for viral replication .
this studies show that the structure of the n - terminal region of protein pb1 is critical for the polymerase complex assembly and replication of the influenza virus . in this work
, we investigate the structural features of n - terminal peptide of protein pb1 to design a potential peptide inhibitor of assembly of a polymerase complex of the influenza virus .
in contrast to studies [ 4 , 7 ] , where the peptide inhibitor interacted with pa and competed for its site of interaction with pb1 , we used the mirror - symmetry motif in the sequence of pb1 to design a peptide that would inhibit pa - pb1 assembly by interaction with the n - terminal part of pb1 .
peptides ' pb1 ( 6 - 13 ) ( tllflkvp ) and pb1 ( 6 - 25 ) ( tllflkvpaqnaisttfpyt ) solid phase peptide synthesis was carried out by 2-chlorotrityl chloride resin according to fmoc-/tbu strategy . for fragments 6 - 25
protein , pb1 target compound was synthesized from fragments divided by the proline residue at position 8 .
the completeness of the reactions was evaluated using the kaiser test for free amino groups .
the purification of the compounds obtained was carried out via preparative reversed - phase hplc .
their purity turned to be not less than 95% ; their structure was confirmed by mass - spectrometry hrms(esi ) .
the study of mobility of the peptides was carried out by the method of molecular dynamics ( md ) using the software package gromacs 4.5.5 ( http://www.gromacs.org/ ) .
md calculation of all the systems was performed in cubic cells with applied periodic boundary conditions .
the temperature of the systems was set to 310 k. the integration step was 2 fs .
the minimum distance from the molecule to the cell wall was 1 nm , which made it possible to avoid the interaction of the peptides in neighboring cells .
cutoff for atom - atom electrostatic and van der waals interactions was 1 nm . long - range electrostatic interactions were calculated by algorithm pme .
circular dichroism ( 194320 nm ) was measured on pb1 ( 6 - 25 ) 1 mg / ml solution .
protein docking was performed using the hex server software ( http://hex.loria.fr/ ) with default parameters ( van der waals plus electrostatic mode ) , the best 10 docking conformations were analyzed .
preparation of samples for electron microscopy was performed according to the standard procedure of negative staining .
a drop ( 20 mkl ) of the test sample diluted to the concentration of 0.2 mm was applied on parafilm .
a 200-celled copper grid with carbon coating was placed on the drop for 15 s. the base plate was washed with distilled water two times for 15 seconds . the sample was contrasted with 1.5% aqueous solution of sodium salt of phosphotungstic acid ( ph 7.4 ) for 15 seconds , then the sample was dried at room temperature .
the study was carried out by electron microscope jeol jem 1100 at the accelerating voltage of 80 kv .
spectra were obtained on laser correlation spectrometer lks-03 ( intox , russia ) ; processing of the spectra was performed using the instrument software .
the analysis of the primary structure of the n - terminal domain of pb1 showed that the site of pb1 ( 6 - 25 ) contains a so - called mirror - symmetry motif ( msm ) ( figure 1 ) .
the frequency of msm occurrence near functional sites seems to exceed the average for the amino acid sequences of natural proteins , and it has been suggested that msm may play a role in the formation of functional sites of proteins , as well as in protein oligomerization [ 8 , 9 ] . the available x - ray crystallographic structure of pa complex with pb1 n - terminal fragment ( amino acids 115 ) shows that , when bound to pa1 , the secondary structure of 115 n - terminal fragment of pb1 is an alpha - helix .
however , at this point , there is no three - dimensional structure of pb1 protein apart from its complex with pa . to investigate the possible structure of the n - terminal domain of the free pb1 , we performed computer modeling of the structure and dynamics of pb1 n - terminal fragment .
an msm - containing peptide matching the residues 6 - 25 of pb1 was chosen for modeling . as a result of the modeling of 3-dimensional structure of the peptide that included 100 ns molecular dynamics simulation in the presence of solvent and by analyzing the trajectories ,
it was found that the peptide was prone to formation of the a beta - hairpin type structure ( figure 2 ) .
the peptide acquired this conformation in the early stages of the simulation , while further simulation resulted only in minor alterations that did not affect the secondary structure of the peptide .
backbone h - bonds l9-y24 and l11-f22 ( pb1 numbering ) were crucial for stabilizing the beta - hairpin conformation .
the study of the secondary structure using circular dichroism ( cd ) ( figure 3 ) revealed that the peptide pb1 ( 6 - 25 ) in solution has a structure similar to the described model of beta - hairpin obtained using molecular dynamics methods ( 8% of alpha - helix , 30% of antiparallel beta - sheet , 25% of beta - turns , and 37% of random coil structure ) .
the beta - type structures appear to , therefore , prevail in the pb1 ( 6 - 25 ) peptide and may represent the native conformation of pb1 n - terminus , while the formation of the alpha - helix observed by x - ray crystallography could likely be the result of pb1 interaction with the polymerase subunit .
the area crucially important for pb1 interaction with pa ( flkv , 47 residues ) , according to the results of the modeling , was located in a beta - sheet , in contrast to the data presented in , where these residues were located in an alpha - helix .
one could suppose that stabilization of the beta - structured state of pb1 n - terminus would result in inhibition of the interaction of this protein with pa , polymerase complex assembly , and reproduction of viral rna . a peptide which would stabilize the beta - hairpin structure of pb1 ( 6 - 25 ) peptide is , therefore , a good candidate for pb1-pa interaction inhibitor .
considering the msm in pb1 ( 6 - 25 ) , we investigated its interactions with pb1 ( 6 - 13 ) peptide that was a half the original peptide . when mixing the solutions of peptides pb1 ( 6 - 13 ) and pb1 ( 6 - 25 ) at high concentrations ( 0.5 mm and higher ) and incubating the solution at room temperature for an hour , turbidity was observed in the sample containing the mixture .
the study of the diluted solution by laser correlation spectroscopy showed that aggregates with the diameter of 5001000 nm ( figure 4 ) are formed .
the study of peptides pb1 ( 6 - 25 ) and pb1 ( 6 - 13 ) and their mixture by electron microscopy confirmed that structured aggregates with the characteristic linear size of about 500 nm were formed in the mixture ( figure 5 ) , but not in the samples containing either one of the peptides .
thus , the ability of peptides pb1 ( 6 - 13 ) and pb1 ( 6 - 25 ) to interact was demonstrated in vitro . it should be noticed that the unsoluble aggregates formed could not reverse into soluble forms after dilution or heating .
the modeling of the 3-dimensional structure of pb1 ( 6 - 13 ) peptide using the methods of molecular dynamics showed that the peptide has linear structure ; that is , it is not flexible and does not form intramolecular bonds .
using protein docking , we make a model of pb1 ( 6 - 25)pb1 ( 6 - 13 ) interaction ( figure 6 ) .
analysis of the model contact map showed that pb1 ( 6 - 13 ) binds to 1425 residues ' fragment of pb1 ( 6 - 25 ) and forms 2 backbone h - bonds , indicating the possible stabilization of pb1 ( 6 - 25 ) beta - hairpin structure by this peptide .
cd spectroscopy study of pb1 ( 6 - 13 ) peptide influence on the secondary structure of pb1 ( 6 - 25 ) showed that , in equimolar ratio on concentrations were near critical ( 0.5 mm ) pb1 ( 6 - 13 ) contributed to increase antiparallel beta - sheets in latter peptide .
we use cd spectroscopy at concentrations which were significantly lower than critical to estimate the dissociation constant kd for pb1 ( 6 - 25)pb1 ( 6 - 13 ) interactions .
the concentrations in the mixtures were calculated using bimolecular reversible binding model with one parameter ( kd ) .
the cd spectra of the mixtures were then modelled using the calculated concentrations by multidimensional linear square fit .
the spectra of pure peptides were also added to data matrix with respective concentration fixed to known values .
total rmse of the model was then calculated , and the dependency of rmse on kd was plotted .
the spectral data were found to be close to degenerate ( small difference of the spectrum of the complex from the linear combination of the components ' spectra ) but the model gives a lower bound for kd , which is located approximately in the range of 0.21 mm .
the known affinity of pa to pb1 ( 1 - 15 ) ( kd~1.6 m ) is much higher than these values . apart from competitive inhibition of pa binding , one can consider other possible mechanisms of pb1 ( 6 - 13 ) action on pb1 protein such as catalysing the change in the secondary structure of pb1 ( 6 - 25 ) from alpha - helix to beta - sheet , or , at higher concentrations , stimulation of unsoluble aggregates ' formation . stabilizing of pb1 n - terminus beta - sheet secondary structure could make it sterically incompartible to pa binding cave and inhibit pba - pb1 complex de novo formation .
we have shown that the mirror - symmetry motif found in the n - terminal region of pb1 protein , which is known to be a part of an alpha - helix in pa - pb1 complex , can likely exist in the beta - conformation in the free pb1 subunit .
peptide pb1 ( 6 - 13 ) , containing half of the mirror - symmetry motif , can interact with pb1 ( 6 - 25 ) , possibly stabilizing its beta - conformation , that at high concentration , leads to formation of insoluble aggregates in vitro . such an ability could make pb1 ( 6 - 13 ) a basis for a potential antiviral agent that would inhibit pb1-pa complex formation by affecting pb1 n - terminus structure . | a mirror - symmetry motif was discovered in the n - terminus of the influenza virus pb1 protein .
structure of peptide comprised of the corresponding part of pb1 ( amino acid residues 6 - 25 ) was investigated by circular dichroism and in silico modeling .
we found that peptide pb1 ( 6 - 25 ) in solution assumes beta - hairpin conformation .
a truncated peptide pb1 ( 6 - 13 ) , containing only half of the mirror - symmetry motif , appeared to stabilize the beta - structure of the original peptide and , at high concentrations , was capable of reacting with peptide to form insoluble aggregates in vitro .
ability of pb1 ( 6 - 13 ) peptide to interact with the n - terminal domain of pb1 protein makes it a potential antiviral agent that inhibits pa - pb1 complex formation by affecting pb1 n - terminus structure . |
cerebral palsy is a condition in which permanent problems affect movements and postural
development due to non progressive lesions in the immature brain .
it is accompanied by
sensory disabilities , cognitive disorders , communication disorders , hypermetamorphosis ,
behavioral disorders , or convulsion1 .
among the types of cerebral palsy
, spastic cerebral palsy is the most common2 and shows motor disorders , such as
spasticity , deep tendon reflex accentuation , and muscle weakening3 . as a result , cerebral palsy patients show kinetic and
kinematic changes as well as diverse forms of gait abnormalities4 .
one major objective in the rehabilitation of cerebral palsy children is gait ability
improvement . to do this ,
therapists apply diverse methods , such as walking aids , drug
treatment , and neuro - developmental treatment5 .
gait analysis is recognized as an important tool in the clinical
assessment and the measurement of treatment outcomes for patients with neuromuscular
disabilities and , more specifically , in cerebral palsy patients undergoing surgery6 .
the purpose of this study was to analyze and compare the gait of spastic diplegia children
with that of normal children by using three - dimensional motion analysis , this will provide
the basic data necessary for therapeutic interventions , such as surgery , neurophysiological
treatment , and assistive device prescription .
children with spastic diplegia type cerebral palsy children aged 612 years were selected
as the experimental group ( cp ) .
criteria for inclusion were a low degree of disorders based
on their medical history of treatment by physical therapists and specialists in the
department of rehabilitation , ability to understand the researcher s instructions , and
ability to walk independently without assistance .
the control group consisted of eight
normal children aged 811 years without any past or current medical history of abnormal gait
( normal ) .
the general characteristics of the subjects were : age , cp 8.250.75 years , normal
9.130.44 years ; height , cp 125.607.05 cm , normal 137.092.78 cm ; weight , cp 30.503.35 kg ,
normal 34.562.79 kg . all participants understood the purpose of this study and provided
their written informed consent prior to participation in accordance with the ethical
principles of the declaration of helsinki . a computer loaded with orthotrak 6.4 ( motion analysis , santa rosa , ca
, usa ) and evart 6.1.1
( motion analysis , santa rosa , ca , usa ) and eight infrared cameras connected to the computer
were used to record the subjects walking motions for three - dimensional gait analysis .
the
data were processed using the orthotrak 6.4 and evart 6.1.1 software . a tester attached 25-mm diameter , round , reflective markers to the subjects lower
extremity segmental points including their pelvis .
these markers were attached to the
subjects , who wore only briefs , after having removed their outerwear and 19 reflective
markers were placed on each subject based on the helen - hays marker set .
the locations of the
markers included the sacrum , asis ( anterior superior iliac spine ) , mid thigh , lateral
femoral epicondyle , mid shank , lateral malleolus , second metatarsal head , and posterior
calcaneus .
the subjects were instructed to look at a mark on the wall while walking so that their line
of sight would not be change , and their walking was measured repeatedly until appropriate
data were obtained .
changes in the maximum angles and minimum angles of individual joints during the stance and
swing phases in the three movement planes sagittal , frontal , and transverse were analyzed
and compared .
the data were statistically processed using the independent sample t - tests for
comparisons between the two groups .
the statistical level was set to p<0.05 , and spss
17.0 version was used for the analysis , and significance was accepted for values of
p<0.05 .
in the sagittal plane , the maximum angles of both the right and left sides of the pelvis
and hip joints in the stance phases and swing phases were found to be significantly greater
among the spastic diplegia children than among the normal children ( p<0.05 ) .
there were
no significant differences in the minimum angles of the right and left sides of the pelvis
and hip joints in the swing phases and stance phases or in the maximum and minimum angles of
the knee and ankle joints in the stance phases or swing phases ( table 1table 1.maximum and minimum angles of the pelvis , hip , knee , and ankle joint motions in
the stance phase and swing phase in the sagittal plane ( angle / unit)jointgroupleftrightmean sepmean seppelvismax stancenormal13.07 1.16 * 13.10 1.21*cp21.59 3.3421.57 3.40min stancenormal9.71 1.4010.47 1.48cp9.46 2.599.22 2.87max swingnormal13.12 1.22 * 13.43
1.03*cp20.28 3.0121.35 3.46min swingnormal10.56 1.1810.67 1.28cp11.06 2.2111.03 2.84hipmax stancenormal30.73 2.98 * 28.93 2.68*cp48.41 7.2642.88 5.62min stancenormal9.61 2.9310.32 3.49cp4.39 10.5417.76 10.92max swingnormal36.89 2.3334.25 2.94*cp52.96 7.1948.73 5.53min swingnormal4.97 2.736.55 3.74cp4.30 10.897.12 10.90kneemax stancenormal35.82 5.3120.86 6.33cp37.83 8.9422.70 5.39min stancenormal1.25 2.535.48 6.30cp13.47 9.0716.14 9.32max swingnormal73.92 8.7256.05 12.97cp62.55 11.0633.99 10.12min swingnormal4.54 3.219.73 24.94cp2.09 6.3828.68 17.94anklemax stancenormal15.05 4.277.69 2.28cp12.29 5.608.64 1.10min stancenormal10.89 2.2712.66 2.35cp15.90 3.4717.71 3.78max swingnormal14.37 8.873.85 2.16cp13.58 8.870.36 1.50min swingnormal16.33 2.6022.54 4.32cp15.48 3.9024.10 5.21*mean
p<0.05 in the frontal plane , there were no significant differences in the maximum and minimum
angles of the left pelvis and the joints of the lower extremity in the swing and stance
phases between the two groups .
there were no significant differences in the maximum and
minimum angles of the right pelvis and the ankle joint in the swing and stance phases , in
the minimum angles of the hip joint in the swing and stance phases , in the knee joint s
maximum angles in the stance phases , or in the maximum and minimum angles in swing phases .
however , significant differences were shown in the maximum angles of the right side of the
hip joint in the stance and swing phases and in the knee joint s minimum angles in stance
phases ( p<0.05 ) ( table 2table 2.maximum and minimum angles of the pelvis , hip , knee , and ankle joint motions in
the stance phase and swing phase in the frontal plane ( angle / unit)jointgroupleftrightmean sepmean seppelvismax stancenormal3.22 0.474.04 0.31cp10.47 10.2015.78 6.74min stancenormal5.40 0.444.88 0.29cp9.25 10.6720.20 7.29max swingnormal4.65 0.355.12 0.32cp10.76 7.927.80 9.30min swingnormal1.23 0.421.05 0.39cp16.18 9.036.86 9.43hipmax stancenormal11.86 3.568.15 1.62*cp6.84 2.900.72 3.09min stancenormal1.41 3.4014.74 1.69cp6.35 2.1214.26 3.66max swingnormal5.81 3.026.34 1.80*cp1.21 2.985.59 4.72min swingnormal3.98 3.3119.24 2.37cp9.60 1.9111.32 7.63kneemax stancenormal4.75 3.0416.49 8.67cp3.81 4.5510.55 5.08min stancenormal6.54 2.241.16 1.38*cp16.93 6.7621.27 8.11max swingnormal10.62 6.0226.23 11.99cp13.02 6.5922.89 10.34min swingnormal10.36 2.023.72 2.60cp9.96 4.277.05 4.47anklemax stancenormal1.63 1.090.53 1.89cp18.19 15.413.63 1.74min stancenormal6.46 2.9610.08 5.03cp9.35 4.086.98 3.28max swingnormal1.49 1.290.56 1.05cp13.48 8.260.23 2.41min swingnormal10.04 3.0315.04 6.94cp9.07 2.2910.24
* p<0.05 in the transverse plane , there were significant differences in the maximum angles of the
left side of the pelvis in the swing and stance phases ( p<0.05 ) .
there were also
significant differences in the maximum and minimum angles of the right pelvis in the stance
phases , and in the minimum angles of the right pelvis in the swing phases ( p<0.05 ) .
there were no significant differences in the maximum and minimum angles of the left / right
hip , knee , or ankle joints in swing and stance phases ( table 3table 3.maximum and minimum angles of the pelvis , hip , knee , and ankle joint motions in
the stance phase and swing phase in the tranverse plane ( angle / unit)jointgroupleftrightmean sepmean seppelvismax stancenormal6.95 0.80 * 7.35 0.61*cp16.52 1.6617.41 4.15min stancenormal8.85
0.954.73 2.01*cp13.34 3.2915.91 1.97max swingnormal6.10 1.00 * 8.29 1.06cp16.38 2.7510.20 3.83min swingnormal7.67 1.024.58 0.74*cp8.72 3.1013.11 2.01hipmax stancenormal6.15 5.4223.62 13.81cp22.30 7.2025.14 16.91min stancenormal5.12 5.7513.27 13.53cp6.29 6.4712.62 17.28max swingnormal5.50 4.5725.40 13.45cp20.42 7.4625.00 17.47min swingnormal6.40 4.7012.73 13.20cp5.01 7.9212.73 17.42kneemax stancenormal0.59 1.790.42 5.93cp3.08 7.8914.46 8.91min stancenormal13.98 2.8715.39 5.11cp23.93 4.6713.28 8.63max swingnormal1.87 3.0311.83 22.40cp1.02 6.2311.97 10.44min swingnormal15.51 2.7742.71 10.11cp21.22 3.8123.01 6.53anklemax stancenormal4.88 5.5812.86 19.49cp23.89 15.8524.64 19.19min stancenormal10.21 7.7228.69 19.89cp10.66 8.0646.17 22.15max swingnormal7.87 4.995.68 17.93cp21.93 9.5630.26
21.58min swingnormal9.60 7.8130.93 23.01cp7.66 7.3945.76 26.10*mean se + , internal rotation ; mean se , external rotation .
mean se + , internal rotation ; mean se , external rotation . *
although the largest movements appear in the sagittal plane during gait analysis , movements
in the frontal plane and in the transverse plane are particularly important in studies of
morbid gaits7 .
therefore , we focused on
comparing differences in gait between normal children and those with spastic diplegia - type
cerebral palsy , and compared maximum angles and minimum angles in the stance phases and
swing phases using kinematic analysis . in kinematic aspects of normal gait , the pelvis moves asynchronously in three movement
planes .
perry ( 1994 ) reported that the pelvis tilts 7 forward in the sagittal plane , moves
4 downward and upward in the frontal plane , and rotates 10 in the transverse plane . in
this study ,
pelvis movements in the sagittal plane showed larger forward tilting in the
spastic diplegia children than in the normal children in both the swing phases and stance
phases .
hemiplegic cerebral palsy children s excessive pelvic tilting has been reported to
be due to the spasticity of the hip joint flexor or the weakening of the hip joint extensor
and the abdominal muscles8 .
pelvis movements in the frontal plane were also found to be greater in the spastic diplegia
children than in the normal children in both the swing phases and stance phases .
this is
consistent with reports results that pelvis angles are much greater in cerebral palsy
children than in normal children in the sagittal , frontal , and transverse planes9 .
these previous studies have reported that
pelvic movements in the transverse plane showed significant differences because cerebral
palsy children perform excessive external rotation of the pelvis compared to normal children
in order to compensate for their excessive internal rotation of the hip joint10 .
perry j reported that the maximum angle of the hip joint in normal gaits was 40 and that
spastic diplegia children showed maximum hip joint angles greater than 40 in the sagittal
plane in the stance phases and swing phases11 .
a study of hemiplegic cerebral palsy children s gaits reported that the spasticity of the
hip joint flexor causes increased forward tilting of the pelvis , which is a cause of
increasing in lumbar lordosis12 . in our
study , too , significant differences were found in the frontal plane and the transverse
plane .
although we consider these differences are thought to be attributable to the
imbalance of asymmetric muscles of the paretic lower extremity and restriction of joint
movement , the precise cause could not be determined .
sutherland reported that spastic cerebral palsy children and patients with severe twitching
of the hamstring muscle tendon showed increases in knee joint flexion during the stance
phases13 .
our results are similar ,
results as the cerebral palsy children group showed increases in the maximum angles of knee
joint flexion compared to the normal group during the stance phases , although the
differences were not significant . based on the results of this study ,
there are differences in the kinematic variables of
children with spastic diplegia and normal children .
we can conclude that spastic diplegia
children have a different gait strategy and pattern from normal children .
primary
abnormalities of children with cp , which arise directly from the damage to the central
nervous system ( e.g. , abnormal muscle tone , abnormal reflex activity , loss of selective
muscle control , spasticity ) , and from secondary effects ( contracture and deformations ) ,
resulting from abnormal bone and muscle growth . due to asymmetric physical movement and
abnormal phasic muscle activity of the paretic lower extremity and the reduction in the
range of motion of both lower extremity joints , postural control and physical balance
ability are impaired in spastic diplegia children .
these children also experience excessive
movements and restrictions during the stance phases and swing phases as compensational
physical reactions compared to normal children , resulting in the different gait strategy and
pattern
. a potential weakness of this study was the small number of participants which
resulted in limits the power of the statistical findings . | [ purpose ] the purpose of this study was to compare joint angles between normal children
and those with spastic diplegia using three - dimensional gait analysis .
[ subjects and
methods ] the study subjects were eight patients with spastic diplegia and eight normal
children .
three - dimensional gait analysis was used for the survey .
the measured gait
variables were the joints of the lower extremity in the sagittal plane , frontal plane , and
transverse planes and the maximum and minimum angles of their stance phase and swing
phases .
[ results ] in the sagittal plane , the maximum angles of both the right and left
pelvis and hip joint in the stance phase and swing phases were significantly greater for
children with spastic diplegia than for normal children . in the stance phase of the right
side of the hip joint ,
the maximum angles of the hip in the swing phase and the knee
joint s minimum angles in the stance phase differed significantly . in the transverse
plane
, there were a significant differences on the left side of the pelvis in the maximum
angles in the swing and stance phases .
there were also significant differences on the
right side pelvis , in the maximum and minimum angles in the stance phase and minimum
angles in the swing phase .
[ conclusion ] children with spastic diplegia employ a different
gait strategy and pattern from normal children . |
the insulin signal transduction pathway in pancreatic -cells is similar to that in most other cell types ( fig .
1 ) . we do not dispute evidence that insulin receptors are expressed in -cells .
moreover , it is generally accepted that some elements in the insulin signal transduction pathways play a critical role for -cell survival , growth , and general well - being ( 14 ) .
perhaps the best example comes from the global insulin receptor substrate ( irs)-1 and irs-2 knockout mouse models , in which irs-2 was shown to play an especially vital role in the ability of -cells to compensate for insulin resistance ( 57 ) .
these landmark studies were a catalyst to change previous thinking in the diabetes research field . before
, the predominant thought was that insulin resistance was the main cause of type 2 diabetes , but it was not widely acknowledged until the 1990s that the onset of type 2 diabetes is marked by a failure of the functional -cell mass to meet the metabolic demand ( 810 ) .
back then , with the realization that irs-2 signaling in -cells could be important , a plethora of studies blossomed to indicate certain downstream elements in irs-2 signaling pathways also play important roles in -cell function and survival ( fig .
its expression is increased by glucose , incretins such as glp-1 , and other factors that increase cytosolic [ ca]i and [ camp]i in -cells ( 1113 ) . by contrast
, it can be downregulated by proinflammatory cytokines , physiological stress , and feedback inhibition of normal irs signaling ( 4,1416 ) .
it is conceivable that the relatively high expression of irs-2 and its quick turnover in -cells ( 13 ) may offset any need for constitutive activity of the insulin receptor , as it does in the liver ( 17 ) . with a controlled upregulation of irs-2
when -cell compensation is needed to maintain glucose homeostasis and downregulation of irs-2 when -cell compensation is not needed , the responsibility for insulin itself to trigger downstream signaling in -cells could be removed and placed more so on glucose , incretins , neuronal connections , and other more physiologically relevant regulators of -cell function .
activation of the irs signaling cascade pathways . a peptide ligand such as insulin or insulin - like growth factor-1 ( igf-1 ) binds to its receptor , activating the intrinsic tyrosine kinase activity of that receptor that then tyrosine phosphorylates ( py ) adaptor molecules such as irs-1 or -2 .
other receptor tyrosine kinases , or receptors that activate tyrosine kinases such as janus kinase ( jak ) , can also activate irs signaling .
this leads to activation of two major signaling cascades , the ras - raf - mitogen - activated protein kinase ( mapk ) pathway ( orange ) and the phosphatidylinositol-3-kinase ( pi3k)/protein kinase - b ( pkb ; also known as akt ) signaling pathway ( green ) . for the ras - raf - mapk pathway , growth factor receptor bound protein-2 ( grb2)/son of sevenless ( sos ) protein complex binds to specific phosphorylated tyrosines on irs-1/2 , activating the gtp / gdp exchange activity of sos , which loads p21 ( ras ) with gtp to activate ras , leading to phosphorylation of the serine / threonine protein kinase raf-1 , which then phosphorylates the mitogen - activated protein kinase kinase ( mek1 ) , which is then activated to phosphorylate the extracellular signal
phospho - activated erk-1/2 can then directly ( or indirectly via phospho - activation of other kinases such as p90 ribosomal serine kinase [ p90 ] ) serine / threonine phosphorylate certain transcription factors , such as cfos and e - twenty - six like transcription factor 1 ( elk-1 ) , to upregulate gene transcription .
phospho - activated erk-1/2 can also phosphorylate mapk interacting kinase ( mnk ) 1 and 2 , leading to phosphorylation activation of the eukaryotic initiation factor-4e
( eif4e ) in a complex also containing eif4a and eif4 g to increase general protein synthesis at the level initiation phase of translational control . for the pi3k / pkb signaling pathway , the p85 regulatory subunit of pi3k docks to other specific phosphorylated tyrosine sites on irs-1/2 that then activates its p110 catalytic activity .
this catalyzes the phosphorylation of phosphatidylinositol-4 , 5-bisphophaste [ pi(4,5)p2 ] to phosphatidylinositol-3 , 4 , 5-trisphophaste [ pi(3,4,5)p3 ] , which then activates 3-phosphoinositide dependent protein kinase-1 ( pdk1 ) .
pdk1 then threonine ( pt ) phosphorylates pkb for pkb activation , which can be amplified by serine phosphorylation ( ps ) of pkb by the target of rapamycin complex-2 ( torc2 ; which includes the protein kinase , mammalian target of rapamycin [ mtor ] and associated proteins rictor and mlst8 ) .
pkb - mediated phosphorylation of the tuberous sclerosis protein-1/2 complex ( tsc1/2 ) inhibits its gtpase activating protein activity to then load the ras homolog enriched in brain ( rheb ) protein with gtp ( rheb ) , leading to activation of the torc1 ( which includes mtor and associated proteins raptor and mlst8 ) .
this includes the eif4e - binding protein-1 ( 4e - bp1 ) that releases it from eif4e binding , enabling eif4e to associate with eif4a and eif4 g in a complex with mnk , where mnk then phosphorylates eif4e to increase rates of protein synthesis translation .
this also shows how the ras / raf / erk and pi3k / pkb signaling pathways can coordinate to give a tight translational control of protein synthesis .
torc1 can also phosphorylate and subsequently activate p70 s6-ribosomal kinase ( p70 ) , which can lead to an increase in the elongation phase of protein synthesis translation .
torc1 also phosphorylates unc-51like kinases-1/2 ( ulk-1/2 ; also known as autophagy gene-1 ) , which results in inhibition of autophagy . among pkb s
other phosphorylation substrates are proteins involved in the apoptotic process such as bcl - antagonist of cell death ( bad ) and x - linked inhibitor of apoptosis protein ( xiap ) , outlining a mechanism whereby pkb is antiapoptotic .
pkb phosphorylation of the transcription factors foxo1 and foxo3a causes their removal from the nucleus and promotes their degradation , causing an inhibition of foxo1/3a - mediated transcription .
phosphorylation of glycogen synthase kinase-3 ( gsk3 ) by pkb inhibits gsk3 activity , resulting in increased glycogen deposition and cell growth . under certain circumstances
, pkb can also influence increases in cell growth by phosphorylating the cell - cycle inhibitor proteins p21 cyclin - dependent kinase inhibitor-1 ( p21 ) and p27 cyclin - dependent - kinase inhibitor ( p27 ) .
pkb can also phosphorylate - inhibit phosphodiesterase-3b ( pde3b ) to elevate intracellular camp ( [ camp]i ) levels .
many of these irs signaling elements have been shown to be expressed and active and play important roles in pancreatic -cells in terms of certain functions , growth , and survival ( rev . in 24 ) , and these are indicated by a yellow highlighted halo . however , despite this growing body of evidence for the necessity of irs-2regulated signaling pathways in -cells , the identity of any physiologically relevant upstream ligand / receptor interaction that triggers irs signal transduction in -cells in vivo has been rather unclear . of course , the insulin / insulin receptor interaction is an attractive candidate , but there are considerations and circumstances that raise significant doubt of an autocrine effect of insulin on -cells .
either the -cells are bathing in a very high concentration of locally secreted insulin , or secreted insulin is rapidly dispensed into the circulation leaving the locality of an islet but then has to complete an entire lap around the body where it is cleared by other tissues before returning to the -cells at a much lower concentration .
prolonged exposure to insulin , and/or high concentrations of insulin in all cells that express the insulin receptor effectively desensitizes the irs signaling pathway downstream of the receptor as well as downregulates expression of insulin receptor itself ( 18 ) .
internalization of the insulin receptor into an endosomal compartment , once insulin is bound contributes to this desensitization reducing the availability of cell - surface insulin receptors ( 19 ) .
some desensitization mechanisms are normal physiological feedback inhibition aimed at preventing potentially harmful effects of prolonged exposure to insulin such as hypoglycemia and/or oncogenesis ( 18 ) ( fig .
2 ) . once the insulin receptor is downregulated and downstream signaling desensitized , some time without insulin is needed to restore normal insulin sensitivity ( 20,21 ) .
considering that the insulin receptor is ubiquitously expressed , such feedback inhibition mechanisms provide a dynamic means of locally controlling insulin receptor activity rather than a more complex regulation of controlling insulin receptor gene expression in a tissue - specific manner .
indeed , some believe that the chronic hyperinsulinemia found in obesity and obesity - linked type 2 diabetes can make a major contribution to the insulin - resistant state because of these desensitization mechanisms ( 18 ) ( fig .
once insulin has activated irs signal transduction pathways in cells , after a period of time there are internal physiological feedback inhibition signals ( indicated by red arrows ) that ensure that the insulin signal is not chronically sustained . under hyperinsulinemic conditions , this
feedback results in chronic desensitization of irs signal transduction and contributes to the insulin - resistant state .
downstream activation of extracellular signal regulated kinases-1 and -2 ( erk-1/2 ) ( as described in fig .
1 ) can lead to erk-1/2 protein kinase mediated serine phosphorylation ( ps ) of irs-1/2 , which results in dissociation of the insulin receptor and irs-1/2 interaction together with irs-1/2 degradation .
3-phosphoinositide - dependent protein kinase-1 ( pdk1 ) activation can result in downstream activation atypical protein kinase - c ( pkc ) isoforms , such as pkc , which can also serine phosphorylate ( ps ) irs-1/2 to promote their degradation , representing another route of delayed feedback inhibition for insulin signal transduction .
in contrast , protein kinase - b ( pkb ; also known as akt ) can serine phosphorylate irs-1/2 at alternative sites to stabilize irs-1/2 tyrosine phosphorylation state and thus enhance downstream signaling .
however , pkb - mediated phosphorylation activation of some of its other substrates can have a more dominant - negative feedback effect on irs signaling .
both target of rapamycin complex-1 ( torc1 ) and p70 s6-ribosomal kinase ( p70 ) ( the latter amplified by pdk1 phosphorylation ) can serine phosphorylate irs-1/2 to promote their degradation , which then dampens irs signaling .
tyrosine phosphatase-2 ( shp2 ) , which upon binding to certain phosphotyrosine residues on irs-1/2 becomes activated and can then remove phosphate from phosphotyrosines on irs-1/2 , thus dampening downstream signaling .
foxo1 and -3a have been shown to be critical factors for driving irs-2 expression under basal conditions , especially foxo3a in -cells ( 15 ) .
but when irs signaling is triggered by insulin , foxo1/3a transcriptional activity is inactivated , resulting in another route of temporal feedback inhibition by decreasing irs-2 expression .
several of these irs signaling feedback mechanisms have indeed been shown to be present in pancreatic -cells ( 24,15,16 ) .
another consideration for feedback inhibition of insulin action is that when insulin binds to its receptor , the insulin / insulin receptor complex is internalized into the cell where it dissociates in an endosomal compartment , allowing the free insulin receptor to return to the surface ( 18,19 ) .
when insulin levels are high , this cycle is biased toward there being minimal insulin receptors on the surface of the cell with the majority being internalized , and acts as an additional physiological mechanism to prevent prolonged activation of irs signal transduction by insulin . under chronic hyperinsulinemia , insulin receptor internalization makes a contribution to insulin resistance .
this long - term hyperinsulinemia also leads to downregulation of insulin receptor gene expression by a mechanism not yet well defined .
the concentration of insulin secreted from the pancreas in vivo in response to physiological stimuli has been estimated by measuring insulin concentrations in the portal vein under normal circumstances and can reach high peak concentrations of 5 nmol / l ( 22 ) .
as such , it is not too far - fetched to assume that if insulin were not efficiently cleared from the local islet milieu its concentration would be at least equally as high , if not higher . considering that significant insulin receptor downregulation in insulin - targeted primary cells
nmol / l ( 23 ) , it is quite possible that the insulin receptor in -cells may be permanently downregulated if insulin is not effectively cleared . in support of this idea ,
recent in vivo studies in normal mice have indicated that despite a marked effect of insulin in the liver to activate molecular targets , such as sterol regulatory element binding protein-1c , there is a negligible , if any , effect of insulin on islet -cells in the very same animal at the same time ( 15 ) . however , an alternative view is that secreted insulin is rapidly cleared from the islet locale .
in favor of this scenario is the fact that pancreatic islets have an extensive microcirculatory network that is required for normal -cell secretory function . in rodents ,
the islet microcirculation favors blood flow away from -cells passing by the other pancreatic endocrine islet cell types ( including glucagon - producing -cells and somatostatin -cells ) on exiting the islet ( 24 ) .
it is thought by some that this unidirectional -cell -cell -cell communication may be involved in coordinate downregulation of glucagon secretion as well as preventing local accumulation of somatostatin to inhibit insulin and glucagon secretion .
although human islet cell architecture is different from that in rodents , the same directional plumbing of the islet microcirculatory network is retained in humans ( 25 ) .
islet -cells are also polarized , which enables them to effectively secrete insulin into the venous islet microcirculation from where it can be readily cleared from the islet milieu ( 26 ) .
if insulin is efficiently dispersed away from the islet then it will be extracted from the circulation by the whole body before returning to the islet via the pancreatic artery at low peripheral picomolar concentrations ( 27 ) .
such a depleted concentration of insulin could be ineffective to transduce irs signaling in -cells , which needs to be at 50 nmol / l as indicated from in vitro experiments using primary islet -cells ( 2,3 ) .
it has been argued that there is always a degree of constitutive ( unregulated ) insulin secretion from normal islet -cells , and this is continually acting as a local autocrine ligand for the insulin receptor ( 13 ) . but
this observation is derived from dedifferentiated tumorigenic -cell lines and transfection of the preproinsulin gene to pituitary cells , and thus it is not necessarily physiologically relevant .
in fact , normal primary islet -cells efficiently sort > 99% of the newly synthesized ( pro)insulin to the regulated pathway and there is negligible , if any , constitutive secretion ( 28 ) .
notwithstanding this , even if there were constitutive insulin secretion from -cells , the same two scenarios described above would still apply for a negligible autocrine effect of insulin on -cells .
both sympathetic and parasympathetic input from the central nervous system ( cns ) can affect -cell function ( 29 ) .
positive parasympathetic neurotransmitter signals , such as acetylcholine ( operating through muscarinic [ m3 ] receptors on -cells ) , vasoactive intestinal polypeptide , and pituitary adenylate cyclase - activating polypeptide ( 31 ) , can enter via the vagus to potentiate glucose - induced insulin secretion ( 30,31 ) .
conversely , some negative effects to inhibit glucose - induced insulin secretion can be transmitted via sympathetic nerves ( 32 ) . in the pancreas , the vast majority of neurons associate with pancreatic islets ( 33 ) . although it has been suggested that the neuronal connections to pancreatic islets are to endothelial cells ( 34 ) , it is nonetheless clear that the cns can have a major influence on the functional regulation on -cells ( 29,32 ) .
indeed , severing the vagus nerve can significantly impair glucose - induced insulin secretion ( 29 ) .
in addition , an insulin - induced feedback inhibition of glucose - induced insulin secretion found in vivo is lost when the pancreas is denervated , including in humans ( 3537 ) .
it has been suggested that a prior exposure to exogenous insulin can enhance subsequent stimulation of endogenous insulin secretion by glucose in humans in vivo ( 38,39 ) .
although this could be consistent with a direct priming effect of insulin on the -cell , an alternative explanation could be that a prior exposure to exogenous insulin downregulates and desensitizes insulin receptor signaling in the brain ( 18 ) .
if so , the insulin - induced feedback inhibition of glucose - induced insulin secretion would be less effective . as a consequence ,
indeed , an inadequate feedback inhibition of insulin on endogenous insulin secretion has been observed in obese / insulin - resistant humans and is thought to play a role in maintaining the persistent hyperinsulinemia under such circumstances ( 36 ) .
although the cns influence on pancreatic -cell function is often underestimated , it is attracting renewed interest .
some investigators even suggest that endogenously produced incretins may , at least in part , exert their effect on -cells via the cns ( 40,41 )
. intriguingly , compensatory increases in -cell mass and function may be somewhat controlled via the cns ( 42,43 ) .
indeed , in vagotomized rats , rates of -cell proliferation decrease by half , which is associated with an 80% reduction in protein kinase - b ( pkb ; also known as akt ) activation , a key element in irs signaling pathways ( fig .
1 ) ( 43 ) . this in turn suggests that the cns may have a degree of control over irs-2regulated signal transduction in -cells .
however , it is noted that direct intracerebroventricular administration of insulin into the cns does not affect circulating insulin levels , but does suppress food intake and decrease body weight ( 44 ) . although this is an intriguing observation , it relies on a pharmacological route for insulin delivery and does not reflect the physiological route by which insulin communicates with the cns via the circulation ( 44 ) . despite the evidence that some aspects of -cell function can be neuronally influenced ,
further research is needed , especially in regard to identifying the specific regions of the cns that directly communicate to the endocrine pancreas .
but in terms of understanding the possible influence of insulin itself on the -cell in vivo , it seems that this may not necessarily be a direct autocrine effect but rather a secondary one mediated in the large part via the cns .
there are now several transgenic rodent models that imply the need for many of the elements of the irs-2 signaling pathway for -cell function , growth and/or survival ( fig .
a critical question is what is the physiologically relevant ligand that activates irs signaling pathways ? is it insulin itself ?
perhaps closely related insulin - like growth factor 1 ( igf-1 ) ? or a combination of both ?
enticing transgenic mouse models in which the insulin receptor and/or the igf-1 receptor genes have been knocked out in an intended -cell specific manner have been presented ( 4547 ) .
intriguingly , deleting the insulin receptor from -cells in this manner ( irko mice ) results in modest glucose intolerance , elevated fasting insulin levels and impaired glucose - induced first - phase insulin secretion ( 45 ) . likewise , deleting the igf-1 receptor by the same technical approach ( igf1r mice ) reveals a similar phenotype ( 46 ) .
cross - breeding these two mouse models deletes the receptors for insulin and igf-1 on -cells ( dko mice ) , rendering a more severe diabetic phenotype with apparent loss of -cell secretory capacity and function , together with elevated glucagon levels in the fed state ( 47 ) .
such unregulated glucagon release would likely be a major contributor to the hyperglycemic state of these double - knockout ( dko ) animals .
these data were very intriguing at the time , but recently the technical approach used to generate these transgenic mouse models has been questioned ( 48,49 ) .
the short form of the rat insulin gene promoter ( rip ) used to drive a cre - recombination to generate the -cell specific knockout of the insulin and/or igf-1 receptor genes is unlikely to be exclusively -cell specific ( 4547 ) . indeed , many of the rips and other -cell transcription factor ( e.g. , pdx1 ) gene promoters can not be considered -cell specific because they also drive quite significant cre - expression in several regions of the brain , including the hypothalamus ( 48,49 ) .
moreover , the rip - cre mice themselves display mild glucose intolerance , apparently owing to cre - impaired first - phase insulin secretion ( 50 ) .
as previously outlined , the cns can have a major influence on metabolic homeostasis , including pancreatic endocrine cell functions involving the -cell .
as such , while it is likely that any potential feedback effect of insulin and/or igf-1 on the -cell is blocked in these animal models , it can not be ruled out that the primary defect driving the phenotype of the irko , igf1r , or dko mice originates in the cns and that the -cells respond secondarily to dysfunctional cns control ( 51 ) . in this regard , there are now multiple studies in which knocking out a gene using a rip - cre or pdx1-cre transgenic approach gave an obese , hyperphagic , or metabolically altered phenotype that has been primarily attributed to deleting that gene in the brain / hypothalamus rather than the -cell ( 48,49 ) . in these instances ,
any apparent -cell defect might be secondary to obesity or changes in metabolic homeostasis controlled by the cns
. however , technology has progressed , and there is now a new transgenic mouse model in which the full - length ( 8 kb ) mouse insulin promoter drives cre - expression , apparently in -cells only ( 46 ) .
future use of this mip - creer mouse to uniquely delete insulin and/or igf-1 receptors in -cells may move toward resolving some of the controversy behind autocrine action of insulin .
in vivo it has been known for more than 70 years that administration of exogenous insulin depletes the pancreas of its insulin stores ( 52 ) .
this is often interpreted as a negative effect of insulin on insulin gene expression ( 2 ) . however ,
this is more likely due to the infused insulin lowering the blood glucose to a hypoglycemic state , which is known to deplete endogenous insulin stores and downregulate insulin gene expression ( 53,54 ) .
insulin gene expression is markedly downregulated under starvation conditions , but then rapidly recovers upon refeeding
a glucose infusion administered to fasted rats is sufficient to drive increases in insulin gene transcription ( 55 ) .
however , if the glucose concentration is clamped in vivo , subsequent insulin infusion to induce hyperinsulinemia can partly reduce insulin gene expression ( 56 ) , but it remains unclear whether this is a direct effect of insulin on the -cell or one acting secondarily via the cns . in hyperglycemic states in vivo ,
insulin gene expression does not appear to drastically vary at the level of the -cells .
although insulin gene expression appears reduced in pancreata from type 2 diabetic rodent models , this can be correlated with loss of -cell mass rather than any relation to hyperinsulinemia ( 57,58 ) .
it should be noted that preproinsulin mrna levels are not only transcriptionally regulated , but also at the level of preproinsulin mrna stability .
indeed , physiologically relevant increases in glucose stabilize preproinsulin mrna , but this does not appear to be mediated by a local autocrine feedback of secreted insulin ( 59 ) . recently however , there has been a series of in vitro studies using transformed -cell lines or isolated islets to suggest , in contrast to established in vivo studies , that there is a positive effect of insulin to drive insulin gene expression ( 2 ) . these in vitro studies have been complemented by experiments using islets isolated from the aforementioned irko and igf1r transgenic mice ( 4547 ) .
however , in these particular mouse models , there could well be a cns influence on the islets of these animals which alters their -cell mass ( 42,43,48,49,51 ) and , as previously observed , preproinsulin mrna levels parallel these changes ( 57 ) .
in short , none of these in vitro studies can readily place the potential of insulin itself directly regulating its own gene expression into a proper physiological context .
there is some indication that elements in the insulin signaling pathway in -cells , such as foxo1 are involved in influencing insulin gene expression in -cells ( 4 ) .
however , whether these elements are directly controlled by an in vivo autocrine feedback action of insulin is not established .
the major regulation of insulin production in normal pancreatic -cells occurs at the translational level ( 60 ) .
fluctuation in glucose concentrations is the main instigator of this control , but it can be supplemented by other nutrients and incretin peptides ( 60 ) . when primary -cells are exposed to a stimulatory glucose concentration there is a 20- to 30-min lag period ( due to recruitment of preproinsulin mrna containing polyribosome complexes to the rough endoplasmic reticulum , the site of proinsulin biosynthesis ) before increases in proinsulin biosynthesis are observed , that can then reach an impressive 10-fold increase by 60 min .
glucose - induced translational control is unique to -cells and specifically directed at proinsulin and the biosynthesis of a subset of 50 secretory granule proteins ( 60 ) .
it ensures that insulin stores in -cells are rapidly replenished after a bout of insulin exocytosis .
the molecular mechanism for specific glucose - induced translational regulation of proinsulin biosynthesis is quite distinct from that for glucose - stimulated insulin secretion ( 60 ) .
for example , unlike ca - dependent regulated insulin secretion , translational control of proinsulin biosynthesis is ca - independent ( 60 ) .
many studies have ruled out an autocrine positive feedback of insulin to drive proinsulin biosynthesis ( 5961 ) , but some have been a proponent of this possibility ( 62,63 ) .
unfortunately , studies that indicate a positive effect of insulin to stimulate proinsulin biosynthesis are questionable because proinsulin biosynthesis was either not measured directly , or if it was only during the initial 30-min lag period after introduction of the stimulus , and as such these measures are inaccurate ( 62,63 ) . without an extended incubation period up to 60 min
, the mechanics for translational control of proinsulin biosynthesis can not be fully appreciated ( 60 ) .
thus , the vast majority of evidence indicates that insulin does not have an autocrine effect on its own production in the -cell .
as previously noted , there is an in vivo temporal negative feedback of insulin on stimulated insulin secretion that is mediated via the cns in humans ( 3537 ) .
however , there is an intriguing suggestion that insulin may also have a positive effect to enhance its own secretion ( 13 ) .
but an alternative explanation could be that the effect was mainly mediated via alleviating the negative feedback of insulin through the cns ( 36 ) , as previously discussed .
yet , the majority of studies arguing for a positive autocrine effect of insulin to drive insulin secretion have been conducted in vitro where central control no longer operates , or in transgenic mouse models where a primary effect of insulin in the cns to which -cells act secondarily can not be ruled out ( 1,2,51 ) .
thus , an in vivo positive autocrine effect of insulin on the -cell remains questionable .
but in our minds , a feed - forward positive effect of insulin on insulin secretion from the -cell does not make sense physiologically .
if this were the case , the more insulin secreted the bigger the autocrine stimulus would be for further insulin secretion , which would forever be increasing with time and eventually have serious deleterious consequences . in this scenario , it would be difficult to see how insulin secretion would be efficiently shut off to critically avoid hypoglycemia .
2 ) , or natural protective insulin resistance ( 18 ) , would be expected to control this , but such built - in off mechanisms take time ( a few hours ) to be effective ( 20,21 ) .
in contrast , it is established that upon removal of a stimulus , such as glucose , insulin secretion returns to basal levels in minutes . as such , in the in vivo physiological context
we have no disagreement that multiple downstream elements of the insulin signal transduction pathway are critical for normal -cell function , growth , survival , and general well - being ( fig .
however , our thoughts and alternative interpretations have led us to believe that autocrine action of insulin is not established , especially when considering the in vivo physiological context .
if insulin is not the appropriate ligand for -cells , then what might be the relevant one ?
we mentioned doubts about this previously , but also note that in obesity / insulin resistance , when -cell mass and function may increase in compensation , igf-1 binding proteins also increase , which would actually decrease the
an intriguing possibility might be igf-2 , which has been proposed to be cosecreted with insulin in response to incretins , and has an autocrine feedback
however , this hypothesis is mostly based on in vitro observations and requires in vivo testing to substantiate .
moreover , a possible autocrine feedback of igf-2 on -cells is also subjected to the same arguments of desensitization made here for insulin , so the jury should still be out on igf-2 for the time being .
another consideration is that a homologous ligand may not be necessary at all , and it is the tight control of irs-2 expression in -cells through the action of many heterologous factors that acts as a gatekeeper to control downstream -cell homeostasis ( 1117 ) .
moreover , the cns may drive compensatory increases in -cell function and mass in response to obesity / insulin resistance ( 42,43 ) .
however , there are also a plethora of other untested growth factor ligand candidates to consider that could act on the -cell which , via their specific receptor , could induce tyrosine kinase activity to engage irs adapter molecules to then transduce downstream signaling ( fig .
the question that insulin is the physiologically relevant molecule responsible for autocrine regulation of the -cell is still open .
doubts and issues remain that are currently difficult to answer convincingly , however as new technologies emerge these could be better addressed experimentally in the near future .
but , for the moment , we prefer use of the term irs signal transduction / signaling pathway rather than insulin signal transduction / signaling pathway in reference to elements of these signaling networks in -cells
. this will avoid the implication that autocrine action of insulin in vivo is established , when in many quarters it is suggested that the concept remains unproven . | in recent years there has been a growing interest in the possibility of a direct autocrine effect of insulin on the pancreatic -cell .
indeed , there have been numerous intriguing articles and several eloquent reviews written on the subject ( 13 ) ; however , the concept is still controversial .
although many in vitro experiments , a few transgenic mouse studies , and some human investigations would be supportive of the notion , there exist different insights , other studies , and circumstantial evidence that question the concept .
therefore , the idea of autocrine action of insulin remains a conundrum .
here we outline a series of thoughts , insights , and alternative interpretations of the available experimental evidence .
we ask , how convincing are these , and what are the confusing issues ?
we agree that there is a clear contribution of certain downstream elements in the insulin signaling pathway for -cell function and survival , but the question of whether insulin itself is actually the physiologically relevant ligand that triggers this signal transduction remains unsettled . |
several studies have shown that subjects suffering from low back pain ( lbp ) frequently show
persistent involvement of the lumbar multifidus muscle . for example , atrophy and fatty
infiltration have been reported1,2,3,4,5,6,7 , reduced activity has been
demonstrated in persistent lbp8,9,10,11 , and fiber transformation from type i to type iic has also
been observed12,13,14,15 .
recovery of multifidus muscle activation and endurance is
considered essential for restoring the proper function of the lumbar muscle core16,17,18 .
european guidelines for the management of
chronic non - specific lbp also recommend supervised exercises as the main treatment for lbp ,
but they do not indicate which exercise is best19 .
physical therapists use different exercises to recruit and strengthen the lumbar multifidus
muscle20 , 21 , but whether they are actually supported by instrumental evidence
demonstrating their ability to change the anatomical characteristics of this muscle is
unknown , particularly in the treatment of lbp - induced atrophy . the purpose of this paper is to illustrate and comment on how exercise can improve the
physical parameters ( tropism ) of the multifidus muscle in patients with specific or
non - specific lbp , using diagnostic imaging as the outcome measure . following to the pico ( problem , intervention , comparison , outcome ) model , we considered
studies of subjects with specific or non - specific lbp that used exercises aimed at
activating the lumbar multifidus muscle and measured its cross - sectional area or thickness
with ultrasound , computed tomography ( ct ) or magnetic resonance imaging ( mri ) .
most
published studies comparing different exercises for the lumbar muscles did not carry out
specific training for the multifidus muscle , and/or did not evaluate the effect of the
exercises on the physiological characteristics of that muscle ; and only a few works have
investigated the most effective exercise for changing the cross - sectional area or
thickness of the multifidus muscle .
the first of these is , a paper of moderate quality ( pedro score = 6)22 by danneels et al.23 , that investigated which type of exercise / muscle contraction is
more effective for multifidus muscle atrophy recovery .
subjects who practiced sports or lumbar muscle
training during the three months prior to the intervention were excluded .
the
cross - sectional area of the multifidus muscle at three levels was measured on ct images
taken by an independent assessor .
the exercises program was conducted for ten weeks , with
a frequency of three sessions per week .
this training was
based on a series of daily activities in various positions , aimed at improving lumbar
dynamic stability in a functional way .
multifidus muscle activation followed the specific
progression of the exercises described by osullivan et al.24 , requiring about 30% of maximal contraction .
this group paired the
stabilization exercises described for group 1 with progressive resistance training in
three standardized exercises ( hip and knee extension in the quadruped position , trunk
extension in the prone position , lower limb lifting in the prone position ) .
each
progressive resistance training exercise was done in a controlled and standardized way , at
the same speed and with the same duration .
this group differed from group 2 only in the
interval between the concentric and eccentric exercises , as five seconds of static
contraction were performed between the two movements .
analysis of the differences between
the groups after the intervention period showed statistically significant differences only
in group 3 at the three levels tested ( p=0.014 , 0.008 and 0.002 respectively ) .
the results
of this study suggest that maintaining static positions between concentric and eccentric
contractions is essential for inducing muscle hypertrophy during 10 weeks of treatment . a
study of equal quality by akbari et al.25 ( pedro score = 6 ) also investigated the effectiveness of two
different exercise programs for the trunk muscles of subjects with chronic lbp .
this
randomized controlled trial compared a motor control program to a general exercise
program .
the study enrolled sixty - three subjects and lasted eight weeks , with twice - weekly
half - hour sessions for both groups .
modifications in the structural characteristics of the
investigated muscles were assessed by measuring the thickness of the lumbar multifidus and
transversus abdominis muscles with an ultrasound device ( b - mode us ) .
group 1 performed
motor control exercises wich were based on the suggestions of osullivan et al.24 , richardson et al.26 , and moseley27 .
it consisted of initial exercises to first isometrically
activate local stabilizers with low loads in the supine , quadruped , sitting and standing
positions .
then , subjects were asked to maintain these contractions for 10 seconds while
breathing normally .
finally , dynamic tasks were introduced ( from the simplest to the most
complex ) , once again maintaining the stabilizers contraction .
group 2 performed general
exercises which implemented abdominal and paravertebral muscle strengthening , based on
mcgill s proposal , which was tailored to individual tissue load capacity12 . at the end of the intervention , both
groups showed increased strength and the motor control exercise program was significantly
better at reducing pain ( p=0.004 ) and , to a lesser extent , in increasing the lumbar
multifidus and transversus abdominis muscle thicknesses .
another pilot study was conducted on five female health professionals affected by
non - specific chronic lbp28 .
a lumbar
stabilization program was carried out for 10 one - hour sessions over 12 weeks and the
subjects were requested to do home exercises twice a week , for about 3040 minutes .
multifidus muscle images were acquired in the paravertebral right and left
areas three times in succession and the arithmetic mean of the measured values was
calculated in order to reduce random error . while there was no evidence of significant
changes in tropism of the transversus abdominis after the intervention , the multifidus
muscle thickness on the more hypotrophic side was augmented in four of the five subjects .
a statistically significant improvement in pain and disability was also found and the
improvements were stable at the two month follow - up .
different results were obtained by willemink et al.29 , who investigated the effect of extensor muscle training on
multifidus muscle morphology by measuring the muscular transverse area via mri .
participants performed progressive resistance training of the isolated lumbar extensors
carried out on a back training device for 12 weeks and continued , more specifically , for
an additional 12 weeks .
participants were instructed to move in a relatively slow and
controlled manner through the full range of lumbar motion ( in approximately 2 seconds from
flexion to extension , and in approximately 3 seconds back to flexion ) , thereby activating
both global and deeper trunk muscles . at the end of this training
, there was a
statistically significant improvement in disability , but it was not accompanied by changes
in the multifidus muscle cross - sectional area .
the study s authors concluded that change
in muscle morphology does not seem to be a determining factor for better functional status
in patients with chronic lbp , at least in the short term .
following to the pico ( problem , intervention , comparison , outcome ) model , we considered
studies of subjects with specific or non - specific lbp that used exercises aimed at
activating the lumbar multifidus muscle and measured its cross - sectional area or thickness
with ultrasound , computed tomography ( ct ) or magnetic resonance imaging ( mri ) .
most
published studies comparing different exercises for the lumbar muscles did not carry out
specific training for the multifidus muscle , and/or did not evaluate the effect of the
exercises on the physiological characteristics of that muscle ; and only a few works have
investigated the most effective exercise for changing the cross - sectional area or
thickness of the multifidus muscle .
the first of these is , a paper of moderate quality ( pedro score = 6)22 by danneels et al.23 , that investigated which type of exercise / muscle contraction is
more effective for multifidus muscle atrophy recovery .
subjects who practiced sports or lumbar muscle
training during the three months prior to the intervention were excluded .
the
cross - sectional area of the multifidus muscle at three levels was measured on ct images
taken by an independent assessor .
the exercises program was conducted for ten weeks , with
a frequency of three sessions per week .
this training was
based on a series of daily activities in various positions , aimed at improving lumbar
dynamic stability in a functional way .
multifidus muscle activation followed the specific
progression of the exercises described by osullivan et al.24 , requiring about 30% of maximal contraction .
this group paired the
stabilization exercises described for group 1 with progressive resistance training in
three standardized exercises ( hip and knee extension in the quadruped position , trunk
extension in the prone position , lower limb lifting in the prone position ) .
each
progressive resistance training exercise was done in a controlled and standardized way , at
the same speed and with the same duration .
this group differed from group 2 only in the
interval between the concentric and eccentric exercises , as five seconds of static
contraction were performed between the two movements .
analysis of the differences between
the groups after the intervention period showed statistically significant differences only
in group 3 at the three levels tested ( p=0.014 , 0.008 and 0.002 respectively ) .
the results
of this study suggest that maintaining static positions between concentric and eccentric
contractions is essential for inducing muscle hypertrophy during 10 weeks of treatment .
a
study of equal quality by akbari et al.25 ( pedro score = 6 ) also investigated the effectiveness of two
different exercise programs for the trunk muscles of subjects with chronic lbp .
this
randomized controlled trial compared a motor control program to a general exercise
program .
the study enrolled sixty - three subjects and lasted eight weeks , with twice - weekly
half - hour sessions for both groups .
modifications in the structural characteristics of the
investigated muscles were assessed by measuring the thickness of the lumbar multifidus and
transversus abdominis muscles with an ultrasound device ( b - mode us ) .
group 1 performed
motor control exercises wich were based on the suggestions of osullivan et al.24 , richardson et al.26 , and moseley27 .
it consisted of initial exercises to first isometrically
activate local stabilizers with low loads in the supine , quadruped , sitting and standing
positions .
then , subjects were asked to maintain these contractions for 10 seconds while
breathing normally .
finally , dynamic tasks were introduced ( from the simplest to the most
complex ) , once again maintaining the stabilizers contraction .
group 2 performed general
exercises which implemented abdominal and paravertebral muscle strengthening , based on
mcgill s proposal , which was tailored to individual tissue load capacity12 . at the end of the intervention , both
groups showed increased strength and the motor control exercise program was significantly
better at reducing pain ( p=0.004 ) and , to a lesser extent , in increasing the lumbar
multifidus and transversus abdominis muscle thicknesses .
another pilot study was conducted on five female health professionals affected by
non - specific chronic lbp28 .
a lumbar
stabilization program was carried out for 10 one - hour sessions over 12 weeks and the
subjects were requested to do home exercises twice a week , for about 3040 minutes .
multifidus muscle images were acquired in the paravertebral right and left
areas three times in succession and the arithmetic mean of the measured values was
calculated in order to reduce random error . while there was no evidence of significant
changes in tropism of the transversus abdominis after the intervention , the multifidus
muscle thickness on the more hypotrophic side was augmented in four of the five subjects .
a statistically significant improvement in pain and disability was also found and the
improvements were stable at the two month follow - up .
different results were obtained by willemink et al.29 , who investigated the effect of extensor muscle training on
multifidus muscle morphology by measuring the muscular transverse area via mri .
participants performed progressive resistance training of the isolated lumbar extensors
carried out on a back training device for 12 weeks and continued , more specifically , for
an additional 12 weeks .
participants were instructed to move in a relatively slow and
controlled manner through the full range of lumbar motion ( in approximately 2 seconds from
flexion to extension , and in approximately 3 seconds back to flexion ) , thereby activating
both global and deeper trunk muscles . at the end of this training , there was a
statistically significant improvement in disability , but it was not accompanied by changes
in the multifidus muscle cross - sectional area .
the study s authors concluded that change
in muscle morphology does not seem to be a determining factor for better functional status
in patients with chronic lbp , at least in the short term .
the assessment of pain , disability and recurrence rate is commonly used to measure the
effectiveness of lbp treatments . unfortunately , these assessments do not allow us to isolate
the individual treatment components that contribute to the result .
moreover , the majority of
studies considered multifidus muscle training within a stabilization program that involved
other muscles ( e.g. , transversus abdominis , internal oblique ) and the changes induced in
this specific muscle were not usually measured .
this literature review is usefully
identified the few studies that have evaluated the effects of specific therapeutic training
on the multifidus muscle using instrumental outcomes ( ultrasound , ct , mri ) . in the first study
, danneels et al.18
showed that it is possible to increase the cross - sectional area of the multifidus muscle ,
with a concomitant decrease in pain , using an exercise protocol progressing from motor
control to increased static and dynamic loads . in the second study ,
akbari et al.24 showed that both specific stabilization
training and generic exercises led to a positive effect on the thickness of the multifidus
muscle , and that stabilization treatment was the more effective of the two .
the third pilot
study28 seems to indicate that
stabilization training promoted the recovery of symmetry of the multifidus muscle between
the affected side and contralateral side .
previous studies have reported multifidus muscle
asymmetry in patients with acute and chronic lbp5 and demonstrated that specific motor control training resolved this
asymmetry by improving most hypotrophic cross - sectional areas30 , 31 .
in contrast , willemink
et al.29 reported that clinical
improvement was not supported by anatomical muscular changes .
similar findings were also
reported by mannion et al.32 for the
transversus abdominis muscle .
this hypothesis is also supported by steiger et al.33 , in a review of the changes in clinical outcomes and changes in
physical function after therapeutic exercises .
these authors did not confirm the
relationship between the effect of exercises and musculoskeletal system changes in patients
with chronic lbp .
the present review was not able to clearly identify which exercise best modifies the
multifidus muscle structure in subjects with lbp , but it did reveal that the multifidus
muscle thickness and/or cross - sectional area may increase when more than one exercise at the
same time and progressing from motor control to increasing static ( overall ) and dynamic
loads .
this training appeared to be more effective than generic exercises at improving
muscle tropism and , when coupled with home treatment , may facilitate recovery from muscular
atrophy .
this short review aimed to provide a more complete picture about the exercises and tropism
of the multifidus muscle in low back pain and contribute to its applicability in
rehabilitation practices .
the data from the studies found provide us with some information
about the type , number of repetitions and exercise methods . | [ purpose ] the purpose of this review was to investigate the types of exercises that can
improve the tropism of the multifidus muscles , based on clinical evidence .
[ methods ]
following to the pico ( problem , intervention , comparison , outcome ) model , we considered
studies of subjects with specific or non - specific lbp that used exercises aimed at
activating the lumbar multifidus muscle and measured its cross - sectional area or thickness
with ultrasound , computed tomography or magnetic resonance imaging .
[ results ] this review
found that most studies compared different types of exercises for lumbar muscles , but
without specifically investigating the multifidus muscle . however , a few studies showed
that the cross - sectional area and thickness of the multifidus muscle can be increased by
activating this muscle , and they progressed from motor control to increased static and
dynamic loads .
[ conclusion ] a review of the literature revealed that specific supervised
and home exercises may improve the symmetry of the multifidus muscle . |
organophosphate compounds ( opcs ) are widely used for pest control in agriculture worldwide which leads to increased risk of human exposure . as a result of easy and widespread availability
they are also often used as suicidal agents in developing countries killing an estimated 300,000 people per annum .
the opcs inhibit the acetyl cholinesterase ( ache ) at nerve synapses leading to accumulation of acetylcholine ( ach ) at neuromuscular junctions causing over - stimulation of ach receptors with varied clinical manifestations .
the cholinesterase inhibitors cross the blood brain barrier and affect the acetylcholine receptors in the brain .
critical illness and several drugs can also alter the functions of neurotransmitters and affect the release of pituitary hormones .
several studies have used both cholinergic agonists and antagonists to demonstrate their effect on these receptors .
effects of opcs on cardiovascular , nervous and respiratory systems are well known but their effect on endocrine system is not properly studied .
hence , this study was planned to evaluate alterations in pituitary - target gland hormones at admission , at discharge following recovery and at three months of follow - up after acute opc poisoning .
this prospective study was conducted in patients with opc poisoning admitted at post graduate institute of medical education and research , chandigarh , india between january 2012 and march 2013 .
of the 46 patients admitted with suspected poisoning , 16 met the inclusion criteria ( age range 18 - 50 years ) with the history and clinical features consistent with opc consumption , documented decrease in plasma cholinesterase activity or presence of the opc in gastric lavage or blood [ figure 1 ] .
patients with the history of consumption of any compound other than opcs , on antidote therapy before hospital admission , prior known endocrine disorder or on any hormone replacements , and pregnant / lactating females were excluded from the study .
the study was approved by the institute 's ethics committee and informed consent was taken from all the subjects .
concert of patient flow venous samples were collected in each patient at admission prior to initiation of treatment , at discharge and 3 months of follow up .
blood samples were analyzed for plasma cholinesterase and pre - treatment levels of serum adenocorticotrphic hormone ( acth ) , cortisol , dehydroepiandrosterone sulphate ( dhea - s ) , prolactin ( prl ) , leutinizing hormone ( lh ) , follicular stimulating hormone ( fsh ) , triidothyronine ( t3 ) , thyroxine ( t4 ) , thyroid stimulating hormone ( tsh ) , insulin like growth factor-1 ( igf-1 ) , estradiol ( e2 , in females ) , testosterone ( t , in males ) .
free triidothyronine ( ft3 ) and free thyroxine ( ft4 ) were estimated form the blood only if required .
serum acth , cortisol , dhea - s , prl , lh , fsh , t3 , t4 , tsh , e2 and t were estimated by electro chemiluminescence immune - assay ( eclia ) using the analyzer elecsys 2010 ( roche , germany ) .
igf-1 ( 150 - 350 ng / ml ) levels were assayed using the drg igf-1 600 elisa kit , catalogue # eia-4140 .
patients in whom there was sub normal level of igf-1/gh and cortisol at third month of follow up were subjected to insulin induced hypoglycaemia ( iih ) to check the integrity of the hypothalamo - pituitary axis .
venous blood samples were analysed for glucose , growth hormone and cortisol at 0 , 30 , 60 , 90 and 120 min .
hypoglycaemia was considered as significant only if plasma glucose was < 40 mg / dl along with signs and symptoms .
the point of occurrence of documented hypoglycaemia was considered as 30 min sample a gh cut off of more than 5 ng / ml and cortisol cut off of 540
all study samples for specific hormones were processed in a single assay to avoid inter assay variability .
nasogastric tube was inserted in all the patients as soon as they were admitted in the hospital for the diagnostic and therapeutic purpose .
qualitative test for opc in stomach contents was based on the basic analytical toxicology by who under international programme on chemical safety by silica gel thin layer chromatography plate with sensitivity to detect 5 mg opc per litre .
the normalcy of data was checked by kolmogorov - smirnov test . the mean and median of the parameters
were obtained and they were compared for any significant difference in the following manner : before treatment and at discharge ; at discharge and at 3 months of opc poisoning ; and before treatment and at 3 months of follow up .
categorical variables were presented in percentages and tested for difference using chi square test and fisher 's exact test .
friedman 's test with post - hoc bonferroni 's correction was used for non - parametric variables whereas repeated measures anova with post - hoc bonferroni 's correction for parametric data .
this prospective study was conducted in patients with opc poisoning admitted at post graduate institute of medical education and research , chandigarh , india between january 2012 and march 2013 .
of the 46 patients admitted with suspected poisoning , 16 met the inclusion criteria ( age range 18 - 50 years ) with the history and clinical features consistent with opc consumption , documented decrease in plasma cholinesterase activity or presence of the opc in gastric lavage or blood [ figure 1 ] .
patients with the history of consumption of any compound other than opcs , on antidote therapy before hospital admission , prior known endocrine disorder or on any hormone replacements , and pregnant / lactating females were excluded from the study .
the study was approved by the institute 's ethics committee and informed consent was taken from all the subjects .
venous samples were collected in each patient at admission prior to initiation of treatment , at discharge and 3 months of follow up .
blood samples were analyzed for plasma cholinesterase and pre - treatment levels of serum adenocorticotrphic hormone ( acth ) , cortisol , dehydroepiandrosterone sulphate ( dhea - s ) , prolactin ( prl ) , leutinizing hormone ( lh ) , follicular stimulating hormone ( fsh ) , triidothyronine ( t3 ) , thyroxine ( t4 ) , thyroid stimulating hormone ( tsh ) , insulin like growth factor-1 ( igf-1 ) , estradiol ( e2 , in females ) , testosterone ( t , in males ) .
free triidothyronine ( ft3 ) and free thyroxine ( ft4 ) were estimated form the blood only if required .
serum acth , cortisol , dhea - s , prl , lh , fsh , t3 , t4 , tsh , e2 and t were estimated by electro chemiluminescence immune - assay ( eclia ) using the analyzer elecsys 2010 ( roche , germany ) .
igf-1 ( 150 - 350 ng / ml ) levels were assayed using the drg igf-1 600 elisa kit , catalogue # eia-4140 .
patients in whom there was sub normal level of igf-1/gh and cortisol at third month of follow up were subjected to insulin induced hypoglycaemia ( iih ) to check the integrity of the hypothalamo - pituitary axis .
venous blood samples were analysed for glucose , growth hormone and cortisol at 0 , 30 , 60 , 90 and 120 min .
hypoglycaemia was considered as significant only if plasma glucose was < 40 mg / dl along with signs and symptoms .
the point of occurrence of documented hypoglycaemia was considered as 30 min sample a gh cut off of more than 5 ng / ml and cortisol cut off of 540
all study samples for specific hormones were processed in a single assay to avoid inter assay variability .
nasogastric tube was inserted in all the patients as soon as they were admitted in the hospital for the diagnostic and therapeutic purpose .
qualitative test for opc in stomach contents was based on the basic analytical toxicology by who under international programme on chemical safety by silica gel thin layer chromatography plate with sensitivity to detect 5 mg opc per litre .
the normalcy of data was checked by kolmogorov - smirnov test . the mean and median of the parameters
were obtained and they were compared for any significant difference in the following manner : before treatment and at discharge ; at discharge and at 3 months of opc poisoning ; and before treatment and at 3 months of follow up .
categorical variables were presented in percentages and tested for difference using chi square test and fisher 's exact test .
friedman 's test with post - hoc bonferroni 's correction was used for non - parametric variables whereas repeated measures anova with post - hoc bonferroni 's correction for parametric data .
the study group initially included 18 patients admitted to the hospital in the stipulated period .
of 16 patients with opc injestion , two were excluded as their gastric lavage did not show any evidence of opc .
finally , eight males and six females were enrolled in the study [ figure 1 ] .
the mean age , duration of hospital stay and glasgow coma scale ( gcs ) of the subjects were 30.1 10.3 years ( range ; 18 to 49 years ) , 9.5 7.6 days ( median ; 4.5 and range ; 2 - 39 days ) 13.5 2.7 ( range ; 6 -15 ) respectively .
the nature of opc was unknown in 5 , dichlorovas in 5 , dimethoate , phorate , monocrotophos and propenofos in one each .
ten patients received only atropine as treatment and remaining 4 received atropine with pralidoxime ( 2-pyridine aldoxime methyl chloride ) .
serum tsh at baseline though within normal range ( 0.7 0.5 ) was lower at the time of admission compared to tsh at 3 months of follow up ( 2.9 2.1 ) ( p = 0.02 ) .
the levels of t3 and t4 did not differ significantly at baseline from that at 3 months .
one patient developed new onset hypothyroidism with very low t4 ( 3.0 g / dl ) and elevated tsh value of ( 6.7 uiu / ml ) [ figure 2a , b and c ] .
the figure displays the distribution of tsh values at baseline ( admission ) , at discharge and three months of follow - up the figure displays the distribution of t3 values at baseline ( admission ) , at discharge and three months of follow - up the figure displays the distribution of t4 values at baseline ( admission ) , at discharge and three months of follow - up there was no significant difference between serum acth at admission to that at recovery and at 3 months follow - up [ table 1 ] .
the levels of serum cortisol were significantly higher at baseline compared to that at 3 months ( p = 0.004 ) . at baseline 11 out of 14 patients had supraphysiological values of cortisol and 4 patients had sub - normal cortisol values . at discharge
only 3 patients had sub normal values of cortisol which recovered at 3 months of follow up . at this juncture 5
hormonal levels of patients at baseline ( admission ) , at discharge and at three months after exposure to organophosphrous compound the figure displaces the distribution of acth values at baseline ( admission ) , at discharge and three months of follow - up the figure displays the distribution of cortisol values at baseline ( admission ) , at discharge and three months of follow - up there was no significant difference in mean serum dhea - s at recovery from acute intoxication after recovery and at 3 months follow up . however , dhea - s was low at admission in 5 patients together with high cortisol but 2 had normal acth . at 3 month
follow up one patient continued to have low dhea - s with high cortisol and low acth while another had low dhea - s with low - normal cortisol and low acth [ figure 3c ] .
the figure displays the distribution of dheas values at baseline ( admission ) , at discharge and three months of follow - up serum prl at baseline was higher than that at 3 months ( p = 0.04 ) .
the serum lh and fsh values were not significant at baseline and on follow up [ figure 4 ] .
serum testosterone level was low in 6 out of 8 male patients at admission which normalised in three of them at discharge . with 3 months of
follow up one among the 6 continued to have low testosterone and another patient had new onset testosterone deficiency [ figure 5a c ] .
in female patients there was no significant difference in serum 17 -estradiol at baseline and at discharge ; however , it was higher at 3 months .
the serum 17 -estradiol was low in 1 out of 6 women at admission which normalised at discharge .
four women at 3 months follow up had above normal 17 -estradiol and all of them were associated with normal fsh and normal to sub - normal lh levels [ figure 5d ] .
the figure displays the distribution of prolactin values at baseline ( admission ) , at discharge and three months of follow - up the figure displays the distribution of lh values at baseline ( admission ) , at discharge and three months of follow - up the figure displays the distribution of fsh values at baseline ( admission ) , at discharge and three months of follow - up the figure displays the distribution of testosterone values at baseline ( admission ) , at discharge and three months of follow - up the figure displays the distribution of estradiiol values at baseline ( admission ) , at discharge and three months of follow - up there was no significant difference in the levels of igf-1 at baseline and at 3 months follow up . however , there was statistically significant difference between igf-1 levels at discharge and at 3 months ( p = 0.01 ) .
seven patients had low igf-1 levels at baseline which recovered in 4 patients with 3 other patients developing new onset deficiency [ figure 6 ] .
1 patient continued to have low level of igf-1 since admission , while others recovered , and one patient who developed igf-1 deficiency at discharge continued to remain so at 3 month of follow up .
both of them failed to have inadequate gh reserve on iih and mri did not reveal any abnormality ( data not shown in the box plot ) .
the figure displays the distribution of igf-1 values at baseline ( admission ) , at discharge and three months of follow - up at baseline the mean random plasma glucose was 138.2 43.5 mg / dl .
out of 14 patients , only one had transient hyperglycaemia with blood glucose more 200 mg / dl which normalised within 8 h of hospitalization and remained normal throughout the study period .
overall 7 patients out of 14 had nine hormonal deficiencies at the end of the study , 4 having sub normal cortisol , two each had testosterone and growth hormone deficiency and only one had t4 deficiency at 3 months follow up .
there was a significant positive co - relation between cholinesterase with baseline igf-1 ( r = 0.83 , p < 0.001 ) , tsh ( r = 0.69 , p = 0.02 ) , acth ( r = 0.74 , p = 0.009 ) .
there was no relationship between levels of acetyl cholinesterase and other hormones at baseline , discharge or at 3 months of follow up .
the duration of hospital stay , atropine and pralidoxime therapy did not influence the hormonal outcome .
the tsh , acth and igf-1 deficiencies at the time of presentation correlated with levels of cholinesterase .
a small subset of patients had varying level of hormonal insufficiency either at diagnosis , recovery or at 3 months of follow up .
the mean serum t3 and tsh were low - normal which were normalized at the time of discharge and at three months follow up .
this pattern of altered thyroid function is similar to those observed during critical illness which is characterized by low t3 , low or low - normal t4 and normal to low normal tsh . in our study ,
the studies by smallridge et al . , and gven et al . , noted similar findings in thyroid function test .
the variation in thyroid function during critical illness may depend on the severity of illness , duration of illness , levels of cytokines and age of the patient .
in addition to the cytokine mediated suppression of tsh in critical illness , it is postulated that cholinergically mediated rise in somatostatin tone may also contribute to the suppression of tsh in patients with opc poisoning .
ultrastructural changes in thyroid tissue has been demonstrated in a study on rats with acute methamidophos poisoning suggesting that certain opc may have direct deleterious effect on thyroid . in our study ,
one patient had biochemical profile suggestive of hypothyroidism at admission and the t4 decreased further with rise in tsh at 3 months of follow up .
the tpo was negative and he had normal urinary iodine and no other identifiable cause of hypothyroidism . in the present study ,
the mean acth and cortisol were high or high normal at baseline that normalised at three months of follow up .
the initial rise in glucocorticoid and acth in patients might have resulted from stress , cholinergically mediated effect on crh and or acth secretion as none had cortisol deficiency at 3 months follow up .
there was an inverse correlation between serum cortisol and dehydroepiandrosterone - sulfate ( dheas ) in our study . at three months of
follow up , out of 5 patients who had poor glucocorticoid reserve three had low dheas and acth suggesting pituitary injury .
other two patients had normal dhea sulphate and two patients developed new onset dhea sulphate deficiency with normal acth and plasma cortisol indicating subtle adrenal insult .
teleologically this is beneficial for the survival and this can be explained by acth stimulated preferential rise in 11-deoxycortisol which acts as a precursor for cortisol synthesis .
the alteration of dhea - cortisol ratio may be defensive rather than mal - adaptive .
circulating prl was normal at baseline and remained so at follow up in all except three who had ingested dichlorvos .
this could be explained by action of acetyl choline on nicotinic receptor on the dopaminergic neurons in arcuate nucleus on lactotroph in pituitary and variable stress response which could be opc specific or consequent to longer duration of stress .
we propose that dichlorvos may have direct action on dopamine pathway or hypothalamic neurons causing elevation of prl levels .
there was no difference in serum lh and fsh at baseline and at follow - up .
when individual patients were analysed two patients had low testosterone on 3 month follow up with normal lh and fsh .
one of them consumed dichlorvas and in others the nature of opc was not known .
seven patients ( 50% ) had low igf-1 with normal gh at admission . at discharge , 6 out of 14 ( 42.8% ) had low igf-1 , two of them continued to have persistently low igf-1 on repeat evaluation at 3 months follow up .
the growth hormone igf-1 axis normalized in all other patients . during acute stress , igf-1 and igfbp levels decreases despite rise in gh .
this acute phase response to stress , representing a peripheral resistance to gh is mediated by cytokines like tumor necrosis factor ( tnf)- , interleukin ( il)-1 and il-6 .
acute rise in ach in the hypothalamic neurons leads to increase in growth hormone ( gh ) .
this forms the pharmacological basis for the gh stimulation test using pyridostigmine to assess the gh reserve in gh deficiency children .
the deficiency on long term follow up could be due to direct pituitary injury in these two patients .
high plasma glucose ( > 200 mg / dl ) was seen in only one of our patients at the time of admission which normalized within 6 h. mean acth , tsh and igf-1 were positively correlated with acetyl cholinesterase which predicts the severity of poisoning .
as the cholinesterase level recovers later , there is lack of co - relation on follow - up .
there was no correlation with atrophine treatment , duration of hospital stay and hormonal insufficiencies .
the probable mechanisms of opc induced delayed hormonal consequences as seen in some of our patients could be explained by complex extensive bio - transformation involving several metabolic systems with simultaneous oxidative injury at number of points utilizing cytochrome p-450 .
human beings have more efficient hydrolytic enzymes than insects thereby meaning more efficient detoxification process .
there could be a lot of inter - individual variation in the detoxification process , dose of opc and nature of opc .
the strength of the current study is prospective nature , evaluating pituitary target gland hormones at admission , on recovery and at 3 months .
furthermore , this is the first study to show that there could be differential effect of opc on pituitary - target gland axis . the limitations of the study are small number of recruited patients , undefined nature of opcs in most , no comparative or control group and relatively early after assessment of hormonal parameters .
we conclude , initial hormonal changes in acute opc poisoning are similar to sick euhormonal syndrome .
these abnormalities disappear in most at recovery and in a small but significant proportion of patients may have hormonal insufficiency . | background : organophosphate compound ( opc ) poisoning is common in the developing countries such as india .
the acute and later effects of opc poisoning on pituitary and target gland hormones is largely unknown.materials and methods : this prospective study was conducted at postgraduate institute of medical education and research between january 2012 and march 2013 .
fourteen patients ( 8 males , age 18 - 50 years ) with acute opc poisoning were included in the study based on the history and clinical features , documented decreased in plasma cholinesterase activity or presence of the opc in gastric lavage / blood samples .
the hormonal parameters were done at baseline , at the time of discharge and at three months of follow-up.results:a total of 14 patients out of 46 with the mean age of 30.1 10.3 years were finally eligible for the study .
hormonal alterations at admission were similar to sick euhormonal syndrome .
overall 7 of them had nine hormonal deficits at three months of follow up , 4 having sub normal basal cortisol level and two each had low testosterone and growth hormone and only one had thyroxine deficiency.conclusion:acute organophosphate poisoning results in endocrine dysfunction akin to sick euhormonal syndrome .
however , in a small subset of patients , varying level of hormonal insufficiency may occur either at admission or later .
these observations need re - validation in a larger group of patients with specific opc . |
its symptomatology could mimic other disease processes such as cholecystitis , diverticulitis , ruptured ovarian cysts and pelvic inflammatory disease .
the diagnosis could be further obscured by underlying undiagnosed anatomical anomalies , for instance intestinal malrotation .
midgut malrotation is a foetal anomaly resulting from the failure of rotation around the axis of the superior mesenteric artery and subsequent retroperitoneal fixation .
it is a rare condition with an approximate incidence of one in 500 live births ( 1 ) .
85% of cases have been estimated to present in the first two weeks of life ( 2 ) .
hence , it is difficult to establish an accurate incidence as this condition can be asymptomatic and undetected until it is discovered in investigations or surgery for other abdominal pathologies in adulthood ( 3 ) .
clinical suspicion of intestinal malrotation declines with age because of the rarity of the disorder .
we report a case of perforated appendicitis in a middle - aged man with an incidental finding of midgut malrotation at the time of diagnostic laparoscopy .
a previously fit and well 56 year old gentleman presented to our emergency department with a two - day history of constant , dull , diffuse central abdominal pain .
he had no significant past medical history but he had a positive family history of colorectal carcinoma .
he is a non - smoker and drinks 20 units of alcohol per week . on examination ,
his observations were t36.7c , pulse 53 beats per minute and blood pressure of 120/75mmhg .
on , palpation , tenderness with guarding , rebound and rigidity was elicited over the epigastrium and left upper quadrant .
his biochemical profile showed raised inflammatory markers white cell count of 12 x10/l , neutrophils of 8.9 x10/l , crp of 62 mg / l and amylase of 40
abdominal film was unremarkable . based on his worsening of symptoms and the clinical findings ,
the findings included a large amount of free pus in the peritoneal cavity , absence of appendix and caecum in the right lower quadrant and significant adhesions in left upper quadrant .
after several attempts in dividing adhesions in the left upper quadrant , the decision was made to convert to open laparotomy . at laparotomy ,
the caecum and a perforated gangrenous appendix was found in the left upper quadrant as shown on the picture in figures 1 and 2 .
operative image showing a perforated gangrenous appendix found in the luq operative image showing a perforated gangrenous appendix found in the luq a malrotation at the jejunal level was found which attributed to the condition .
post - operatively , the patient had paralytic ileus and mild wound infection which resolved with conservative management and discharged on the 7 day .
the process occurs between the fourth and twelfth week of foetal life . at four weeks
, the embryological gut exists as a single tube , suspended anterior to the superior mesenteric artery ( sma ) .
the midgut enlarges rapidly beyond the capacity of the peritoneal cavity , herniating into the umbilical cord at six weeks . within the cord
this brings the third and fourth parts of the duodenum across to the left of the midline , behind the superior mesenteric artery .
the mid - gut returns to the abdomen at the tenth and twelfth week . during this time
, it continues to rotate a further 180 counterclockwise , bringing the ascending colon to the right side of the abdomen with the caecum lying immediately below the liver .
the caecum descends into its normal position in the right iliac fossa , pulling the colon with it .
the third and fourth part of duodenum is fixed in the retroperitoneum , supported by the ligament of treitz .
when any of the above sequent of events fails , the duodenojejunal flexure may freely hang from the foregut , at the right side of the abdomen .
the caecum may be abnormally mobile or lying in the left upper quadrant as seen in our case report ( 4 ) .
this may result in dangerous conditions like intestinal obstruction or intestinal ischaemia of secondary to volvulus .
diagnosis of intestinal malrotation is often an incidental finding in barium studies , angiographies , and computer tomography scans as found in various literature ( 5 ) .
presently , no reported cases of laparoscopically - diagnosed intestinal malrotation is found in the literature . in conclusion , appendicitis is a common surgical condition with various clinical presentations . in cases
where peritonism is elicited elsewhere other than the right iliac fossa , clinicians could bear in mind the possibility of underlying intestinal malrotation , as this could be the first presentation of this rare congenital condition . | appendicitis is a common surgical condition with various clinical presentations .
the diagnosis could be obscured by underlying undiagnosed anatomical anomalies like intestinal malrotation .
intestinal malrotation is a rare foetal anomaly resulting from an incomplete , or failure of midgut rotation and fixation .
85% of cases have been estimated to present in the first two weeks of life .
presentation at adulthood is rare . in cases where
peritonism is elicited elsewhere other than the right iliac fossa , clinicians should bear in mind the possibility of underlying intestinal malrotation , as this could be the first presentation of this rare congenital condition . |
internal hernias comprise 1% of hernias , 8% of which are through the foramen of winslow into the lesser sac .
this is the third reported case describing true gastric outlet obstruction secondary to caecal herniation through the lesser foramen .
it is common for this type of herniation to initially be misinterpreted as a gastric outlet obstruction . in this case
, we observed a temporal progression from the treated gastric outlet obstruction to ongoing bowel obstruction caused by caecal herniation into the lesser sac .
a 69-year - old female presented with a 1-day history of acute onset left upper quadrant ( luq ) pain .
she had normal bowel opening at presentation and described feeling sweaty and cold on systems enquiry .
she had a previous history of hypertension and hysterectomy . on examination , she was severely tender with a localized 1515 cm smooth , tympanic mass in the luq , otherwise the abdomen was soft and non - tender with normal bowel sounds ( bs ) .
clinically , she was in atrial fibrillation with fast ventricular response ( af ) at a rate of 170 bpm with bp 180/100 .
all other observations were normal . computed tomography ( ct ) imaging demonstrated gastric outlet obstruction with collapsed small bowel loops , initially interpreted as possible gastric duplication cyst .
management with nasogastric ( ng ) tube decompressed the stomach but she remained in refractory fast af despite metoprolol and amiodarone loading .
upper gi endoscopy demonstrated fundal and proximal body congestion with uninterrupted scope passage into the duodenum .
high ng tube aspirates continued despite resolution of the large luq mass , and she developed generalized abdominal distension with hypoactive bs .
repeat ct scan demonstrated caecal herniation into the lesser sac and distended loops of obstructed small bowel , with normal appearances of the stomach .
the patient proceeded to exploratory laparotomy , which found small bowel obstruction caused by caecal volvulus and herniation through the foramen of winslow .
the caecum was decompressed in the lesser sac via the appendiceal orifice and a limited right hemicolectomy performed with primary anastomosis .
she made a steady and uncomplicated recovery to discharge on the eighth post - operative day .
internal hernias are uncommon comprising 1% of all hernias , of these ~8% are into the lesser sac [ 2 , 3 ] , with paraduodenal ( 53% ) , pericaecal ( 13% ) and transmesenteric ( 8% ) forming the majority of other internal hernias .
there are nearly 150 cases of hernia into the lesser sac described in the literature . of these ,
small bowel ( 60% ) and ascending colon ( 2530% ) are the most common .
this is the third reported case to describe gastric outlet obstruction in the context of herniation through the lesser foramen , and herniation through the hepatogastric ligament .
we feel this case is unique as it provides insight into the temporal progression of these presentations , from both a clinical and radiological perspective .
a common clinical presentation is that of obstruction with abdominal distension , epigastric pain , nausea and vomiting .
patients may describe curling into a ball or leaning forward as a relieving factor , which corresponds with an increase in size of the opening into the lesser sac lessening pressure on the obstructed viscus . owing to the presentation often mimicking gastric outlet obstruction
several authors have described characteristic x - ray findings that can aide identification , but ct remains the modality of choice owing to speed and reconstruction capabilities . in this case , we proceeded straight to ct , based on the likely complexity of the intra - abdominal pathology .
characteristic ct findings described by patel and newton can be retrospectively applied to this case : air - fluid levels in the lesser sac with distended bowel loops in the sub - hepatic space and absence of ascending colon in its normal anatomical position .
once diagnosis has been established prompt surgical management is essential with mortality approaching 50% even with treatment , excess mortality has been attributed to delays in instigating surgical management . in this case , we proceeded to laparotomy , but an alternative approach is with minimally invasive surgery .
this case highlights the presentation of caecal herniation into the lesser sac . often initially mimicking gastric outlet obstruction
internal herniation remains an uncommon but important differential diagnosis in patients presenting with bowel obstruction ( figs 12 ) .
figure 1:ct abdomen coronal reconstruction demonstrating obstructed stomach with obstructed and dilated caecum present in lesser sac.figure 2:axial section of ct abdomen demonstrating ng tube in stomach , with successful decompression of obstructed gastric outlet , however obstructed caecum clearly visible within lesser sac .
ct abdomen coronal reconstruction demonstrating obstructed stomach with obstructed and dilated caecum present in lesser sac .
axial section of ct abdomen demonstrating ng tube in stomach , with successful decompression of obstructed gastric outlet , however obstructed caecum clearly visible within lesser sac .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
copies of the written consent are available for review by the editor - in - chief of this journal on request . | abstractinternal hernias comprise 1% of hernias , 8% of which are through the foramen of winslow into the lesser sac .
these hernias can mimic gastric outlet obstruction and cause associated morbidity . in this case
, we describe a caecal herniation into the lesser sac presenting as true gastric outlet obstruction in a 69-year - old female .
initial computed tomography ( ct ) imaging demonstrated a distended stomach with collapsed small bowel representing likely gastric outlet obstruction .
nasogastric tube insertion decompressed the stomach but the clinical picture progressed to that of small bowel obstruction with generalized abdominal distension and hypoactive bowel sounds .
repeat ct demonstrated caecal herniation into the lesser sac .
this was confirmed at exploratory laparotomy with the caecum found in the lesser sac via the foramen of winslow .
the caecum was grossly ischaemic with patchy necrosis .
a limited right hemicolectomy was performed .
the patient made an uncomplicated recovery and was discharged on the eighth post - operative day . |
the leukodystrophies are a heterogeneous group of genetic diseases involving dysmyelination of brain white matter as a result of substrate accumulation due to enzymatic defects .
recognition of leukodystrophies has been revolutionized by mri technology because of its increased sensitivity compared to ct scan and because of its ability in some cases to reveal disease - specific features that can lead to a diagnosis .
the onset of clinical disease is often insidious or intermittent and their signs and symptoms are often non - specific .
these symptoms may include delay in attaining milestones , growth retardation , seizure , spasticity , ataxia , or movement disorders .
some clinical features may be more specific to one or more disorders and can help orient the diagnostic investigations .
an example would be the presence of macrocephaly , which is typically seen in alexander , canavan , and megalencephalic leukoencephalopathy with subcortical cysts ( mlc ) . here , we report a 6-year old palestinian girl who presented at the age of 4 years with absence seizure that responded to valproate therapy .
a 6-year - old palestinian girl was born to non - consanguineous parents after uneventful pregnancy and labor .
she had normal early development . at the age of 4 years , she developed episodes of sudden loss of activities and unresponsiveness to voice , each lasting only for few seconds followed by altered awareness for few minutes then she resumed her activities .
the episodes were noted by the parents several times in 6 months . on examination ,
she had normal neurological examination including normal gait , cranial nerves , muscle strength and tone , deep tendon reflexes , and planter response .
urine organic acid chromatography , plasma amino acid chromatography , and very long - chain fatty acids ( vlcfa ) were also normal .
cerebral mri showed diffuse symmetric high signal intensity in the periventricular white matter [ figure 1 ] .
t2-weighted mr images showing symmetric confluent high signal intensities of the periventricular white matter the seizures resolved after several weeks of treatment with valproic acid .
co - enzyme q10 and b vitamins were also supplemented for the hope of treating one of several rare genetic vitamin - responsive disorders .
t2-weighted mr images obtained after 8 months of treatment with b vitamins and co - enzyme q10 showing the same high signal intensities of periventricular white matter at last follow up at the age of 6 years , the patient had normal development and neurological examination including gait , cranial nerves , muscle tone , strength , deep tendon reflexes , and coordination .
in recent years , the use of magnetic resonance techniques has facilitated the characterization of several new forms of white matter disorders in children .
a pattern recognition program was used to identify cases with common features among the unclassified leukoencephalopathies , in which 7 major categories could be distinguished and 2 new entities were described : megalencephalic leukoencephalopathy with cysts ( mlc ) and vanishing white matter disease ( vwmd ) . demyelinating leukodystrophies are characterized by prominent hyperintensity of the white matter in t2-weighted images and prominent hypointensity in t1-weighted images compared with gray - white matter structures .
the clinical presentation of the different leukodystrophies is often similar for a given age group .
neurological symptoms of leukodystrophies consist of progressive motor symptoms and changes in cognition and language , but recalcitrant seizures are an unusual feature of disorders mainly affecting brain white matter .
some clinical features may be more specific to one or more disorders and can help orient the investigations as macrocephaly in alexander disease , canavan disease , mlc , and vwmd .
rare presentations of childhood leukodystrophy of unknown origin were reported including inattention and behavioral problems resembling bipolar disorder .
the adolescent or adult presentations are usually characterized by cognitive regression and psychiatric manifestations while the motor symptoms are typically more subtle .
the patient we present in this article has typical demyelinating leukodystrophy presenting with seizure as the sole manifestation , which , to our knowledge , has not been described in previous reports .
neurometabolic workup excluded many entities in this patient such as l2-hydroxyglutaric aciduria , adrenoleukodystrophy , metachromatic leukodystrophy , and canavan disease .
serum biotinidase enzyme assay was also normal excluding this entity as a rare cause of white matter disease .
alexander disease was not considered as a diagnostic possibility in the patient due to the absence of macrocephaly and the lack of its typical recognition pattern on cerebral mri .
our patient has a leukodystrophy with a distinct phenotype further expanding the clinical spectrum and adding to the increasingly recognized evidence that the clinical features of childhood leukodystrophy are extremely variable . | leukoencephalopathy refers to any disease of the white matter including hereditary as well as acquired and toxic causes .
inherited leukodystrophies are diseases of myelin including abnormal myelin development , hypomyelination , or degeneration of myelin .
we report a 6-year old female who presented with absence seizure at the age of 4 years .
cerebral magnetic resonance imaging ( mri ) of the brain showed bilateral periventricular confluent high signal intensity .
the seizure responded to anticonvulsant therapy , and the clinical course was characterized by normal development and neurological examination . |
bureaucracy has consequences . in 2004 , time magazine attributed the nearly 400,000 european
researchers in the us to two key factors funds and bureaucracy .
although many researchers were attracted by money and opportunity , many more were
escaping constraints that faced them at home .
the european union ( eu ) framework programmes
( fps ) should have provided a fresh opportunity , but their bureaucracy often surpassed the
already cumbersome mechanisms present in many european countries . in the early 2000s , even
finding project calls on the commission 's research website was extremely difficult .
once
calls finally started appearing in a standard place , the associated documentation purporting
to help applicants often comprised more pages than a phd thesis .
the bureaucracy in european science funding has been criticized so often that the message
has finally seeped through . in particular ,
concerns about the adverse impact on smaller
research institutes and small and medium enterprises ( smes ) have been accepted .
submissions will be faster , shorter and follow standardized practices . additionally ,
meaningless timesheets for those employed full - time on eu projects will be abolished and
reimbursement of indirect costs will be simplified .
private
partnerships , the commission could also simplify its sub - contracting rules to encourage
entrepreneurial dynamism .
research increasingly requires innovative small and medium
enterprises to provide bespoke software and hardware , an interaction which stimulates niche
developments in european industry .
many small companies waste scarce time compiling bids which fail and
investigators are bound to strangers who present the lowest bid regardless of passion or
competence beyond fulfilling minimal requirements . for this reason , we recommend that sub - contracting should not require compulsory tendering
where the sums fall below one of the european commission ( ec ) procurement thresholds for
public sector contracting authorities ( 130,000/200,000 ) .
of course , safeguards are needed
but this could be covered by governance arrangements already in place .
these projects have
been accepted , complete with budgetary approval , so costing has already occurred .
the
selection should now concern quality , which the project team are best placed to judge .
projects should be encouraged to advertise widely ( an online virtual market place for
relevant businesses and projects would be a valuable resource ) , but ultimately the projects
should have power to select and negotiate .
the supposed risks associated with trusting
research teams to choose their business partners are far outweighed by the speed ,
effectiveness , dynamism and opportunities gained by unshackling companies and projects alike
from this costly bureaucratic burden .
the us national institutes of health 's reporter website has an online interface detailing
the $ 30bn per annum of research funded .
it is possible to drill down by geography , year or
subject area to access what is funded .
the ec had no such well - developed interface nor
detailed subject area classification for its fp5 and fp6 projects .
to analyse health - related
research funded by the eu , we had to manually classify over 4700 projects .
there are clear benefits to mapping scientific research , particularly in the health domain
where research funding can be cross - referenced with measures such as burden of disease or
patient demand .
previous research has indicated that in many health research funding
schemes , investment does not reflect societal need . to the commission 's credit , they have recognized this and horizon 2020 will address
big societal questions such as health and sustainability .
additionally , a detailed online hierarchical categorization of eu investments allows
national funders to see collaboration opportunities or gaps , and it can shed light on areas
of research that might not withstand wider scrutiny .
we identified some 35 million spent on
antioxidants with many of the research abstracts implying health benefits . sadly , after
initial high - level excitement ( and despite continued marketing ) , it has been known since the
1980s that supplemental antioxidants are ineffective in cancer prevention and probably
dangerous .
finally , an online database of projects with an analysis interface might resolve
the 4 billion mismatch we documented between what the eu had claimed to spend under fp5 and
fp6 ( 34bn ) and what their official databases showed ( 30bn ) .
perhaps the most important recommendations we can make relate to new trends in science
informatics that could increase productivity dramatically .
the two key issues are making
projects fully digital object identifier ( doi ) networked and open access to research
databases .
the crossref collaboration of
journals has developed a doi system to provide unique identities for scholarly articles
which can then be linked electronically .
useful metadata can added to paper dois ( year , mesh
terms ) , or metrics calculated for groups of paper dois ( e.g. journal / author citation
indices ) .
an obvious next step would be to create dois for individual researchers ( analogous
to the researcher ids of the canadian institute for health research ) , dois for funded
projects and dois for project databases .
dealing first with individual dois , rather than setting up a new eu - only system the current
effort to establish global researcher dois should be harnessed .
orcid was launched in
october 2012 ( http://about.orcid.org/ ) , backed by an impressive list of
academic bodies .
orcid provides researchers with a unique i d and associates this i d with a
regularly updated list of publications ( much like thomson reuters researcherid the code
upon which orcid is based ) .
so long as orcid provides an appropriate application programming interface , the eu
could automatically send a list of researcher ids to orcid at any time and obtain a list of
up - to - date publications for them .
similarly , to access outputs associated with any project ,
project dois should be linked to publication dois .
currently , anyone can enter a us grant
code in pubmed to retrieve that project 's output .
further , this can be automated en masse
via pubmed 's application programming interface : eutilities .
if the eu were to have unique
grant codes and a contract with pubmed to register those codes ( as do other major funders ) ,
the entire up - to - date list of publications associated with any set of grant codes could be
used to populate a database almost instantly .
this would certainly be superior to their
recent painstaking exercise that involved sending questionnaires to 12,000 researchers , then
analysing 22% of those .
finally , the ec should mandate that all projects funded should provide , as a deliverable ,
any databases which they compile during the project . the case for open
data from publically
funded research has been advocated frequently to enable validation of results , use in meta - analysis , novel unforeseen exploitation ,
and mitigation of publication bias .
the idea that raw data are a public good has led funding
bodies in the uk , such as the mrc and the wellcome trust , to advocate for open data .
however , a new gold standard would be created should the eu mandate delivery of all data ,
supply reviewers to check for third party usability , then provide all databases in a central
searchable repository .
there are already established database archiving services such as
figshare and datacite that could cater for an eu account .
the ec 's impact assessment of health research projects published in november 2011 produced
alarming findings .
participation rates ( representation on projects ) and the percentage of
total eu research funding each region receives varied markedly between member states ( table 1 ) .
the original 15 member
states had received 34 times more health research funding under fp7 than the 12 newest
members .
that difference can not be explained by the 3.8 times larger population of the eu-15
members , nor their 13.3 times greater combined gdp , nor even their 12.8 times greater
contribution to the eu budget .
worse , both
the participation rate and the funding have dropped since fp6 , and the eu-12 gets less money
per participation than the rest of the world group ( which includes africa , asia , etc . ) ,
despite contributing financially to the common pool.table 1.distribution of participation in eu research projects and shares of total funding
received by geographical region.fp6 lifescihealthfp7 healthparticipation ( % ) eu contribution ( % ) participation ( % ) eu contribution ( % ) eu-1583.388.478.185.0eu-126.23.05.82.5associated countries6.86.66.97.0rest of the world3.72.09.25.5total100100100100fp : framework programme ; eu : european union ; eu-15 : original 15 member states ;
eu-12 : newest member states ; associated countries : switzerland , israel , norway ,
iceland who contribute to the science budget according to their gdps ; rest of the
world : all other countries ( they can participate but can not lead projects and do not
contribute financially ) .
distribution of participation in eu research projects and shares of total funding
received by geographical region .
fp : framework programme ; eu : european union ; eu-15 : original 15 member states ;
eu-12 : newest member states ; associated countries : switzerland , israel , norway ,
iceland who contribute to the science budget according to their gdps ; rest of the
world : all other countries ( they can participate but can not lead projects and do not
contribute financially ) .
so who is to blame for this state of affairs ? it could be that research infrastructure in
these countries is weak , evidence for which is their lack of participation in major conferences .
the net salary earned on an eu project in
slovenia is only a third of what the same researcher would receive in london .
each time
project proposals are written , all researchers must use local salaries , so that the bulk of
the budget moves to northwest europe , reflecting the higher salaries there . to believe
the
differences reflect living costs ignores the financial hardship of eastern european
researchers .
eu project salary compensation offers no respite , rather it reinforces the
tilted playing field and fuels the brain drain .
the participation to money ratios ( table 1 ) are substantially explained
by this salary difference .
although in march 2012 the eu regional policy commissioner
announced the need to prioritize the issue of east to west brain drain , this issue is barely mentioned in any of the impact assessments or proposals for
horizon 2020 .
our proposition is to pay researchers in new member states the same salary as in western
europe . paying equitably
would let eastern europe use its competitive advantage of
marginally lower living costs to retain and even attract top researchers , so that principal
investigators can assimilate critical masses of young eager talent .
additionally , it
encourages many more applications to eu projects and forces local funders to match those
compensation rates .
this is what eu science stimulation should be . and what is the cost ?
table 1 indicates that doubling all salaries to eastern european researchers would increase
the budget by only 2.5% . | over the past 15 years , the european union ( eu ) has spent around 80 billion on science
research via framework programmes ( fp5 , fp6 and fp7 ) . in 2014 ,
a new programme , horizon
2020 , will likely invest another 70 billion over 6 years .
health research has been a
major part : between 12% and 17% was spent on official fp5 and fp6 health research lines ,
although our work categorizing all eu science projects puts the health - related investment
proportion nearer to 20% . here
, we compare our analyses and experiences with the european
commission 's own impact assessments and plans that inform the horizon 2020 programme .
much
is moving in the right direction but some key gaps are overlooked .
we discuss four areas :
red tape , what to fund , harnessing informatics and neglect of eastern europe . |
although substantial progress has been made in the awareness , prevention , and treatment of cardiovascular disease in women in the united states ( usa ) over the past 10 years , women 's lifetime risk of cardiovascular disease is high .
adverse trends in cardiovascular disease risk factors are a growing concern , partly due to an ongoing increase in average body weight , with nearly two of every three women in the united states ( usa ) over 20 years of age now being classified as overweight or obese .
data from the national health and nutrition survey ( nhanes ) collected from a series of cross - sectional national representative health surveys suggest that obesity prevalence among women in the usa has been stable for more than 10 years and that cardiovascular disease risk factors have been declining in the population , particularly among those with overweight or obesity [ 3 , 4 ] . yet
, nhanes data also show that the prevalence of obesity and metabolic syndrome is markedly higher in rural women than urban residents [ 5 , 6 ] .
metabolic syndrome is a designation given to individuals who have a cluster of risk factors characterized by abdominal obesity and disorders of lipid and carbohydrate metabolism that predispose individuals to cardiovascular disease and type ii diabetes [ 79 ] .
the prevalence of metabolic syndrome components varies between populations due to differences in genetic heterogeneity and variations in lifestyles .
rural midwestern women are recognized as part of a distinct group in the us population who have documented health disparities , with poorer self - reported health , higher rates of sedentary behavior and obesity , lower fitness , and higher risk of other cardiovascular risk disease factors , compared to their urban counterparts [ 5 , 6 , 1013 ] .
the reported findings related to the influence of rurality in terms of geographic locations and associated lifestyles with metabolic syndrome is inconsistent , reinforcing the need to understand the unique health risks for specific rural populations [ 11 , 14 , 15 ] . identifying risk factors for and methods to improve metabolic
health is an important public health issue , especially for this vulnerable rural population , as metabolic syndrome may be a stronger predictor for future risk of cardiovascular disease risk in women than in men .
agencies within the us department of health and human services , such as the national institutes of health ( nih ) and the agency for healthcare quality and safety have designated women in underserved rural communities as priority populations for targeted research to address health disparities , especially as related to cardiovascular disease risk prevention [ 1719 ] .
in addition to obesity , cross - sectional studies also show low cardiorespiratory fitness being a risk factor associated with metabolic syndrome ; however , the relationship between fitness and fatness with metabolic syndrome is complex .
higher cardiorespiratory fitness is associated with more favorable metabolic health even among obese individuals , yet the relative importance of fitness regardless of body composition remains unclear [ 2024 ] .
a prospective longitudinal study of 3,148 healthy adults , ages 18 or older , by lee and colleagues found that maintaining or improving fitness from baseline to an average follow - up of 6.6 year , appeared to attenuate , but not eliminate , some of the negative effects of fat gain as related to the risk of developing metabolic syndrome , and that lowering body fat from baseline lowered the increased risk of developing metabolic syndrome associated with fitness loss .
discrepancies among findings related to the relative combined contributions of cardiorespiratory fitness and fatness with metabolic syndrome could be due to the use of different methods for measuring fitness and fatness .
studies frequently use self - reported physical activity which may be more prone to overestimation , particularly in sedentary populations such as rural women , rather than objective measures of cardiorespiratory fitness .
fatness is most frequently defined as being above a certain measure of body mass index ( bmi ) , because bmi is an easy to assess , low cost , and convenient measure .
recent literature suggests that as obesity management is becoming a larger public health priority , the detection of obesity may need to include measurement of percent body fat , as the nih bmi - based classification ( 30 kg / m ) may significantly underestimate the prevalence of obesity when compared to percent body fat [ 2730 ] .
blew and colleagues suggest that a lower cut - score of bmi ( 25 kg / m ) , referred to as revised bmi in this paper , may be superior for diagnosing obesity in postmenopausal women .
fitness and fatness as risk factors may vary in adult populations , in part due to differences in race / ethnicity or other factors .
we did not find studies that specifically examined objective measures of fitness and percent body fat or their associations in the targeted health disparities group of rural midwestern us midlife and older women , especially women with prehypertension .
individuals with prehypertension , defined by the joint national committee on the prevention , detection , and treatment of high blood pressure ( jnc 7 ) as a systolic blood pressure of 120139 mmhg and/or diastolic blood pressure of 8089 mmhg , are at greater risk for developing hypertension .
individuals having blood pressures in the upper half of the prehypertensive range ( 130135/8589 mmhg ) have more than a 2-fold increase in relative risk from cardiovascular disease compared to those with normal blood pressure .
this study investigated the fitness , fatness , and metabolic syndrome in an understudied population of rural midwestern us midlife and older women who were prehypertensive and enrolled in the wellness for women : dashing toward health community - based clinical trial . specifically , the purposes of this cross - sectional study were to ( 1 ) describe estimated cardiorespiratory fitness , bmi , percent body fat , and metabolic syndrome in midlife and older rural women with prehypertension , and ( 2 ) investigate associations of fitness and/or fatness with the occurrence of metabolic syndrome .
we anticipated that women enrolled in this dashing toward health clinical trial might have a high prevalence of metabolic syndrome , as all had documented blood pressures in the prehypertensive range and were not taking any antihypertensive medication .
this study cohort included rural women with prehypertension , ages 4069 , who were enrolled in the wellness for women : dashing towards health community - based randomized - controlled trial .
briefly , this community - based clinical trial compared the effectiveness of two theory - based tailored 12-month interventions for promoting healthy eating and activity using different delivery methods ( internet or mailed printed materials ) to standard advice only , with the goal of facilitating a reduction in blood pressure among rural women with prehypertension who were not candidates for drug therapy .
recruitment methods included advertisements for women to participate in free community - based blood pressure screenings and participation by having a booth at local health fairs , craft shows , and other social events .
women with blood pressure readings at screening sites that were from 10 mmhg below to 10 mmhg above the prehypertension systolic range or five mm hg below to five mmhg above the prehypertension diastolic range were told briefly about the study .
if interested , women were asked to provide their phone and address for mailing study information and for a follow - up screening interview to establish eligibility for the study . to confirm prehypertensive status for inclusion in the study , women attended two visits one week apart to assess blood pressure by research nurses at our local research office . written informed consent was obtained from all subjects prior to participation .
the institutional review board of the university of nebraska medical center approved this study ( approval no .
other inclusion criteria included being able to walk one - mile without an assistive device or oxygen and answering no to all questions on the physical activity readiness questionnaire ( par - q ) or obtaining medical clearance from their physician to participate .
women were excluded from the study if they were taking antihypertensive medication , including diuretics , or systemic glucocorticoids , or if they reported consuming more than 14 alcoholic drinks per week .
women were excluded if they were unable to walk one mile continuously without stopping to rest and/or presented with other physical or medical restrictions that would preclude following the jnc 7 recommendations for moderate physical activity and healthy eating .
the women were primarily non - hispanic white ( 97.2% ) , married ( 83% ) , employed outside of the home full- or part - time ( 79% ) , had some education beyond high school ( 82% ) , and lived in a household with $ 40,000 or more income ( 71.2% ) ( see table 1 ) .
only 16 ( 5.5% ) of the participants had type ii diabetes , of which nine of the 16 had metabolic syndrome .
cross - sectional baseline data was examined from women enrolled in the wellness for women : dashing toward health community - based clinical trial .
participants were scheduled for two visits scheduled one week apart at a centralized research office located within 70 miles of their homes . for the second visit ,
a calibrated mercury sphygmomanometer with an appropriate size cuff for each woman was used following standardized ausculatory methods . at each visit
, at least two blood pressure measurements were obtained , separated by at least 30 seconds .
the blood pressure was the mean of the two measurements that were within 5 mmhg for both systolic and diastolic measures .
the baseline blood pressure recorded was the mean of the two final blood pressure measurements across the 2 visits .
participants were asked to avoid caffeine , exercise , and smoking for at least 30 minutes prior to a measure .
blood specimens were drawn after a 12-hour fast to determine high - density lipoprotein ( hdl - c ) , triglycerides , and fasting glucose following a standardized protocol to be used for calculating the presence of metabolic syndrome . the tanita model ( tbf-215 , tanita corporation of america , inc .
, arlington heights , il 60005 - 9824 ) was used in this field trial to measure height , weight , and percent body fat following the manufacturer 's recommendations .
each woman was measured at least two times until two exact measures of height were obtained .
as the bioelectrical impedance analysis system methodology for determining percent body fat is sensitive to hydration status , women were asked to fast within 4 hours of the test , not exercise within 12 hours of the test , avoid alcohol or diuretics before testing , and to void the bladder within 30 minutes of the test .
although the bioelectrical impedance method for estimating body fat percentage underestimates the percent body fat compared to the underwater weighing and dual x - ray absorptiometry ( dxa ) methods , this method nevertheless was appropriate for the overall community - based clinical trial because it is a safe , non - invasive , and reliable clinical method shown to detect similar changes in percent body fat during weight loss in obese women that were comparable to the magnitude of change as determined via dxa [ 3640 ] .
waist circumference was measured by placing a tape in a horizontal plane around the abdomen at the level of the iliac crest .
the tape was snug and parallel to the floor but was held without skin compression .
the measurement was taken at the end of expiration , with the average of two trials recorded [ 41 , 42 ] .
cardiorespiratory fitness was estimated using the submaximal 1-mile walk test , a safe and adaptable test for the women in this community - based trial , whereby each woman was asked to walk as fast as possible over a 1-mile indoor track .
estimated vo2max was calculated using weight , age , gender , total walk time , and 15-second post - activity heart rate in the equation validated for older women .
metabolic syndrome was defined in this study according to the third report of the national cholesterol education program expert panel on detection , evaluation , and treatment of high blood cholesterol in adults , adult treatment panel iii ( ncep atp iii ) which includes three or more of the following : waist circumference > 88 centimeters ; systolic blood pressure of 130 mmhg or diastolic blood pressure of 85 mmhg ; fasting plasma glucose 100 mg / dl or on medication for high blood glucose ; hdl cholesterol < 50 mg / dl ; or triglycerides of 150 mg / dl .
the ncep atp iii guidelines include women on antihypertensive medication as a criterion ; however , eligibility criteria for this study excluded women on any antihypertensive medication so this was not an issue .
ibm spss statistics , version 19 for windows ( spss inc , chicago il ) was used to analyse the data . to address the first purpose of this paper , descriptive statistics were used to describe the sample , and a test
was used to compare women with and without metabolic syndrome with respect to demographic characteristics ( e.g. , education , employment ) , self - reported health , estimated cardiorespiratory fitness , bmi , percent body fat , and the five criteria comprising metabolic syndrome . to address the second purpose of this paper , logistic regression
was used to predict the occurrence of metabolic syndrome from percent body fat and cardiorespiratory fitness as continuous variables , alone and in combination .
all models were adjusted for age , income , and education . because the literature indicates that cardiorespiratory fitness may influence metabolic health regardless of fatness
, we fit a hierarchical model with step 1 including percent body fat only , step 2 adding the estimated vo2max , and at step 3 adding the product of percent body fat and estimated vo2max in order to evaluate the possibility that the effect of one might depend on the level of the other ( interaction ) .
we also used a second method to examine the combined effects of cardiorespiratory fitness and body composition to predict occurrence of metabolic syndrome from the fitness and fatness categories using logistic regression where women were grouped into four categories of ( 1 ) fit and not fat , ( 2 ) fit and fat , ( 3 ) not fit and not fat , and ( 4 ) not fit and fat , using literature cited cut - scores for fit and fat .
we repeated this analysis using both percent fat and bmi , as practitioners in the field are likely to find bmi a more easily implemented method of determining obesity in rural patients / clients than is the percent body fat .
( 25 ml / kg / min ) or fit ( > 25 ml / kg / min ) , consistent with age - appropriate guidelines by the american college of sports medicine ( acsm ) where the cut - score selected was closest to the mean age of our population .
fat and those with < 30% were considered not fat consistent with the literature for midlife and older women . for bmi
, we used the revised cut - score of bmi 25 kg / m to categorize women as fat and < 25 kg / m to categorize women as not fat as recommended by blew and colleagues , rather than the nih bmi cut- score ( 30 kg / m ) , as this lower cut - score is purported to better correlate with percent body fat to define obesity in postmenopausal women .
fit / not fat and unfit / not fat had metabolic syndrome regardless of which measure of body composition was used to create the categories , we omitted these two categories from the logistic regression analyses to avoid the estimation problems that such structural zeros cause .
all logistic regression models were adjusted for age , education , and household income . in all analyses ,
of the 289 rural women , 31% ( n = 90 ) of women met the criteria for metabolic syndrome , of which 70% were classified as obese by nih bmi standards ( 30 kg / m ) , and all were classified as obese using the revised bmi score ( 25 kg / m ) and the percent body fat cut - score ( 30% ) , respectively .
none of the 18.3% ( n = 53 ) classified as normal weight by the revised bmi ( 2024.9 kg / m ) or as having < 30% body fat ( 26.6% ; n = 53 ) met the definition of metabolic syndrome .
table 1 highlights the characteristics of the total sample and women categorized with and without metabolic syndrome .
overall , 40.1% ( n = 116 ) of women reported their general health as very poor to fair , with 51% ( n = 46 ) of women with metabolic syndrome reporting very poor to fair general health .
differences were observed in estimated fitness categories , bmi , percent body fat , and all five criteria for metabolic syndrome between women categorized with and without metabolic syndrome , with women classified with metabolic syndrome having less desirable results .
rates of obesity in the total sample was 41.8% ( n = 127 ) , 81.7% ( n = 236 ) and 94% ( n = 273 ) as defined by nih based bmi , the revised bmi , and percent body fat , respectively
.
table 2 presents the results of the logistic regression models , adjusted for age , income and education , predicting occurrence of metabolic syndrome from body percent body fat and fitness , alone and in combination .
higher body fat percentage ( or = 1.21 , 95% ci = 1.141.29 , p <
fitness category considered alone also was a significant predictor , with the odds of having metabolic syndrome 7% lower with higher fitness ( or = 0.93 , 95% ci = 0.900.97 , p = 0.001 ) .
with body fat percentage , addition of the estimated vo2max and/or the interaction of percent body fat and fitness did not significantly improve the fit of the model .
unfit / fat and fit / fat categories using the two body composition methods . when using body fat percentage , being in the fit group reduced the odds of metabolic syndrome by approximately 75% compared to being in the unfit group ( or = 0.25 , 95% ci = 0.120.52 , p < 0.001 ) .
a similar pattern was observed when the revised bmi obesity cut - score as appropriate for postmenopausal women , with a reduction in odds of metabolic syndrome by 59% ( or = 0.41 , ci = 0.190.87 , p = 0.02 ) .
this study explored the associations between fitness and fatness with the presence of metabolic syndrome in an understudied population of midlife and older rural us midwestern women with prehypertension . because the cohort consisted of women with documented prehypertension , we anticipated these women might have a high prevalence of metabolic syndrome .
though rural populations differ from each other , our findings that 31% of this cohort had metabolic syndrome was similar to the work of vaughan and associates who reported that 33% of rural australian women ages 2574 years were classified as having metabolic syndrome . however , our prevalence of metabolic syndrome ( 31% ) was much less than the 40.2% reported for rural women across the usa from the 19992006 nhanes data , perhaps in part due to our inclusion / exclusion criteria which included women able to walk one - mile without assistance and excluded women with hypertension .
while the overall cohort had a high percentage ( 40.1% ) who reported their quality of health being very poor to fair , the percentage of women with metabolic syndrome who rated their health as very poor to fair was much higher ( 51% ) .
this is consistent with findings from other rural us midwestern women . of those with metabolic syndrome ,
the finding of a high prevalence of both obesity and low estimated cardiorespiratory fitness are of concern , as the evidence demonstrates that obesity , particularly abdominal obesity , is a core component of metabolic syndrome and that a minimal level of cardiorespiratory fitness is important for overall health .
our results are consistent with the work of shah and braverman who compared bmi to a direct determination of percent body fat using duel energy x - ray absorptiometry ( dxa ) with findings that nih - based bmi standards for defining obesity significantly underestimated the prevalence of obesity , especially in women with advancing age .
although we used the bioelectrical impedance method for estimating percent body fat which has a large standard of error estimation of 3.5 to 5.0% and tends to underestimate the percent body fat compared to the gold standard of dual - x - ray absorptiometry ( dxa ) , we felt that examining body composition using bioelectrical impedance analysis for percent body fat would further inform this study of rural women [ 37 , 38 ] .
we did attempt to reduce the risk of measurement error by having the participants follow pre - test control conditions that affect hydration levels .
as noted by shah and braverman , bmi may ignore the influence of sarcopenic obesity in aging women .
for this reason , several groups of researchers have attempted to identify new cut - points for bmi that would better categorize individuals as obese , with studies suggesting obesity cut points for women should range from 24 to 25.5 kg / m [ 27 , 29 , 30 ] .
our results also suggested the nih bmi cut - score ( 30 kg / m ) for obesity misclassified 50.5% of women who were defined as obese by percent body fat ; however , the revised bmi cut - score resulted in a more similar percentage of women classified as obese ( 81.6% ; n = 236 ) as those classified obese by body fat ( 94.5% ; n = 273 ) . using a revised cut - score of 25 kg / m to better categorize women as fat
fit / fat women ( see table 3 ) had lower odds of prevalent metabolic syndrome similar to that when using percent body fat in the analysis .
rural practitioners may want to consider using this revised bmi score to define obesity in midlife and older women .
while obesity is highly associated with a cluster of metabolic abnormalities , current studies suggest that 2030% of obese individuals appear to maintain a favorable metabolic profile , perhaps due to having a higher cardiorespiratory fitness level .
aging has been associated with a decrease in lean mass and an increase in percent body fat , both which have been identified as contributing to the development of metabolic syndrome .
studied women ages 5779 with results that indicated low cardiorespiratory fitness could be considered a feature of metabolic syndrome .
they used a more accurate measure of fitness , symptom - limited maximal exercise stress test on a cycle ergometer , in contrast to our 1-mile walk test which provided an estimate of cardiorespiratory fitness .
our findings were similar to hassinen and colleagues in that when examining the association of fitness alone with the presence of metabolic syndrome , women with higher fitness had 7% lower odds of having metabolic syndrome and fitness was associated with lower odds of metabolic syndrome when classified as
fit / fat by either the percent body fat cut - score or the revised bmi cut - score .
when we entered both % body fat and fitness as continuous variables , fitness was not significant .
our finding is also similar to hassinen and associates who found that abdominal obesity as measured by waist circumference markedly weakened the association between cardiorespiratory fitness and metabolic syndrome .
our study is unique in that it included successful recruitment of a large sample of midlife and older rural women with prehypertension , an understudied population that is recognized for having health disparities .
this study included complete data for all variables and included two body composition measures ( percent body fat in addition to bmi ) and an objective measure of estimated cardiorespiratory fitness .
the cross - sectional design limited any interpretation of causality . because recruitment used a convenience sample of female volunteers , the women in this study
may not have been representative of , or generalizable to , the overall population of women of that region .
there was potential for misclassification of both fitness and fatness due to the limitations of measurement methods selected for use in this community - based clinical trial ( percent body fat via bioelectrical impedance and fitness via the 1-mile submaximal field test ) as opposed to more direct , complex laboratory studies of dxa for percent body fat and a maximal , graded exercise test for fitness .
our women may not have been representative of the population - at - large as they were not hypertensive , able to walk at least one mile unassisted , and were volunteers seeking a lifestyle intervention to reduce blood pressure .
in addition , we did not examine medications that might impact weight nor did we collect the proportion of women on statins or other lipid - lowering agents . in conclusion , these rural women with prehypertension had a high prevalence of metabolic syndrome , placing them at high risk for cardiovascular disease .
metabolic syndrome was associated with a high prevalence of obesity , lower estimated cardiorespiratory fitness , and more self - reports of poor to fair health status .
nih bmi - based classifications appeared to underestimate obesity in this population whereas the revised bmi cut - score produced results that were similar to the percent fat cut - scores for defining obesity .
rural practitioners may find using a bmi cut - score of 25 kg / m to be useful in defining obesity in this population .
there is an indication that fitness may be important to reducing the risk of metabolic syndrome , though the presence , and potentially the degree of fatness , may reduce this association . | background .
this study investigated the associations of fitness and fatness with metabolic syndrome in rural women , part of a recognized us health disparities group . methods .
fitness , percentage body fat , bmi , and metabolic syndrome criteria were assessed at baseline in 289 rural women with prehypertension , ages 4069 , enrolled in a healthy eating and activity community - based clinical trial for reducing blood pressure .
results .
ninety ( 31% ) women had metabolic syndrome , of which 70% were obese by bmi ( 30 kg / m2 ) , 100% by percentage body fat ( 30% ) , and 100% by revised bmi standards ( 25 kg / m2 ) cited in current literature .
hierarchical logistic regression models , adjusted for age , income , and education , revealed that higher percentage body fat ( p <
0.001 ) was associated with greater prevalence of metabolic syndrome . alone , higher fitness lowered the odds of metabolic syndrome by 7% ( p < 0.001 ) , but it did not lower the odds significantly beyond the effects of body fat . when dichotomized into
fit and unfit groups , women categorized as fat had lower odds of metabolic syndrome if they were fit by 75% and 59% , for percentage body fat and revised bmi , respectively .
conclusion . among rural women with prehypertension ,
obesity and fitness were associated with metabolic syndrome .
obesity defined as 25 kg / m2 produced results more consistent with percentage body fat as compared to the 30 kg / m2 definition . |
os objetivos do presente estudo foram avaliar as concentraes de flor ( f ) em leites integrais , desnatados e achocolatados disponveis comercialmente no brasil e estimar a ingesto diria de f a partir destas fontes .
as concentraes de f foram determinadas em 23 marcas de leite , aps difuso facilitada por hmds , usando um eletrodo on - especfico para f. a ingesto provvel de f por kg de peso corporal foi estimada , baseando - se em volumes sugeridos de consumo de frmulas infantis , para crianas entre 1 e 12 meses de idade .
as concentraes de f variaram entre 0,02 e 1,6 g / ml para todas as marcas analisadas .
os leites integrais e desnatados apresentaram as menores concentraes de f , variando entre 0,02 e 0,07 g / ml .
com relao aos leites achocolatados , trs marcas apresentaram concentraes de f acima de 0,5 g / ml . algumas marcas de leites achocolatados excederam a dose de flor reconhecida como o limite mximo de exposio a fim de se evitar o desenvolvimento da fluorose dentria , sem se considerar outras fontes de ingesto de f.
as altas concentraes de f encontradas em algumas marcas de leites achocolatados no presente estudo indicam que muitos produtos podem contribuir significativamente para a ingesto total de f , reforando a necessidade de se avaliar o contedo de f em alimentos e bebidas consumidos por crianas pequenas .
fluoride is the most clinically used mean of reducing dental caries . besides the decline in tooth decay ,
the widespread use of fluoride has contributed to an increase in the prevalence of dental fluorosis3 . since dental fluorosis results of excessive ingestion of fluoride during enamel formation
, all sources that contribute to the total intake of this ion should be considered to evaluate the risk of development of dental fluorosis . regarding infants
, the major dietary intake constitutes the breast milk , cow 's milk and infant milk formulations , in which the fluoride content varies widely4,12,15,21 .
milk is a universal food for newborns and growing mammals and contains all of the essential nutrients for their development and growth12 .
a decline in breast feeding has been noted , whereby a considerable number of infants are weaned early and artificially fed before they are a month old15 .
thus , the concentration of fluoride in cow 's milk and infant formula plays an important role in the supply of fluoride to infants . although cow 's milk is reported to have low fluoride concentrations9,15,18 , some studies related wide variations in fluoride concentrations in dairy milks9,12,14,15 . in addition ,
thus , the aim of the present study was to evaluate the fluoride concentrations in whole , defatted and chocolate milks commercially available in brazil and to estimate the daily fluoride intake from these sources .
twenty - three brands of uht ( ultra high temperature ) milks were purchased in supermarkets of bauru , so paulo state , brazil , in october 2004 .
they were divided into three groups , as follows : gi chocolate milks , gii whole milks and giii defatted milks .
the division of the brands into the groups , as well as the manufacturers , is presented in table 1 .
the boxes of milks were opened on the day of the analysis and 1 ml of milk was used for fluoride analysis .
fluoride determinations were carried out after overnight hmds - facilitated diffusion , as described by taves19 , using the ion - specific electrode ( orion research , cambridge , ma , usa , model 9609 ) .
a set of standards ( ranging between 0.0253.200 ppm f ) was prepared , using serial dilution from a 100 ppm naf stock solution ( orion # 940907 ) and diffused in triplicate in the same way as the samples .
the millivoltage potentials were converted to g f using a standard curve with a coefficient correlation of r>0.999 .
table 1 presents all brands of cow 's milk analyzed , divided into the three groups ( chocolate , whole and defatted milks ) , the manufacturers , as well as the fluoride concentrations determined for all brands of milk ( expressed in g / ml ) .
all whole and defatted milks had low fluoride concentrations , ranging from 0.02 to 0.07 g / ml f. among chocolate milks , we found higher fluoride concentrations , ranging from 0.04 to 1.6 g / ml . three brands of chocolate milks had fluoride concentrations above 0.5 g / ml ( nestl nescau , toddy pronto and toddynho vitaminado ) .
average infant body masses and suggested volumes of formula consumption for infants from one to twelve months of age are shown in table 2 . using these data , the possible daily fluoride ingestion per kg body mass , presented in table 3 ,
daily fluoride intake ( mg / kg body weight ) from whole and defatted milks ranged from 0.003 0.0110 , 0.0020.008 and 0.0020.008 , while daily fluoride intake from chocolate milks ranged from 0.0060.240 , 0.005 0.192 and 0.0040.173 , respectively for infants aging 1 , 6 and 12 months .
in the present study , whole and defatted milks had low fluoride concentrations . the values found ( 0.02 - 0.07 g / ml ) are in accordance with other previous reports8,12,21 .
these low fluoride levels were expected because fluoride is poorly transported from plasma to milk , and concentrations of fluoride in milk remain low even when the intake of fluoride by the mammal is high18 . analyzing fluoride concentrations in whole and
defatted milks in relation to daily fluoride intake per kilogram of body mass ( table 3 ) , it can be concluded that the use of such products is safe , concerning the dosages believed to increase the risk of development of dental fluorosis2 . among the different types of milk analyzed in this study , these products may be the most consumed . regarding chocolate milks , fluoride concentrations varied widely among the brands analyzed .
three brands of chocolate milks had fluoride concentrations above 0.5 g / ml : nescau ( nestl ) had 0.5 g / ml f , while toddy pronto and toddynho vitaminado ( both from quaker ) , had 1.6 g / ml f. we had already found high fluoride levels in chocolate milks6,14 , but we were not able to identify the possible source of fluoride .
powdered chocolate was previously analyzed ( data not shown ) and we found low fluoride levels ( ranging between 0.05 and 0.09 mg / g ) , excluding cocoa as the possible fluoride source . when analyzing the values found for all brands of chocolate milks in the present study , we can not explain the differences among fluoride levels of the brands of chocolate milks evaluated .
we have already analyzed foods and beverages ( including toddynho ) produced in three different dates , and the differences among the dates of production were small and not statistically significant6 .
this does not allow the precise estimation of their contribution to the total daily fluoride intake .
it is considered that the optimal range of fluoride intake is 0.05 - 0.07 mg f / kg body weight / day2,9 .
taking into account average body masses and suggested volumes of formula consumption for infants of one , six and twelve months of age ( table 3 ) , it is observed that some brands of chocolate milks exceeded the upper limit of daily fluoride intake ( nestl nescau ) , or even exceeded the threshold level for development of dental fluorosis ( toddy pronto and toddynho vitaminado ) .
the bioavailability of milk is another point to be considered . due to the high calcium concentrations in milk
, there is a possibility of diminution of fluoride absorption from gastro - intestinal tract18,20,22 .
thus , the calculations made for these products could be overestimated . on the other hand ,
milk is also rich in fats , what are known to increase the lag time of the food or beverage in the stomach20,22 .
although milk is known to interfere with the rate of fluoride absorption , it is demonstrated that 67 to 82% of total fluoride in milk is absorbed17 .
our special attention concerns children older than 11 months , since this period coincides with the beginning of the development of the permanent teeth crowns . even for brands
that did not exceed the upper optimal limit of fluoride intake , the risk of development of dental fluorosis exists , because children at this age are fed with other foods than milk , ingesting fluoride from water , beikost and formulas , etc .
the ingestion of these sources with other fluoride - rich products is a possibility that must be considered .
it seems likely that fluoride intake from beikost rarely averages more than 0.02 mg / kg body weight / day9 .
mean daily fluoride intake from water by itself up to 9 months of age is usually < 0.05 mg11 .
powdered or liquid concentrated infant formulas , as well as soymilk , appear to have high fluoride concentrations3,4,5 .
it is estimated that the fluoride intake of children aging 6 and 12 months from powdered milk - based formulas reconstituted with optimally fluoridated water is 0.145 and 0.116 mg / kg body weight / day , respectively9 , thus exceeding the optimal range of fluoride intake2 .
it must be also considered that some parents start to use fluoride dentifrice when the first teeth erupt and it is known that fluoride ingestion from dentifrice is inversely related to the age of the child4,16 . however , a recent study by brofitt , et al.1 has shown that the mean fluoride ingestion from dentifrice is negligible during the first twelve months . taking these situations together , total fluoride intake will be higher and probably will exceed the upper limit recommended for fluoride intake .
taking into account that enamel fluorosis can occur following either an acute or chronic exposure to fluoride during tooth formation7 this assumes a greater relevance , showing the importance of monitoring fluoride intake by small children . in this sense
, dairy companies should consider providing information to the public on fluoride content of all dairy products .
in addition , public health measures should be taken , informing the consumer with respect to fluoride ingestion and its adverse effects .
the high fluoride concentrations found in some brands of chocolate milks in the present study , together with many others around the world , indicate that many products may be important contributors to the total fluoride intake , reinforcing the need of assaying fluoride content of foods and beverages consumed by small children . | objectives : the aims of the present study were to evaluate the fluoride ( f ) concentrations in whole , defatted and chocolate milks commercially available in brazil and to estimate the daily f intake from these sources.material and methods : f concentrations were determined for 23 brands of milks , after hmds - facilitated diffusion , using a f ion - specific electrode .
possible f ingestion per kg body weight was estimated , based on suggested volumes of formula consumption , for infants aging 1 to 12 months.results:f concentrations ranged from 0.02 to 1.6 g / ml f for all brands analyzed .
whole and defatted milks had the lowest f concentrations , ranging from 0.02 to 0.07 g / ml .
with respect to chocolate milks , three brands had f concentrations above 0.5 g / ml . some brands of chocolate milks exceeded the dose regarded as the threshold level for the development of dental fluorosis , without taking into account other sources of fluoride intake.conclusion:the high fluoride concentrations found in some brands of chocolate milks in the present study indicate that many products may be important contributors to the total fluoride intake , reinforcing the need of assaying fluoride content of foods and beverages consumed by small children . |
the mode of action of many drugs in clinical use for the treatment of cancer , genetic disorders , and viral diseases is believed to be based on their binding to nucleic acids and subsequent modification of the genetic material [ 1 , 2 ] .
the discovery of the double helical structure of deoxyribonucleic acid ( dna ) by watson and crick in 1953 and the subsequent revelations relating to the structural features to the basic functional properties of dna such as replication and transcription have proven the utility of dna as a potential target for the development of biologically active agents [ 4 , 5 ] .
ribonucleic acid ( rna ) also play essential role in normal biological process as well as in the progression of many diseases that led to growing interests in exploiting in designing of new class of therapeutics [ 68 ] .
a number of small organic molecules are endowed with cytotoxic properties that are exerted through highly specific but noncovalent and reversible interactions with nucleic acid structures [ 913 ] .
intercalation occurs when planar aromatic molecules are sandwiched between two adjacent base pairs of nucleic acids .
nonintercalative binding occurs in the major or minor grooves of nucleic acids without inserting any part of the binding molecule between base pairs .
the physical and molecular basis of binding of natural alkaloids to nucleic acids has been a subject of extensive study in the recent past [ 1417 ] .
elucidation of the nature , strength and energetics of these interactions may reveal the molecular basis of many antitumor and antiviral natural products and provide helpful guidance for rational drug design of new potential chemotherapeutic agents .
the intercalating ligands constitute one of the most widely studied groups as they form important class of compounds for cancer therapy . in this context , considerable attention has been paid to naturally occurring protoberberine and benzophenanthridine alkaloids for their low toxicity and wide range of biological activities that include antimicrobial , antileukemic , anticancer , and topoisomerase inhibitory activities [ 1926 ] .
this review focuses on the complex formation of two groups of isoquinoline alkaloids with various polymorphic nucleic acid structures in relation to their anticancer activities .
these alkaloids ( figure 1 ) include berberine , palmatine , and coralyne of the protoberberine group , sanguinarine of the benzophenanthridine group , and some of their derivatives .
it is generally recognized that the most sensitive target for the inhibition of rapidly proliferating cancer cells are those molecules involved in aspects of dna function , especially dna itself .
it is , therefore , not surprising that most clinically useful anticancer compounds act against such targets . in this context
, significant attention has been focused on the dna binding properties of protoberberine group of alkaloids that constitute an important class of isoquinoline alkaloids widely present as active constituents of folk medicines used world over for centuries .
several protoberberine alkaloids and their derivatives have been investigated as potential antitumor agents , among which berberine , palmatine , and coralyne ( a synthetic analogue of berberine ) represent the most intensively studied molecules .
the physicochemical properties of protoberberine alkaloids presented in table 1 provide an understanding of the similarity and differences in their chemical structures ( figure 1 ) and properties .
the basic skeleton of the quaternary protoberberine moiety is 5,6-dihydro[a , g ] quinolizinium . in berberine , there is an addition of 2,3-methylenedioxy and 9,10 dimethoxy group to the above basic structure and thus the structure of berberine becomes 7,8,13,13a - tetrahydro-9,10-dimethoxy and 2,3 methylenedioxy berberinium . on the other hand , palmatine has the same tetracyclic structure ( 7,8,13,13a - tetradehydro-9,10-dimethoxy-2,3 , methylene dioxy berberinium ) as berberine but differs by the nature of the substituents at position 2,3 on the benzo ring ; being dimethoxy for palmatine .
similarly coralyne has the basic structure with a methyl group attached to the c8 position .
notably , while berberine and palmatine have buckled structures , coralyne has a planar structure . in aqueous condition ,
the absorption spectra of berberine and palmatine are similar while that of coralyne is remarkably different .
both berberine and palmatine have weak intrinsic fluorescence emission spectra while coralyne has a strong fluorescence emission spectrum in aqueous and alcoholic media .
berberine , palmatine , and coralyne are achiral molecules as evidenced from circular dichroism spectroscopy .
it exhibited ph dependent structural equilibrium between the iminium ( charged ) and the alkanolamine ( neutral ) forms with a pka value of 7.4 .
sanguinarine existed almost exclusively as iminium and alkanolamine forms in the ph rang 1.06.0 and 8.511.0 , respectively .
one interesting point to be noted is that the fluorescence of alkanolamine form is more intense than that of iminium form .
the most convenient tool for detecting the complex formation between nucleic acids and small molecules is absorbance spectra . like many other small molecules that bind nucleic acids , berberine has by virtue of heterocyclic structure visible absorption band where dna does not contribute and hence is convenient to monitor the binding .
the complex formation essentially involves bathochromic and hypochromic effect on its absorption spectrum with clear isosbestic points indicating the onset of equilibrium binding .
the bathochromic shift of the spectrum of the free alkaloid on interaction with dna indicates the binding of individual alkaloid to dna substrate , that is , no aggregation prevails .
moreover , such red shift presumably arises from the strong intermolecular interaction involving the effective overlapping of the electronic cloud of the base pairs of dna with that of the alkaloid .
again , the less polar environment of the bound ligand can cause the hypochromic effect on the free absorption spectrum of the ligand and intercalative positions are hydrophobic in nature .
a large number of studies reported the complexation of berberine with calf thymus dna possibly by a mechanism of partial intercalation [ 15 , 16 ] and references therein .
[ 30 , 3638 ] have first demonstrated the base and sequence specificity of berberine from studies with several naturally occurring and synthetic dnas of differing base composition and sequences as evidenced from hypochromism and bathochromism in the absorption spectrum ( figure 2 ) , remarkable enhancement of steady - state fluorescence emission intensity , stabilization of dna against thermal denaturation , and perturbation in the circular dichroism spectrum .
berberine increased the contour length of sonicated rod like duplex b - dna depending on base composition and induced the unwinding - rewinding process of covalently closed superhelical dna with an unwinding angle of 13. using high - field nmr , saran et al .
first proposed that berberine binds to double helical calf thymus dna by a mechanism of partial intercalation in which the planar isoquinoline moiety remained intercalated between base pairs .
the partial intercalation model was further substantiated from several analytical techniques that included absorption , fluorescence , isothermal titration calorimetry , differential scanning calorimetry , electrospray ionization mass spectroscopy , in combination with computer molecular modeling studies [ 27 , 4043 ] . however , a fluorescence spectral study of berberine with calf thymus dna and a high - resolution hnmr and uv spectroscopy in conjunction with molecular modeling studies on berberine binding to various short oligonucleotide duplexes ( b - form ) favoured a groove binding geometry .
this suggested that berberine is located in the minor groove with a preference for at sequences , lying with the convex side on the helix groove where the positively charged nitrogen atom is close to the negative ionic surface of the oligonucleotide helices .
zhu et al . supported the interaction of berberine with b - dna by partial intercalation in which isoquinoline part intercalated in presence of -cyclodextrin .
however , chen et al . studied the interaction of berberine with several double stranded oligonucleotides [ d(aagaattctt)2 , d(aaggatcctt)2 , and d(aagcatgctt)2 using electrospray ionization mass ( esi - ms ) and fluorescence spectroscopic techniques to establish the sequence selectivity and preference of binding to these structures .
the results from esi - ms and fluorescence titration experiments indicated that the sequence selectivity of berberine was not significant and remarkable at rich dna binding preference was not observed .
it is known that alternating gc polymer and its methylated analogue structures assumes left - handed conformation ( z - dna ) in high molar salt ( na , k ) , in low molar divalent cations ( ca , mg , ni ) , micromolar concentration of hexamine cobalt chloride , and millimolar concentration of polyamines . in order to analyze the binding of berberine to z - form dna , kumar et al .
reported that z - dna structure remained invariant in the presence of berberine up to a nucleotide phosphate /alkaloid
molar ratio of 0.8 and suggested that berberine neither bound to z - form dna nor converted the z - dna to the bound b - form structure like other intercalators .
the molecular architecture of the h - from dna appears to be unique , stable left - handed structure with hoogsteen base pairs , is distinctly different from the right - handed b - dna and left - handed z - dna [ 15 , 16 ] .
the binding of berberine to h - dna was found to be non - cooperative resulting in perturbation of the circular dichroism spectrum and generation of extrinsic circular dichroism bands with opposite sign and magnitude compared to the b - form dna .
it was suggested that berberine could be used as a probe to detect the alternation of structural handedness due to protonation and may potentiate its use in regulatory roles for biological functions .
the formation of intermolecular triplex offers a means of achieving sequence specific recognition of duplex dna forming specific hydrogen bonds .
although the third strand oligonucleotide possesses specific sequence recognition properties , the binding is not so strong which results in a relatively low stability of the triplex structure .
the instability of triple helical structures under normal physiological conditions is a critical limitation that restricts their uses in vivo .
several intercalating ligands have now been reported that can preferentially stabilize triplex structures [ 5055 ] .
ren and chaires have shown that berberine preferentially and very strongly bound to txa.t triplex compared to at duplex using novel competition dialysis and thermal melting experiments.recently das et al
. demonstrated that berberine stabilized the hoogsteen base - paired third strand of txa.t triplex without affecting the watson - crick strands as revealed from thermal melting ( figure 3(a ) ) and circular dichroic studies ( figure 3(b ) ) and proposed that it binds to the third strand presumably by mechanism of intercalation .
thermodynamics of berberine - triplex complexation revealed that the binding process was exothermic and entropy driven favouring a tight binding at the intercalation site . in recent years , recognition that certain sequences promote formation of higher - order structures of dna has revealed new targets for chemotherapy [ 5860 ] .
these structures can form the association of four single strands containing tracts of guanine bases ( tetramer ) , and can also arise within one or two strands containing several of these sequences as the strands fold back upon themselves . in order to understand the potential of quadruplex dna as a chemotherapeutic target ,
guanine rich sequences with the ability to form quadruplex structures are found in telomeres at the end of eukaryotic chromosomes ( ttaggg repeats in humans ) as well as in promoter regions of genes and in recombination sites [ 61 , 62 ] .
the observations that telomerase is expressed in malignant but not normal tissues led to the paradigm that drugs that interfere with the action of telomerase could be expected to cause the death of cancer cells .
if interactions of telomeric proteins with specific sequences of telomeric dna are lost , this also leads to cell death by apoptosis .
this suggests that compounds that bind to dna and inhibit protein binding would cause the rapid death of tumor cells .
compounds that stabilize quadruplex dna , presumably preventing interactions with proteins have been shown in vitro to inhibit telomerase .
recent studies show that berberine inhibits telomere elongation and binds strongly to various g - quadruplex dna structures and inhibits telomerase activity [ 50 , 67 ] .
thus berberine may be used as a target to probe for g - quadruplex dna for its development as potential antitumor drugs .
palmatine is structurally similar to the well - known isoquinoline alkaloid berberine . to evaluate the affinity and mode of binding to b - dna , kluza et al . and
bhadra et al . reported that addition of calf thymus dna caused marked changes in the absorption spectrum of palmatine ( figure 4 ) with strong hypochromic and bathochromic shifts , with well - resolved isosbestic points , and with increased thermal melting temperature to form stable complexes with b - dna .
further from electric linear dichroism studies , it was interpreted that palmatine binds either by an intercalation or by an external stacking parallel to the base pairs . however , an earlier study by yu et al . proposed a nonintercalative binding of palmatine .
recently , the author 's laboratory demonstrated the base and sequence - dependent binding of palmatine and established that palmatine formed a partial intercalation complex with all the polymers except the homo and heteropolymer of gc , with which it formed a nonintercalative complex .
the binding of palmatine to calf thymus dna was further characterized by thermodynamic studies using isothermal titration calorimetry and the binding process was exothermic and entropy - driven which favour the intercalative mode of binding .
it does not bind to z - dna nor does it convert z - form to b - form .
to date there is no report on the binding of palmatine to higher - order dna structures , triplexes , and quadruplexes and study in this direction is awaited .
another important alkaloid of protoberberine group is the synthetic molecule coralyne , which was found to be potentially capable of intercalating with b - dna [ 32 , 73 , 74 ] and the results led to the assumption that the dna binding may be correlated with biological activity .
it was reported that at a lower molar ratio of coralyne to b - dna , an intercalating complex by stacking along the backbone of dna , while at higher molar ratios , a dna - induced aggregation occurred .
although a wealth of data [ 32 , 73 , 74 ] is presently available regarding the complex formation between coralyne and b - dna , the interpretation of these data needs to be performed with caution as coralyne exhibits a high tendency of aggregation in aqueous buffer even at very low concentrations .
maiti et al . first addressed this problem and showed that aqueous solutions containing 30% ( v / v ) ethanol suppressed the aggregation of coralyne and allowed spectrophotometric titration of a fully monomeric form with calf thymus dna .
the base and sequence specificity of coralyne ( monomeric ) on binding to various naturally occurring and synthetic b - dna were studied by spectrophotometric and spectrofluorimetric titrations , circular dichroic spectroscopy , thermal melting , isothermal titration calorimetry , and viscometric studies .
the results of spectrophotometric and calorimetric studies of a fully monomeric form of coralyne with calf thymus dna is presented in figures 5(a ) and 5(b ) , respectively .
coralyne increased the contour length of sonicated rod like duplex b - dna and induced the unwinding and rewinding process of covalently closed superhelical b - dna with an unwinding angle of 21. these experiments revealed that monomeric coralyne binds to b - dna by a mechanism of intercalation with a relatively high specificity toward alternating gc sequences .
comparative spectroscopic and thermodynamics binding parameters for the interaction of isoquinoline alkaloids with calf thymus dna are presented in tables 2 and 3 , respectively .
recently , it has been reported [ 28 , 75 ] that coralyne , unlike other classical intercalators , binds to z- form dna without converting the bound form to the b - form bound structure as revealed from circular dichroic spectroscopy .
studies indicated that coralyne could be used as a probe to detect the z - form structure in vivo .
coralyne showed strong perturbation of circular dichroic spectrum of h - form dna ( figure 6 ) .
however , the mode and mechanism of binding of coralyne - h - dna complexation could not be revealed from these studies .
in addition , coralyne was shown to bind to parallel triplex dna but it does not bind to antiparallel triplex dna .
it was concluded from early experiments using thermal denaturation analysis and spectrophotometric titration with txa.t and cxg.c containing dna triplexes that coralyne does not exhibit significant sequence selectivity during triplex complexation
. a later study , however , using various techniques like dnase foot printing , thermal denaturation analysis , spectrophotometric titration and circular dichroic , as well as nmr spectroscopy , showed a preferential intercalation of coralyne into txa.t triplexes , whereas in cxg.c triplexes , the coralyne molecule was only partially intercalated due to the electronic repulsion between the cationic alkaloid and protonated cytosine .
the high propensity of coralyne to stabilize triplex dna has been demonstrated by hud et al .
[ 55 , 82 ] from the observation that duplex poly(dt)poly(da ) is transformed into a mixture of poly(dt)xpoly(da)poly(dt ) triplex and single stranded poly(da ) in presence of coralyne at slightly elevated temperature ( t > 37c ) .
this disproportionation reaction of double - stranded dna has been rationalized in terms of the formation of a highly stable intercalation complex between coralyne and triplex dna .
further , they demonstrated that coralyne may act as a template for the formation of an antiparallel dna triplex from homoadenine sequences .
it was shown that coralyne has the ability to induce and stabilize various g - quadruplex structures including human g - quadruplex as well as its efficiency in inhibiting telomerase .
the thermal stabilization of a preformed intermolecular g - quadruplex structure shows that coralyne and berberine is able to stabilize rather than to induce g - quadruplex intermolecular structure . in both cases , coralyne is the most active component possibly due to its fully aromatic core and it is the most efficient telomerase inhibitor . among the benzophenanthridine alkaloids ,
sanguinarine , an isoquinoline plant alkaloid , is known to generate a wide variety of biochemical and pharmacological effects including its use in cancer prevention .
the physical and molecular basis of the binding of sanguinarine with canonical b - dna were first demonstrated by maiti et al .
[ 33 , 34 , 8488 ] using various physicochemical techniques like absorption , fluorescence , circular dichroism , thermal melting , viscometry , calorimetry , and nmr spectroscopy . on binding to b - dna structures , the absorption spectrum of sanguinarine underwent bathochromic and hypochromic changes exhibiting clear isosbestic points ( figure 7(a ) ) ,
the steady - state fluorescence emission intensity quenched , thermal melting temperature of b - dna shifted to higher temperatures and there were strong perturbations of circular dichroic pattern of b - dna .
sanguinarine induced the unwinding process of covalently closed superhelical plasmid b - dna with an unwinding angle of 27. the binding of sanguinarine to calf thymus dna was also investigated by hnmr and all the proton resonances in the molecule were assigned using a combination of 2d cosy , nosey , and rosey techniques .
all these experiments conclusively suggested that it binds b - dna by a mechanism of intercalation .
the sequence selective binding of sanguinarine to b - dna was also studied by waring group using dnase foot printing technique and a mixed nucleotide sequence binding for sanguinarine - dna complex formation was proposed .
recent calorimetric studies of sanguinarine calf thymus dna complexation ( figure 7(b ) ) showed the binding process to be exothermic and enthalpy driven .
binding and thermodynamic parameters of sanguinarine calf thymus dna complexation obtained from isothermal titration calorimetry are presented in table 3 .
a recent study showed that sanguinarine also binds to single pyrimidine bulges in hairpin sequences .
the interaction of sanguinarine with z - dna structure was first studied by das et al . .
it was demonstrated that on addition of the alkaloid to the z - form structure , a direct conversion of the z - form toward the characteristic right - handed bound complex without forming any intermediate forms was observed .
sanguinarine bound strongly to b - form in a noncooperative manner while its binding to z - form was highly cooperative and 2 - 3 base pairs of z - form switched to the right - handed form for each bound sanguinarine molecule .
the interaction of sanguinarine with h - form showed that it does not bind to h - form structure , but it converted the structure to the bound right - handed b - form [ 94 , 95 ] . here also approximately 2 - 3 base pairs of the h - form switched to the right - handed bound b - form for each bound molecule of sanguinarine .
first demonstrated that sanguinarine binds to poly(dt)poly(da)xpoly(dt ) but only weakly to poly(dc)poly(dg)xpoly(dc ) more quantitatively and reported that sanguinarine binds to triplexes by intercalation in a noncooperative manner and it stabilized the hoogsteen base paired third strand of dna compared to their watson - crick strands .
thermodynamic study revealed that the process of binding of sanguinarine to poly(dc)poly(dg)xpoly(dc ) triplex was exothermic and enthalpy - driven while that to poly(dt)poly(da)xpoly(dt ) triplex was exothermic and entropy - driven .
the binding constants and thermodynamic parameters of berberine , coralyne , and sanguinarine complexation to various triplexes are presented in table 4 .
reported that sanguinarine can form a complex with g - quadruplex telomere dna as revealed from dna polymerase stop assay , uv melting , electrospray mass spectroscopy , and circular dichroism studies which may potentiate a strategy for structure - based anticancer drug design targeting human telomeres .
rnas are versatile molecules that are now known to play an essential role in normal biological process and in the progression of several serious human diseases like hiv and hepatitis c virus ( hcv ) , and so forth which has led to growing interest in exploiting rna as a cellular target for therapeutic drug design .
the recent discovery of a number of micro rnas and the knowledge of their functions further reiterated the critical and diverse roles played by rna in cell biology [ 98 , 99 ] .
ribosomal rna ( r - rna ) has long been known to be the receptor for antibiotics and antisense oligonucleotides are being used to downregulate gene expression .
new drugs could potentially be designed to bind to unique structural regions in mrna and thereby regulate gene expression .
virtually all mrnas formed in eukaryotic cells have a poly(a ) tail of approximately 200250 adenosines at the 3'end .
the long poly(a ) tail is an important determinant of mrna stability and maturation , and is essential for the initiation of transcription [ 101 , 102 ] and the production of alternate forms of proteins [ 103 , 104 ] .
the structure of poly(a ) has been established to exist as a single - stranded helix stabilized by pair - wise stacking interaction between adjacent adenines at physiological ph and temperature [ 105 , 106 ] .
poly(a ) polymerase ( pap ) catalyses 3'-end poly(a ) synthesis , participates in an endonucleolytic cleavage step and is one of the key factors in polyadenylation of the 3'end of mrna .
neo - pap , a recently identified human pap , is significantly overexpressed in human cancer cells in comparison to its expression in normal or virally transformed cells [ 107 , 108 ] and may represent a tumor - specific target .
small molecules capable of recognizing and binding to the mrna - poly(a ) tail might interfere with full processing of mrna by pap and would represent a new type of potential therapeutic agent . in this context ,
several years ago maiti et al . first observed a preferential binding of berberine to poly(a ) structure over b - dna and trna and reported that the binding affinity of berberine to poly(a ) was approximately 12 times higher than b - dna and approximately 100 times higher than t - rna molecules .
recently , they investigated the mode , mechanism , and energetics of berberine binding to poly(a ) structure in details .
it was suggested that berberine binds strongly to single - stranded poly(a ) structure by a mechanism of partial intercalation and the binding was found to be endothermic and entropy driven .
studied the interaction of palmatine with poly(a ) using various biophysical techniques and their results also showed a strong affinity of palmatine to poly(a ) .
the energetics of this strong binding of palmatine to poly(a ) was found to be exothermic and enthalpy - driven .
. showed that coralyne binds to single - stranded poly(a ) helix and induced the formation of a self - structure in poly(a ) .
depending on solution conditions such as ph , salt and temperature , single - stranded poly(a ) can adopt a parallel double stranded conformation formed by the pairing of adenine bases at the n1 position [ 111 , 112 ] .
the possibility of the formation of such a double stranded poly(a ) in eukaryotic cells was hypothesized to result in the termination of the polyadenelation process .
small molecules that could bind strongly to single - stranded poly(a ) may be useful as potential leads for controlling the mrna - poly(a ) chain elongation and in turn mrna degradation as well as the conversion of single - stranded poly(a ) to double stranded poly(a ) that may open up an opportunity for modulating nucleic acid structure - functions .
the plant alkaloids berberine and palmatine exhibit stronger binding to single stranded poly(a ) , but do not induce any self - structure formation in poly(a ) helix [ 109 , 114 ] .
binding of coralyne to the poly(a ) helix leading to self structure formation was further supported by giri and kumar and the binding process was found to be predominantly exothermic and enthalpy - driven with a stoichiometry of one coralyne per four adenines bases .
sanguinarine was also found to bind very strongly to single stranded poly(a ) [ 71 , 78 ] and induce unique self - structure in poly(a ) .
the alkaloid binding process was exothermic and predominantly enthalpy - driven as revealed from isothermal titration calorimetry .
comparative circular dichroic spectrum of poly(a ) treated with various alkaloids are shown in figure 8 .
spectroscopic studies showed that among the four alkaloids , the affinity of binding of berberine , palmatine , and coralyne to poly(a ) helix is one order lower than that of sanguinarine .
thus , the very strong binding of sanguinarine to poly(a ) leading to self structure formation may open up a new avenue for the use of this natural product in modulating the gene expression and development of anticancer agent .
topoisomerases are nuclear enzymes that act on the topology of dna . it was first discovered by wang .
there are three main types of dna topology ; supercoiling , knotting , and catenation .
when outside of replication or transcription , dna needs to be kept as compact as possible and these three states help this cause .
however , when transcription or replication occurs , dna needs to be free and these states seriously hinder the process .
( topo i ) changes the degree of supercoiling of dna by causing single strand breaks and religation whereas topoisomerase ii ( topo ii ) causes knotting , catenation and decatenation of dna by passing the double - stranded dna helix through a transient double - stranded break and then resealing in the strand break [ 118 , 119 ] .
the difference roles of dna topo i and ii may indicate an opposing pair of roles in the regulation of dna supercoiling . both activities are especially crucial during dna transcription and replication , when the dna helix must be unwound to allow proper function of large enzymatic machinery .
recently , two more topoisomerases have been discovered and these are namely topoisomerase iii and topoisomerase iv .
topoisomerase iii enzymes are implicated in regulation of recombination events while topoisomerase iv has roles in the process of segregating newly replicated chromosomes .
dna topoisomerases topo i and topo ii represent the molecular target of many anticancer drugs .
fewer topo i inhibitors have been discovered than topo ii inhibitors and a large number of them including the anthracyclines have been used as anticancer agents before identification of topo ii as their target .
these inhibitors are chemotherapy agents designed to interfere with the action of topoisomerase enzymes that control the changes in dna structure by catalyzing the breakage and rejoining of the phosphodiester backbone of dna strands during the normal cell cycle . in recent years , topoisomerase inhibitors have become popular targets for cancer chemotherapy treatments .
it is thought that topoisomerase inhibitors block the ligation step of the cell cycle , and that topo i and topo ii inhibitors interfere with the transcription and replication of dna by upsetting proper dna supercoiling .
topo i has been established as an attractive molecular target for anticancer drugs , primarily due to the discovery of camptothecin ( cpt ) a naturally occurring plant alkaloid for its successful development as an anticancer drug with a unique mode of action , that is , inhibition of dna topo i , which added an entirely new dimension to the fields of chemotherapy .
thus , developing compounds that are capable of poisoning the topoisomerases have emerged as an attractive approach for the search of novel cancer chemotherapeutic agents [ 122126 ] . for drugs [ 127 , 128 ] that target topo ii intercalation into the host dna duplex
to understand the role that ligand - dna interactions play in topo i poisoning li et al .
have characterized and cross correlated the topo i poisoning and dna binding properties of various families of structurally related dna binding protoberberine alkaloids ( figure 9 ) .
these four analogues differ with respect to the chemical substituents on their a- and d - rings .
their dna binding data reveal that protoberberine alkaloids intercalate into duplex dna while unwinding the host dna by 13. in addition , the data showed that modification of the d - ring alter both dna binding affinity and topo i poisoning activity .
the modifications of the a - ring on the other hand have a negligible impact on dna binding affinity but exerted a profound influence on topo i poisoning activity .
these structure - activity relationships are consistent with a model for topo i poisoning by protoberberines in which both alkaloid - dna and alkaloid - enzyme interactions are necessary for stabilization of the ternary ( topo i - dna - alkaloid ) cleavable complex ( figure 10 ) .
it is suggested that an ensemble of both alkaloid - dna and alkaloid - enzyme interaction may be of general importance for alkaloid - induced poisoning of topo i. berberine , palmatine , and several of its derivatives were shown to act as mammalian topo i and topo ii inhibitors although weakly , while coralyne and many of its derivatives were found to be strong inhibitors of dna topo i .
both berberine and palmatine did not affect the dna cleavage relegation reaction catalyzed by topoisomerases .
this lack of effect most likely accounts for the presence of a methoxy substituent at 9-position , which prevents hydrogen bonding interactions with guanine residues .
the most significant aspect of coralyne is its ability to inhibit dna relaxation in a fashion significantly similar to the most potent antitumor alkaloid cpt .
wang et al . demonstrated that coralyne and several of its derivatives ( figure 11 ) including the partially saturated derivative , 5,6-dihydrocoralyne ( c2 ) were tested for their ability to induce single - stranded cleavage by formation of ternary complexes with plasmid dna and topo i. it was observed that all the derivatives ( c1c11 ) induced cleavage in plasmid dna but are not as effective as coralyne ( c1 ) or 5,6-dihydrocoralyne ( c2 ) and stabilize the complex between topo i and dna .
however , coralyne ( c1 ) was more effective than 5,6-dihydrocoralyne ( c2 ) at lower concentration .
in a subsequent study , pilch et al . confirmed that 8-desmethylcoralyne ( c3 ) and 5,6-dihydro-8-desmethylcoralyne ( c11 ) may act as topo i poison , whereas no topo ii poisoning properties were observed .
more recent immunocytochemical studies in mcf-7 breast cancer cells showed that sanguinarine induces a striking disruption of normal cyclin and topo ii trafficking which inhibited the proliferation of transformed cells .
although great attention is currently focused in unraveling the molecular , cellular , and genetic process that lead to cancer , this knowledge has not been translated into effective new cures for the disease .
recent report of american cancer society ( december 2007 ) showed that 7.6 million people died from various types of cancers . apart from humans , other animals and plants may also be affected by cancer . in general , cancer defines diseases , in which abnormal cells divide without control that have the capacity to invade surrounding normal tissue , can spread to other parts of the body through the blood and lymph systems and kill the host in which it originates . for the last several years , it was observed that the incidence of some types of cancer increases with age . therefore , as the life span of humans has increased , the incidence of neoplastic diseases has also increased .
carcinoma is the cancer that begins in the skin or in tissues that line or cover internal organs .
sarcoma is the cancer that begins in bone , cartilage , fat , muscle , blood vessels , or other connective or supportive tissue .
leukemia is the cancer that starts in blood - forming tissue , such as bone marrow , and causes large numbers of abnormal blood cells either produce or enter the blood .
lymphoma and multiple myeloma are cancers that begin in the cells of the immune system .
central nervous system diseases are cancers that begin in the tissues of the brain and spinal cord .
adrenocortical carcinoma disease is cancer that forms in the outer layer of tissues of adrenal glands .
breast cancer forms in tissues of the breast , usually the ducts and lobules of women .
fibroids are common tumors of the uterus ( womb ) found in about 20% of young women and they form ball - like and may vary in size from pin to very large size .
lung cancer is a disease that forms in tissues of the lung , usually in the cells living air passages .
lung cancer is one of the leading causes of cancer - related death throughout the world . despite of the advances in current therapeutic approaches for lung cancer , including surgery , radiotherapy , and chemotherapy , the overall survival rate of the patients with lung cancer has been static for the past two decades .
improvement in curability and survival is dependent on advances in early detection , surgery , radiotherapy , and chemotherapy ; but once widespread metastatic disease has become established , chemotherapy is a central component of management .
chemotherapy is the treatment of cancer with drugs ( anticancer drugs ) that can destroy cancer cells . in current usage
, the term chemotherapy usually refers to cytotoxic agents that affect rapidly dividing cells in general .
several antitumor antibiotics and plant alkaloids are the more recent clinically effective antitumor cytotoxic drugs , which act by disrupting the structure of duplex b - dna , thereby affecting their interaction with proteins that are important in dna replication , repair , or transcription .
investigation into the mechanism of action of plant - derived compounds with low toxicity remains an important approach in the search for new and more effective anticancer agents .
protoberberine and benzophenanthridine group of alkaloids possessing broad range of biological activity [ 138146 ] are widely distributed in several botanical families and are used in traditional chinese and ayurvedic medicine for many centuries .
berberine is found in the roots of rhizomes of several families of plants ; goldenseas , coptin , oregon grapes , barberry , tree turmeric , yerba mansa , among others and possess antitumor activities [ 147 , 148 ] .
it exhibits potent in vitro antitumor activity against sarcoma s180 cells with inhibition of the biosynthesis of dna , rna , and proteins .
it was evident that berberine interfered with all these biochemical process ( table 5 ) .
however , the synthesis of rna and protein was most sensitive to the alkaloid at 40 ug / ml . dna and protein biosynthesis were examined further by incorporation of thymidine [ h ] into dna and glycine [ c ] into protein in s180 cells in vitro and dose response curves was shown in figure 12 .
creasey reported that the in vivo 30-minutes treatment with berberine on tumor bearing mice ( 10 mg / kg ) before injection of thymidine [ h ] or glycine [ c ] had only minimal effects on the incorporation of these precursors into dna and protein , respectively , and inhibition was found to be only 17% for protein and 14% for dna .
however , when cells were harvested 30 minutes after treatment with berberine and then incubated with glycine [ c ] and thymine [ h ] in vitro incorporation into protein and dna ( figure 12 ) was reduced by 85% and 45% , respectively .
kuo et al . observed an increase of g2/m phase population in berberine - treated hl60 cells .
it modulates expression of mdr1 gene product in track cancer cells and was identified as a potential inhibitor for caspase 3 , a major apoptosis effector .
it was also reported that berberine downregulates k - ras 2 gene expression in human embryonic carcinoma cells .
wu et al . demonstrated that berberine - induced apoptosis of human leukemia hl60 cells with downregulation of nucleophosmin / b23 and telomerase activity .
linked its cytotoxic activity to a duel topo i and topo ii poisoning activity . for management of skin cancers ,
montena et al . for the first time demonstrated in vitro treatment of human epidermoid carcinoma a431 cells with berberine that decreased cell viability , induced cell death in dose - dependent manner which was associated with an increase in g1 arrest and suggested berberine to be used as chemotherapeutic drug against human epidermoid carcinoma a431 cells .
the effect of berberine on human malignant brain tumor esopharyngeal cancer and human leukemic and human colon cancer cell lines was tested , where a significant killing effect was achieved [ 156 , 157 ] .
subsequently , szeto et al . investigated the cytotoxicity and the dna damage effect on npc / hk-1 cells after berberine treatment and evaluated the possibility of dna repair in this cell line .
their studies showed that berberine , at 5200 m induced cell death in a dose dependent manner .
treatment of cells with 200 m berberine for 5 hours yielded 60% drop of cell viability ( figure 13).the comet assay was employed after berberine treatment ( 5 to 200 m ) .
the damaged cells were not able to be repaired as indicated by the increase in tail dna content ( figure 14 ) .
furthermore , berberine was observed mainly in the cytoplasmic region of npc cells at 2 hours of incubation at 200 m as examined under a fluorescent microscope ( figure 15 ) .
again , berberine has been found to be near the nuclear region and able to bind with dna and may inhibit dna topoisomerase ii .
therefore , inhibition of dna repair ability and stability may also contribute to the cytotoxic effect on the cancer cells .
the dna damaging effect and the inhibition of dna repair contributed to the cytotoxic activity in the cancer cells may be part of the mechanisms of action of berberine on cancer cells .
berberine also displayed alterations in cell cycle progression , dna synthesis , cell proliferation , and cell death in melanoma cells k1735-m2 mouse and wm 793 human melanoma malignant cell lines which were correlated with the concentration and intracellular distribution of the alkaloid and suggested that it may be used in clinical development of a cytotoxic compound .
moreover , berberine was shown to inhibit telomere elongation , and bind to various human g - quadruplex structures inhibit telomerase .
berberine has been found to be useful for the treatment of lung cancer patients and the correlation between the overexpression of ap - i and lymphatic metastasis has been reported .
combined treatment with berberine and an anticancer drug camptothecin analogue , cpt - ii resulted in a marked inhibition of the tumor growth at the implantation site and of lymphatic metastasis , as compared with either treatment alone .
berberine also exhibited dose - dependent antiproliferative effects of the human promonocytic u937 cells and induced significant changes in mitochondrial membrane potential of the tumor cells .
in a recent study , the effects of berberine on the human promyclocytic leukemia hl60 cells and on the murine mylomonocytic leukemia weh1 - 3 cells suggested berberine induced g2/m arrest in both the cells via the inhibition of cyclin b1 and the promotion of wee1 .
the treatment of human prostate cancer cells ( pc-3 ) with berberine revealed that berberine - mediated cell death of human prostate cancer cells was regulated by reactive oxygen species and suggested that berberine may be considered as a promising therapeutic candidate for prostate cancer .
berberine was assessed for the anti - human cytomegalovirus ( hcmv ) activity using the plaque assay .
it was suggested that berberine inhibited the replication of hcmv that was presumed to interfere with intracellular events after virus penetration into the host cells and before viral dna synthesis .
berberine showed inhibition of reverse transcriptase activity of rna tumor viruses and the results were correlated with its antileukemic activity .
the enzyme cyclooxygenase - z ( cox-2 ) is abundantly expressed in colon cancer cells and plays a key role in colon tumerogensis .
compounds inhibiting cox-2 transcriptional activity have therefore potentially a chemopreventive property against colon tumor formation .
fukuda et al . have reported that berberine effectively inhibits cox-2 transcriptional activity in colon cancer cells in a dose - dependent manner at concentrations higher than 0.3 m . the inhibitory activity of berberine on cox-2 promotor activity is not simply due to the direct inhibition of macromolecular synthesis in the cells .
in addition , reactive oxygen intermediates play a role in the expression of cox-2 and cox-2 promotor activity is regulated by interactions of diverse transcription factors and intracellular messages .
it has been reported that cox-2 overexpression is associated with the acquisition of resistance to apoptosis in intestinal epithelial cells .
it is likely that inhibition of cox-2 promotor activity may be associated with antitumor and cytostatic / cytocidal effects of berberine .
palmatine has been found in the roots , rhizomes , and stem bark of many species of berberidaceae , papaveraceae , fumararaace , menispermaceae , rutaceae , raunculaece , and other plant family .
palmatine has been used as folk medicine for centuries due to its immense pharmacological properties .
the medicinal plants containing palmatine have been used in the treatment of jaundice , dysentery , hypertension , inflammation , and liver diseases [ 174 , 175 ] .
coralyne has been shown to exhibit significant antileukemic activity against both p388 and l1210 leukemia cells in mice [ 74 , 176 ] .
although its antitumor activity is not very high , coralyne represents a promising lead structure because of its low toxicity [ 177 , 178 ] .
it was shown that irradiation of superhelical plasmid dna in the presence of coralyne leads to an efficient single - stranded cleavage even upon exposure to sunlight . thus photodamaging
again , the photoinduced cleavage may be used for the introduction of dna lesions in triplex and quadruplex dna because coralyne exhibits a remarkable propensity to bind to these higher - order dna structures [ 67 , 68 , 81 ] . in particular , the photoinduced damage of the triplex dna may be of interest to those therapeutic approaches in which the selective attack of triplex dna is the key process .
the quaternary benzophenanthridine alkaloids which are mainly distributed in the plants of the papaveraceae , rutaceae , fumariaceae families have been the focus of increasing attention for their diverse biological activities . among this group of alkaloids , sanguinarine possesses potent anticancer [ 179182 ] , promising apoptosis activities , as well as cytotoxic effects .
in fact , the plants containing sanguinarine have been widely used as chinese medicines and folk medicines for the treatment of human cancer and other diseases . in the uk and
usa , sanguinarine has been in use in mouthwashes and toothpastes to inhibit dental plaque and inhibit gingival health .
the potential anticancer activities of sanguinarine may be attributed to its binding of chromosomal dna .
studies have revealed that in physiological conditions , both iminium and alkanolamine forms of the alkaloid exist because they all exhibit ph - dependent structural equilibrium between these two forms [ 87 , 88 , 185 ] .
alkanolamine form was reported to have larger cellular availability than iminium form due to its greater lipophilicity .
maiti et al . studied the interaction of sanguinarine with b - dna under various environmental conditions ie salt molarity , ph , etc . and suggested that sanguinarine may be useful as a potential antitumoral agent .
this proposal was found to be true when an in vitro study of cytotoxicity of sanguinarine using cancer cell lines and primary cells from oral human tissues established the potential use of sanguinarine for the inhibition of growth of s - g gingival epithelial cells and kb carcinoma cells , and correlated with its complex formation with dna .
studied the effect of sanguinarine on proliferation of the s - g cells exposed for 1 to 3 days as shown in figure 16 .
it was noted that linear increase in growth was significantly affected with increasing concentration of sanguinarine and no growth was observed after 3 days of exposure to sanguinarine at 5.1 m or greater concentration .
as assessed by dna quantification , s - g - cells maintained continuously inhibition of growth increasing concentration of sanguinarine from 1.7 m to 8.5 m may reflect the ability of sanguinarine to intercalate dna .
sanguinarine has been reported to possess the potential for selective / preferential elimination of cancer cells without affecting normal cells [ 180 , 187 ] .
the study of differential effects of the alkaloid upon cell viability , dna damage , and nucleus integrity in mouse primary spleen cells and mouse lymphocytic leukemic cells l1210 showed that sanguinarine produces dose - dependent increase in dna damage and cytotoxicity in both cells .
in contrast to primary mouse spleen cells , leukemic l1210 cells showed slightly higher sensitivity to sanguinarine , suggesting that cytotoxic and dna damaging effects of sanguinarine are more selective against mouse leukemic cells and primary mouse spleen cells .
investigated the possible mechanism through which sanguinarine exerts its antiproliferative action in cultured rat glioblastoma cells and suggested the induction of apoptosis to be due to the downregulation of the extra cellular signal regulated kinase and akt signaling pathway .
the effect of sanguinarine on the human cells line hela ( cervix carcinoma cells ) has been studied and the results showed a strong killing effect of sanguinarine which was explained as a consequence of its easy penetration through cell membrane owing to nonpolar pesudobase formation and to a high degree of molecular planarity .
primary effusion lymphoma [ pel ] is an incurable , aggressive b - cell malignancy that develops rapid resistance to conventional chemotherapy .
it was established that sanguinarine inhibits cell proliferation and induces apoptosis in a dose dependent manner in several pel cell lines and suggested that the alkaloid is a potent inducer of apoptosis of pel cells via upregulation of death receptor 5 and raise the possibility that sanguinarine may be of value in the development of novel therapeutic approaches for the treatment of pel cells .
to investigate sanguinarine sensitivity in neuroendocrine tumor cell lines ( human pancreatic carcinoid and human bronchial carcinoid cells ) , larsson et al .
reported that these tumor cell lines are very much sensitive to sanguinarine , which may be used , in clinical trials and show therapeutic effect in patients with neuroendocrine tumors .
a recent study showed that treatment of human breast cancer mda-231 cells with sanguinarine induced remarkable apoptosis accompanying the generation of reactive oxygen species leading to cell death .
very recently matkar et al . studied the effect of sanguinarine in human colon cancer cell - line and showed its ability to cause dna single and double stranded break as well as increased level of 8-oxodeoxyguanosine and reported dna damage consistent with colon cancer cell death .
alkaloids of plant origin are natural products that exhibit a myriad of biological and pharmacological properties . in view of their extensive occurrence in nature and significantly low toxicities , prospective development and use of these alkaloids as
this review has focused on nucleic acid binding properties and topoisomerase inhibitory activities of some important representatives of the protoberberine ( berberine , palmatine , and coralyne ) and benzophenanthridine ( sanguinarine ) groups of alkaloids and their derivatives in relation to their role in cancer .
a large amount of published data suggests that all these alkaloids bind to various polymorphic dna and also to single stranded poly(a ) structures as evidenced from diverse biophysical techniques .
berberine and palmatine bind to the b - form dna by partial intercalation . on the other hand ,
berberine binds to left - handed hoogsteen base paired h - form dna suggesting the effective use of berberine as sensitive probes to detect handedness change in dna on protonation but it does not bind to left - handed watson - crick base paired z - form .
coralyne on the other hand exhibits strong binding to duplex b - form dna and single stranded poly(a ) structure by mechanism of intercalation as well as it induces self - structure formation on binding to single stranded poly(a ) .
again , berberine and coralyne have the ability to induce and stabilize dna triplexes and g - quadruplex structures ( including human g - quadruplex ) as well . their efficiency in inhibiting telomerase and topoisomerase activity may potentiate their use as potential anticancer drugs .
the alkaloid sanguinarine is known for its anticancer / antitumor properties and possesses the potential for selective / preferential elimination of cancer cells .
it binds to b - form duplex dna and single stranded poly(a ) structure very strongly by intercalation and it induces the conversion of single stranded poly(a ) to double - stranded poly(a ) on binding .
sanguinarine does not bind to z - form or h - form but it converts both z - form and h - form dna to bound b - form structure .
it also formed a strong complex with triplex dna that may lead to its use in triplex - targeted cancer therapy .
sanguinarine has been shown to stabilized the intramolecular dna g - quadruplex formed in human teleomeric sequence ( tandem d(ttaggg ) repeats ) and to inhibit the activity of telomerase and teleomere maintenance .
the striking aspect here is that the specific binding of all these alkaloids to single - stranded adenine regions of the mrna - poly(a ) tail could interfere in mrna processing by human pap to be significantly overexpressed in human cancer cells and may emerge as new type of rna based therapeutic agents for cancer diseases .
thus , the differential binding of these alkaloids to various polymorphic nucleic acid conformations with specific biological functions may potentiate their use in nucleic acid - targeted drug design that in turn may convey some specific meaning for their regulatory roles in neoplastic diseases . | bioactive alkaloids occupy an important position in applied chemistry and play an indispensable role in medicinal chemistry . amongst them , isoquinoline alkaloids like berberine , palmatine and coralyne of protoberberine group , sanguinarine of the benzophenanthridine group , and their derivatives represent an important class of molecules for their broad range of clinical and pharmacological utility . in view of their extensive occurrence in various plant species and significantly low toxicities , prospective development and use of these alkaloids as effective anticancer agents are matters of great current interest .
this review has focused on the interaction of these alkaloids with polymorphic nucleic acid structures ( b - form , a - form , z - form , hl - form , triple helical form , quadruplex form ) and their topoisomerase inhibitory activity reported by several research groups using various biophysical techniques like spectrophotometry , spectrofluorimetry , thermal melting , circular dichroism , nmr spectroscopy , electrospray ionization mass spectroscopy , viscosity , isothermal titration calorimetry , differential scanning calorimetry , molecular modeling studies , and so forth , to elucidate their mode and mechanism of action for structure - activity relationships .
the dna binding of the planar sanguinarine and coralyne are found to be stronger and thermodynamically more favoured compared to the buckled structure of berberine and palmatine and correlate well with the intercalative mechanism of sanguinarine and coralyne and the partial intercalation by berberine and palmatine .
nucleic acid binding properties are also interpreted in relation to their anticancer activity . |
within hcfa 's office of the actuary , the office of national health statistics ( onhs ) is charged with the responsibility for maintaining the national health accounts ( nha ) .
these accounts form the structure for maintaining health care expenditure information for the united states . in response to a request by the 1993 president 's task force on health care reform for estimates of health care spending by states , onhs initiated a project to update estimates of state - specific health spending to current periods .
the purpose behind this effort was twofold : to create an analytical tool that could be used to identify differences among states in patterns and levels of spending , and to create statistics that could be useful in addressing global budgeting issues for health care reform .
as a first step in this process , onhs created state estimates of personal health care spending , where state represents the location of the provider of service ( levit et al . , 1995 ) .
in other words , these estimates show the amount of revenues received by providers of a state , regardless of the residency of the patients using the services .
this provider - state definition is driven by data available for estimating purposes that is usually collected from providers based on their location .
the provider - based estimates are useful for measuring demand for health care in the state ; however , they fail to address one important issue : how do health care costs per person vary from state to state ? because state spending estimates based on location of provider do not accurately represent
spending by persons residing in that state , calculating per capita spending estimates using expenditures by state of provider along with resident population will produce inaccurate results . to address this issue ,
onhs is refining the state estimates so that they reflect health care expenditures made by the population of a state rather than revenues received by the state 's providers .
the adjustment process requires the creation of complete interstate flow matrices that track service - specific expenditures incurred by medicare beneficiaries of each state in every other state .
medicare interstate flow matrices are also intended to serve as the building blocks for estimating border - crossing patterns and expenditures per capita for non - medicare beneficiaries residing in a state .
although spending for medicare services by residence state is available , similar data for the non - medicare population is not .
thus , medicare flows will provide a basis for calculating non - medicare flows , the work on which has already started .
the data adjusted for border crossing are expected to greatly enhance the usefulness of state estimates by providing states with tools to evaluate and assess the spending patterns of their residents for health care as a whole and by type of service . despite the waning of the comprehensive national health care reform effort and the accompanying need to address global budgeting issues , per capita spending estimates by location of beneficiary residence
these estimates are also useful for evaluating the effectiveness of individual state health reform initiatives by providing information on expenditures incurred by the residents for different services , spending growth over time , and the impact of policy changes on spending patterns .
the adjustment for border crossing is especially important because studies indicate that patients traveling long distances use more resources and incur higher hospital charges than do local patients ( welch , larson , and welch , 1993 ) .
thus , per capita expenditure will be grossly overstated for a state that is a major net exporter of services if the high - cost out - of - state cases are not excluded . for example , per capita medicare spending for the district of columbia would be 43 percent higher , had no border - crossing adjustment been done .
border crossing is a much - discussed subject area and is a part of the broader research on health service markets and identification of health service areas .
literature regarding the distance between patients and providers and the impact of distance on health care utilization can be traced back many years ( kleinman and makuc , 1983 ; shannon , bashshur , and metzner , 1969 ) .
prior research on border crossing often used a health service area as the unit of analysis , defined either by geographic boundaries or by market shares of providers ( garnick et al .
, 1988 ; morrisey , sloan , and valvona , 1988 ) . only a few of these studies focused on interstate border crossing ( buczko , 1992 ; holahan and zuckerman , 1993 ; u.s .
many of the previous studies analyzed travel patterns of patients using the utilization data of beneficiaries and primarily focused on inpatient hospital care ( hogan , 1988 ; bronstein and morrisey , 1990 ; buczko , 1992 ; mcguirk and porell , 1984 ) , physician services ( holahan and zuckerman , 1993 ) , and ambulatory care ( kleinman and makuc , 1983 ; makuc , kleinman , and pierre , 1985 ) .
this article presents the results of a pioneering effort by hcfa to measure interstate border crossing for each of 10 individual types of services used by medicare beneficiaries by analyzing the expenditure data at the beneficiary level .
the study 's findings are based on the data base created as a result of this effort , which is the only nationwide data base covering a broad array of services .
this analysis summarizes the out - of - state spending patterns of all medicare beneficiaries , based on the interstate flow of expenditure data developed for calendar year 1991 .
the analysis provides state - specific data on medicare expenditures for medicare - covered services by state of provider and by state of beneficiary residence .
the study also provides estimates of per capita medicare expenditure by state and analyzes interstate differences in expenditure flows by computing rates of inflow and outflow of medicare expenditure in each state .
for the medicare population , the data on expenditures for health care services are obtained from the medicare data files known as the national claims history ( nch ) files .
these files contain records of transactions between the medicare program and providers of health care services . among the information items recorded
are allowed charges , medicare payment amounts , and number and type of services provided .
the calendar year 1991 data base was used as the primary source for computing medicare interstate flows for 1991 .
nch files were used to process claims data for inpatient and outpatient hospital services , skilled nursing facility ( snf ) services , home health agency ( hha ) services , hospice care , independently billing laboratory services , freestanding end stage renal disease ( esrd ) facilities , and physician and part b supplier services .
selection criteria were established and adjustments were made to the data to estimate the interstate spending pattern of medicare beneficiaries .
first , the state of the provider and the state of the beneficiary residence were determined , based respectively on the first two digits of the institutional claim number and the social security administration ( ssa ) standard state codes identifying geographic area of residence . for physician and part
b supplier services , the provider state was determined using one or a combination of methods , e.g. , the carrier number for carriers servicing all or part of a single state , pricing locality codes for multistate carriers , and provider zip codes .
second , medicare payment amounts for institutional claims from prospective payment system ( pps ) hospitals were calculated by adding the result of per diem passthrough amounts times the number of medicare covered days , plus covered charges for organ acquisition , to the provider payment amounts recorded in the institutional claims . physician / supplier records contained the allowed charge amount , which was used as a proxy for program payments to providers or beneficiaries .
for this , a crosswalk was developed between the medicare bill types and nha type - of - service categories .
the bill - type variable , which is the primary means used to allocate institutional data into nha categories , was determined from the provider number facility - type code and the claim service - classification type code . for physician and part
b suppliers , the provider specialty code was used to categorize claims from physicians , other professionals , and durable medical equipment suppliers .
tables 1 - 6 present summarized information on interstate flows of medicare expenditure that resulted from hcfa 's study . the tables
follow the format usually presented in the national health expenditure ( nhe ) studies ( levit et al . , 1994 ) , where services are grouped together into nha categories according to the establishments providing services .
these establishments are defined by the standard industrial classification coding system ( executive office of the president , 1987 ) .
for example , expenditures for inpatient and outpatient hospital services are included in expenditures for hospital care .
similarly , expenditures for care provided in hospital - based nursing homes are included in hospital care .
expenditures for a few medicare service categories are not presented separately but are included in the broader nhe group ( e.g. , independently billing laboratory services are included under expenditures for physician services ) .
table 1 provides data on medicare personal health care expenditure amounts for 1991 by economic region and state of provider .
table 2 shows the results of converting the estimates based on provider location of table 1 into estimates based on state of beneficiary residence . in preparing this conversion ,
these categories represent medicare types of services , including inpatient hospital care , outpatient hospital care , physician services , nursing home care , independent laboratory services , other professional services , services provided by freestanding kidney dialysis facilities , home health care , durable medical equipment supplies , and hospice services .
a set of flow factors was calculated for each service type , based on the expenditure incurred by medicare beneficiaries in their home state and in each of the other states in which they received services .
these interstate flow matrices provide the basis for residence - based adjustments made in table 2 to the provider - based data in table 1 . in preparing the adjusted data , the flow ratios for each individual medicare service category
have been applied to the corresponding provider - based estimates and then collapsed into nha categories mentioned earlier . in some cases ,
for example , although it belongs to the hospital service , hospital - based nursing home estimates in table 2 are derived by using the interstate flow factors for the nursing home category . similarly , although not shown separately , the physician estimates include independent laboratory expenditures , which are derived by using a separate set of adjustment factors ( or flow ratios ) .
home health care is split into hospital - based ( included under outpatient hospital care in the tables ) and non - hospital - based categories in either case , however , the residence - based adjustment is made using flow ratios derived for total home health care .
an underlying assumption in this study is that medicare enrollees in health maintenance organizations ( hmos ) have similar travel patterns and health care expenditures to individuals covered by fee - for - service ( ffs ) plans .
a major constraint in analyzing expenditure patterns for hmo enrollees is that no billing data exist for services received by medicare hmo patients .
the extent of border crossing by this group depends largely on the location of the provider network and other constraints on the choice of the providers .
thus , no definite conclusion can be reached on the border - crossing pattern by medicare hmo enrollees . for lack of data and evidence supporting an alternative hypothesis ,
hmo enrollees were assumed to have out - of - state spending patterns similar to enrollees under ffs plans .
this assumption , however , does not significantly impact the analysis , because hmos are a , small part of total medicare ( in 1991 , about 6 percent of enrollment and 5 percent of expenditures ) .
moreover , for states with the largest hmo enrollment , e.g. , california ( accounting for approximately 40 percent of total medicare hmo enrollment in the nation in 1991 ) , the issue is probably insignificant , as border crossing for health care services by medicare beneficiaries in general in that state is very small . to show the relationship between provider - based and residence - based estimates , table 3 contains a set of ratios calculated by dividing medicare expenditures by state of beneficiary residence by the corresponding expenditure by the provider state .
the ratios , called net flow ratios ( nfrs ) , measure the extent to which a state is a net importer or net exporter of services both overall and by types of service .
these states provide more services to out - of - state residents than the corresponding services their residents receive out of state . in contrast , the states with net flow ratios greater than 1 are net importers of services .
regional data presented in table 3 indicate that the new england , plains , southeast , southwest , and far west regions are net exporters of services in the aggregate , and the remaining regions are net importers .
the table indicates that except in the plains region , which exports a net amount of 3 percent of services ( nfr equals 0.9716 ) , all other exporting regions export a net amount of less than 1 percent .
the rocky mountains and great lakes regions import a net amount of about 2 percent of their services ( nfr values are 1.0219 and 1.0245 , respectively ) . for the mideast region ,
the services produced are nearly identical with services consumed by residents of that region , resulting in an nfr value close to unity ( 1.0008 ) . in general , nfrs for regions are usually close to 1 overall and for most services .
whether a state is a net importer or net exporter of services depends on a variety of factors representing both supply and demand .
studies investigating the border - crossing issues for inpatient hospitals and for physician services identify a few of these factors .
the supply indicators that have been found to have significant impacts on border crossing for inpatient care are availability of physicians , inpatient beds , specialists , and specialized services ( buczko , 1992 ) .
demand is determined by a variety of factors , such as sociodemographic characteristics , population size , health status , and the complexity of illness .
although distance is found to be largely associated with severity of illness in several studies ( adams et al . , 1991
; welch , larson , and welch , 1993 ) , it can be a deterrent to hospital choice , particularly for older medicare beneficiaries ( adams et al . , 1991 ) .
states with large proportions of very elderly medicare beneficiaries ( 85 years of age or over ) , such as nebraska , may tend to have lower nfrs ( 0.9784 ) , because there is evidence to suggest that the very old do not travel extensively , particularly for hospitalization ( hogan , 1988 ; adams et al .
on the other hand , studies also indicate that rural medicare beneficiaries , particularly those with complex diagnoses , tend to travel more to urban hospitals with a large scope of services and with teaching status ( adams et al . , 1991 ) .
rural states , such as wyoming and idaho , are found to be major importers of services ( with nfr values of 1.3362 and 1.2126 , respectively ) , and large urbanized states with teaching hospitals , such as massachusetts , tend to be major exporters ( nfr equals 0.9695 ) for most services .
the location of urban areas within a state also plays a major role in determining its service area .
states with urban areas on borders ( e.g. , tennessee ) are likely to experience more border crossing for routine services . the states providing highly specialized services are major exporters of these services , because studies indicate that border crossing tends to be greater for high - technology services than for routine visits to physician offices ( holahan and zuckerman , 1993 ) . some states , such as florida and arizona , have nfrs of less than 1 as a result of large seasonal inflows of out - of - state patients ( buczko , 1994 ) .
tables 4 and 5 explain the variation in nfrs by state more clearly by computing the inflow rate and the outflow rate of medicare expenditures by states .
the outflow rate is the percent of out - of - state medicare expenditures incurred by residents of a state , and the inflow rate is the percent of medicare expenditures incurred by out - of - state residents in the provider state .
the inflow rate is calculated as medicare expenditures incurred by out - of - state residents in the provider state as a proportion of total medicare revenues received by that state 's providers ( table 4 ) . the outflow rate , on the other hand ,
is computed as medicare expenditures incurred by state residents outside the state as a proportion of total medicare expenditures incurred by that state 's residents ( table 5 ) .
an outflow of expenditures indicates import of services , and an inflow of expenditures implies export of services .
the states with higher outflow than inflow rates are net importers of services , with nfr values of greater than 1 .
the weighted average inflow and outflow rates of expenditures for all areas together are the same , indicating no net flow occurring overall .
the inflow and outflow rates of expenditure for all services together are highest in the plains region ( 12.75 and 10.20 percent , respectively ) and lowest in the far west ( 3.91 and 2.96 percent , respectively ) .
the regions having a net outflow of medicare expenditures are the mideast , great lakes , and rocky mountains .
the states showing the highest and the lowest outflow rates of expenditures , respectively , are wyoming ( 29.75 percent ) and california ( 1.79 percent ) .
the states with the highest inflow rates are the district of columbia , north dakota , and minnesota .
generally , high - spending states ( e.g. , pennsylvania , california , florida , texas , minnesota , massachusetts ) are those with high inflow rates .
a few of the states retaining most of the funds spent by their residents ( more than 95 percent ) are tennessee , louisiana , massachusetts , pennsylvania , texas , north carolina , california , and hawaii . the rural states ( e.g. , alaska , wyoming , idaho , vermont , new hampshire ) experience a high proportion of out - of - state spending by their residents ( greater than 20 percent ) because services are not conveniently found within the states .
the states that have large population bases often tend to have lower rates of inflow and outflow because these states have the population density to support large health service establishments . a large amount of exporting and importing of services is observed in states with large cities near their borders .
for example , missouri experiences a large inflow of expenditures because of the proximity of st .
louis , and illinois patients go out of state from the border city of east st .
ranked by services , the lowest amount of border crossing ( indicated by both average outflow and inflow rates ) is observed in the category of home health care ( 2.73 percent ) . because home health services are mostly used by the home - bound elderly ( helbing , sangl , and silverman , 1993 ) , and the agencies delivering the services are in most cases licensed by the state ( intergovernmental health policy project , 1993 ) , most home health service areas are limited by state boundaries .
longer travel time is also likely to be an impediment to access , particularly in rural areas , because this raises service delivery costs ( kenney , 1993 ) .
the highest rate of border crossing is observed in the area of medical durable supplies ( 20.85 percent ) .
the large interstate flows for medical durables appear to indicate that there is no fixed local market area for these services .
the major components of medical durables paid by medicare include prostheses , orthotics , wheelchairs , oxygen , and oxygen supplies .
the concentration of wholesale distributors in certain regions and telemarketing may be contributing factors causing large interstate buying and selling of these products .
moreover , medicare claims data from several medical supply companies reflect centralized billing offices located outside the state where services are actually rendered , contributing to ambiguity in correctly identifying the location of the provider .
the data for hospital care and physician services show that out - of - state spending for these two areas falls between the two extremes .
the border crossing occurring in the use of inpatient hospital care is 6.72 percent and that for physician services is 7.20 percent in 1991 .
tables 4 and 5 indicate that , in general , medicare out - of - state expenditures are similar for inpatient hospital care and for physician services , indicating similarity of forces driving border crossing across these services .
the outflow rate exceeds the inflow rate in the mideast , great lakes , and rocky mountain regions for both types of service expenditures .
the reverse pattern is observed in other regions , except in new england , where the outflow rate corresponds to the inflow rate for physician services and is lower than the inflow rate for inpatient hospital services .
in addition to serving as the tool to track down expenditure flows across states , the major purpose of developing border - crossing measures for medicare spending is to provide adjustment factors so that valid computation of per capita expenditures is possible . without the adjustment for interstate border crossing ,
estimates of state spending per person could be produced only by using ( 1 ) expenditures by location of provider and ( 2 ) population by location of beneficiary residence .
general accounting office , 1992 ) suggest that state transfers of health services are not statistically significant in explaining differences in state spending levels .
however , the implication of the border - crossing adjustment for individual states can not be overstated in light of its role in accurately identifying the spending levels for its residents .
for example , the border - crossing adjustment lowered medicare expenditures by 13 percent for north dakota and raised them by 34 percent in wyoming in 1991 ( table 3 ) .
similarly , the district of columbia provides extensive health care services to persons residing in maryland and virginia . without the adjustment ,
spending per person in the district of columbia would be grossly overestimated , and spending in maryland and virginia could be understated , if not offset by other border - crossing flows of health care spending .
table 6 provides estimates of per capita expenditures by type of service after dividing expenditures for resident beneficiaries in each state by the population incurring these expenditures represented by the total number of medicare enrollees for 1991 . because the nha categories are grouped in such a way that both part a and part b enrollees are eligible to receive most services , a combined total including enrollees in either or both categories is used as the denominator in computing per enrollee expenditures for different services
. a comparison of per enrollee expenditures provides the background for a more meaningful analysis of the variation in expenditures across states for each service because it controls for the difference in the population size .
the border - crossing adjustment raises per capita expenditures for states with nfrs greater than 1 and lowers the same for states with nfrs less than 1 , relative to estimates produced using provider - based expenditures without the adjustment .
the per capita expenditure data in table 6 show that the highest spending per medicare enrollee is for inpatient hospital services ( $ 1,868 ) , followed by physician services ( $ 900 ) , home health care ( $ 122 ) , and other professional care ( $ 117 ) .
overall , the mideast region spends the most per enrollee ( $ 3,852 ) , followed by the far west ( $ 3,809 ) and new england ( $ 3,618 ) .
many states with lower per capita spending are in the rocky mountain and south regions . some of the high - cost states are california , the district of columbia , maryland , massachusetts , and louisiana .
although state - to - state variations in per capita spending were observed , the spending was within 10 percent of the u.s .
studies investigating the causes of interstate variation in total spending indicate that factors such as state differences in personal income , the supply of health care resources ( including the number of physicians and hospital and nursing home beds per capita ) , the concentration of hospital services in urban areas , and health status explained more than 80 percent of the difference in health spending among states ( u.s .
the state rankings in personal income per capita influence health spending . the states with high per capita income ( e.g.
, the district of columbia , massachusetts , maryland , new jersey ) generally spend more per capita .
the opposite is true for states with low per capita income ( e.g. , idaho , utah ) .
the states with relatively high urban populations generally have high costs of care and , consequently , relatively high per capita expenditures ( the district of columbia , massachusetts , florida , connecticut , new york , new jersey , etc . ) .
the average payment per urban enrollee was found to be approximately 17 percent higher than that for rural beneficiaries ( health care financing administration , 1995 ) .
the states with more health care resources tend to experience higher spending by their residents ( such as california , with a high physician - to - population ratio ) .
other factors , such as age and sex composition of medicare enrollees and their health and disability status ( helbing , sangl , and silverman , 1993 ) , might serve as important determinants of per capita variation in medicare spending .
states with poor health status of the residents ( e.g. , georgia , louisiana , and alabama ) tend to spend more per capita than states with better health status ( e.g. , utah , idaho , minnesota , oregon , and hawaii ) ( prospective payment assessment commission , 1995 ) .
in addition , factors such as provider practice patterns , managed - care market penetration , and provider resource costs are also important in explaining regional differences in per capita spending . the high resource costs in louisiana ( 17 percent above the u.s .
average ) might partly explain the high per capita spending in that state ( prospective payment assessment commission , 1995 ) .
the growth of managed care in recent years might also have slowed the growth of per capita spending in states with high hmo enrollment however , evidence is still mixed as to the effect of managed care on growth of expenditures . by analyzing the difference between per capita expenditures with and without adjusting for border crossing ,
several characteristics are observed : the effect of border crossing is found to be very large ( 9 - 34 percent ) for certain states , such as wyoming , idaho , alaska , and new mexico . for states such as minnesota , north dakota , and the district of columbia , per capita spending estimates decline by 10 - 30 percent as a result of this adjustment even for services such as home health care , for which there is very little border crossing , the effect on some states ( e.g. , new mexico , west virginia , the district of columbia , virginia , south dakota ) is quite substantial ( 8 - 18 percent increase ) . for services such as durable medical equipment ,
a maximum difference of more than 200 percent is also observed ( table 3 ) . as a whole
, the border - crossing adjustment reduces the variability of per capita expenditures across states : the coefficient of variation declines from 22 to 15 percent as a result of using the residence - based estimates to calculate the per capita expenditure .
the adjustment for border crossing has the greatest impact ( measured by mean percent difference between adjusted and unadjusted per capita expenditures ) on per capita expenditures for laboratory services and durable medical supplies , but only a small impact on home health care , outpatient hospital care , and inpatient hospital services . ranked by the average size of the impact , the lowest- to highest - ranking medicare categories
are : home health care ( 0.58 percent ) , outpatient hospital care ( 0.75 percent ) , inpatient hospital care ( 0.98 percent ) , other professional care ( 1.77 percent ) , physician services ( 1.80 percent ) , hospice care ( 3.20 percent ) , nursing homes ( 3.24 percent ) , esrd services ( 4.90 percent ) , medical durables ( 19.0 percent ) , and independent laboratory services ( 51.0 percent ) .
the impact is also found to vary substantially across states . in terms of the variability of impact ( measured by the coefficient of variation of percent differences between adjusted and unadjusted per capita expenditure ) ,
the lowest- to highest - ranking medicare categories respectively are : nursing homes , medical durables , independent laboratory services , other professional care , esrd services , hospice , physician services , home health care , outpatient hospital care , and inpatient hospital care .
that is , the interstate fluctuations of impact are found to be highest for inpatient hospital services and lowest for nursing homes .
the highest variability for hospital services is an indication of the wide fluctuation among states in how border crossing impacts per capita expenditures for this service , although the low average value of the impact for this service indicates that some of the positive and negative impacts cancel out . for services such as independent laboratory and medical durables , on the other hand ,
these findings indicate the sensitivity of the state estimates , especially for hospital services , to the border - crossing adjustment and highlight the significance of making this adjustment .
table 1 provides data on medicare personal health care expenditure amounts for 1991 by economic region and state of provider .
table 2 shows the results of converting the estimates based on provider location of table 1 into estimates based on state of beneficiary residence . in preparing this conversion ,
these categories represent medicare types of services , including inpatient hospital care , outpatient hospital care , physician services , nursing home care , independent laboratory services , other professional services , services provided by freestanding kidney dialysis facilities , home health care , durable medical equipment supplies , and hospice services .
a set of flow factors was calculated for each service type , based on the expenditure incurred by medicare beneficiaries in their home state and in each of the other states in which they received services .
these interstate flow matrices provide the basis for residence - based adjustments made in table 2 to the provider - based data in table 1 . in preparing the adjusted data , the flow ratios for each individual medicare service category
have been applied to the corresponding provider - based estimates and then collapsed into nha categories mentioned earlier . in some cases ,
for example , although it belongs to the hospital service , hospital - based nursing home estimates in table 2 are derived by using the interstate flow factors for the nursing home category . similarly , although not shown separately , the physician estimates include independent laboratory expenditures , which are derived by using a separate set of adjustment factors ( or flow ratios ) .
home health care is split into hospital - based ( included under outpatient hospital care in the tables ) and non - hospital - based categories in either case , however , the residence - based adjustment is made using flow ratios derived for total home health care .
an underlying assumption in this study is that medicare enrollees in health maintenance organizations ( hmos ) have similar travel patterns and health care expenditures to individuals covered by fee - for - service ( ffs ) plans .
a major constraint in analyzing expenditure patterns for hmo enrollees is that no billing data exist for services received by medicare hmo patients .
the extent of border crossing by this group depends largely on the location of the provider network and other constraints on the choice of the providers .
thus , no definite conclusion can be reached on the border - crossing pattern by medicare hmo enrollees . for lack of data and evidence supporting an alternative hypothesis ,
hmo enrollees were assumed to have out - of - state spending patterns similar to enrollees under ffs plans .
this assumption , however , does not significantly impact the analysis , because hmos are a , small part of total medicare ( in 1991 , about 6 percent of enrollment and 5 percent of expenditures ) .
moreover , for states with the largest hmo enrollment , e.g. , california ( accounting for approximately 40 percent of total medicare hmo enrollment in the nation in 1991 ) , the issue is probably insignificant , as border crossing for health care services by medicare beneficiaries in general in that state is very small .
to show the relationship between provider - based and residence - based estimates , table 3 contains a set of ratios calculated by dividing medicare expenditures by state of beneficiary residence by the corresponding expenditure by the provider state .
the ratios , called net flow ratios ( nfrs ) , measure the extent to which a state is a net importer or net exporter of services both overall and by types of service . states that are net exporters of services have nfrs less than 1 .
these states provide more services to out - of - state residents than the corresponding services their residents receive out of state .
in contrast , the states with net flow ratios greater than 1 are net importers of services .
regional data presented in table 3 indicate that the new england , plains , southeast , southwest , and far west regions are net exporters of services in the aggregate , and the remaining regions are net importers .
the table indicates that except in the plains region , which exports a net amount of 3 percent of services ( nfr equals 0.9716 ) , all other exporting regions export a net amount of less than 1 percent .
the rocky mountains and great lakes regions import a net amount of about 2 percent of their services ( nfr values are 1.0219 and 1.0245 , respectively ) .
for the mideast region , the services produced are nearly identical with services consumed by residents of that region , resulting in an nfr value close to unity ( 1.0008 ) . in general , nfrs for regions
whether a state is a net importer or net exporter of services depends on a variety of factors representing both supply and demand .
studies investigating the border - crossing issues for inpatient hospitals and for physician services identify a few of these factors .
the supply indicators that have been found to have significant impacts on border crossing for inpatient care are availability of physicians , inpatient beds , specialists , and specialized services ( buczko , 1992 ) .
demand is determined by a variety of factors , such as sociodemographic characteristics , population size , health status , and the complexity of illness . although distance is found to be largely associated with severity of illness in several studies ( adams et al . , 1991 ;
welch , larson , and welch , 1993 ) , it can be a deterrent to hospital choice , particularly for older medicare beneficiaries ( adams et al . , 1991 ) .
states with large proportions of very elderly medicare beneficiaries ( 85 years of age or over ) , such as nebraska , may tend to have lower nfrs ( 0.9784 ) , because there is evidence to suggest that the very old do not travel extensively , particularly for hospitalization ( hogan , 1988 ; adams et al .
on the other hand , studies also indicate that rural medicare beneficiaries , particularly those with complex diagnoses , tend to travel more to urban hospitals with a large scope of services and with teaching status ( adams et al . ,
rural states , such as wyoming and idaho , are found to be major importers of services ( with nfr values of 1.3362 and 1.2126 , respectively ) , and large urbanized states with teaching hospitals , such as massachusetts , tend to be major exporters ( nfr equals 0.9695 ) for most services .
the location of urban areas within a state also plays a major role in determining its service area .
states with urban areas on borders ( e.g. , tennessee ) are likely to experience more border crossing for routine services .
the states providing highly specialized services are major exporters of these services , because studies indicate that border crossing tends to be greater for high - technology services than for routine visits to physician offices ( holahan and zuckerman , 1993 ) .
some states , such as florida and arizona , have nfrs of less than 1 as a result of large seasonal inflows of out - of - state patients ( buczko , 1994 ) .
tables 4 and 5 explain the variation in nfrs by state more clearly by computing the inflow rate and the outflow rate of medicare expenditures by states .
the outflow rate is the percent of out - of - state medicare expenditures incurred by residents of a state , and the inflow rate is the percent of medicare expenditures incurred by out - of - state residents in the provider state .
the inflow rate is calculated as medicare expenditures incurred by out - of - state residents in the provider state as a proportion of total medicare revenues received by that state 's providers ( table 4 ) .
the outflow rate , on the other hand , is computed as medicare expenditures incurred by state residents outside the state as a proportion of total medicare expenditures incurred by that state 's residents ( table 5 ) .
an outflow of expenditures indicates import of services , and an inflow of expenditures implies export of services .
the states with higher outflow than inflow rates are net importers of services , with nfr values of greater than 1 .
the weighted average inflow and outflow rates of expenditures for all areas together are the same , indicating no net flow occurring overall .
the inflow and outflow rates of expenditure for all services together are highest in the plains region ( 12.75 and 10.20 percent , respectively ) and lowest in the far west ( 3.91 and 2.96 percent , respectively ) . in both regions , however , the inflow rate exceeds the outflow rate .
the regions having a net outflow of medicare expenditures are the mideast , great lakes , and rocky mountains .
the states showing the highest and the lowest outflow rates of expenditures , respectively , are wyoming ( 29.75 percent ) and california ( 1.79 percent ) .
the states with the highest inflow rates are the district of columbia , north dakota , and minnesota . generally , high - spending states ( e.g. , pennsylvania , california , florida , texas , minnesota , massachusetts ) are those with high inflow rates .
a few of the states retaining most of the funds spent by their residents ( more than 95 percent ) are tennessee , louisiana , massachusetts , pennsylvania , texas , north carolina , california , and hawaii .
the rural states ( e.g. , alaska , wyoming , idaho , vermont , new hampshire ) experience a high proportion of out - of - state spending by their residents ( greater than 20 percent ) because services are not conveniently found within the states .
the states that have large population bases often tend to have lower rates of inflow and outflow because these states have the population density to support large health service establishments .
a large amount of exporting and importing of services is observed in states with large cities near their borders .
for example , missouri experiences a large inflow of expenditures because of the proximity of st .
louis , and illinois patients go out of state from the border city of east st .
ranked by services , the lowest amount of border crossing ( indicated by both average outflow and inflow rates ) is observed in the category of home health care ( 2.73 percent ) . because home health services are mostly used by the home - bound elderly ( helbing , sangl , and silverman , 1993 ) , and the agencies delivering the services are in most cases licensed by the state ( intergovernmental health policy project , 1993 ) , most home health service areas are limited by state boundaries .
longer travel time is also likely to be an impediment to access , particularly in rural areas , because this raises service delivery costs ( kenney , 1993 ) .
the highest rate of border crossing is observed in the area of medical durable supplies ( 20.85 percent ) .
the large interstate flows for medical durables appear to indicate that there is no fixed local market area for these services .
the major components of medical durables paid by medicare include prostheses , orthotics , wheelchairs , oxygen , and oxygen supplies .
the concentration of wholesale distributors in certain regions and telemarketing may be contributing factors causing large interstate buying and selling of these products .
moreover , medicare claims data from several medical supply companies reflect centralized billing offices located outside the state where services are actually rendered , contributing to ambiguity in correctly identifying the location of the provider . the data for hospital care and physician services show that out - of - state spending for these two areas falls between the two extremes .
the border crossing occurring in the use of inpatient hospital care is 6.72 percent and that for physician services is 7.20 percent in 1991 .
tables 4 and 5 indicate that , in general , medicare out - of - state expenditures are similar for inpatient hospital care and for physician services , indicating similarity of forces driving border crossing across these services .
the outflow rate exceeds the inflow rate in the mideast , great lakes , and rocky mountain regions for both types of service expenditures .
the reverse pattern is observed in other regions , except in new england , where the outflow rate corresponds to the inflow rate for physician services and is lower than the inflow rate for inpatient hospital services .
in addition to serving as the tool to track down expenditure flows across states , the major purpose of developing border - crossing measures for medicare spending is to provide adjustment factors so that valid computation of per capita expenditures is possible . without the adjustment for interstate border crossing ,
estimates of state spending per person could be produced only by using ( 1 ) expenditures by location of provider and ( 2 ) population by location of beneficiary residence .
general accounting office , 1992 ) suggest that state transfers of health services are not statistically significant in explaining differences in state spending levels .
however , the implication of the border - crossing adjustment for individual states can not be overstated in light of its role in accurately identifying the spending levels for its residents .
for example , the border - crossing adjustment lowered medicare expenditures by 13 percent for north dakota and raised them by 34 percent in wyoming in 1991 ( table 3 ) .
similarly , the district of columbia provides extensive health care services to persons residing in maryland and virginia . without the adjustment ,
spending per person in the district of columbia would be grossly overestimated , and spending in maryland and virginia could be understated , if not offset by other border - crossing flows of health care spending .
table 6 provides estimates of per capita expenditures by type of service after dividing expenditures for resident beneficiaries in each state by the population incurring these expenditures represented by the total number of medicare enrollees for 1991 . because the nha categories are grouped in such a way that both part a and part b enrollees are eligible to receive most services , a combined total including enrollees in either or both categories is used as the denominator in computing per enrollee expenditures for different services .
a comparison of per enrollee expenditures provides the background for a more meaningful analysis of the variation in expenditures across states for each service because it controls for the difference in the population size .
the border - crossing adjustment raises per capita expenditures for states with nfrs greater than 1 and lowers the same for states with nfrs less than 1 , relative to estimates produced using provider - based expenditures without the adjustment .
the per capita expenditure data in table 6 show that the highest spending per medicare enrollee is for inpatient hospital services ( $ 1,868 ) , followed by physician services ( $ 900 ) , home health care ( $ 122 ) , and other professional care ( $ 117 ) .
overall , the mideast region spends the most per enrollee ( $ 3,852 ) , followed by the far west ( $ 3,809 ) and new england ( $ 3,618 ) .
. some of the high - cost states are california , the district of columbia , maryland , massachusetts , and louisiana .
although state - to - state variations in per capita spending were observed , the spending was within 10 percent of the u.s .
studies investigating the causes of interstate variation in total spending indicate that factors such as state differences in personal income , the supply of health care resources ( including the number of physicians and hospital and nursing home beds per capita ) , the concentration of hospital services in urban areas , and health status explained more than 80 percent of the difference in health spending among states ( u.s .
the states with high per capita income ( e.g. , the district of columbia , massachusetts , maryland , new jersey ) generally spend more per capita .
the opposite is true for states with low per capita income ( e.g. , idaho , utah ) .
the states with relatively high urban populations generally have high costs of care and , consequently , relatively high per capita expenditures ( the district of columbia , massachusetts , florida , connecticut , new york , new jersey , etc . ) .
the average payment per urban enrollee was found to be approximately 17 percent higher than that for rural beneficiaries ( health care financing administration , 1995 ) .
the states with more health care resources tend to experience higher spending by their residents ( such as california , with a high physician - to - population ratio ) .
other factors , such as age and sex composition of medicare enrollees and their health and disability status ( helbing , sangl , and silverman , 1993 ) , might serve as important determinants of per capita variation in medicare spending .
states with poor health status of the residents ( e.g. , georgia , louisiana , and alabama ) tend to spend more per capita than states with better health status ( e.g. , utah , idaho , minnesota , oregon , and hawaii ) ( prospective payment assessment commission , 1995 ) .
in addition , factors such as provider practice patterns , managed - care market penetration , and provider resource costs are also important in explaining regional differences in per capita spending . the high resource costs in louisiana ( 17 percent above the u.s .
average ) might partly explain the high per capita spending in that state ( prospective payment assessment commission , 1995 ) .
the growth of managed care in recent years might also have slowed the growth of per capita spending in states with high hmo enrollment however , evidence is still mixed as to the effect of managed care on growth of expenditures . by analyzing the difference between per capita expenditures with and without adjusting for border crossing , several characteristics are observed : the effect of border crossing is found to be very large ( 9 - 34 percent ) for certain states , such as wyoming , idaho , alaska , and new mexico . for states such as minnesota , north dakota , and the district of columbia , per capita spending estimates decline by 10 - 30 percent as a result of this adjustment even for services such as home health care , for which there is very little border crossing , the effect on some states ( e.g. , new mexico , west virginia , the district of columbia , virginia , south dakota ) is quite substantial ( 8 - 18 percent increase ) . for services such as durable medical equipment ,
a maximum difference of more than 200 percent is also observed ( table 3 ) . as a whole
, the border - crossing adjustment reduces the variability of per capita expenditures across states : the coefficient of variation declines from 22 to 15 percent as a result of using the residence - based estimates to calculate the per capita expenditure .
the adjustment for border crossing has the greatest impact ( measured by mean percent difference between adjusted and unadjusted per capita expenditures ) on per capita expenditures for laboratory services and durable medical supplies , but only a small impact on home health care , outpatient hospital care , and inpatient hospital services . ranked by the average size of the impact , the lowest- to highest - ranking medicare categories
are : home health care ( 0.58 percent ) , outpatient hospital care ( 0.75 percent ) , inpatient hospital care ( 0.98 percent ) , other professional care ( 1.77 percent ) , physician services ( 1.80 percent ) , hospice care ( 3.20 percent ) , nursing homes ( 3.24 percent ) , esrd services ( 4.90 percent ) , medical durables ( 19.0 percent ) , and independent laboratory services ( 51.0 percent ) .
the impact is also found to vary substantially across states . in terms of the variability of impact ( measured by the coefficient of variation of percent differences between adjusted and unadjusted per capita expenditure ) ,
the lowest- to highest - ranking medicare categories respectively are : nursing homes , medical durables , independent laboratory services , other professional care , esrd services , hospice , physician services , home health care , outpatient hospital care , and inpatient hospital care .
that is , the interstate fluctuations of impact are found to be highest for inpatient hospital services and lowest for nursing homes .
the highest variability for hospital services is an indication of the wide fluctuation among states in how border crossing impacts per capita expenditures for this service , although the low average value of the impact for this service indicates that some of the positive and negative impacts cancel out . for services such as independent laboratory and medical durables , on the other hand ,
these findings indicate the sensitivity of the state estimates , especially for hospital services , to the border - crossing adjustment and highlight the significance of making this adjustment .
this analysis indicates the extent to which the border - crossing adjustment influences medicare spending estimates by states .
it should be noted that border crossing is one of several factors that explain differences in spending levels .
other factors , such as differences in personal income , the supply of health care resources , the concentration of hospital services in urban areas , and health status , are often found to be as significant in explaining differences in per capita spending levels ( u.s .
the reason why the impact of the border - crossing adjustment is not visible at the level of average comparison is that positive and negative impacts
however , from the perspective of national and state policymaking based on interstate comparisons , the impact of this adjustment on individual state spending estimates can not be overemphasized .
the next step includes the development of flow matrices for non - medicare beneficiaries for hospital and physician services , by applying a service - mix adjustment developed from two private data bases to medicare flows .
work will also continue on updating flow matrices to reflect expenditures for 1992 and 1993 for both medicare and non - medicare patients
. this update will provide adjustment factors for 1992 and 1993 to the state estimates already published ( levit et al .
the goal of this project is to enhance the methods of producing estimates of health care expenditures per capita and design a system in which state location of population and expenditure estimates are identical .
this project will also enable hcfa to determine the stability of these flow matrices over time .
the results of this work will be used to generate a time series of interstate flow ratios to adjust expenditures from the provider - state location to the beneficiary - residence state .
hcfa will also explore the feasibility of studying border crossing for geographic areas smaller than the state . | as the first step in a pioneering effort by the health care financing administration ( hcfa ) to measure interstate border crossing for services used by both medicare and non - medicare beneficiaries , the authors study the spending behavior of medicare beneficiaries for 10 medicare - covered services . based on interstate flow - of - expenditure data developed for calendar year 1991 ,
the authors analyze the spending patterns of state residents by studying the inflow and outflow rates and the net flow ratios of expenditures incurred by medicare patients .
the report also provides per capita expenditure estimates with residence - based adjustments and evaluates the impact of the border - crossing adjustment for individual services and states . |
bereavement is a common term used to describe having lost someone important or significant through death .
this intense emotion or distress following bereavement is referred to as grief , or the emotional reaction to bereavement .
grief is complex , and incorporates multiple symptoms such as anger and sadness , and varies by the individual , by culture , and across time .
although the experience of grief varies by individual , some individuals experience complicated bereavement processes , more commonly known as complicated grief ( cg ) . while most people experience bereavement over the course of their lifetime , cg is a chronic and debilitating consequence of bereavement that is associated with clinically significant distress and impairment . establishing typical norms for time spent grieving and
the intensity of these symptoms is difficult , and may vary for individuals of different backgrounds and cultures .
cg is thought to occur in 10% of individuals who have been bereaved , and affects people worldwide .
individuals from all countries and cultures are affected by cg , and it has been noted that further investigation is needed to understand the specific role of cultural factors in cg .
although the specific role of cultural factors such as religion , language , and beliefs on the bereavement process may not be known , one can understand the important role culture has in understanding bereavement .
in fact , the diagnostic and statistical manual of mental disorders ( dsm)-iv notes the importance of culture in understanding an individual 's duration and expression of bereavement .
it has been argued that no studies or ideas surrounding grief are truly viewed without a cultural lens . in this sense ,
this trajectory may involve the individual seeking meaning in grief , and constructing new meanings about who they are and who they must be following bereavement .
it is important to truly understand the culturally based meanings and assumptive worlds in which individuals exist before attempting to make sense of their bereavement experience .
schemata , or assumptions , can be shattered by a traumatic change such as death , which results in grief for the bereaved individual . in this case
, individuals must relearn themselves and the world cognitively following their loss , before they can change and advance emotionally .
western values , which view grieving as largely an individual experience , are not shared by all cultures .
rosenblatt discusses the idea of bereavement in a cross - cultural perspective , and highlights that for some people grief is a shared experience , which is shaped by the community 's members .
other studies have also identified grief to be a collective experience , with more concern about the group and less about individual levels of coping .
the experience of grief must be viewed using a wider cultural framework allowing for the examination of different cultural responses to grief . disentangling culture and grief
is a preliminary step in beginning to understand how people grieve , and what kinds of supports they need .
although this research gap exists , it can be hypothesized that aboriginal populations may be at increased risk for cg , given a variety of factors including increased rates of all - cause mortality and death by suicide .
first nations people in north america face concurrent stressors and hardships , including adverse childhood events , poverty , unemployment , and witnessing traumatic events such as violence and homicide .
first nations people also have a past history of multiple stressors resulting from the effects of colonization and forced assimilation ; a significant example being residential school placement , where aboriginal children were forced to leave their homes and were separated from their culture , families , and communities .
this acculturation resulted in cultural oppression , damaged social support , loss of tradition , and experiences of physical , sexual , and emotional abuse for many first nations children .
australian aboriginal people have also experienced intergenerational transmission of violence , abuse , and loss .
this loss of culture and high rates of traumatic events may place aboriginal individuals at increased risk for suicide , as well as cg resulting from traumatic loss and suicide bereavement . while little information is available on aboriginal populations and cg , some authors have discussed the concept of a soul wound or historical trauma and their impact on health and grief .
duran et al discuss the soul wound , and the symptoms that sometimes accompany such trauma .
for example , symptoms of pain , suffering , guilt , and psychological stress have been thought to reflect survivor syndrome , or outcomes resulting from colonialism .
brave heart also argues that aboriginal populations such as the lakota ( teton sioux ) experience impaired grief , and that this grief results from massive cumulative traumas . it may be that impaired grief and cg share or reflect similar concepts and characteristics .
brave heart defines impaired grief as resulting from the prohibition of indigenous spiritual practices , which inhibits the culturally specific ways or modes of working through normative grief .
this cultural bereavement can lead to poor health outcomes such as posttraumatic stress disorder and anxiety disorders .
brave heart also discusses the traditional bereavement or grief process of the lakota , and highlights traditional mourning practices , including visible signs of grief , in that close relatives cut their hair to symbolize the emotional pain of losing the loved one . because the bereaved were identified by short hair , their community treated them respectfully and recognized the mourning process . during this time ,
spirit - keeping ceremonies are also conducted , enabling the grief process for a year after the death . releasing of the spirit and wiping of the tears ceremonies are also held to help resolve grief and to welcome the bereaved into their community . due to the effects of historical trauma , brave heart argues that the lakota were not able to resolve their grief , and experienced impaired grief .
while impaired and complicated grief may share similarities in that grief responses are complex , it is understandable how the prohibition of spiritual practices and high rates of traumatic events may place first nations individuals at increased risk for poor health outcomes .
suicide rates are an important and serious health outcome and public health issue that are linked to cg , and are extremely high for first nations people .
suicide rates among first nations individuals are between 3 and 6 times that of the general population .
canadian first nations suicide rates are higher than in the general population in both the united states and canada . in a population - based examination of the province of manitoba , individuals living in northern communities ( largely consisting of first nations individuals ) were also at increased likelihood for suicide and suicide attempts . the link between complicated grief and increased risk for suicidal behavior has also been demonstrated , as well as the increased likelihood for cg among individuals bereaved by suicide .
although these relationships have not been examined in first nations populations , the role of acculturation and multiple traumas may play a role in these relationships .
chandler and lalonde highlight the role of cultural continuity as a protective factor against youth suicide in first nations .
residential school survivors have been found to experience adverse outcomes such as poor mental health , substance abuse , and criminal activity .
although first nations individuals have higher suicide rates compared with other non - first nations groups , these rates may vary depending on the community as well as individuals in the community .
a study by bolton et al analyzed two american indian samples and found that american indians from northern plains and southwest tribes were less likely to have suicidal thoughts as compared with the us general population .
the authors also found that the northern plains tribe had a greater likelihood of attempting suicide . although some differences were found between tribes , overall american indian samples were at greater risk for suicide attempts than the us general population . in another study examining suicidal behavior in first nations ,
mota et al investigated the correlates of suicidal behavior among a manitoba sample of first nations adolescents .
the goal of this study was to examine the individual , friend / family , and community / tribe correlates of suicidal behavior among on - reserve adolescents .
the authors found that several factors were found to increase the likelihood of suicidal behavior including female sex , depressed mood , abuse or fear of abuse , hospital stay , and substance use . in this study ,
perceived community caring played a protective role , in that adolescents were less likely to engage in suicidal behavior if they perceived their community as caring , as determined by a combination of factors regarding community life .
although further investigation is needed to understand the role of community support and suicidal behavior in first nations , these two studies suggest that the community may play an important role in understanding suicide , as well as the bereavement experience among some first nations populations .
despite the impact of colonization on aboriginal peoples and collective traumas , aboriginal cultures have endured . understanding the role of healing and bereavement in aboriginal populations necessitates the inclusion of cultural and healing traditions ( figure 1 ) .
kirmayer investigates the symbolic aspects of healing among individuals of different cultures . using metaphor theory
, the author argues that one can understand the course of healing by examining metaphorical concepts .
kirmayer discusses a hierarchy of metaphors where there are multiple modes of healing and mediating processes interacting with many levels , such as society and the environment , the community , family , and biological factors .
while this hierarchy of metaphors allows a thorough examination of the factors that may impact the course of bereavement in diverse populations , it can be tailored to help understand grief and bereavement in diverse aboriginal populations ( figure 2 ) .
future research , nevertheless , is needed to examine bereavement among aboriginal populations , including the development of theoretical models that best explains cg as an outcome of bereavement .
such studies must include full partnership with aboriginal communities to help identify risk factors for cg , understand the role of culture among these communities , as well as identify interventions to reduce poor health outcomes such as suicidal behavior . in manitoba , canada ,
the team is comprised of international experts , researchers , and community members , and aims to advance the understanding of effective suicide - prevention strategies for first nations people .
guidance from community members is an essential component of this research team , and is a necessary element of future research in the area of cg . given the paucity of research examining the dimensions of complicated grief in aboriginal populations , more research is required .
this research must consider the role of culture , intergenerational trauma , and traditional healing practices in order to address this important public health issue . | to date there have been no studies examining complicated grief ( cg ) in aboriginal populations .
although this research gap exists , it can be hypothesized that aboriginal populations may be at increased risk for cg , given a variety of factors , including increased rates of all - cause mortality and death by suicide .
aboriginal people also have a past history of multiple stressors resulting from the effects of colonization and forced assimilation , a significant example being residential school placement .
this loss of culture and high rates of traumatic events may place aboriginal individuals at increased risk for suicide , as well as cg resulting from traumatic loss and suicide bereavement .
studies are needed to examine cg in aboriginal populations .
these studies must include cooperation with aboriginal communities to help identify risk factors for cg , understand the role of culture among these communities , and identify interventions to reduce poor health outcomes such as suicidal behavior . |
image - guided tumour ablation is one of the exciting and promising developments in radiology .
radiofrequency ablation ( rfa ) is used in many centres to treat hepato - cellular carcinoma ( hcc ) and hepatic metastases as a complementary method to liver resection .
there is interest in using magnetic resonance ( mr ) imaging to guide the tumour targeting and to perform a post - procedural imaging evaluation based on the different mr contrast mechanisms .
most importantly , mri is the only imaging modality that can provide quantitative and high spatial resolution real time monitoring of the temperature changes ( and hence thermal dose ) evolution in the heated area .
the value of temperature and thermal dose maps as a means of determining the cut - off point ( or endpoint ) for the application of power has not yet been demonstrated clinically , even though there is a sustained interest in this method .
the interest in mr thermometry stems from the hypothesis that real - time monitoring of rfa via mr thermometry should enable the operator to actually follow the evolution of the thermal coagulation necrosis .
this would represent a significant improvement over relying on empirical settings for the electrical power and duration for the rfa procedure . as opposed to global measures such as impedance measures
, the temperature and associated thermal dose maps should provide a local description of the temperature and the state of thermal coagulation of the tissue .
unlike thermocouples inserted in the electrode tip that can measure the temperature at a few points only , mr thermometry can calculate the temperature changes at each pixel .
the derived thermal dose at every pixel could thus be monitored by the operator in real - time , and should provide reliable criteria for deciding when the entire tumour plus a safety margin of 10 mm have been completely necrosed via thermal coagulation ( the endpoint of rfa ) .
damage to adjacent structures such as hepatic veins and bile ducts can also be prevented .
one of the main challenges of rf thermal ablation of liver tumours remains the incomplete ablation typically observed in the vicinity of large vessels which act as important heat sinks due to convection .
the proximity to a large vessel can locally prevent the onset of thermal coagulation , as demonstrated via histology in both animal and patient studies .
in such situations , local temperature and thermal dose real - time information should help the operator decide whether to continue the application of power , add more electrodes ( when multi - polar configurations are possible ) , or reposition one or more of the existing electrodes .
some of the earlier mr monitoring of rfa was performed under open - bore low - field mr systems with the t1 temperature measurement method . however , t1 exhibits not only temperature dependence but also a dependence on structural tissue changes , such as coagulation and tissue drying .
thus , t1 monitoring is only a qualitative indication that something is happening in the heating zone , and empirical recipes need to be used to be able to draw conclusions about the state of the tissue during the monitoring .
the advent of closed - bore larger diameter high - field ( 1.5 t ) interventional mr systems has made it possible to perform rfa inside 1.5 t systems and thus implement the proton resonance frequency ( prf ) mr thermometry method .
prf at 1.5 t has superior sensitivity to the t1 method and depends only on temperature and not on tissue structural changes ( tissue structural changes may only reflect on a loss of snr in the heated region which leads to a loss of temperature accuracy ) .
the prf method at 1.5 t is promising , and provided that the susceptibility map remains constant , fat saturation is robust , and respiratory motion is accounted for , can give a real quantitative measure of the temperature increments at each pixel with high spatial and temporal resolution .
although the principles of real time monitoring of rfa of liver tissue with the prf mr thermometry method at 1.5 t are well established and have been demonstrated in the liver ex - vivo and in animal studies , very few clinical results have been published .
this is due to several technical challenges , such as rf interferences that degrade the mr images , continuous respiratory motion of the liver leading to intra - scan artifacts , large temperature errors in the presence of inter - scan motion ( unless corrected by image registration algorithms ) , and large susceptibility artifacts of the rf electrodes which block the useful information around the active zone of the electrode .
the various types of electrodes used for rfa are described in the proposal for updating the terminology for rfa electrodes by mulier et al .
the susceptibility artifacts are a persistent problem for rf electrodes , regardless of the manufacturer . despite the lack of results for an integral mr - monitored patient rfa procedure
, progress has been made on several aspects of mr thermometry with prf at 1.5 t , as well as on targeting and post - evaluation imaging with mr .
the goal of this review is to discuss some of the recent confirmations of the role of mri in the targeting , intra - procedural monitoring , and post - procedural evaluation of rfa of liver tumours with mri , and give some indication of potential future trends . in order to understand the challenges of rf ablation with mr
guidance and mr monitoring , several subsequent steps of the procedures must be distinguished , namely targeting , monitoring and controlling , and post - procedural assessment .
the targeting of the tumours has been performed under ultrasound ( us ) or computed tomography ( ct ) guidance , but mr - guided electrode placement may provide advantages and may be , in some cases , the only clinical option as some liver tumours are not visible under us or ct . the feasibility of rf electrode placement with sequential t1 images ( not real - time ) was demonstrated for both open - bore 0.23 t systems and closed - bore 1.5 t systems .
the major drawback of mr - guided targeting has so far been the long procedure time .
fluoroscopic pulse sequences and in - room mr - compatible screens has significantly decreased the electrode placement time . for mr targeting
, the conspicuity of lesions may be enhanced occasionally by the administration of hepato - specific contrast materials such as mangafodipir - trisodium ( mn - dpdp ; teslascan , ge healthcare ) .
initial clinical results at our institution at 1.5 t ( magnetom espree , siemens medical solutions , erlangen , germany ) with a radial interleaved real - time acquisition with the ability to adjust the imaging plane interactively during the scan indicate that mr may supersede all other modalities for image - guided targeting ( ultrasound , ct ) thanks to : multi - planar double oblique targeting associated with high spatial resolution , speed , reliability , and lack of ionizing radiation .
the first four images in fig . 1 show selected frames from the advancement of an mr - compatible rf electrode ( celon ag , teltow , germany ) in the liver of a patient placed under general anaesthesia , 20 min after the administration of mn - dpdp .
the update rate provided by the balanced steady state free precession ( b - ssfp or truefisp ) radial k - space interactive pulse sequence ( irttt , siemens medical solutions , erlangen , germany ) is of the order of 300 ms , and spatial resolution of 1.6 1.6 mm .
the targeting was immediately followed by a high resolution t1-weighted volume interpolated breath hold examination ( vibe ) mr control ( fig .
1 ) to confirm the accuracy of the needle placement before applying the rf power and thus starting the tissue thermal coagulation .
figure 1liver metastases targeting under mri real - time guidance .
this procedure necessitated two parallel electrodes placed close to the outer boundary of the tumour on both sides of the tumour ( the electrical current was circulating between the active zones on the two electrode tips ) .
therefore , the target was not the centre of the tumour , but the region just outside its boundary .
four different stages of the advancement of the first electrode are shown from left to right ( a d ) .
the targeting was assessed with a high - resolution vibe t1-weighted image ( e ) which confirmed the correct final position of the two rf electrodes . note
that whereas the actual diameter of each mr - compatible bipolar rf electrode is 2 mm , the width of the susceptibility artifact is of the order of 812 mm ( depending on the sequence te and on the orientation of the needle with respect to b0 ) .
this procedure necessitated two parallel electrodes placed close to the outer boundary of the tumour on both sides of the tumour ( the electrical current was circulating between the active zones on the two electrode tips ) .
therefore , the target was not the centre of the tumour , but the region just outside its boundary .
four different stages of the advancement of the first electrode are shown from left to right ( a d ) .
the targeting was assessed with a high - resolution vibe t1-weighted image ( e ) which confirmed the correct final position of the two rf electrodes . note
that whereas the actual diameter of each mr - compatible bipolar rf electrode is 2 mm , the width of the susceptibility artifact is of the order of 812 mm ( depending on the sequence te and on the orientation of the needle with respect to b0 ) . for treatment of large tumours
( diameter > 3 cm ) or tumours close to major blood vessels , placement of multiple rf electrodes may be necessary , and this is possible when a multi - polar rf generator mode is available .
the new interactive imaging capabilities of interventional mr systems should enable the placement of three electrodes within a reasonable time ( of the order of 30 min ) .
mr - compatible ultrasound systems may have a complementary role for the initial insertion of the electrode especially when the space for needle placement inside the closed bore is limited . in the future ,
monitoring and controlling refer to the continuous ( or sequential ) viewing of temperature and thermal effects occurring in the ablation zone during application of power and to the intra - procedural use of image - based feedback to modify the procedure ( i.e. change electrode position or adapt energy delivery algorithm ) .
the determination of the optimal endpoint from temperature and thermal dose maps represents the main goal of monitoring .
when rf ablation and mr thermometry are performed simultaneously , the mr images show severe rf interference due to the harmonics of the rf generator ( operating at a nominal frequency of the order of 500 khz ) that fall within the reception window of the mr receive chain ( centred around the 63.7 mhz carrier ) . in the past
, intermittent power application and mr imaging was proposed for rfa , but this leads to implementation problems , lengthens the procedure , and may render it more inefficient .
the use of hardware filtering inserted in the transmission line from the generator to the rf electrode to eliminate interfering harmonics and thus restore acceptable image snr , permitting simultaneous rfa and mr thermometry , was demonstrated for 0.5 t and 1.5 t animal studies . at our institution , we were also able to restore the snr of the mr images by introducing hardware filtering in the transmission line from the rf generator to the rf electrode and thus perform truly simultaneous rfa and mr thermometry at 1.5 t. t1 , diffusion , and spectroscopy - based mr thermometry methods are theoretically possible but have problems for real - time temperature monitoring .
the t1 method is not appropriate as it combines effects of temperature and tissue structural changes .
the method of choice at 1.5 t remains prf , as it provides linearity with temperature and tissue-independence.with the prf method , a time series of phase images are acquired during the heating using spoiled gradient - echo based acquisitions . for such sequences , the temperature sensitivity can be optimized by setting the echo time te equal to the t2 * of the liver tissue .
the dynamic ( time ) series of phase images acquired during the rf ablation procedure can be used to calculate the increments in temperature by subtracting the phase of the initial ( reference ) phase image from each phase image in the dynamic series , according to eq .
( 1 ) :
( 1 )
where = 0.01 ppm/c is the temperature - dependent water chemical shift , is the gyromagnetic ratio and b0 is the field strength .
the thermal maps resulting from this type of post - processing are usually displayed superimposed on the magnitude images .
recently , it has been shown that multi - acquisition and multi - echo balanced steady state free precession sequences could also be used to perform prf mr thermometry , with a potential for increased temperature sensitivity .
with prf , in immobile tissue at 1.5 t , the typical performance of single mr thermometry is less than 1c temperature standard deviation ( i.e. temperature noise ) , the temporal resolution is less than 1 s , and spatial resolution is about 2 2 mm .
lipid signal suppression is necessary as the temperature - dependent chemical shift of lipid hydrogen atoms is nearly zero , and it is usually achieved by water - selective slice excitation with binomial pulses . for rf ablation ,
as the electrode is fixed with respect to the imaged tissue , the susceptibility field map remains unchanged throughout the experiment and therefore susceptibility map corrections are not needed .
however , the hardware - related global phase drift during the procedure needs to be eliminated . finally , as the method is based on subtracting a reference phase image from the current phase image , robust respiratory triggering for the acquisition and residual motion image post - processing corrections are needed to achieve correct temperature measurements .
respiratory triggering will evidently reduce the temporal resolution of thermometry to one temperature update per respiratory cycle period at each slice .
preliminary animal study results at our institution also indicate that in - vivo mr thermometry with the prf method at 1.5 t during simultaneous rfa is feasible .
power was applied via a bipolar mr - compatible cooled electrode , and the imaging was performed with a gradient - echo - echo planar hybrid sequence with respiratory triggering .
the phase images were used to calculate the temperature increments according to the prf method , and the thermal dose was also calculated .
( a ) representative thermal map calculated and displayed in real - time superimposed on the magnitude mr images : blue , + 5c ; green , + 10c ; red , + 15c and above .
( b ) final thermal dose map ( red indicates pixels where at least one lethal thermal dose has been reached ) .
( c ) gd - eob - enhanced t1-weighted image showing the thermal coagulation zone immediately after the rf ablation .
( d ) histological aspect of the ablation zone also shows the cooling effect of the inferior vena cava .
( a ) representative thermal map calculated and displayed in real - time superimposed on the magnitude mr images : blue , + 5c ; green , + 10c ; red , + 15c and above .
( b ) final thermal dose map ( red indicates pixels where at least one lethal thermal dose has been reached ) . a base temperature of 37c was assumed .
( c ) gd - eob - enhanced t1-weighted image showing the thermal coagulation zone immediately after the rf ablation .
( d ) histological aspect of the ablation zone also shows the cooling effect of the inferior vena cava .
the classical thermal dose calculation is based on an empirical model proposed by sapareto et al . in this model , which results by applying a few simplifications to the arrhenius equation for tissue damage ,
a thermal dose equivalent at 43c is calculated as follows :
( 2 )
where t43 is the thermal dose in equivalent minutes at 43c , t the average temperature during t and c is a constant resulting from the arrhenius model and approximated to 0.5 above 43c and 0.25 below 43c . at 43c ,
empirical studies show that it will take 240 min to cause cell necrosis via protein denaturizing and thermal coagulation .
the thermal dose concept enables us to compare the effect of an arbitrary temperature time evolution to the equivalent effect of hypothetically exposing the same tissue to a constant temperature of 43c for a certain duration t43 .
( 2 ) allows t43 to be calculated precisely . in typical rfa thermal dose real - time monitoring software tools ,
if t43 is superior to 240 for a given pixel , the pixel becomes red on the thermal dose map and it is assumed to correspond to necrosed tissue ( this is also illustrated in our preliminary in vivo animal study in fig .
2b ) . for liver tissue , the correlation between the prediction of thermal coagulation region size by thermal dose ( assuming a lethal value of t43 = 240 ) and by histology was performed ex - vivo , and in - vivo in animal studies , but this type of correlation will remain difficult for patients , with few cases undergoing resection of a liver lobe containing a previously ablated lesion .
also , we should note that the sapareto model and the lethal t43 value of 240 were developed for hyperthermia treatments with temperatures around 43c , whereas the tissue heating with rfa is much more rapid and can reach very high temperatures .
we note that whenever temperatures of about 57c are reached the sapareto model predicts essentially instantaneous coagulation .
thus , binary threshold methods based exclusively on temperature isotherms are not in contradiction with the thermal dose model , but they may be only partially correct as they only take into account pixels that have reached temperatures above 57c ( or some other fixed threshold ) for the zone of thermal coagulation .
however , depending on the temperature time evolution , tissue regions that have seen varying temperatures between 43c and 57c ( for the typical procedure durations between 10 and 30 min ) may also accumulate enough thermal dose to become thermally coagulated .
for such regions temperature isotherms recorded at a fixed time instant may not be sufficient by themselves to predict thermal coagulation .
it appears that the value of 240 equivalent minutes at 43c as lethal thermal dose has some predictive value , but it needs further validation for use as a complete ablation criterion for rfa of liver tumours .
the predictive value for complete coagulation for both thermal dose and binary threshold methods is difficult to evaluate in - vivo in patient studies due to the errors in the identification of the actual boundary of the coagulation zone from t1- and t2-weighted post - procedural imaging . a bovine liver study relying on mr imaging only for the identification of the ablated zone found the lethal t43 to be 340 ( and not the typical 240 ) equivalent minutes at 43c , but with large error bounds .
ideally , the thermal dose map predictive value and the post - procedural mr imaging predictive value need to be established by comparisons against histology .
the histological study resulting from patient rfa followed within 6 weeks by resection of malignant liver tumours indicated that the boundary between viable and non - viable cells is sharply delimited .
generally , the number of rfa patients undergoing a subsequent resection of the ablated zone is limited , and thus such validation data may also be limited .
animal studies are important in confirming the predictive value of the lethal thermal dose by using histology as the reference .
a study of rfa ablation in rabbit liver has indicated that the thermal dose maps have an average predictive precision of about 1 mm through a comparison with histological measurements performed after 8 days .
the lethal thermal dose may not be the same for cells of different types of tissue and thus it may exhibit a dependence on the tumour type .
another source of error for patient studies is the unknown base temperature of the liver , typically assumed to be equal to 37c .
monitoring and controlling refer to the continuous ( or sequential ) viewing of temperature and thermal effects occurring in the ablation zone during application of power and to the intra - procedural use of image - based feedback to modify the procedure ( i.e. change electrode position or adapt energy delivery algorithm ) .
the determination of the optimal endpoint from temperature and thermal dose maps represents the main goal of monitoring .
when rf ablation and mr thermometry are performed simultaneously , the mr images show severe rf interference due to the harmonics of the rf generator ( operating at a nominal frequency of the order of 500 khz ) that fall within the reception window of the mr receive chain ( centred around the 63.7 mhz carrier ) . in the past
, intermittent power application and mr imaging was proposed for rfa , but this leads to implementation problems , lengthens the procedure , and may render it more inefficient .
the use of hardware filtering inserted in the transmission line from the generator to the rf electrode to eliminate interfering harmonics and thus restore acceptable image snr , permitting simultaneous rfa and mr thermometry , was demonstrated for 0.5 t and 1.5 t animal studies . at our institution , we were also able to restore the snr of the mr images by introducing hardware filtering in the transmission line from the rf generator to the rf electrode and thus perform truly simultaneous rfa and mr thermometry at 1.5 t. t1 , diffusion , and spectroscopy - based mr thermometry methods are theoretically possible but have problems for real - time temperature monitoring .
the t1 method is not appropriate as it combines effects of temperature and tissue structural changes .
the method of choice at 1.5 t remains prf , as it provides linearity with temperature and tissue-independence.with the prf method , a time series of phase images are acquired during the heating using spoiled gradient - echo based acquisitions . for such sequences , the temperature sensitivity can be optimized by setting the echo time te equal to the t2 * of the liver tissue .
the dynamic ( time ) series of phase images acquired during the rf ablation procedure can be used to calculate the increments in temperature by subtracting the phase of the initial ( reference ) phase image from each phase image in the dynamic series , according to eq .
( 1 ) :
( 1 )
where = 0.01 ppm/c is the temperature - dependent water chemical shift , is the gyromagnetic ratio and b0 is the field strength .
the thermal maps resulting from this type of post - processing are usually displayed superimposed on the magnitude images .
recently , it has been shown that multi - acquisition and multi - echo balanced steady state free precession sequences could also be used to perform prf mr thermometry , with a potential for increased temperature sensitivity .
with prf , in immobile tissue at 1.5 t , the typical performance of single mr thermometry is less than 1c temperature standard deviation ( i.e. temperature noise ) , the temporal resolution is less than 1 s , and spatial resolution is about 2 2 mm .
lipid signal suppression is necessary as the temperature - dependent chemical shift of lipid hydrogen atoms is nearly zero , and it is usually achieved by water - selective slice excitation with binomial pulses . for rf ablation ,
as the electrode is fixed with respect to the imaged tissue , the susceptibility field map remains unchanged throughout the experiment and therefore susceptibility map corrections are not needed .
however , the hardware - related global phase drift during the procedure needs to be eliminated . finally , as the method is based on subtracting a reference phase image from the current phase image , robust respiratory triggering for the acquisition and residual motion image post - processing corrections are needed to achieve correct temperature measurements .
respiratory triggering will evidently reduce the temporal resolution of thermometry to one temperature update per respiratory cycle period at each slice .
preliminary animal study results at our institution also indicate that in - vivo mr thermometry with the prf method at 1.5 t during simultaneous rfa is feasible .
power was applied via a bipolar mr - compatible cooled electrode , and the imaging was performed with a gradient - echo - echo planar hybrid sequence with respiratory triggering .
the phase images were used to calculate the temperature increments according to the prf method , and the thermal dose was also calculated .
( a ) representative thermal map calculated and displayed in real - time superimposed on the magnitude mr images : blue , + 5c ; green , + 10c ; red , + 15c and above .
( b ) final thermal dose map ( red indicates pixels where at least one lethal thermal dose has been reached ) .
( c ) gd - eob - enhanced t1-weighted image showing the thermal coagulation zone immediately after the rf ablation .
( d ) histological aspect of the ablation zone also shows the cooling effect of the inferior vena cava .
( a ) representative thermal map calculated and displayed in real - time superimposed on the magnitude mr images : blue , + 5c ; green , + 10c ; red , + 15c and above .
( b ) final thermal dose map ( red indicates pixels where at least one lethal thermal dose has been reached ) . a base temperature of 37c was assumed .
( c ) gd - eob - enhanced t1-weighted image showing the thermal coagulation zone immediately after the rf ablation .
( d ) histological aspect of the ablation zone also shows the cooling effect of the inferior vena cava .
the classical thermal dose calculation is based on an empirical model proposed by sapareto et al . in this model , which results by applying a few simplifications to the arrhenius equation for tissue damage ,
a thermal dose equivalent at 43c is calculated as follows :
( 2 )
where t43 is the thermal dose in equivalent minutes at 43c , t the average temperature during t and c is a constant resulting from the arrhenius model and approximated to 0.5 above 43c and 0.25 below 43c . at 43c ,
empirical studies show that it will take 240 min to cause cell necrosis via protein denaturizing and thermal coagulation .
the thermal dose concept enables us to compare the effect of an arbitrary temperature time evolution to the equivalent effect of hypothetically exposing the same tissue to a constant temperature of 43c for a certain duration t43 .
( 2 ) allows t43 to be calculated precisely . in typical rfa thermal dose real - time monitoring software tools ,
if t43 is superior to 240 for a given pixel , the pixel becomes red on the thermal dose map and it is assumed to correspond to necrosed tissue ( this is also illustrated in our preliminary in vivo animal study in fig .
, the correlation between the prediction of thermal coagulation region size by thermal dose ( assuming a lethal value of t43 = 240 ) and by histology was performed ex - vivo , and in - vivo in animal studies , but this type of correlation will remain difficult for patients , with few cases undergoing resection of a liver lobe containing a previously ablated lesion . also , we should note that the sapareto model and the lethal t43 value of 240 were developed for hyperthermia treatments with temperatures around 43c , whereas the tissue heating with rfa is much more rapid and can reach very high temperatures .
we note that whenever temperatures of about 57c are reached the sapareto model predicts essentially instantaneous coagulation .
thus , binary threshold methods based exclusively on temperature isotherms are not in contradiction with the thermal dose model , but they may be only partially correct as they only take into account pixels that have reached temperatures above 57c ( or some other fixed threshold ) for the zone of thermal coagulation
. however , depending on the temperature time evolution , tissue regions that have seen varying temperatures between 43c and 57c ( for the typical procedure durations between 10 and 30 min ) may also accumulate enough thermal dose to become thermally coagulated .
for such regions temperature isotherms recorded at a fixed time instant may not be sufficient by themselves to predict thermal coagulation .
it appears that the value of 240 equivalent minutes at 43c as lethal thermal dose has some predictive value , but it needs further validation for use as a complete ablation criterion for rfa of liver tumours .
the predictive value for complete coagulation for both thermal dose and binary threshold methods is difficult to evaluate in - vivo in patient studies due to the errors in the identification of the actual boundary of the coagulation zone from t1- and t2-weighted post - procedural imaging . a bovine liver study relying on mr imaging only for the identification of the ablated zone found the lethal t43 to be 340 ( and not the typical 240 ) equivalent minutes at 43c , but with large error bounds .
ideally , the thermal dose map predictive value and the post - procedural mr imaging predictive value need to be established by comparisons against histology .
the histological study resulting from patient rfa followed within 6 weeks by resection of malignant liver tumours indicated that the boundary between viable and non - viable cells is sharply delimited .
generally , the number of rfa patients undergoing a subsequent resection of the ablated zone is limited , and thus such validation data may also be limited .
animal studies are important in confirming the predictive value of the lethal thermal dose by using histology as the reference .
a study of rfa ablation in rabbit liver has indicated that the thermal dose maps have an average predictive precision of about 1 mm through a comparison with histological measurements performed after 8 days .
. the lethal thermal dose may not be the same for cells of different types of tissue and thus it may exhibit a dependence on the tumour type .
another source of error for patient studies is the unknown base temperature of the liver , typically assumed to be equal to 37c .
the ultimate goal of the mr monitoring with thermal dose maps is to confirm complete tumour ablation ( including the 10 mm safety margin ) and thus the endpoint of the procedure .
however , the reliability of the thermal dose as a predictor of complete ablation still needs to be validated in patient studies .
thus , at the present time , post - procedural imaging remains necessary for the confirmation of complete tumour coverage , and is often achieved in clinical routine via a ct scan .
another possibility is to perform a robust mr - image based protocol for the evaluation of the rfa procedure success immediately after the rfa procedure , while the patient is still inside the mr system .
t1 , t2 , perfusion and diffusion imaging may all play a role as they all reflect the tissue thermal coagulation as a change of contrast .
first , the shape and size of the resulting coagulation volume needs to be characterized .
a standardized description terminology for the size and geometry of the thermal coagulation volumes has been proposed , including description of the index tumour , the ablation zone , and the ablation margin .
second , and more importantly , the post - procedural mr imaging should be sufficient to indicate the success of the procedure in terms of complete tumour coverage .
the tissue structural changes will reflect on the signal intensity for the different image contrasts .
on t2-weighted images , the hyperintense region corresponding to the tumour on pre - procedural imaging is expected to become isointense with the parenchyma on post - procedural imaging after complete thermal coagulation . in reality , the necrosis zone is surrounded in the early phase after ablation by a zone in which phenomena such as oedema , haemorrhage and inflammation lead to signal hyperintensity on t2-weighted images . this makes it difficult to visually identify the exact location of the border between coagulation necrosis and viable cells from t2-weighted images with limited spatial resolution .
therefore , the t2-weighted imaging in the early phase is not sufficient by itself to confirm the success of the rf ablation procedure .
hepato - specific contrast materials may help differentiate peri - ablational enhancement in the haemorrhage zone from residual tumour . for the identification of residual ( incompletely ablated ) tumour ,
dynamic gd enhancement may play a role , and in particular a double - contrast method where iron - oxide particles are administered to render the viable parenchyma hypo - intense prior to dynamic gd - enhanced imaging has been proposed to identify incompletely ablated tumour regions .
also , new experimental intravascular mr contrast agents are investigated as potential indicators of differences in blood vessel density between ablated and residual tumour areas .
finally , diffusion imaging may play a role in characterizing the post - ablation region as the coagulated region will exhibit very low apparent diffusion coefficient ( adc ) values , whereas the haemorrhage zone will exhibit higher adc than the rest of the parenchyma .
the most used criterion remains the t2-weighted evaluation , whereby pre - treatment hyperintense tumour regions are expected to become slightly hypointense or isointense with the liver tissue after the procedure .
illustrates a case of pre- and post - procedural t2-weighted images at our institution .
figure 3changes in signal in the ablation area . left : pre - ablation respiratory - triggered t2-weighted haste images showing a 3-month - old hypointense necrosis from a previous rf ablation of a liver metastases ( b ) and a hyperintense local tumour recurrence to the left of the old ablation zone ( a ) .
right : image acquired with the same sequence immediately after the rfa procedure showing hypointensity in region a as well . note the hyperintense rim corresponding to haemorrhage .
changes in signal in the ablation area . left : pre - ablation respiratory - triggered t2-weighted haste images showing a 3-month - old hypointense necrosis from a previous rf ablation of a liver metastases ( b ) and a hyperintense local tumour recurrence to the left of the old ablation zone ( a ) .
right : image acquired with the same sequence immediately after the rfa procedure showing hypointensity in region a as well .
mr imaging could enhance the success of rf ablation of liver tumours due to its potential to provide accurate targeting , monitoring , and post - procedural evaluation . for targeting , the feasibility of rapid and accurate electrode placement during real - time mr imaging has been demonstrated . for thermometry monitoring and control , the main remaining challenges are related to the motion of the liver due to respiration , the large electrode susceptibility artifacts , and the determination of a correct lethal thermal dose for in - vivo tumour and parenchyma cells .
even though the ability to perform simultaneous rf ablation and mr acquisition with appropriate rf filtering has already been demonstrated , further improvements in the filtering scheme and the generator design may be possible to minimize interference and increase the image snr in all configurations . mr thermometry and thermal dose mapping in real time will remain essential for visualizing areas with incomplete coagulation due to perfusion heat sink effects . for post - treatment assessment , mr should be able to distinguish between the different regions of an ablation zone more accurately than other imaging modalities .
the vision is to use exclusively mri at all stages of the rf ablation procedure and move towards an integrated mr - guided and monitored rfa procedure
. the advantage would be the ability to combine and superimpose the following information for quantification with an integrated clinical evaluation tool : pre - procedural tumour and neighbouring vessel segmentation with associated 3d rendering and volumetric measures ; 3d thermal dose maps predicting the extent of thermal coagulation ; and post - procedural 3d imaging with multiple contrast mechanisms .
| abstractthere is a sustained interest in using magnetic resonance ( mr ) thermometry to monitor the radiofrequency ablation of liver tumours as a means of visualizing the progress of the thermal coagulation and deciding the optimal end - point . despite numerous technical challenges ,
important progress has been made and demonstrated in animal studies .
in addition to mr thermometry , mr can now be used for the guidance of the tumour targeting with fluoroscopic rapid image acquisition , and it can provide several contrast mechanisms for post - procedural assessment of the extent of the thermal coagulation zone .
challenges of in vivo simultaneous mr thermometry implementation and the current limitations of the thermal dose model for the estimation of the extent of the thermal coagulation zone are discussed .
mr imaging could enhance the success of rf ablation of liver tumours due to its potential to provide accurate targeting , monitoring , and post - procedural evaluation . |
blood flow rate ( bfr ) < 300 ml / min often is used to define hemodialysis catheter dysfunction , including in the national kidney foundation 's ( nkf ) kidney disease outcomes quality initiative ( kdoqi ) vascular access guidelines [ 1 , 2 ] , and in many research studies . causes of catheter dysfunction include mechanical kinking , malpositioning of the catheter tip , thrombus accumulation , and growth of a fibrin sheath .
early dysfunction , which has been defined as occurring within the first two weeks of placement , is most often , but not exclusively , caused by mechanical problems . delayed or late dysfunction is typically caused by thrombus accumulation , with or without the presence of a fibrin sheath , and is considered to be the most likely cause of low bfr overall [ 47 ] .
other definitions of catheter dysfunction reported in the literature include frequent arterial and venous pressure alarms , poor conductance , and poor dialysis efficiency based on urea reduction ratio or kt / v calculations : these have been applied when evaluating the impact of catheter dysfunction on clinical outcomes , economic expenditures , and patient quality of life [ 811 ] .
however , the impact of catheter dysfunction using a bfr threshold , such as in the nkf - kdoqi guidelines , has received less attention .
one notable exception is a recent study examining the relationship between hemodialysis catheter bfr and dialysis adequacy in a cohort of 259 patients at two university - based centers .
the premise for this study was that since the nkf - kdoqi blood flow threshold for catheter dysfunction was opinion based and has been interpreted to mean that maintaining bfr > 300 ml / min is necessary for adequate dialysis , it is important to better understand the association between bfr and dialysis adequacy .
the study found that mean bfrs < 300 ml / min were not commonly associated with dialysis inadequacy , leading the authors to conclude that strict adherence to the guideline could result in a significant number of unnecessary interventions . to our knowledge , however ,
the impact of hemodialysis catheter bfr < 300 ml / min on dialysis and other medical services has not been evaluated .
the objective of this study was to identify medical service utilization , including missed sessions , access - related procedures , and all - cause hospitalizations , associated with dialysis catheter dysfunction defined according to a bfr threshold < 300 ml / min .
we conducted a retrospective cohort study using data from davita inc . and the united states renal data system ( usrds ) .
the davita clinical data warehouse is a repository for detailed demographic , treatment , medication , and laboratory information .
information is available for each patient 's individual dialysis sessions , allowing the investigator to reconstruct detailed longitudinal treatment histories .
the usrds is a national data system that collects , analyzes , and distributes information about end - stage renal disease ( esrd ) .
the dataset includes the centers for medicare and medicaid services ' ( cms ) , renal beneficiary and utilization system ( rebus ) , and the esrd standard analysis files ( saf ) .
rebus contains demographic , diagnosis , and treatment history information for all medicare beneficiaries with esrd , a point prevalent cohort of approximately 570,000 in the second quarter of 2009 .
the safs contain 100% of part a and part b institutional claims and part b physician supplier claims for these patients .
the dataset used in this project consisted of a point prevalent dialysis patient population in august 2004 , with a maximum followup period through december 31 , 2006 .
patients were included in this study based on the following criteria : they had at least eight continuous weeks of hemodialysis exclusively through a catheter between august 1 , 2004 , and december 31 , 2006 ; in the first eight weeks of catheter dialysis , they did not have a gap between two consecutive outpatient dialysis sessions > 30 days in which they were not hospitalized ; they had both part a and part b medicare coverage during the entire catheter dialysis period ; they did not have a kidney transplant during the entire catheter dialysis period ; at least 95% of their catheter dialysis sessions had actual and planned bfrs between 100 ml / min and 500 ml / min ; they were alive and in the dataset for at least 90 days following the first catheter dialysis session ( figure 1 ) .
planned and actual bfr values < 100 ml / min or > 500 ml / min were set to missing to minimize the potential impact of coding errors . in the final cohort , 99.9% of bfr values were within this range .
patients were followed from their first catheter dialysis session ( defined as their index date ) , to their last catheter dialysis session that was uninterrupted by either a change in access or dialysis modality .
this was defined as their observation period . for each patient included in the study
, we reconstructed a longitudinal history of catheter dialysis and medical resource use during their observation period .
reasons for reaching the end of the observation period were ( i ) death , if the patient died on or before december 31 , 2006 , and if the last catheter dialysis session was within 30 days of death , ( ii ) end of data ( censored ) , if the last catheter dialysis session was within 30 days of december 31 , 2006 , or ( iii ) change in access type or modality , if the last observed catheter dialysis session was not due to either death or the end of data .
catheter dysfunction was defined as actual bfr < 300 ml / min despite a planned bfr 300 ml / min .
a slight modification of the nkf - kdoqi vascular access guideline was adopted to eliminate misclassification of catheter dysfunction where the intent , as indicated by planned bfr , was to provide bfr < 300 ml / min .
the outcome variables in this study were dialysis run time ( in minutes ) , missed dialysis session due to access problems , access - related procedures , and all - cause hospitalization .
the davita data contained a record for each missed session , which included the date and the reason for the missed session , including access problems .
access - related procedures were identified using health care common procedure coding system ( hcpcs ) codes from medicare claims .
limited definition of access - related procedures consisted of the following : injection for catheter evaluation with fluoroscopy ( 36598 ) ; thrombolytic declotting of catheter ( 36593 ) ; mechanical removal of clot ( 36596 ) ; mechanical removal of intraluminal ( intracatheter ) obstructive material ( 75902 ) ; injection of tpa
the expanded definition also included tunneled catheter exchange or replacement ( 36581 ) and the combination of removal of tunneled catheter ( 36589 ) plus tunneled catheter insertion ( 36558 ) .
we used two different approaches to analyze associations between catheter dysfunction sessions and economic outcomes : case - crossover and multivariate - repeated measures analysis . in the case - crossover analyses ( figure 2 )
, we included only patients who had the event of interest ( e.g. , missed dialysis session due to access problem ) .
for each of these patients , we identified the first event at least six sessions following the beginning of catheter dialysis .
as shown in figure 2 , we divided the six dialysis sessions immediately preceding the event into two exposure periods : the case period was defined as sessions 13 immediately preceding the event , and the control period was defined as sessions 46 preceding the event . for each patient
, we identified the presence of at least one session with catheter dysfunction within each of the two periods , case and control .
patients were then divided into four groups , as illustrated in the two - by - two table within figure 2 : catheter dysfunction in both periods ( labeled a in the two - by - two table ) ; no catheter dysfunction in either period ( d ) ; catheter dysfunction in the case but not the control period ( c ) ; catheter dysfunction in the control but not the case period ( b ) .
the odds of the exposure ( in this case , the presence of at least one catheter dysfunction session ) being associated with the outcome of interest is defined as the ratio of the count of patients with a catheter dysfunction session in the case , but not the control , period divided by the count of patients with a catheter dysfunction session in the control , but not the case , period ( c / b in figure 2 ) . as
patients serve as their own controls , there is no need to adjust this ratio for covariables .
we conducted a sensitivity analysis , where we changed the lengths of the case and control periods from three sessions each to one session each .
multivariate - repeated measures analyses [ 16 , 17 ] were used as the second approach .
all patients in the catheter dialysis cohort were included in these analyses , as were all of their sessions .
these analyses used individual dialysis sessions as the repeated measures and incorporated as covariables patient age , gender , race , underlying cause of renal failure , esrd network , dialysis vintage , charlson comorbidity index , days since the start of catheter dialysis , and whether the patient could have started catheter dialysis prior to the beginning of the observation window . each dialysis session included a binomial outcome variable indicating whether the outcome , for example , missed session due to access problems , occurred between the date of the current session and the date of the following session .
therefore , the repeated measures analysis was designed to measure the relative odds of the outcome occurring before the following dialysis session , among those with versus without catheter dysfunction in the previous session .
< 300 ml / min versus 300 ml / min , the reference category ) , we created five intervals of bfr ( 100<150 ml / min ; 150<200 ml / min ; 200<250 ml / min ; 250<300 ml / min ; and 300 ml / min , the reference category ) and then repeated all the multivariate analyses using this specification instead of the dichotomous specification as the independent variable for catheter dysfunction . in these analyses
analysis file construction , descriptive analyses , and the case - crossover analyses were performed in sas ( version 9.1.3 ) .
of 44,470 patients in the combined davita usrds database , 9,707 ( 22% ) met all the inclusion criteria ( figure 1 ) .
the average age of the cohort was 62 years ( range 18102 ) , 53% were female gender , 40% were black race , 46% had diabetes , 28% had hypertension recorded as their underlying cause of renal failure , and 63% had a charlson comorbidity index of 2 ( table 1 ) .
the average duration of esrd prior to entering the cohort was 40 months ( range 0367 ) . the median duration of catheter dialysis was 190 days ( interquartile range 108386 days ) .
reasons for the end of catheter dialysis were as follows : 7,476 ( 77% ) switched to another dialysis access type or modality and 1,068 ( 11% ) died while receiving catheter dialysis .
there were 1,163 ( 12% ) patients who were alive and on catheter dialysis at the end of the data ( 12/31/06 ) .
the cohort accounted for 1,075,701 catheter dialysis sessions over the entire observation period ( mean 111 ; median 73 ; range 7502 ) and 218,166 sessions during the first eight weeks of catheter dialysis ( mean 22 ; median 24 ; range 348 ; mean per week 3 sessions ) .
there were 1,074,966 ( 99.9% of 1,075,701 ) sessions with a planned bfr between 100 and 500 ml / min : 400 ml / min ( 38% ) ; 350 ml / min ( 33% ) ; 300 ml / min ( 13% ) ; < 300 ml / min ( 1% ) .
in contrast to planned bfr , 7% ( 77,628 ) had an actual bfr < 300 ml / min .
< 300 ml / min despite planned bfr 300 ml / min ( catheter dysfunction ) and 6,331 ( 65.2% ) patients had at least one session with catheter dysfunction ( mean = 11.1 sessions ; median = 5 sessions ) . in the baseline case - crossover analysis ( table 2 ) , using three case sessions and three control sessions , catheter dysfunction was associated with increased odds of a missed session due to access problems , an access - related procedure ( both limited and expanded definition ) , and either a missed session due to access problems or an access - related procedure . catheter dysfunction was associated with increased odds of all - cause hospitalization in the sensitivity analysis using one case and one control session , but not in the baseline analysis . in the multivariate - repeated measures analysis that included catheter dysfunction specified as a dichotomous variable ( bfr < 300 ml / min versus 300 ml / min , the reference category , table 3 ) , bfr < 300 ml / min was associated with increased odds of a missed session due to access problems , access - related procedure ( limited and expanded definition ) , missed session or access - related procedure , and all - cause hospitalization ( odds ratio 1.10 ; p = 0.001 ) .
the association between catheter dysfunction and dialysis run time in the same session was not significant . in the multivariate - repeated measures analysis that included five intervals of bfr ( 100<150 ml / min ; 150<200 ml / min
; 200<250 ml / min ; 250<300 ml / min ; 300 ml / min , the reference category ) and included dialysis run time in minutes as an independent variable ( table 4 ) , lower bfr was associated with increased odds of all the outcomes . with the exception of the odds ratio for hospitalization at bfr 250<300 ml / min ,
using davita and usrds data , we were able to identify a large cohort of almost 10,000 dialysis patients who received exclusively catheter dialysis for at least eight weeks between august 2004 and december 2006 .
the median duration of catheter dialysis was longer than 26 weeks , and most patients switched to a different type of access or modality before december 2006 , so we were able to document precisely the end of uninterrupted catheter use .
the proportion of patients receiving catheter dialysis in this study is consistent with current point prevalent estimates of chronic catheter use in the dialysis population overall .
we found that approximately 70,000 sessions , or 7% of those with both a planned and an actual bfr value present , had an actual bfr < 300 ml / min despite a planned bfr 300 ml / min , a definition of catheter dysfunction that closely approximates nkf - kdoqi vascular access guidelines .
this illustrates that catheter dysfunction defined according to the bfr threshold is a common problem , affecting approximately one in 14 sessions or one patient session every month .
almost two thirds of all patients had at least one session with catheter dysfunction , and more than 25% of these had at least 12 such sessions , suggesting that catheter dysfunction may be an ongoing problem in some patients . using two different analytic approaches , case - crossover and multivariate - repeated measures analysis
, we found strong associations between the presence of catheter dysfunction and increased risk of a missed session due to access problems , access - related procedures , a missed session due to access problems or access procedure , and all - cause hospitalization . in multivariate - repeated measures analysis ,
when we changed the specification of the catheter dysfunction independent variable from dichotomous ( bfr < 300 ml / min versus 300 ml / min ) to one that included multiple levels ( 100<150 ml / min ; 150<200 ml / min ; 200<250 ml / min ; 250<300 ml / min ; 300 ml / min ) and included dialysis run time in minutes as an additional independent variable , we found strong associations between the level of catheter dysfunction and the odds of the outcome .
each of these analytic approaches has strengths and limitations . in the case - crossover approach
, we included only patients who had the outcome of interest ( e.g. , missed session due to access problems ) .
further , we considered only the first such event for each patient , ignoring those that may have occurred later in the patient 's catheter dialysis history .
one important advantage of the case - crossover approach is that patients serve as their own controls because the risk of exposure , in this instance catheter dysfunction in at least one of three sessions before the event , is compared during two different periods for each individual patient .
consequently , there is no need to adjust for many of the factors that can confound associations between exposure and outcomes .
case - crossover designs are not immune to problems of confounding , however , as temporal changes within patients can confound comparisons between the control and the case period .
we do not believe this was a significant issue in our study since the case and control periods were limited to a maximum of three dialysis sessions each . in the multivariate - repeated measures approach , we included all patients , all catheter sessions , including those defined as having dysfunction , and all outcomes events of interest during the observation period .
one limitation of this approach is that since it includes both patients with and without at least one session with catheter dysfunction , it is necessary to adjust for differences between patients that may confound observed associations between catheter dysfunction and medical services .
it is possible , therefore , that observed differences in patterns of medical services between those with versus without catheter dysfunction may reflect unobserved differences in patient characteristics .
first , our data source , as in most retrospective analyses , lacked some clinical variables that would have strengthened the study .
for example , although we sought to characterize catheter dysfunction according to the most recent nkf - kdoqi guidelines as failure to attain and maintain an extracorporeal blood flow of 300 ml / min or greater at a prepump arterial pressure more negative than 250 mm hg , we did not have access to information on prepump arterial pressure or whether or not the patients had undergone line reversal in our dataset .
although this limited our ability to exactly replicate the nkf - kdoqi criteria , the inclusion of blood flow represents a significant clinical focus in assessing a catheter 's ability to provide an adequate dialysis treatment . additionally , we were unable to specifically identify the brand of catheter , the precise handling techniques , or the use of locking solution for each catheter session although presumably most dialysis sessions would have been conducted under standardized guidelines as described by davita clinical policies and procedures .
another limitation of the data is that we did not have access to a variable indicating dialysis center and included dialysis network in lieu of center . also , to limit false - positive catheter dysfunction , we restricted our definition to those sessions with actual bfr < 300 ml / min and planned bfr 300 ml / min , and we set to missing actual and planned bfr values < 100 ml / min . as a result , we have almost certainly underestimated the number of sessions with catheter dysfunction , and possibly also the effect of catheter dysfunction on utilization of medical services .
these differences may , in part , account for the discrepancy between the proportion of sessions with catheter dysfunction in this study and the proportion previously reported by cms .
second , by requiring patients to have at least eight weeks of catheter dialysis , and to have survived at least 90 days following the start of dialysis , we have excluded patients who were on catheter dialysis for shorter periods of time or who died within 90 days of beginning catheter dialysis . to the extent that catheter dysfunction is more common sooner after placement , by excluding patients with short - term catheter dialysis , we may have underestimated the overall rate of dysfunction .
catheter dysfunction due to mechanical reasons , which is known to occur sooner rather than later after placement , may be disproportionately underrepresented .
also , if death during the first 90 days after catheter placement is related to serious complications of catheter dysfunction , such as bloodstream infection , by requiring at least 90 days ' survival , we may have underestimated the impact of catheter dysfunction on the use of medical services , in particular , on all - cause hospitalization .
third , our study population consisted of a point prevalent cohort of patients who were receiving dialysis services at davita in august 2004 , and who , by definition , had their first esrd service before or during that month .
we did not have access to detailed dialysis session data , including type of dialysis , prior to august 2004 .
consequently , we could not determine the actual start of catheter dialysis for those who had their first esrd service before the beginning of the davita data and their first documented catheter dialysis session in august 2004 .
it is highly likely , however , that some of these patients began catheter dialysis months before entering our cohort .
again , to the extent that catheter dysfunction occurs sooner rather than later following placement , had we been able to observe all patients from the start of catheter dialysis , we may have observed a higher rate of dysfunction , and different patterns of medical services .
finally , we were unable to determine whether the disruptions to dialysis services and the utilization of other medical services we observed were necessary to ensure adequate dialysis or an unnecessary response based only on reaching the bfr threshold in the vascular access guidelines .
dialysis facilities have strong financial incentives to operate at full capacity , and any missed dialysis session may represent a loss of revenue for the facility . also , it seems unlikely that low bfr alone would be sufficient to result in hospitalization .
however , it is more difficult to draw conclusions regarding other medical services observed in this study , especially in light of the study by moist and colleagues that showed bfr < 300 ml / min is not an accurate predictor of dialysis inadequacy .
in spite of these limitations , which we believe may have resulted in underestimating both the rate of catheter dysfunction and the strength of the associations between catheter dysfunction and use of medical services , we found that catheter dysfunction defined according to nkf - kdoqi is common and results in disruptions in the provision of dialysis services and utilization of additional medical services .
efforts should continue to reduce chronic catheter use , and to minimize the clinical and economic consequences of catheter dysfunction through early detection and intervention .
further research would be required to determine the direct impact of the vascular access guideline on unnecessary procedures .
our study should be repeated in other countries , where the epidemiology and outcomes of catheter dysfunction may differ from the usa . also ,
conducting this study in patients with arteriovenous fistula or graft would help validate our findings on inadequate bfr and outcomes . | practice guidelines define hemodialysis catheter dysfunction as blood flow rate ( bfr ) < 300 ml / min .
we conducted a study using data from davita and the united states renal data system to evaluate the impact of catheter dysfunction on dialysis and other medical services .
patients were included if they had 8 consecutive weeks of catheter dialysis between 8/2004 and 12/2006 .
actual bfr < 300 ml / min despite planned bfr 300 ml / min was used to define catheter dysfunction during each dialysis session . among 9,707 patients , the average age was 62,53% were female , and
40% were black .
the median duration of catheter dialysis was 190 days , and the cohort accounted for 1,075,701 catheter dialysis sessions .
there were 70,361 sessions with catheter dysfunction , and 6,33 1 ( 65.2% ) patients had at least one session with catheter dysfunction . in multivariate repeated measures analysis , catheter dysfunction was associated with increased odds of missing a dialysis session due to access problems ( odds ratio [ or ] 2.50 ; p < 0.001 ) , having an access - related procedure ( or 2.10 ; p < 0.001 ) , and being hospitalized ( or 1.10 ; p = 0.001 ) .
catheter dysfunction defined according to nkf vascular access guidelines results in disruptions of dialysis treatment and increased use of other medical services . |
a seventy year - old woman presenting gradual evolution pruritus gets worse recently . at clinical examination ,
both labia were observed as white plates , dispersed , irregular borders and sanded surface , suggestive of high - grade intraepithelial vulvar neoplasia .
she was undergone to a vulvar polyp that revealed a high - grade vulvar intraepithelial neoplasia , so it was decided superficial vulvectomy .
the pathological examination revealed vulvar intraepithelial squamous neoplasia of high - grade , without evidence of invasion of the underlying stroma with signs of hpv infection , diffusely involving both lips , which developed in condyloma plan context , coexisting neuroendocrine carcinoma of high - grade small cell with lymphatic tumour embolization .
discrete parakeratosis and dense inflammatory infiltrate , predominantly lymphocytic , was observed in both lips .
the neuroendocrine lesion is a mass of intermediate size cells with high nucleus / cytoplasm ratio , hyperchromatic nucleus , some pleomorphism , with necrosis and figures of mitoses . the lesion is surrounded by inflammatory infiltrate mononucleate ( figure 1a ) .
the immunohistochemistry was positive for ae1/ae3 , ck7 , ema , cam5.2 and berep4 , and negative for ck5 , p63 , ck20 and ttf1 .
it is also observed immunostaining for neuroendocrine markers synaptophysin and chromogranin a ( figure 1c and d ) , with a proliferative activity assessed by ki67 about 100% .
pathological examination of the surgical specimen revealed squamous intraepithelial lesion of low grade , with no evidence of residual carcinoma or neuroendocrine tumour lymphatic embolization , and solid metastasis in 1 of the 6 right inguinal nodes of neuroendocrine carcinoma small cell without contralateral inguinal lymph node metastasis ( figure 2 ) .
neuroendocrine tumours have an aggressive behaviour and are a diagnostic and clinical challenge , due to their rarity and the lack of standardized therapeutic approaches .
they are rarely found in gynaecology , however they comprise approximately 2% of all tumours of the gynaecological tract .
most of them occur in the cervix , but they can also arise in other sites including vulva , vagina , uterus , and ovary .
these tumours are a heterogeneous group of neoplasms that show various histologic findings and biologic behaviours .
neuroendocrine small cell carcinoma is included in the group of high - grade neuroendocrine carcinomas .
small cell carcinoma , arising in other organs has the same histopathologic characteristics , as the ones originated in the gynaecologic tract .
recent genomic analyses of small cell carcinoma of the lung have revealed potential driver genomic alterations .
some authors believe that the comprehensive genomic characterization of gynaecologic small cell carcinomas may lead to the identification of markers , that result in an improvement of diagnostic reproducibility of small cell carcinomas of the gynaecologic tract and of molecular aberrations , that may be exploited therapeutically in subgroups of the disease .
the uterine cervix is the most frequent place in the female genital tract of this cancer and high - risk human papillomavirus ( hpv ) infection .
regardless of the location , small cell carcinoma in the gynaecologic tract displays an aggressive clinical behaviour , with few - reported long - term survivors .
this tumour has been observed in women with vulvar intraepithelial neoplasia or squamous cell carcinoma .
the small - cell carcinomas of the vagina and vulva need to be distinguished from merkel cell cancers , a neuroendocrine carcinoma of the skin .
attention to the histologic features of neuroendocrine differentiation and the immunohistochemical staining of neuroendocrine markers is necessary to reach a correct diagnosis .
microscopically , they are composed of small , oval - spindle cells , which are arranged like a sheet , trabecular or nested pattern . rosette - like or acinar formation may be seen .
the cells have high nuclear to cytoplasmic ratio , scanty cytoplasm , and hyperchromatic nuclei with inconspicuous nucleoli .
small foci of squamous and/or glandular differentiation can be seen but they usually are less than 5 - 10% of the total volume of the tumour .
immunohistochemistry : neuroendocrine differentiation can be proved with neuroendocrine markers , such as , chromogranin s , synaptophysin , cd56 , cd57 , neuron specific enolase , protein gene product 9.5 and synaptic vesicle protein 2 .
these markers recognize antigens that are expressed independently of the specific hormones secreted by neuroendocrine cells .
chromogranin - a , synaptophysin and cd56 are the most commonly used neuroendocrine markers in most practices .
reactivity for neuron - specific enolase and cd56 is usually present , although it is known to be a less specific neuroendocrine marker , compared to chromogranin and synaptophysin .
the frequent coexistence of neuroendocrine carcinoma and epithelial tumours alltogether with the monoclonality of the two components implies a common cellu lar origin of the neuroendocrine and epithelial components .
better survival is associated with fewer genetic aberrations , but tumour - related mortality occurs in approximately one - third of patients .
neuroendocrine tumours show highly aggressive clinical behaviour , regardless of the site of origin . despite the potential differences in etiology and
risk factors , small cell carcinoma from different sites of the gynaecologic tract , have similar morphologic appearances and clinical behaviour .
early stage disease has varied treatment approaches , based on the site of malignancy , but systemic chemotherapy with or without radiation , plays a role in the adjuvant setting to mitigate the risk of recurrence .
similar to small - cell cancers arising in other sites , it appears that the regional therapy is not a sufficient treatment , for this tumour . | neuroendocrine tumours are rare in the gynaecologic tract , comprising approximately 2% of all gynaecological tumours .
they have an aggressive behaviour and are a diagnostic and clinical challenge , due to their rarity and the lack of standardized therapeutic approaches .
there are a few case reports .
it is defined as a high - grade carcinoma exhibiting neuroendocrine differentiation .
the authors describe the case of a 70-year - old woman , with vulvar neuroendocrine small cell carcinoma after superficial vulvectomy .
the patient was submitted to a surgery with wide local excision and adjuvant radiation therapy .
a review of the literature on this topic is also presented . |
priapism constitutes a urologic emergency in which persistent erection occurs that is not accompanied by sexual desire , usually lasting for more than 6 h and typically involving only the corpora cavernosa .
priapism has been associated with many known different disease states and some situations that constitute risk associations for the condition .
it is estimated that idiopathic category accounts for as many as half of all documented cases .
pick disease is a lysosomal storage disease caused by the deficiency of sphingomyelinase enzyme leading to the accumulation of sphingomyelin in the cells of the macrophage monocyte system . to our knowledge
this unique case of priapism associated with niemann pick disease ( sphingolipid accumulation ) represents the first such case reported in the literature .
a 15-year - old boy presented with persistent painful erection of 46 h duration . the patient had woken up in the early morning with pain .
he had experienced a similar episode of painful erection 1 month back , which resolved spontaneously within 4 h. there was no history of perineal trauma or sickle cell anemia .
physical examination revealed a poorly nourished adolescent male with pallor , clubbing , and generalized lymphadenopathy ( bilateral cervical , axillary , and inguinal ) .
genitourinary examination showed an erect viable penis , edema of proximal shaft of penis , and adjacent scrotum [ figure 1 ] .
erect viable penis with edema of proximal shaft and adjacent scrotum laboratory evaluation revealed a white blood count of 13.9 10/l , hematocrit 43.6% , and platelet count 356 10/l , which were within normal limits .
corporeal blood gas analysis showed a ph of 6.86 , paco2 88 mmhg , and pao2 26 mmhg .
biopsy of the lymph node from the cervical area showed infiltration of paracortical and cortical areas by foam cells , which are large cells with abundant pale pink vacuolated cytoplasm with small pyknotic nucleolus placed centrally as well as eccentrically [ figure 2 ] .
bone marrow aspiration showed large histiocytes with abundant vacuolated cytoplasm ( sea - blue histiocytes ) characteristic of neimann - pick cells suggestive of metabolic storage disorder [ figure 3 ] .
lymph node biopsy showing infi ltration of paracortical and cortical areas by foam cells ( h and e , reduced from 100 ) bone marrow aspiration showing large histiocytes with abundant vacuolated cytoplasm ( h and e , reduced from 100 ) he was managed with cavernous aspiration and irrigation with saline followed by 100 g phenylephrine injected into corpus cavernosum for a total of 4 doses , 10 - 15 min apart .
various etiologies of priapism have been described , including sickle cell disease , hematologic malignancies , penile metastases , perineal trauma , total parenteral nutrition , intracavernous injection therapy for erectile dysfunction , and oral medications . nevertheless , many cases of priapism are classified as idiopathic .
pick disease is an inherited lysosomal metabolic storage disease resulting from the deficient activity of the lysosomal hydrolase acid sphingomyelinase leading to lysosomal accumulation of sphingomyelin .
sphingomyelin is the major lipid that accumulates constituting about 70% of total phospholipid fraction ( normal 5%-20% ) .
the pathologic hallmark of niemann pick disease is the characteristic lipid - laden foam cell often referred to as the niemann pick cell in the bone marrow .
enzymatic evaluation by measuring the acid sphingomyelinase activity level in peripheral leukocytes , cultured fibroblasts , and/ or lymphoblasts will confirm the diagnosis by showing markedly decreased enzymatic activity .
although the exact pathogenesis of the priapism associated with niemann pick disease is not clear , possible mechanism could be accumulation of foam cells causing sluggish blood flow , hypercoagulability and venous outlet obstruction of corpora cavernosa .
since many cases of priapism have unidentifiable etiology , we consider that this could be one of the causes .
occurrence of priapism led to the diagnosis of niemann pick disease in this patient .
this boy had erection lasting 46 h long in duration , usually aspiration fails in such prolonged priapism and a shunt is required .
perhaps success of aspiration in this case could be attributed to the young age of the patient having protective action and hence irreversible damage did not occur despite long duration of priapism . | a 15-year - old boy presented with priapism of 46 h duration .
there was no known cause of priapism detected in him . during evaluation , biopsy of the lymph node and bone marrow aspiration detected patient
to be having niemann pick disease .
he was managed with cavernous aspiration , saline irrigation , and intracavernosal phenylephrine injection .
although priapism is associated with many known diseases , about half of the cases are of idiopathic origin .
we are reporting the first case of priapism associated with niemann pick disease in the literature . |
the duodenum is a common location for intestinal obstruction in the newborn requiring surgical intervention .
intrinsic obstruction ranges from complete atresia with a gap to membranous web and to stenosis .
in contrast to small intestinal atresia , ontogeny is thought to be secondary to failure of luminal recanalization .
treatment often consists of operative duodenoplasty or bypass with a duodenoduodenostomy and has traditionally been performed through a laparotomy ; however , laparoscopic approach has previously been shown to be a viable option .
overall mortality for this congenital anomaly has improved ; moreover , associated medical conditions are now responsible for most deaths .
prolonged postoperative enteral feeding intolerance continues to challenge surgeons caring for this cohort of patients . since
early institution of nutritional support is critical in postoperative newborns , identification of patients likely to require alternative nutritional support is likely of benefit to this subset of patients .
therefore , our aim was to describe the short term nutritional outcomes and investigate risk factors leading to delayed establishment of full enteral nutrition in a modern cohort of patients with duodenal atresia at a single - institution .
following approval from our institutional review board ( irb number m08 - 08 - 0381 ) , we performed a retrospective review of records from 1998 to 2012 utilizing icd-9 code for small intestine atresia ( 751.1 ) at boston children 's hospital ( bch ) .
patients were included if they had a primary surgical repair or surgical treatment for a complication of a previous repair of a newborn intrinsic duodenal obstruction during this 14-year period .
we reviewed the following variables : gender , gestational age ( ga ) , birth weight , apgar 1-minute score , apgar 5-minute score , and radiographic findings such as proximal dilatation , duodenal anatomy , technique of primary surgical repair , malrotation , annular pancreas , congenital heart disease ( chd ) , down 's syndrome , and esophageal atresia with or without tracheoesophageal fistula ( ea / tef ) .
kaplan - meier product - limit analysis with greenwood estimators was used to calculate median time to full enteral nutrition , with the log - rank test to compare area under the survival curves .
covariates were evaluated by univariate and multivariate analysis using the cox proportional - hazards model to identify independent predictors with the likelihood ratio test to assess significance and log - minus log plot to verify the assumption of proportional hazards over time .
hazard ratios ( hr ) and 95% confidence intervals ( ci ) were determined for significant multivariate predictors of time to full enteral nutrition .
statistical analysis was performed using ibm spss statistics ( version 21.0 , ibm , armonk , ny ) .
power analysis indicated that the sample sizes of patients with potential risk factors provided 80% power for detecting a significant hazard ratio of 0.75 in delayed transition to full enteral nutrition ( version 7.0 , nquery advisor , statistical solutions , saugus , ma ) .
a total of 87 patients were found to have an intrinsic duodenal obstruction and met inclusion criteria .
median age was 37 weeks ( interquartile range [ iqr ] : 3539 weeks ) .
median apgar scores were 8 at 1 and 5 minutes ( iqr : 7 - 8 and 8 - 9 , resp . ) .
proximal bowel dilatation was documented in 19% ( 16/86 ) of the patients ( table 1 ) . a majority of the duodenal anatomy encountered was atresia ( 63% ) , followed by webs ( 24% ) and stenosis ( 13% ) .
concurrent congenital anomalies included chd ( 55% ) and down syndrome ( 38% ) ( table 1 ) .
chd was further subdivided into the following types encountered : 9 ( 19% ) pda ; 21 ( 44% ) asds and/or vsds ; 7 ( 15% ) tetralogy of fallot ( tof ) ; 2 ( 4% ) coarctation of the aorta ; 6 ( 12% ) hypoplastic hearts or pulmonary arteries ; 2 ( 4% ) complete av canal ( cavc ) ; and 1 ( 2% ) bicuspid aortic valves .
choice of operation , as well as whether it was performed open or laparoscopically , was dictated by the choice of the operating surgeon .
five ( 6% or 5/86 ) of the operations were performed laparoscopically , while the remaining 81 were performed through a laparotomy .
the techniques described for the primary duodenal repair included 58 duodenoduodenostomies , 6 duodenojejunostomies , 1 gastroduodenostomy , and 15 duodenoplasty repairs ( with or without web excision ) , and 6 patients had web excision without duodenoplasty .
one patient was not repaired given that she died two days into her hospital course from complex cardiac complications .
six ( 7% or 6/86 ) patients were found to have a second , distal , concurrent , and intestinal obstruction at the time of their proximal duodenal obstruction repair .
fifty - six percent of the operating surgeons performed a maneuver to detect a concurrent obstruction , while no maneuver was noted in the remaining 44% of operative notes .
these maneuvers included inspection alone ( 30% ) , passage of small transanastomotic catheter ( 22% ) , and passage of transanastomotic catheter with small fluid bolus ( 3% ) .
as a result of these maneuvers , the following anomalies were identified ; two patients had a distal duodenal web , two had a second duodenal atresia , one had a jejunal atresia , and one had multiple distal atresias in both the duodenum and the jejunum .
seven patients did not receive pn ; six patients never received pn and one patient never had the opportunity to receive pn .
the cohort that received pn required a median of 10 days on pn ( iqr : 612 days ) . the median time to 100% en was 12 days ( iqr : 917 days ) , excluding the one patient who died 2 days into her hospital course .
the median time to 100% en in those patients who never received pn was 7 days ( range 526 days ) .
71 out of the 84 patients were transitioned to 100% oral feeds prior to discharge at a median of 13 days ( iqr : 1021 days ( table 2 ) .
their median time on pn was 9 days ( iqr : 516 days ) , with a full range 065 days , versus the larger cohort ( p = .29 , mann - whitney u - test ) .
regarding time to 100% enteral nutrition , the median time for those with feeding tubes was 9 days ( iqr : 722 d ) , with full range 263 days , versus the larger cohort ( p = .28 , mann - whitney u - test ) .
those included gender , ga 35 weeks , birth weight , apgar 1 scores , duodenal anatomy , proximal bowel dilatation , technique of primary surgical repair , presence of feeding tube , malrotation , annular pancreas , chd , down 's syndrome , and ea / tef ( table 3 ) . of these ,
ga 35 weeks , birth weight , atresia anatomy , surgical repair , malrotation , and chd were found to be significantly associated with delayed progression to full enteral feeding on univariate analysis .
.001 ) , chd ( p = .02 ) , and malrotation ( p = .03 ) .
variables such as birth weight , atresia anatomy , and surgical repair were not found to be independent predictors of delayed progression to full enteral feeding .
patients were then further classified based on the presence or absence of chd and then stratified by ga . among patients without chd who had ga > 35 weeks ( n = 30 ) the median time to 100% en progression was 10 days ( iqr : 812 days ) , whereas if ga 35 weeks ( n = 9 ) the median time to progression was 16 days ( iqr : 1120 days ) ( p = .003 , log - rank test = 9.12 ) ( figure 1(a ) ) . among patients with chd who had ga
> 35 weeks ( n = 27 ) , the median time to progression was 11 days ( iqr : 814 days ) , whereas for those with ga 35 weeks ( n = 20 ) the median time to progression was 23 days ( iqr : 1530 days ) ( p < .001 , log - rank test = 16.21 ) ( figure 1(b ) ) .
the second patient , born with a hypoplastic left heart and aortic coarctation , died of uncontrollable pulmonary hypertension shortly following an open duodenal atresia repair .
the third patient , born with tetralogy of fallot ( tof ) and a chromosome 12 deletion , died almost one year following surgery while being in the hospital for a cardiac procedure . among the 84 survivors , the median followup was 20 months ( iqr : 548 months ) .
there were 13 ( 15% ) major surgical complications following initial operative experience ( table 4 ) .
the 13 surgical complications included six bowel obstructions , four anastomotic leaks , two late - strictures , and one incisional hernia .
twenty - four percent ( 10/42 patients ) occurred between 1998 and 2005 versus 3% ( 3/44 ) between 2006 and 2012 ( p = .04 ) .
the earlier cohort excluded one patient from analysis as she was an early mortality and was never repaired .
the medical complications included 2 episodes of line sepsis and one episode of necrotizing enterocolitis ( nec ) .
treatment of the medical complications was as follows : ( 1 ) the two episodes of line sepsis were treated with antibiotics ; and ( 2 ) the one episode of nec was treated with a 14-day course of antibiotics and bowel rest .
this study describes a cohort of 87 patients who were treated for intrinsic duodenal obstruction in the newborn period at a single institution . this may be one of the largest series of repairs as compared to other previously published series .
six patients ( 7% ) did have a concurrent second intestinal obstruction , while 13 patients had intraoperative nasojejunal transanastomotic feeding tubes placed without anastomotic complications .
variables that had previously been associated with delayed transition to full enteral feedings were not important in our study , including proximal bowel dilatation , duodenal anatomy , or technique of primary surgical repair .
we concluded that the independent predictors for longer postoperative progression to full enteral nutrition were prematurity , chd , and malrotation in our cohort . in this cohort of patients ,
the mortality rate across 14 years was 3% , which is comparable to the approximately 4% early operative mortality reported in the available literature [ 1 , 2 ] .
presence of complex cardiac conditions , down 's syndrome , and other intestinal atresia and trachea - esophageal fistula has been reported to place these patients at higher risk of late morbidity and mortality .
55% of patients in this cohort had chd , while 38% patients had down 's syndrome .
contrary to the findings of worse outcomes with associated down 's syndrome , no such association was elucidated in this cohort .
recent literature has suggested that the repair of duodenal atresia can be effectively and safely performed laparoscopically [ 1115 ] .
our study only had 5 laparoscopic operations performed ; however , many laparoscopic - based studies are suggesting an early return of bowel function congruent with laparoscopic procedures in general .
for example , spilde et al . showed that the length of postoperative hospitalization ( 20.1 versus 12.9 days ; p = .01 ) , time to initial feeding ( 11.3 versus 5.4 days ; p = .002 ) , and time to full oral intake ( 16.9 versus 9 days ; p = .007 ) were shorter in the group undergoing laparoscopic repair versus open repair .
as reported in this study , slightly prolonged transition time to full enteral feeding may be in part due to a greater proportion of patients undergoing open operative approach versus an inherent component of the study cohort , including the initiation of pn in 80 ( 92% ) patients within the cohort .
similar to a recently published study , the small subset of patients ( n = 6 ) in our study who never received pn had a faster transition to full enteral nutrition , 7 days versus 12 days for the entire cohort .
the two variables though , that had the greatest influence overall , were prematurity and presence of chd ; both were noted to be associated with increased odds of delayed transition to full enteral nutrition and outweighed all other variables . even with this being a retrospective study and there being an absence of a standardized approach to feeding advancement , we do believe that there is the potential of significant benefit of early institution of appropriate nutritional support and using these risk factors to tailor the appropriate nutritional regimen . from a surgical perspective , several important findings also arose from this study .
first , intraoperatively , six patients ( 7% ) were noted to have a second , distal , concurrent , and intestinal obstruction , representing a higher rate of concurrent intestinal obstruction as compared to ( 13% ) the rate reported in the available literature [ 2 , 13 ] .
this does suggest that the evaluation for presence of concurrent obstruction may be worthwhile to avoid the morbidity associated with a missed lesion .
other major complications did include line sepsis in two patients ; however , there was not an increased rate in the highest risk groups [ 10 , 16 ] .
for example , it appeared that between the two epochs 19982005 and 20062012 , there was a significant decrease in the number of associated in - hospital complications ( p = .04 ) in the time period between 2006 and 2012 .
this may be due in part to both a learning curve and a higher volume of cases performed , which is congruent with more recent studies but further studies are needed .
lastly , none of the patients in our cohort who had received a transanastomotic tube developed a leak or developed a stricture and had a slightly decreased transition to full enteral feeds .
more recent studies have shown that use of a transanastomotic feeding tube decreases not only the postoperative time to initiation of enteral nutrition , but also the time to full enteral nutrition delivered proximal to the anastomosis [ 1821 ] .
there was a trend in decreased number of days within our subset of patients who received transanastomotic feeding tubes but did not remain on multivariate analysis .
we do believe that with a larger number of patients and a standardized placement of these transanastomotic feeding tubes , we would anticipate a significant difference in transition to full enteral nutrition .
another limitation was that there was no overt standardization for repair guidelines ( open versus laparoscopic or type of repair employed ) , or whether a patient received a transanastomotic feeding tube versus an access line for nutrition .
however , these patients were followed postoperatively with minimal loss to follow - up and no significant long - term complications .
based on our findings , prematurity , chd , and malrotation predict a longer time to reach full enteral nutrition ; therefore , supportive nutrition is suggested .
furthermore , we did not see an associated increase in leak rate in the small subset of patients who received transanastomotic feeding tubes . given the aforementioned findings and limitations , further studies are needed to elucidate a standardized methodology for nutritional support during clinical management of these patients in the setting of single or multiple intestinal obstructions . in the absence of prospectively collected data , these risk factors can be used to potentially identify patients with a greater risk of delayed transition to full enteral nutrition and therefore tailor their nutritional support regimen accordingly . | background .
a common site for neonatal intestinal obstruction is the duodenum .
delayed establishment of enteral nutritional autonomy continues to challenge surgeons and , since early institution of nutritional support is critical in postoperative newborns , identification of patients likely to require alternative nutritional support may improve their outcomes .
therefore , we aimed to investigate risk factors leading to delayed establishment of full enteral nutrition in these patients . methods .
87 patients who were surgically treated for intrinsic duodenal obstructions from 1998 to 2012 were reviewed .
variables were tested as potential risk factors .
median time to full enteral nutrition was estimated using the kaplan - meier method .
independent risk factors of delayed transition were identified using the multivariate cox proportional hazards regression model .
results .
median time to transition to full enteral nutrition was 12 days ( interquartile range : 917 days ) .
multivariate cox analysis identified three significant risk factors for delayed enteral nutrition : gestational age ( ga ) 35 weeks ( p < .001 ) , congenital heart disease ( chd ) ( p = .02 ) , and malrotation ( p = .03 ) .
conclusions .
chd and prematurity are most commonly associated with delayed transition to full enteral nutrition .
thus , in these patients , supportive nutrition should strongly be considered pending enteral nutritional autonomy . |
primary intraosseous odontogenic squamous cell carcinoma ( pioscc ) is a rare and malignant tumor .
the etiology seems to be related to the malignant degeneration of embryological remains . in this line ,
epithelial rests of malassez , dental lamina and epithelium of the dental follicle represent potential suspects . according to the 2005 ,
the world health organization classification of tumors , three subtypes of pioscc exist :
solid tumor that invades the marrow spaces and provokes osseous absorptionsquamous cell carcinoma ( scc ) originated from the lining of an odontogenic cyst .
specifically , this subgroup includes two variants : keratocystic odontogenic tumor and odontogenic cystsscc associated with other benign epithelial odontogenic tumors .
solid tumor that invades the marrow spaces and provokes osseous absorption squamous cell carcinoma ( scc ) originated from the lining of an odontogenic cyst .
specifically , this subgroup includes two variants : keratocystic odontogenic tumor and odontogenic cysts scc associated with other benign epithelial odontogenic tumors .
the diagnostic criteria of pioscc included undamaged oral mucosa and absence of distant metastasis during the follow - up .
moreover , the switch from the epithelium of the cyst to scc is essential for a proper diagnosis .
differential diagnosis includes alveolar carcinomas , jaw metastases from other locations , odontogenic tumors and tumors of the maxillary sinus . in this sense ,
in fact , due to the rarity of this disease , there are no established treatment protocols .
the adequate surgical resection remains the gold standard for the treating this tumor . in this report
hence , this case underlines the importance of routine monitoring of patients presenting large inflammatory cyst .
the article describes a case of pioscc of the mandible affecting a 77-year - old man .
more in detail , the patient was referred at maxillofacial outpatient department of granada university hospital by his general practitioner .
in fact , the patient reported a progressive painless swelling in the left side of mandible .
oral and extraoral examination evidenced an abnormal mandibular ballooning in the left side . in view of that , an orthopantomography and a computed tomography ( ct ) scan of the cervicofacial area were performed for allowing the proper diagnosis .
panoramic radiography evidenced the presence of a large cyst in the left molar region and the lack of several tooth .
ct scan evidenced an aggressive lesion invading body and ascendant branch of the jaw [ figures 13 ] . after analyzing this data , anamnesis and physical examination
patient referred a tooth extraction of tooth 38 , 3 years ago because of the presence of a pericoronal cyst .
after due consideration of all data , we staged cancer as ct3n0m0 . in the oncological committee of our hospital
, we decided to treat the case with a wide surgical resection , neck dissection and postoperative radiotherapy .
postoperative examination confirmed the diagnosis of pioscc poorly differentiated [ figure 4 ] with resection margins free .
three - dimensional computed tomography image showing an intraosseous odontogenic squamous cell carcinoma of the mandible . at the physical examination ,
the patient presented a bone consistency with swelling of vestibular region in the left side of the jaw axial computed tomography image of mandibular intraosseous odontogenic squamous cell carcinoma .
the lesion affects the body and the ascendant branch of the mandible in the left side orthopantomogram of mandibular intraosseous odontogenic squamous cell carcinoma histopathological image of mandibular intraosseous odontogenic squamous cell carcinoma with h&e stain
however , the majority ( 50% ) are well - grade malignant tumors . the median age of presentation is between the sixth and eight decade of life and it occurs more often in males .
according to the literature , the time diagnosis is often delayed due to the absence of symptoms provoked by pathology . in this line ,
a review of 30 cases evidenced that pioscc showed the following forms of presentation : asymptomatic ( 26,7% ) , asymptomatic mass ( 26.7% ) , mass ( 16.7% ) , pain ( 13.3% ) , nerve involvement ( 10% ) and expansion ( 6.7% ) .
thus , it is possible that the tumor could have been confused with a pericoronal cyst .
panoramic x - ray and ct scan are essentials for a proper diagnosis and analysis of the tumor extension .
a number of other lesions should be considered in the differential diagnosis such as alveolar carcinomas , jaw metastases , odontogenic tumors and maxillary sinus tumors .
moreover , several reports suggest that postoperative radiotherapy could be helpful to improve the overall survival .
we treat the case with surgical resection , neck dissection and postoperative radiotherapy . considering the wide surgical margins and lack of evidence about the effectiveness of chemotherapy
resuming , this case underlines the importance of routine follow - up of patients presenting large inflammatory cyst .
| primary intraosseous odontogenic squamous cell carcinoma ( pioscc ) is a rare tumor .
the incidence is low , and approximately 200 cases are reported in literature .
the etiology is associated with the malignant degeneration of embryological remains .
differential diagnosis includes alveolar carcinomas , jaw metastases from other locations , odontogenic tumors , and tumors of the maxillary sinus .
however , the diagnosis could be delayed due to the absence of symptoms in early stages .
surgery represents the first choice treatment . whereas , postoperative radiotherapy could be helpful to improve the overall survival .
the prognosis is generally poor . in this report
, we describe the case of a 77-year - old man accidentally diagnosed pioscc .
the patient referred a tooth extraction and cystectomy 3 years before .
the pathological examination of the cyst tissue evidenced an inflammatory cyst .
however , no patient follow - up was performed .
hence , is important to stress that routine monitoring of patients affected by large inflammatory cysts of the jaw bones should be mandatory . |
cerebral microbleeds ( cmbs ) are defined as small perivascular hemosiderin deposits . with the widespread use of magnetic resonance imaging ( mri ) , cmbs are more commonly detected . in studies using sensitive mri technique ,
the prevalence of cmbs is as high as 11.1%23.5% in community - dwelling elderly people . prevalence is higher in patients with ischemic stroke ( 40% ) and spontaneous intracerebral hemorrhage ( ich ) (
ethnicity seems to play a role in the prevalence of cmb , with higher prevalence in participants from asian origin .
cmbs are related to age , hypertension , leukoaraiosis , amyloid angiopathy , atrial fibrillation , and some genes .
findings of cmbs , especially in stroke patients , may be important . some stroke patients will have a recurrent stroke , and intravenous thrombolysis is the standard treatment in eligible patients within 4.5 h of stroke onset .
a recent meta - analysis showed that the presence of cmbs on pretreatment mri scans in patients with acute ischemic stroke increased risk of symptomatic ich after intravenous thrombolysis ( odds ratio [ or ] 2.87 , 95% confidence interval [ ci ] 1.764.69 , p < 0.0001 ) .
however , the authors concluded that although the presence of cmbs increased the risk of ich after thrombolytic treatment , detecting cmbs should not prevent thrombolytic treatment based on present evidence .
the purpose of this study is to look for the prevalence of cmbs and associated factors in thai patients with ischemic stroke .
patients who presented with acute ischemic stroke and had mri and magnetic resonance angiography ( mra ) during january
mri was performed using a 1.5 tesla philips achieva xr scanner with a neurovascular 16-channel radiofrequency head coil .
a t2 * -weighted gradient - recalled echo ( gre ) sequence ( tr 700 ms , te 23 ms , and flip angle of 18 ) with a 5-mm slice thickness and 1 mm gap was used to define hemorrhage .
small ( < 10 mm ) , hypointense , well - demarcated , rounded lesions on gre were used to define cmbs .
if the hypointense lesions on gre were larger than 10 mm , these were defined as ich . if the hypointense lesions on gre were within the infarct lesions , these were defined as hemorrhagic transformation [ figure 1 ] .
fazekas scale ( 03 ) was used to classify severity of white matter lesions ; with higher score , more confluent of the lesions .
fazekas 0 : none or a single punctate white matter hyperintensity lesion , fazekas 1 : multiple punctate lesions , fazekas 2 : beginning confluency of lesions ( bridging ) , and fazekas 3 : large confluent lesions [ figure 2 ] .
fazekas was scored on transverse t2/fluid attenuation inversion recovery images ( tr 11,000 ms , te 140 ms ) .
( a ) cerebral microbleed at rightside of pons from gradient - recalled echo ; ( b ) intracerebral hemorrhage at bilateral putaminal hemorrhages from gradient - recalled echo ; ( c ) ( diffusion - weighted imaging ) ; ( d ) ( gradient - recalled echo ) acute hemorrhagic infarct at left middle cerebral artery territory ( t2/fluid attenuation inversion recovery images ) ; ( a ) fazekas scale 0 ; ( b ) fazekas 1 ; ( c ) .
fazekas 2 ; ( d ) fazekas 3 all patients with acute ischemic stroke were treated per standard care protocol .
stroke subtypes were classified by toast ( trial of org 10172 in acute stroke treatment ) criteria : large - artery atherosclerosis ( laa ) , cardioembolism ( ce ) , small - artery occlusion ( sao ) , stroke of other determined cause , and stroke of undetermined cause .
severity of stroke was evaluated by the national institutes of health stroke scale ( nihss ) .
baseline characteristics of patients , stroke subtypes , antithrombotic medications , and all imaging data were collected and studied .
the data were presented as a mean for the continuous variables and a percentage ( number ) for the dichotomous variables .
we compared the demographics and vascular risk factors in the stroke patients with and without cmb using a student 's t - test ( for the continuous variables ) and the chi - square test ( for the proportions ) .
the multivariate analyses were performed by the inclusion of the prespecified factors that were associated with cmb in the univariate analysis .
ich and hemorrhagic transformation were found in 12 ( 6% ) and 41 ( 21% ) patients , respectively .
baseline characteristics of the patients in the study in the subgroup of patients who received intravenous thrombolysis ( 83 patients ) , hemorrhagic transformation and ich did not occur more frequently in patients with cmb than in those without cmb ( 23.5% vs. 31.8% , p = 0.512 ) . in patients with confluent white matter lesions ( fazekas 3 )
, cmbs were found in 16 patients ( 43% ) and located at cortico - subcortical area ( 6 patients , 37.5% ) , infratentorial area ( 4 patients , 25% ) , and both cortico - subcortical and deep cerebral hemisphere ( 6 patients , 37.5% ) .
cmbs were related to older age , hypertension , some stroke subtypes ( laa , sao ) , and higher fazekas scale [ table 2 ] .
however , multivariate analysis showed that only hypertension ( or 3.05 , 95% ci 1.078.68 , p = 0.037 ) and moderate - to - severe white matter lesions ( fazekas 23 , or 7.61 , 95% ci 3.0618.95 , p <
baseline characteristics of patients with and without cerebral microbleeds there was association between the number of cmbs and severity of white matter lesions or the presence of hypertension as higher number of cmbs related to more severe white matter lesions ( p < 0.001 ) and the presence of hypertension ( p = 0.001 ) [ table 3 ] .
correlation between number of cmbs and severity of white matter lesions / hypertension in a subgroup of patients who were on single antiplatelet , there was no significant difference in the presence of cmb and type of antiplatelet ( aspirin ; 23% ( 27/116 ) vs. clopidogrel ; 20% ( 2/10 ) , p = 0.495 ) .
the prevalence of cmb was 20% in this study , which was lower than those reports from japanese studies but closed a report from a chinese study .
ethnicity seems to be significant with higher prevalence of cmbs among participants of asian origin ( 43% ) compared with western participants .
cmbs were found in 24% of 458 chinese patients with ischemic stroke in a study .
cmbs were related to older age , hypertension , stroke subtypes ( laa , sao ) , and leukoaraiosis in our study and in other studies .
the finding that the prevalence of cmbs increased with age was reported in several studies in adult participants without stroke . in patients with ischemic stroke
kato et al . studied 213 patients with stroke and reported higher prevalence of cmbs in patients with lacunar infarct ( 62% ) than those with cardioembolic infarct ( 30% ) and atherothrombotic infarct ( 21% ) .
another study from japan found that cmbs were associated with atherothrombotic ( 5/22 , 23% ) and lacunar ( 7/31 , 23% ) but not in cardioembolic stroke ( 0/13 , 0% ) . in our study ,
clinical characteristics of the patients , results of cardiac investigations and mra were used to classify stroke subtypes .
cmbs were found in patients with laa ( 53% ) and sao ( 28% ) more than those with ce ( 15% ) .
accumulation of ischemic injuries in brain parenchyma is thought to differ in atherothrombotic stroke and cardioembolic stroke , suggesting that the relationship of cmbs and stroke subtype in turn may reflect a different degree of fragility of vascular walls .
our study revealed that severe white matter lesions ( fazekas 3 ) were related to the presence of cmbs .
strong associations of cmbs with confluent white matter lesions , leukoaraiosis , and lacunes were reported , all of which are known to be associated with chronic hypertension .
the association of antithrombotic treatment and prevalence of cmbs is still debated as some studies showed the relationship but not in others .
our study did not show a significant difference between antithrombotic uses in patients with or without cmbs .
although no patient receiving novel oral anticoagulation ( noac ) had cmbs , only a small number of patients used noac in the study .
however , all mri studies were performed after acute ischemic stroke , so most patients were on antithrombotic drugs .
this may not accurately investigate the effect of antithrombotic medications on the presence of cmbs .
performed a systemic review and meta - analysis to assess whether the presence of cmbs on pretreatment mri scans in patients with acute ischemic stroke treated with thrombolysis is associated with the increased risk of symptomatic ich . in this meta - analysis ,
ich after thrombolysis was higher compared to those without cmbs ( 8.5% vs. 3.9% , or2.26 , 95% ci 1.463.49 , p < 0.0001 ) . in our study , although hemorrhagic transformation occurred more often in patients after intravenous thrombolysis as compared to those not having thrombolytic treatment ( 30% vs. 13.7% ) , the hemorrhagic transformation rate was not different in patients with or without cmbs ( 23.5% vs. 31.8% , p = 0.512 ) .
patients with ischemic stroke were included in the study if they had mri / mra during the study period .
most patients did not have pretreatment mri , so the effect of antithrombotic drugs on the incidence of cmb could not be answered .
however , this was the first study looking for cmbs in thai patients with ischemic stroke .
in our study , cmbs were found in 20% of thai patients with ischemic stroke .
| background : with the widespread use of magnetic resonance imaging ( mri ) , cerebral microbleeds ( cmbs ) are commonly detected .
ethnicity seems to play a role in the prevalence of cmb , with higher prevalence in participants from asian origin .
the purpose of the study is to look for the prevalence of cmbs and associated factors in thai patients with ischemic stroke.methods:patients with acute ischemic stroke who had mri and magnetic resonance angiography during january august 2014 were included in the study . t2
* -weighted gradient - recalled echo was used to define cmbs .
baseline characteristics , stroke subtypes , and severity of white matter lesions were compared between patients with and without cmbs.results:two hundred patients were included in the study .
mean age of the patients was 61-year - old .
mean national institutes of health stroke scale was 8 .
the prevalence of cmbs was 20% ( 39/200 patients ) .
hypertension ( odds ratio [ or ] 3.05 , 95% confidence interval [ ci ] 1.078.68 , p = 0.037 ) , and moderate - to - severe white matter lesions ( fazekas 23 , or 7.61 , 95% ci 3.0618.95 , p <
0.001 ) were related to the presence of cmbs.conclusions:cmbs were found in 20% of patients with ischemic stroke , which was lower than those reported from japanese studies but comparable to a chinese study .
cmbs were associated with hypertension and severity of the white matter lesions . |
dermatitis herpetiformis ( dh ) is one of the subepidermal autoimmune bullous diseases ( abd ) characterized by skin and intestinal lesions .
diagnosis of dh is established based on the results of direct immunofluorescence test ( dif ) and the presence of circulating iga antibodies directed against endomysium and/or tissue and epidermal transglutaminase ( ttg , etg ) [ 2 , 3 ] .
skin lesions in dh are histologically characterized by neutrophilic infiltrate leading to destruction of basement membrane zone ( bmz ) proteins , anchoring fibers , and blister formation [ 46 ] .
bullous pemphigoid ( bp ) is a blistering disease , characterized by inflammatory infiltrate in the dermis , presence of igg and c3 deposits along the basement membrane zone , and circulating igg autoantibodies .
autoantibodies binding to autoantigens ( bpag1 and bpag2 ) localized in the basement membrane of the epidermis activate a series of immunological and enzymatic phenomena leading to destruction of bmz components and anchoring fibers and blister formation as in dh [ 7 , 8 ] .
ultrastructural studies confirmed also the presence of intensive inflammatory infiltrate at dermoepidermal junction , as well as destruction of components of extracellular matrix in bp and dh [ 911 ] . in the available literature , there are few reports on the expression of procoagulant factors , abnormal activation of coagulation process , and their role in the creation of blisters .
recently , the dominant role of eosinophils in the inflammatory infiltrate in bullous diseases is postulated .
therefore , the fact that they might be an important source of procoagulant tissue factor ( tf ) gives the scientific basis to consider its contribution of this process in the pathogenesis of lesions in the subepidermal bullous diseases .
recent studies have shown an increased risk of fatal thrombotic events in patients with bp treated with glucocorticoids .
confirmation of these complications is the increased concentration of d dimers and prothrombin in the serum of patients in the active stage of the disease .
therefore , it is important to define the role of procoagulant factors in the destruction of the basement membrane in pemphigoid and activity of eosinophils and mediators of these cells in skin lesions and normal looking skin as well as in serum in the active phase of the disease . under physiological conditions , there is a balance between the coagulation and fibrinolysis , but in pathological states this balance might be deteriorated . in bullous diseases inflammation
interleukin-5 , produced mainly by th2 lymphocytes , is a key factor in the differentiation and activation of eosinophils [ 1416 ] .
eosinophils , the dominant inflammatory cells in bp , seem to be a major source of intravascular tissue factor .
coagulation pathway might cause the activation of adhesion molecules expression and subsequent release inflammatory mediators and proteolytic enzymes [ 10 , 19 ] .
recent experimental studies have also shown that these processes may also activate procoagulant factors that cause the development of blood clots , which are the most common cause of complications in patients with bp .
tf , in addition to well - documented prothrombotic properties , plays an important role in the inflammatory process .
constitutive expression of tf is present on fibroblasts and epithelial cells , while on monocytes and macrophages it is induced by antigen - antibody complexes and complement activation products and proteins as well as by inflammatory cytokines [ 21 , 22 ] .
the goal of this study was to assess the activity of procoagulant factors and their impact on the inflammatory infiltrate in bp and dh before treatment .
the data might be an important therapeutic indication allowing for alleviating the high mortality rate among patients with pemphigoid treated with corticosteroids and confirming a new trend in the treatment of bp .
the study group involved 41 patients , including 27 patients with bp ( 15 women and 12 men , age range : 58 to 84 years , mean age : 68.5 years ) and 14 patients with dh ( 6 women and 8 men , age range : 18 to 70 years , mean age : 49.8 years ) in the active phase of the disease .
the control group consisted of 20 healthy subjects ( 5 women and 5 men , age range : 5080 years , mean age : 71.6 for bp patients and 6 women and 4 men , age range : 1949 years , mean age : 42.0 years for dh patients ) . in dh patients
direct immunofluorescence tests revealed the presence of granular deposits of iga in skin papillae and indirect immunofluorescence tests were positive for igaema in all the patients ( titer 1 : 401 : 640 , median 1 : 80 ) . anti - tissue transglutaminase antibodies measured using an immunoassay were present in 7/10 cases ( median : 5.1 iu / ml ( range : 0.0186.3 ; iu / ml ) ) .
the bp patients were at an active stage of the disease and the average score on bpdai ( bullous pemphigoid disease activity index ) was 38 11 .
in all the patients direct immunofluorescence test revealed igg / c3 linear deposits along bmz . in salt split test deposits were observed in epidermal part of the blister . using indirect immunofluorescence test
elisa test showed the anti - nc16 autoantibodies present in serum of 11 out of 14 patients .
all the patients signed informed consent before entering the study and the study protocol ( # rnn/93/03/ke ) was approved by the local ethical committee of medical university of lodz . from all patients in the study group and the healthy control group the blood
skin biopsies from skin lesions were taken for histopathological examination in the study groups and from the healthy skin in the neck area in control group .
paraffin - embedded sections were used for routine h+e staining and for immunohistochemistry with dako envision detection system using immunoperoxidase method .
the following primary monoclonal antibodies were used : anti - tissue factor obtained from american diagnostica inc . , greenwich , ct , usa , according to manufacturers ' instruction .
histological morphometry was performed by means of image analysis system consisting of a pc computer equipped with a pentagram graphical tablet , indeo fast card , produced by indeo ( taiwan ) , and color tv camera panasonic ( japan ) coupled to a carl zeiss microscope ( germany ) .
the tf positive cells were counted in a sequence of 710 consecutive computer images of 400x high power fields , 0.0047 mm each .
the results were expressed as percentages of tf positive cells of all lymphocytes determined by their morphology .
tf levels were measured in plasma in all patients and healthy controls undergoing skin biopsy .
five ml of venous blood was drawn from the ulnary vein and after centrifugation serum was stored at 20c for an immunoassay .
the enzyme - linked immunoassays were used to measure tf - imubind tissue factor elisa kit ( american diagnostica inc . ) .
d dimers levels were performed , using the optical test sta compact ( roche diagnostics ) ; fibrinogen levels were measured using chronometric method of clauss and turbidity method ; coagulogram was done according to the standard procedure ; dd ( d dimers ) , f2 ( factor ii ) , f5 ( factor v ) , f7 ( factor vii ) , and venom ( snake venom factor v activator ) were assessed by destiny plus in bp patients ( horiba abx ) .
total protein from frozen skin samples was extracted in ripa protein extraction buffer , supplemented with protease inhibitor cocktail ( sigma - aldrich , st .
the lysate was centrifuged at 14,000 g for 20 min and the pellet was discarded .
protein concentrations were determined by the bca protein assay kit ( pierce thermo scientific , usa ) .
electrophoresis was performed in sds - nupage gels ( life technologies , california ) , subsequently transferred to a nitrocellulose .
the membrane was blocked and incubated with the mouse monoclonal anti - tf ( merck millipore , billerica , usa ) . afterwards
the membrane was washed with tbst and incubated in pbst containing the goat anti - mouse igg secondary antibodies conjugated with alkaline phosphatase ( merck millipore , usa ) .
the bands were developed using bcip / nbt alkaline phosphatase substrate ( merck millipore , billerica , usa ) , analyzed with imagej 1.34s software ( wayne rasband , national institutes of health , bethesda ) , and expressed as optical density ( od ) .
differences between groups were tested using unpaired student 's t - test proceeded by the evaluation of normality .
results were considered statistically significant if p < 0.05 . the data of immunoblotting were analyzed with student 's t - test and mann - whitney u test .
tf expression was demonstrated in cells in the inflammatory infiltrates ( eosinophils ) and keratinocytes and in the blister fluid ( figure 1 ) . in patients with bp , the expression of tf
was also shown in the perilesional skin ( in 17 out of 27 individuals ) ( figure 2 ) . in patients with bp , in skin lesions ,
a high expression of tf has been demonstrated mainly in keratinocytes of the basal layer ( in 20 out of 27 biopsies ) and also in other layers of the epidermis ( in 19 out of 27 individuals ) , but expression insensitivity was lower .
morphometric analysis showed a higher expression of tf in skin lesions than in perilesional skin in patients with bp ( p < 0.02 ) ( table 1 ) .
studies of patients with dh did not show a significant expression of tf either in pathologically changed tissues ( tf was present in 3 out of 14 samples ) or in perilesional skin ( figure 3 ) .
tf protein expression was also found in biopsy specimens from healthy individuals ( in 2 out of 14 ) .
therefore , the tf expression , in healthy group , was significantly lower than in the lesions in bp and comparable to expression in dh lesions . utilizing an immunoblotting technique and specific antibodies against tf we found significantly higher expression of tf in bp patients as compared to dh patients ( p < 0.05 ) and as compared to healthy controls ( p < 0.05 ) as shown in figures 4 and 5 .
the mean tf plasma level in bp patients was 847.2 402.7 pg / ml as compared to patients with dh ( 285.7 187.7
pg / ml for bp and 221.9 153.3 pg / ml for dh .
these results indicate a statistically significant higher tf plasma concentration in patients with bp as compared to dh patients ' and the control subjects ( p < 0.001 ) . prior to treatment
, the d dimers levels in patients with bp were within the upper limits of the reference values ( in 20 out of 27 patients ) .
the mean d dimers level was 0.49 0.4 mg / ml ( whereas the reference value range was 0.332.9 mg / ml ) . in 7 patients these levels were elevated and were between 1.2 and 2.9 mg / ml . these values were higher as compared to dh patients and the control group .
the mean d dimers values in dh patients were 0.31 0.19 mg / ml and were within the reference value . in the control group of patients ,
used as a reference group for bp patients , d dimers levels were within normal limits ( the mean for control group was 0.29 0.3 mg / ml ) . in the control group of patients
used as a reference group for patients with dh , d dimers levels were within normal limits ; the mean was 0.34 0.6 mg / ml . therefore , there is a statistically significant difference in d dimers levels between the dh and bp patients ( p < 0.05 ) .
there is also a statistically significant difference in d dimers levels between bp patients and the control group ( p < 0.05 ) . in
bp patients coagulation parameters were as follows : prothrombin time mean of 13.1 0.2 sec ( reference value : 1218 sec ) , mean prothrombin index of 85.9% 0.8% ( reference range : 70100% ) , mean inr of 1.04 0.4 ( reference range 0.91.30 ) , and mean aptt of 31 s 1.0 sec ( reference range : 2636 sec . ) . in patients with dh coagulation parameters were as follows : mean prothrombin time of 15.3 0.5 sec , mean prothrombin index of 78.9% 0.6% , mean inr values of 1.28 0.3 , mean aptt of 29 sec 1.0 sec ( reference range : 2636 sec ) , and average values of 29 s 1.0 sec .
in the control group used for comparison with bp patients , coagulation parameters were as follows : mean prothrombin time of 14.2 0.2 sec , mean prothrombin index of 88.7% 0.4 , mean inr of 1.17 0.5 , and mean aptt of 30 sec 1.0 sec . in the control group used for comparison with dh patients , coagulation parameters were as follows : mean prothrombin time of 14.9 0.5 sec , mean prothrombin index of 98.7% 0.2 , mean inr of 1.21 0.4 , and mean aptt of 28 sec .
there is no difference in coagulation parameters between bp and dh groups and examined groups and healthy individuals .
the platelets levels in all groups were within the reference range ( bp patients : 284.0 96.9 10/l ; dh patients : 187.0 58.4 10/l ; and control group : 254.0 79 10/l ) .
in bp patients , mean fibrinogen level was 4.50 0.18 g / l as compared to 2.60 0.3
dh patients mean fibrinogen concentration was 2.80 0.14 g / l as compared to 2.45 0.26
there is a statistically significant difference ( p < 0.05 ) between the group of patients with dh and bp , as well as between bp patients and the control group ( p < 0.05 ) .
the d dimers levels were significantly higher in the bp patient group as compared to controls ( 312 ng / ml versus 96 ng / ml ) ( p < 0.01 ) .
all blood coagulation factors like f2 , f5 , f7 , and venom in bp and dh patients and control group were within the reference values .
the following correlations were found in bp patients between the following : f2 and dd ( r = 0.3419 , p = 0.003 ) ; f5 and dd ( r = 0.2074 , p = 0.005 ) ; dd and venom ( r = + 0.4120 , p = 0.0111 ) ; f5 and f2 ( r = + 0.4258 , p = 0.0031 ) ; f7 and f2 ( r
similarly , they were found between the following : venom and f2 ( r = 0.5517 , p = 0.0001 ) ; venom and f5 ( r = 0.3985 , p = 0.0054 ) ; venom and f7 ( r = 0.4424 , p = 0.0016 ) ( figure 6 ) .
eosinophils in response to many stimuli secrete many proteins including eosinophil cationic protein ( ecp ) .
elevated ecp prolongs the clotting time , due to its antagonism with heparin and streptokinase .
another protein , playing an important role in coagulation process , is tissue factor ( tf ) , also produced by eosinophils .
glucocorticoids do not stop the release of these proteins , but immunosuppressive agents such as cyclosporine inhibit the secretion of ecp from eosinophils .
evaluated the tf expression in skin biopsies in bp patients and showed that immunohistochemistry revealed strong tf reactivity in bp skin , and colocalization studies confirmed eosinophils as a source of tf .
our results indicate a statistically significant difference in the value of tf between the group of patients with dh and bp and bp and the control group .
elevated levels of the prothrombin fragment f1 + 2 and both plasma and blister fluid of bp patients in the active phase of the disease were reported . during the remission ,
the concentration of the prothrombin fragment f1 + 2 correlated with the concentration of immunoglobulins directed against the bp180 antigen .
tf is the main activator of extrinsic coagulation pathway , which in addition to well - documented prothrombotic properties plays an important role in the inflammatory process .
expression of tf present in fibroblasts , epithelial and endothelial cells , and macrophages is induced by antigen - antibody complexes , complement activation products , proteins , and proinflammatory cytokines .
our study showed low level tf expression in dh patients and healthy subjects , confirming its constitutive production .
emphasized the role of tnf and il-6 , which are increased in bp , in the induction of tf expression .
the eosinophilic infiltration in bp skin might provoke an elevation of tf concentration in this disease .
morphometric analysis showed a higher expression of tf in skin lesion than in perilesional skin in patients with bp opposite to dh and control biopsies . in our study
, we confirmed that kind of influx has important role in activation of tf in skin .
the inflammatory and neoplastic processes of coagulation and fibrinolysis system are activated in response to the local infiltration of inflammatory cells and increased vascular permeability [ 18 , 22 ] .
tf increases the influx of monocytes to the sites of inflammation [ 25 , 26 ] , and its induction in monocytes may depend on a direct interaction with t cells .
several studies have confirmed the effect of the inflammatory process in the excessive production of tissue factor [ 2730 ] .
both in the case of pemphigoid and dh in our earlier studies , we showed in skin lesions increased expression of proinflammatory cytokines which may activate the production of tf .
however , only in bp , expression was increased in both : skin lesions and blood .
this is probably related to eosinophilic infiltration in bp and production by these cells procoagulant factors .
the product of activation of tf , thrombin , acts by increasing vascular permeability for inflammatory cells and causing the release of interleukin production and activation of adhesion molecules . in our earlier studies , we confirmed the increased expression of adhesins and mmps , which may be an effect of action of tf in bp . in the aspect of described results , participation of activation markers of eosinophils , mast cells activation , and coagulation factors in the pathogenesis of some subepidermal blistering diseases
abnormal expression of procoagulant factors in the tissue may be responsible for the inflammatory process and blister formation in bp . according to literature [ 32 , 33 ] , up to 25% of the cells in the dh influx are eosinophils .
in addition , myeloperoxidase , ecp , and major basic protein secreted by eosinophils were found in sera of patients with dh , which may indicate the production of inflammatory mediators by eosinophils in dh . the small tf expression in our study in dh patients
may be related to the fact that the presence of a small population of eosinophils in the inflammatory infiltrates in these patients .
this study showed that the group of bp patients revealed the normal mean d dimer concentration , however the individual results were within the upper limit of the reference range , and some patients had elevated d dimer levels .
however , in the evaluation by the destiny plus system , d dimer concentration was significantly higher in the bp patient group as compared to controls .
it should be noted that these values were determined prior to treatment with corticosteroids , which exacerbate prothrombotic processes .
the values of d dimer in patients with dh were normal and similar to the levels in healthy subjects .
the treatment of patients with bp , especially the elderly , with local steroids or antihistamines and immunomodulators has been postulated [ 34 , 35 ] .
therefore , it seems important to indicate the need to change the current treatment of patients with bp with the standard treatment of corticosteroid into drugs that do not increase the risk of thrombotic complications .
, we use many drugs acting through different mechanisms . because of the key role of autoimmune phenomena , most commonly used drugs are glucocorticosteroids ( gcs ) , a class of drugs having a particularly strong impact on the homeostatic system .
effects of thrombogenicity related to gcs might be connected with their direct influence on the coagulation system and the metabolic disturbances linked to the nature of the disease .
our study also showed elevated levels of fibrinogen in patients with bp compared to the patient with dh and control group .
g / l is seen as the value that causes an increased risk of thrombosis and thus an increased risk of ischemic heart disease or a stroke [ 38 , 39 ] .
the main cause of death in patients with pemphigoid is the complications of steroid treatment , which is considered to be the most effective in this disease [ 4042 ] .
underline that embolic complications with severe bacterial infections are the most common side effects in the course of corticosteroid overall in the age group above 70 years .
other authors also confirm the good results of local therapy in the treatment of bullous pemphigoid and reducing the incidence of adverse events compared with patients treated with oral steroids .
so far , there are no data on the expression of procoagulant factors and abnormal activation of coagulation process in the formation of blisters .
however , the dominant role of eosinophils in the inflammatory infiltration in bp was recently revealed and the fact that they secrete tissue factor gives the scientific basis to consider the contribution of this process in the pathogenesis of lesions in the subepidermal bullous diseases .
demonstration of elevated d dimer and prothrombin in patients in the active phase of the disease indicates the need to assess the suitability of patients for thromboembolic complications .
an increased risk of death due to the stroke in patients with bp treated with glucocorticoids might confirm these findings . despite these risks , the latest consensus on the treatment of pemphigus and of bullous pemphigoid still recommends the use of corticosteroids in the treatment of bp .
our results may be useful in clinical practice and objective determination of the degree of disease activity as well as an important therapeutic indication which allows reducing the high mortality rate among patients with pemphigoid treated with corticosteroid and confirming a trend in the treatment of this disease with antihistamines and nonsteroidal immunomodulators . | dermatitis herpetiformis ( dh ) and bullous pemphigoid ( bp ) are skin diseases associated with eosinophilic and neutrophilic infiltrations . although chemokines are critical for the selective accumulation and activation of various leukocyte subsets in the inflammatory process , there are few findings concerning inflammatory cells and production of coagulation factors in blistering diseases .
skin biopsies were taken from 14 patients with dh , 27 with bp , and 20 control subjects . the localization and expression of tissue factor ( tf ) in skin lesions and perilesional skin
were studied by immunohistochemistry and confirmed by western blot .
moreover the plasma concentrations of tf were measured by immunoassays .
d dimers , fibrinogen , and selected coagulation parameters were measured by routine methods .
expression of tf in the epidermis and in inflammatory influxed cells in dermis was detected in skin biopsies from bp patients .
examined tf expression was detected in perilesional skin of all bp patients too .
the expression of tf was not observed in biopsies from healthy people and dh patients .
the findings of the study show an increased expression of tissue factor in the lesional and perilesional skin of patients with bullous pemphigoid .
the difference in chemokine pattern expression and variations in the cellular infiltration in bp and dh cause variable expression of tf . |
preservation of hard tissues close to the living state is essential for understanding of cellular and subcellular structures and functions .
the cutting of thin sections by ordinary methods is impossible in the case of tissues such as teeth , bone , teratomas containing bony tissue , lesions that have become partly calcified , odontomes and bony lesions .
such tissues must be treated to remove calcium phosphate by a process known as decalcification
decalcification of hard tissue is one of the most technique-0sensitive procedures in the histopathology laboratory .
it is of special significance in oral pathology as decalcification of bone and teeth is a routinely required procedure .
decalcification is carried out by chemical agents , either with acids to form soluble calcium salts or chelating agents that bind to calcium ions . in the manual method of decalcification ,
hard tissues are placed in a decalcifying agent at room temperature with changes of the solution at regular intervals till the end point is reached .
microwave decalcification is a novel technique compared to the manual method . in this method ,
hard tissues are placed in the decalcifying agent in a microwave oven for intermittent periods with regular changes of the solution till the end point is reached .
microwave irradiation has been shown to speed up the process of decalcification significantly from days to hours .
balaton and loget ( 1989 ) have reported that the decalcification of bone is accelerated about 10 times compared with that at ambient temperature .
the aim of the present study was to determine and compare routine decalcification with microwave decalcification of bone and teeth using nitric acid , formic acid and edta with respect to speed of decalcification , preservation of tissue structure and staining efficacy .
premolars extracted for orthodontic reasons and sections of mandibular condyle from specimens of resected mandibles from the archives of the department of oral and maxillofacial pathology were used in the study . thirty premolar teeth and 30 pieces of condyles were decalcified by both routine and microwave method using three decalcifying solutions ( five condyle pieces and five teeth in each ) .
the three solutions were dilute nitric acid ( 5% ) , formic acid ( 5% ) and ethylene diamine tetra acetic acid ( 14% ) .
these solutions were chosen as they are commonly used and easily available . a domestic microwave oven ( lg intellowave , model 1911he ) with a fixed rotary plate , maximum power output of 700 w and input voltage 230 v-50 hz was used . a glass beaker containing 100 ml of distilled water
this was replaced by100 ml fresh distilled water and irradiated to maintain the temperature at around 41 - 43c .
this took 14 s. the glass beaker was placed at different points in the oven while irradiating it to determine the best position of the specimen during microwave decalcification , since the microwave oven used had a constant timing but not a constant temperature [ figure 1a and b ] .
( b ) housing for microwave oven with outlet for fumes all the specimens were fixed in 10% neutral buffered formalin fixative and then washed in water for about 30 min before decalcification .
was then suspended in a coplin jar with the help of a thread in approximately 100 ml of decalcifying agent for decalcification .
the decalcifying solutions were changed and ph and temperature of the solutions were recorded on a daily basis . in the manual decalcification procedure , the solution was changed once in 3 days and the ph and temperature were recorded on a daily basis . in the microwave technique ,
the specimens were irradiated for eight cycles of 8 s each ( at 1-h intervals ) per day for formic and nitric acids , and eight cycles of 10 s each at 1-h intervals per day for edta so that the temperature of all three decalcifying solutions was maintained at around 41 - 43c .
the decalcifying solution was changed every day and the end - point was ascertained on a daily basis using the calcium oxalate method ( clayden 1952 ) . after ensuring complete decalcification , the tissues were washed using distilled water for 30 min , following which the specimens were subjected to manual tissue processing .
the condyle pieces were taken whole and teeth were each cut into two halves , both longitudinally and transversely .
after processing , the tissues were embedded in paraffin and were sectioned to a thickness of 7 - 8 m using the soft tissue microtome .
the sections were then stained by harris hematoxylin and eosin y. the stained sections of decalcified bone and teeth were assessed for the quality of staining and preservation of tissue details as objectively as possible .
the following parameters were recorded for each specimen :
missing osteocytes from the lacunae
missing osteocytes from the lacunae
shrinkage of pulp away from the dentinal walldamage to odontoblastic layer
shrinkage of pulp away from the dentinal wall damage to odontoblastic layer
yellow discoloration of specimentearing and crumpling of sectionpatchy staining .
yellow discoloration of specimen tearing and crumpling of section the data obtained from the above observations were entered and analyzed using statistical package for social sciences ( spss ) version 10.5 ibm .
premolars extracted for orthodontic reasons and sections of mandibular condyle from specimens of resected mandibles from the archives of the department of oral and maxillofacial pathology were used in the study . thirty premolar teeth and 30 pieces of condyles were decalcified by both routine and microwave method using three decalcifying solutions ( five condyle pieces and five teeth in each ) .
the three solutions were dilute nitric acid ( 5% ) , formic acid ( 5% ) and ethylene diamine tetra acetic acid ( 14% ) .
a domestic microwave oven ( lg intellowave , model 1911he ) with a fixed rotary plate , maximum power output of 700 w and input voltage 230 v-50 hz was used . a glass beaker containing 100 ml of distilled water
this was replaced by100 ml fresh distilled water and irradiated to maintain the temperature at around 41 - 43c .
this took 14 s. the glass beaker was placed at different points in the oven while irradiating it to determine the best position of the specimen during microwave decalcification , since the microwave oven used had a constant timing but not a constant temperature [ figure 1a and b ] .
( b ) housing for microwave oven with outlet for fumes all the specimens were fixed in 10% neutral buffered formalin fixative and then washed in water for about 30 min before decalcification .
was then suspended in a coplin jar with the help of a thread in approximately 100 ml of decalcifying agent for decalcification .
the decalcifying solutions were changed and ph and temperature of the solutions were recorded on a daily basis . in the manual decalcification procedure , the solution was changed once in 3 days and the ph and temperature were recorded on a daily basis . in the microwave technique ,
the specimens were irradiated for eight cycles of 8 s each ( at 1-h intervals ) per day for formic and nitric acids , and eight cycles of 10 s each at 1-h intervals per day for edta so that the temperature of all three decalcifying solutions was maintained at around 41 - 43c .
the decalcifying solution was changed every day and the end - point was ascertained on a daily basis using the calcium oxalate method ( clayden 1952 ) .
after ensuring complete decalcification , the tissues were washed using distilled water for 30 min , following which the specimens were subjected to manual tissue processing .
the condyle pieces were taken whole and teeth were each cut into two halves , both longitudinally and transversely .
after processing , the tissues were embedded in paraffin and were sectioned to a thickness of 7 - 8 m using the soft tissue microtome .
the sections were then stained by harris hematoxylin and eosin y. the stained sections of decalcified bone and teeth were assessed for the quality of staining and preservation of tissue details as objectively as possible .
the following parameters were recorded for each specimen :
missing osteocytes from the lacunae
missing osteocytes from the lacunae
shrinkage of pulp away from the dentinal walldamage to odontoblastic layer
shrinkage of pulp away from the dentinal wall damage to odontoblastic layer
yellow discoloration of specimentearing and crumpling of sectionpatchy staining .
yellow discoloration of specimen tearing and crumpling of section the data obtained from the above observations were entered and analyzed using statistical package for social sciences ( spss ) version 10.5 ibm .
the decalcification of condyles in nitric acid , formic acid and edta took 22 , 33 and 57 days , respectively for complete decalcification by the manual method ; whereas , the microwave oven technique took 2 , 6 and 9 days , respectively [ graph 1a ] . ( a ) comparison of duration of condyle decalcification by both methods ( b ) comparison of duration of tooth decalcification by both methods the decalcification of premolars in nitric acid , formic acid and edta took 35 , 42 and 85 days , respectively for complete decalcification whereas the microwave oven technique took 4 , 9 and 20 days , respectively [ graph 1b ] . the stained sections of decalcified bone and teeth
the presence of osteocytes within their lacunae [ graph 2a ] was not statistically significant ; but the percentages show there is better preservation of tissue in the microwave oven method compared to routine method with all three decalcifying agents [ figure 2 ] .
( a ) distribution of osteocytes visible in the lacunae in bone sections ( b ) distribution of pulp shrinkage in tooth sections ( a ) histologic section of bone decalcified by manual method using 5% nitric acid ( h&e stain , 100 ) .
( b ) histologic section of bone decalcified by microwave method using 5% nitric acid ( h&e stain , 100 ) shrinkage of the pulp away from dentinal wall [ graph 2b ] was observed to be 80% for all three decalcifying agents in the routine method . in the microwave method , this feature was seen in 80% , 100% and 100% in nitric acid , formic acid and edta , respectively .
the presence of damaged odontoblastic layer [ graph 3a ] was minimal ( 20% ) in the samples treated with nitric acid by microwave method when compared to routine method ( 80% ) .
though it was also not found to be statistically significant , the percentages show tissue preservation to be better in the microwave method as compared to routine method .
all the teeth samples treated with formic acid and edta showed no damage to the odontoblastic layer [ figure 3 ] ( a ) distribution of damaged odontoblastic layer in tooth sections ( b ) distribution of yellow discoloration in bone and tooth sections ( a ) histologic section of tooth decalcified by manual method using edta ( h&e stain , 100 ) ( b ) histologic section of tooth decalcified by microwave method using edta ( h&e stain , 100 ) yellow discoloration of the condyle and teeth specimens [ graph 3b ] was present in 5% nitric and absent in formic acid and edta irrespective of the method used .
the presence of patchy staining [ graph 4 ] was 100% in nitric acid decalcification by manual method compared to 40% in microwave method .
this difference was statistically significant and shows staining efficacy to be better in the microwave method compared to routine method using nitric acid .
but in formic acid and edta , all the samples showed patchy staining in both the methods .
it takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process . a new method using microwave oven
the choice of decalcifying agent and method is largely dictated by the urgency of the procedure .
the potential application of microwave energy in histotechnology was first recognized by mayers ( 1970 ) .
this form of nonionizing radiation produces alternating electromagnetic fields that result in the rotation of dipolar molecules such as water and the polar side chains of proteins through 180 c at the rate of 2.45 billion cycles / second .
the molecular kinetics so induced result in the generation of energy flux which continue until radiation ceases .
the idea of using microwaves to decrease the time for decalcification of temporal bones was originally introduced by hellstrom and nilsson ( 1992 ) for rat cochleas .
more recently , microwaves have been demonstrated to be useful in reducing the time needed for decalcification in edta of dense , primate temporal bones ( madden and henson , 1997 ) .
the energy produced by microwaves generated in a domestic oven interacts with dipolar molecules by imparting kinetic energy and altering the electric fields .
this energy induces a dielectric field leading to a rapid oscillation of dipolar molecules at about 180c , generating heat that is rapidly distributed homogeneously within the tissue .
pitol et al , ( 2007 ) showed there was a 30-fold increase in decalcification speed compared to the traditional method when the material was irradiated in a microwave oven .
however , balaton and loget ( 1989 ) reported that the decalcification of bone is accelerated about 10 times in the microwave oven compared with that at ambient temperature .
pitol et al . , used a domestic microwave oven for decalcification of rat bone using edta 8.5% solution and showed a reduction of experiment time from 45 days in the conventional method to 48 h in the microwave - aided method . in our study
14% edta solution took 57 days in the conventional method and 9 days using microwaves for complete decalcification of the condyle .
our decalcification of condyles in routine nitric acid took 22 days ; whereas , the microwave oven technique took 2 days .
our decalcification of condyle in routine 5% formic acid took 33 days ; whereas , the microwave oven technique took 6 days .
impressions of histological sections are subjective and affected by many variables , for example , fixation , processing , cutting technique , staining timings , etc .
it is important for histological analysis that the sections be obtained with the least possible alterations during processing .
damaged odontoblastic layer , shrinkage of pulp away from dentinal wall , and presence of osteocytes within the lacunae may be affected by fixation as well as by the choice of decalcifying agent .
strong acids are faster in their action than edta and formic acid , but need to be monitored closely as they carry an increased risk of tissue damage due to hydrolysis of proteins , which can result in maceration or the dissolution of the soft tissue components , with possible complete loss of histological detail . in our study
the presence of osteocytes within the lacunae was observed in all the specimens decalcified using microwave method with all the three decalcifying agents , whereas they were completely absent in nitric acid and partially absent in formic acid and edta specimens decalcified by routine method .
shrinkage of pulp away from dentinal wall in teeth may be affected by fixation and processing techniques as well as by decalcifying agents . in our study ,
shrinkage of pulp away from dentinal wall in teeth was seen in both the methods using all the decalcifying agents , with statistically insignificant results .
strong acids such as nitric acid can decalcify rapidly , but cause serious deterioration of stainability reviewed in stevens et al , ( 1990 ) and callis and sterchi ( 1998 ) . using chelating agents such as edta for
decalcification in edta has little or no effect on tissues other than on the bone mineral itself .
it only binds with calcium ions and gradually depletes the crystal size of the outer layer of the hydroxyapatite crystal . however , the major disadvantage is that decalcification by edta proceeds only slowly , with incubation times up to several weeks depending on the extent of mineralization . in our study ,
damaged odontoblastic layer in teeth was seen in most of the specimens in the routine nitric acid method ; whereas , the microwave method showed significant absence of damage to the odontoblastic layer in teeth . in case of formic acid and edta , both methods showed absence of damage to the odontoblastic layer in teeth .
the study in this criterion demonstrates that by microwave method one can preserve the tissue details almost precisely .
moore ( 1994 ) noted that the yellow staining of tissue which results from prolonged decalcification with nitric acid solutions may detract from the macroscopic appearance , but does not affect the histological examination since the color leaches from the specimen during processing .
stanley ( 1983 ) hypothesized that the specimen decalcified in nitric acid underwent a spontaneous yellow discoloration due to formation of nitrous oxide , which was responsible for the damage to the tissues . in our study , we observed the yellowish discoloration in all the specimens decalcified by nitric acid by both methods and the discoloration was totally absent in the specimens decalcified by formic acid and edta in both the methods . regarding patchy staining , the microwave nitric acid method shows a significant reduction in presence of patchy staining when compared to the routine method .
the other observation made in this study is that the overall staining of the tissue was similar in both the techniques except in the routine nitric acid method which showed inadequate nuclear staining .
the overall histological impression in the microwave method compared to routine method was significantly better .
cellular structures could be well - appreciated in all the sections of edta and formic acid .
a larger sample size would have given more conclusive resultsthe times taken for decalcification are likely to be different if used on different weights of bone and other teeth than premolars , as the decalcification time is dependent on the size and structural density of the hard tissuesince we have used a domestic microwave oven , our recording of the temperatures may have been only approximate .
a larger sample size would have given more conclusive results the times taken for decalcification are likely to be different if used on different weights of bone and other teeth than premolars , as the decalcification time is dependent on the size and structural density of the hard tissue since we have used a domestic microwave oven , our recording of the temperatures may have been only approximate .
a larger sample size would have given more conclusive resultsthe times taken for decalcification are likely to be different if used on different weights of bone and other teeth than premolars , as the decalcification time is dependent on the size and structural density of the hard tissuesince we have used a domestic microwave oven , our recording of the temperatures may have been only approximate .
a larger sample size would have given more conclusive results the times taken for decalcification are likely to be different if used on different weights of bone and other teeth than premolars , as the decalcification time is dependent on the size and structural density of the hard tissue since we have used a domestic microwave oven , our recording of the temperatures may have been only approximate .
the present study provided an insight into routine and microwave decalcification of bone and teeth and led to the following conclusions : the microwave method decalcifies both bone and teeth much faster than the routine method and in both the techniques nitric acid shows faster decalcifying ability followed by formic acid , and then edta .
tissue preservation and staining efficacy is poor using nitric acid in the routine method compared with the microwave method ; whereas , it is good using both formic acid and edta in both routine and microwave methods .
thus , it is evident that microwave decalcification is faster than routine decalcification using all three decalcifying agents .
there is no statistically significant difference between all the three decalcifying agents where preservation of tissue structure and staining efficacy are concerned irrespective of the methods used , except for patchy staining in the condyle and teeth using nitric acid , which shows a statistically significant difference between the routine and microwave methods . | introduction : the microwave oven has been used quite often for tissue processing , but there are very few studies describing its use in decalcification of bone or teeth . in this study
we have attempted to decalcify bone and teeth using a microwave oven and compare the process and results with conventional decalcification methods.aims and objectives : the objectives of the study were to determine and compare routine decalcification with microwave decalcification of bone and teeth using 5% nitric acid , 5% formic acid , and 14% ethylenediaminetetraacetic acid ( edta ) with respect to speed of decalcification , preservation of tissue structure and staining efficacy.materials and methods : in our study the total sample size used for both routine and microwave decalcification was 30 premolar teeth and 30 pieces of condyles .
the three solutions were dilute nitric acid ( 5% ) , formic acid ( 5% ) , and edta ( 14% ) .
each set consisting of the same type of premolars and condyles in each of the three decalcifying solutions were used in both manual method and microwave method.results:the results in the present study confirmed the fact that the microwave method using nitric acid was indeed the fastest decalcifying method needing just about 2 days for condyle and 4 days for premolars , compared with routine decalcification .
the results also showed that the overall histological picture was good with edta and formic acid irrespective of the methods used . in the routine method ,
nitric acid gave poor cellular detail when compared with microwave method.conclusion:with our study we conclude that microwave oven decalcification is faster than routine decalcification irrespective of the decalcifying agents used .
the tissue preservation and staining efficacy was good in microwave nitric acid decalcification compared to routine nitric acid decalcification .
both formic acid and edta show good tissue preservation and staining efficacy irrespective of the method used . |
the ttm trial randomized 950 adult comatose patients resuscitated after out - of - hospital cardiac arrest of presumed cardiac cause from november 2010 to january 2013 .
the trial protocol was approved by the ethics committees in the participating countries and registered at clinicaltrials.gov ( nct01020916 ) .
sedation was mandatory during temperature management and tapered at normothermia if not needed for intensive care reasons or treatment of status epilepticus .
a routine eeg was performed during office hours 1236 hours after rewarming in patients who were still comatose , typically corresponding to 4872 hours after the cardiac arrest or later if this period coincided with a weekend .
the detailed rationale of the present eeg study , including the prespecified hypotheses , was published previously .
all 36 intensive care units in europe and australia participating in the ttm trial performed routine eegs .
for this eeg study , we included all patients from sites that had an eeg system allowing eeg data export including notations on reactivity testing .
sedation , antiepileptic medication , and level of consciousness were documented prospectively . at 72 hours after rewarming , a physician blinded to target temperature , but not to the local eeg report , performed a neurologic evaluation and recommended continuation or withdrawal of life - sustaining therapy ( wlst ) .
persisting deep coma ( glasgow coma scale : motor 12 ) was a criterion of poor prognosis and allowed wlst if combined with either bilateral loss of somatosensory evoked potentials ( ssep ) n20 responses or treatment - refractory status epilepticus .
treatment refractory was defined as unresponsive to propofol , midazolam , or thiopental in combination with at least 1 iv antiepileptic substance for at least 24 hours .
in addition , a clinical finding of early status myoclonus , regardless of eeg correlate , in combination with bilateral loss of n20 potentials after rewarming was a criterion of poor prognosis
. in patients not fulfilling these criteria , continued active intensive care was protocolized and patients reevaluated daily .
the details and rationale of the prognostication algorithm and how wlst was implemented in the trial was published .
eegs were retrieved as electronic data format files to a central eeg database and analyzed using the acns standardized terminology , independently by 4 eeg specialists ( sweden : e.w .
the eeg specialists were blinded to all clinical data including outcome and reported eeg findings through a web - based electronic case report form structured to assure complete data .
the eegs were full - length ( > 20 minutes ) and at least 16 eeg channels were used .
based on the findings regarding periodic or rhythmic patterns , background pattern , and reactivity , the eegs were classified according to the prespecified hypotheses into the following categories : highly malignant eeg ( figure 1)suppressed background without dischargessuppressed background with continuous periodic dischargesburst - suppression background with or without dischargesmalignant eegmalignant periodic or rhythmic patterns ( abundant periodic discharges ; abundant rhythmic polyspike-/spike-/sharp - and - wave ; unequivocal electrographic seizure)malignant background ( discontinuous background ; low - voltage background ; reversed anterior - posterior gradient)unreactive eeg ( absence of background reactivity or only stimulus - induced discharges)benign eeg ( absence of all malignant features stated above ) highly malignant eeg ( figure 1 ) suppressed background without discharges suppressed background with continuous periodic discharges burst - suppression background with or without discharges malignant periodic or rhythmic patterns ( abundant periodic discharges ; abundant rhythmic polyspike-/spike-/sharp - and - wave ; unequivocal electrographic seizure ) malignant background ( discontinuous background ; low - voltage background ; reversed anterior - posterior gradient ) unreactive eeg ( absence of background reactivity or only stimulus - induced discharges ) benign eeg ( absence of all malignant features stated above ) highly malignant patterns used in the study defined according to the standardized eeg terminology by the american clinical neurophysiology society .
( a ) suppressed background ( amplitude < 10 v , 100% of the recording ) without discharges .
( c ) burst - suppression ( periods of suppression with amplitude < 10 v constituting > 50% of the recording ) without discharges .
surviving patients attended a follow - up 180 days after their cardiac arrest by a blinded assessor .
good neurologic outcome was defined as cpc 12 ( no or moderate disability ) at follow - up or at any time during the hospital stay .
poor outcome was defined as best achieved cpc of 35 ( severely disabled , comatose , or deceased ) .
prognostic ability ( specificity , sensitivity ) was calculated for the highly malignant patterns ( primary hypothesis ) and the other prespecified patterns ( secondary hypotheses ) and is presented as the mode value for the 4 interpreters , i.e. , the pattern that 2 or more interpreters have reported , with 95% confidence intervals ( ci ) . if 2 interpreters reported 1 pattern and the other 2 interpreters reported another , the most malignant pattern was assigned . for comparisons among temperature groups , sedation , and eeg patterns , we applied generalized linear mixed models including interpretations from all 4 eeg specialists . statistical analysis was performed using r , version 3.0.2 .
the trial protocol was approved by the ethics committees in the participating countries and registered at clinicaltrials.gov ( nct01020916 ) .
sedation was mandatory during temperature management and tapered at normothermia if not needed for intensive care reasons or treatment of status epilepticus .
a routine eeg was performed during office hours 1236 hours after rewarming in patients who were still comatose , typically corresponding to 4872 hours after the cardiac arrest or later if this period coincided with a weekend .
the detailed rationale of the present eeg study , including the prespecified hypotheses , was published previously .
all 36 intensive care units in europe and australia participating in the ttm trial performed routine eegs .
for this eeg study , we included all patients from sites that had an eeg system allowing eeg data export including notations on reactivity testing . patients who awoke or died before the recommended time point of eeg were excluded .
sedation , antiepileptic medication , and level of consciousness were documented prospectively . at 72 hours after rewarming , a physician blinded to target temperature , but not to the local eeg report , performed a neurologic evaluation and recommended continuation or withdrawal of life - sustaining therapy ( wlst ) . persisting deep coma ( glasgow coma scale : motor 12 ) was a criterion of poor prognosis and allowed wlst if combined with either bilateral loss of somatosensory evoked potentials ( ssep ) n20 responses or treatment - refractory status epilepticus .
treatment refractory was defined as unresponsive to propofol , midazolam , or thiopental in combination with at least 1 iv antiepileptic substance for at least 24 hours .
in addition , a clinical finding of early status myoclonus , regardless of eeg correlate , in combination with bilateral loss of n20 potentials after rewarming was a criterion of poor prognosis
. in patients not fulfilling these criteria , continued active intensive care was protocolized and patients reevaluated daily .
the details and rationale of the prognostication algorithm and how wlst was implemented in the trial was published .
eegs were retrieved as electronic data format files to a central eeg database and analyzed using the acns standardized terminology , independently by 4 eeg specialists ( sweden : e.w .
the eeg specialists were blinded to all clinical data including outcome and reported eeg findings through a web - based electronic case report form structured to assure complete data .
the eegs were full - length ( > 20 minutes ) and at least 16 eeg channels were used .
based on the findings regarding periodic or rhythmic patterns , background pattern , and reactivity , the eegs were classified according to the prespecified hypotheses into the following categories : highly malignant eeg ( figure 1)suppressed background without dischargessuppressed background with continuous periodic dischargesburst - suppression background with or without dischargesmalignant eegmalignant periodic or rhythmic patterns ( abundant periodic discharges ; abundant rhythmic polyspike-/spike-/sharp - and - wave ; unequivocal electrographic seizure)malignant background ( discontinuous background ; low - voltage background ; reversed anterior - posterior gradient)unreactive eeg ( absence of background reactivity or only stimulus - induced discharges)benign eeg ( absence of all malignant features stated above ) highly malignant eeg ( figure 1 ) suppressed background without discharges suppressed background with continuous periodic discharges burst - suppression background with or without discharges malignant periodic or rhythmic patterns ( abundant periodic discharges ; abundant rhythmic polyspike-/spike-/sharp - and - wave ; unequivocal electrographic seizure ) malignant background ( discontinuous background ; low - voltage background ; reversed anterior - posterior gradient ) unreactive eeg ( absence of background reactivity or only stimulus - induced discharges ) benign eeg ( absence of all malignant features stated above ) highly malignant patterns used in the study defined according to the standardized eeg terminology by the american clinical neurophysiology society .
( a ) suppressed background ( amplitude < 10 v , 100% of the recording ) without discharges .
( c ) burst - suppression ( periods of suppression with amplitude < 10 v constituting > 50% of the recording ) without discharges .
surviving patients attended a follow - up 180 days after their cardiac arrest by a blinded assessor .
good neurologic outcome was defined as cpc 12 ( no or moderate disability ) at follow - up or at any time during the hospital stay .
poor outcome was defined as best achieved cpc of 35 ( severely disabled , comatose , or deceased ) .
prognostic ability ( specificity , sensitivity ) was calculated for the highly malignant patterns ( primary hypothesis ) and the other prespecified patterns ( secondary hypotheses ) and is presented as the mode value for the 4 interpreters , i.e. , the pattern that 2 or more interpreters have reported , with 95% confidence intervals ( ci ) .
if 2 interpreters reported 1 pattern and the other 2 interpreters reported another , the most malignant pattern was assigned . for comparisons among temperature groups , sedation , and eeg patterns , we applied generalized linear mixed models including interpretations from all 4 eeg specialists . statistical analysis was performed using r , version 3.0.2 .
eight study sites randomized 202 patients to targeted temperature management at 33c ( n = 103 ) or 36c ( n = 99 ) .
a routine eeg was recorded at a median 77 hours ( interquartile range 53102 ) after cardiac arrest in 103 patients still comatose after rewarming .
exclusion and inclusion in the present study is described in figure 2 . in 99 of the 202 randomized patients ,
69 of whom awoke and 22 died before the recommended time point of prognostication , eegs were not performed .
the recommended time point of prognostication was 72 hours after rewarming , corresponding to approximately 108 hours after the cardiac arrest .
the most probable cause of death according to the treating physician is reported ( cerebral , multiorgan failure , or cardiovascular ) .
eight study sites were included since they had an eeg system that allowed export of eeg data that included notations regarding testing of reactivity .
eight patients with prolonged coma ( > 5 days ) did not have an eeg .
one of these was brain dead and 2 had absent n20 potentials . in the remaining 5 patients ,
two of these patients did not have wlst and eventually recovered to cpc 3 , 1 died on day 5 of cerebral cause without wlst , 1 died on day 6 due to multiorgan failure without wlst , and 1 died on day 5 of cerebral cause after wlst .
patient characteristics characteristics of the patients on the day of eeg registration are presented in table 2 . at this time
clinical seizures occurred during the eeg recording in 16% , mostly myoclonus , and 35% of patients had antiepileptic treatment .
patient characteristics on the day of eeg recording ( n = 103 ) the prognostic ability of the highly malignant patterns is presented in table 3 ( primary hypotheses ) .
a highly malignant pattern was reported by the majority of the 4 interpreters ( mode value ) in 38 patients ( 37% ) and all had a poor neurologic outcome .
the sensitivity for a highly malignant pattern to predict a poor outcome was 50% ( mode value ) .
sensitivity varied among the 4 interpreters but specificity was 100% for all individual interpreters ( table e-1 on the neurology web site at neurology.org ) .
ability of highly malignant and malignant patterns to predict poor outcome the prognostic ability of the malignant features is presented in table 3 .
a malignant eeg , defined as presence of at least one malignant feature and thus also including the highly malignant patterns , was reported in 89 patients ( 86% ) .
the sensitivity to predict a poor outcome was 99% with a specificity of 48% ( mode values ) .
regarding the subcategories of malignant features , any malignant periodic or rhythmic pattern showed a higher specificity ( 100% ) to predict a poor outcome compared to any malignant background pattern ( 74% ) or a nonreactive eeg ( 70% ) .
an isolated finding of a single malignant feature occurred in 30 patients , 17 of whom had a good outcome . if at least 2 of the 3 malignant subcategories were present in the same eeg , the specificity to predict a poor outcome increased significantly to 96% ( p < 0.001 ) , while sensitivity decreased to 76% ( p < 0.001 ) .
fourteen patients ( 14% ) had a benign eeg , defined as a lack of all malignant features .
a benign eeg was found in 1% ( 95% ci 0%7% ) of the 76 patients with a poor outcome and in 48% ( 95% ci 31%66% ) of the 27 patients with a good outcome .
eight patients ( 8% ) had a benign and reactive eeg , defined as absence of all malignant features and presence of reactivity to either sound or pain stimuli , and none of these patients had a poor outcome .
a benign and reactive eeg was found in none ( 95% ci 0%5% ) of the patients with a poor outcome and in 30% ( 95% ci 16%49% ) of the patients with a good outcome .
the ability for a benign eeg to predict good outcome did not significantly differ compared to that of a benign and reactive eeg ( p = 0.08 ) in this small sample size .
there were no significant differences between patients treated with 33c compared to 36c regarding prevalence or prognostic ability of highly malignant eeg ( prevalence p = 0.58 ; sensitivity p = 0.58 ; specificity p = not applicable ) or malignant eeg ( prevalence p = 0.17 ; sensitivity p = 0.64 ; specificity p = 0.81 ) .
ongoing or residual sedation affected the level of consciousness in 56% of the patients on the day of the routine eeg , according to the treating physician . in 36% , sedation was ongoing during recording of the eeg .
there were no significant differences regarding prognostic ability of highly malignant eeg ( sensitivity p = 0.81 ; specificity p = not applicable ) or malignant eeg ( sensitivity p = 0.44 ; specificity p = 0.21 ) between patients with ongoing sedation and those without ongoing sedation .
the prognostic ability of the highly malignant patterns is presented in table 3 ( primary hypotheses ) .
a highly malignant pattern was reported by the majority of the 4 interpreters ( mode value ) in 38 patients ( 37% ) and all had a poor neurologic outcome .
the sensitivity for a highly malignant pattern to predict a poor outcome was 50% ( mode value ) .
sensitivity varied among the 4 interpreters but specificity was 100% for all individual interpreters ( table e-1 on the neurology web site at neurology.org ) .
a malignant eeg , defined as presence of at least one malignant feature and thus also including the highly malignant patterns , was reported in 89 patients ( 86% ) .
the sensitivity to predict a poor outcome was 99% with a specificity of 48% ( mode values ) .
regarding the subcategories of malignant features , any malignant periodic or rhythmic pattern showed a higher specificity ( 100% ) to predict a poor outcome compared to any malignant background pattern ( 74% ) or a nonreactive eeg ( 70% ) .
an isolated finding of a single malignant feature occurred in 30 patients , 17 of whom had a good outcome . if at least 2 of the 3 malignant subcategories were present in the same eeg , the specificity to predict a poor outcome increased significantly to 96% ( p < 0.001 ) , while sensitivity decreased to 76% ( p < 0.001 ) .
fourteen patients ( 14% ) had a benign eeg , defined as a lack of all malignant features .
a benign eeg was found in 1% ( 95% ci 0%7% ) of the 76 patients with a poor outcome and in 48% ( 95% ci 31%66% ) of the 27 patients with a good outcome .
eight patients ( 8% ) had a benign and reactive eeg , defined as absence of all malignant features and presence of reactivity to either sound or pain stimuli , and none of these patients had a poor outcome .
a benign and reactive eeg was found in none ( 95% ci 0%5% ) of the patients with a poor outcome and in 30% ( 95% ci 16%49% ) of the patients with a good outcome .
the ability for a benign eeg to predict good outcome did not significantly differ compared to that of a benign and reactive eeg ( p = 0.08 ) in this small sample size .
there were no significant differences between patients treated with 33c compared to 36c regarding prevalence or prognostic ability of highly malignant eeg ( prevalence p = 0.58 ; sensitivity p = 0.58 ; specificity p = not applicable ) or malignant eeg ( prevalence p = 0.17 ; sensitivity p = 0.64 ; specificity p = 0.81 ) .
ongoing or residual sedation affected the level of consciousness in 56% of the patients on the day of the routine eeg , according to the treating physician . in 36% , sedation was ongoing during recording of the eeg .
there were no significant differences regarding prognostic ability of highly malignant eeg ( sensitivity p = 0.81 ; specificity p = not applicable ) or malignant eeg ( sensitivity p = 0.44 ; specificity p = 0.21 ) between patients with ongoing sedation and those without ongoing sedation .
eeg is a standard investigation to assess prognosis after cardiac arrest , but its reliability may be limited by lack of consensus on definitions of malignant patterns and by interrater variability .
we found that the proposed highly malignant eeg patterns reliably predicted a poor outcome in our cohort of comatose post cardiac arrest patients .
these patterns were previously found to have substantial interrater and intrarater reliability and the sensitivity to detect patients with poor prognosis was 50% , exceeding that of ssep and absent ocular reflexes .
since routine eeg is a generally available , noninvasive examination , these findings , if verified in a different cohort , may enhance the safe use of eeg as a key component in decisions on continuation or withdrawal of life - sustaining therapy . nevertheless , it is strongly advisable to combine the eeg findings with other tests to perform a multimodal prognostication .
standardization of eeg interpretation is critical for reproducibility of clinical studies , meta - analyses , and application of these results into everyday clinical practice .
therefore , the use of an international well - defined terminology , such as the acns eeg terminology , is strongly recommended .
the highly malignant patterns used in this study were strictly defined according to the acns eeg terminology and adhere closely to previously described malignant patterns .
our secondary hypothesis that a malignant eeg should nearly always be associated with a poor outcome was discarded due to lack of specificity and considerable variability between interpreters .
our results therefore discourage the use of isolated findings of a malignant feature in decisions on wlst
. combination of at least 2 malignant features from diverse malignant subcategories significantly increased the specificity to 96% , indicating that such combinations , if confirmed in another cohort , may be of use to predict a poor prognosis . low - voltage background ( most background activity < 20 v ) was a common pattern with several false - positive predictions , in contrast to the highly malignant pattern of suppressed background ( all background activity < 10 v ) , which had a specificity of 100% for predicting poor outcome .
this is of considerable importance since 20 v has been used as a limit of the background voltage for predicting a poor outcome .
our results highlight that the degree of discontinuity is an important predictor of prognosis . a discontinuous background ( suppression periods constituting 10%49% ) resulted in some false - positives ( for 2 individual interpreters ) while a burst - suppression background ( suppression periods constituting 50%99% ) was a reliable predictor of poor prognosis for all 4 interpreters .
this is in concordance with a recent retrospective single - center study using the same standardized acns terminology that found a zero false - positive rate for burst - suppression to predict a poor outcome .
however , that study also reported no false - positives for a low - voltage background 24 hours after cardiac arrest or later , which was not the case in our study using 4 interpreters from different centers .
a proposed malignant pattern is the alpha - theta coma pattern , which has a nonreactive background with a reversed anterior - posterior gradient ( pathologic distribution of the background activity ) .
a reversed anterior - posterior gradient could per se not reliably predict a poor outcome in our study .
there is no consensus on the definition of postanoxic electrographic seizure activity , but several investigators using different definitions have shown a strong correlation to poor outcome .
all 5 patients who had unequivocal electrographic seizure activity during the limited time period of the routine eeg had a poor outcome in this study .
in contrast , abundant periodic discharges were not inevitably associated with a poor outcome for the individual interpreters ( table e-1 ) , but no patients with periodic discharges on a suppressed background ( < 10 v ) according to any interpreter had a good outcome .
the treatment of epileptiform activity was not defined in the protocol and not included in the intervention of this trial .
whether patients with electrographic status epilepticus may benefit from antiepileptic medication is unclear and currently addressed in a separate randomized trial .
since continuous eeg monitoring was not available in most centers of this study , it is likely that some patients with intermittent electrographic seizures were missed .
it is well - known that eeg activity is affected by sedation and in sufficient doses it may cause burst - suppression , but whether sedation in clinically used doses affects the prognostic value of malignant eeg patterns is unknown . lacking detailed data on the amount of sedation used , we found that the majority of patients were still considered affected by sedation by the reporting physician at the time of eeg , which is close to the recommended time point for neurologic prognostication in recent guidelines .
however , the predictive ability of our malignant patterns was not significantly affected by ongoing sedation . background reactivity was previously found to have strong prognostic implications in several single - center studies .
it is therefore not surprising that the specificity for an unreactive eeg varied considerably among our interpreters .
false predictions of a poor outcome ranged from none to the majority of patients among the interpreters .
this might reflect different traditions in assessing reactivity in different centers , since the interpreter who had the highest specificity also had the lowest sensitivity and vice versa . a more conservative approach when stating absence of reactivity by the former interpreter compared to the latter
more strict definitions , standardized stimuli , video recordings , bedside assessments , use of muscle relaxants , or quantitative measurements could possibly improve agreement and specificity .
we termed eegs that lacked any malignant features as benign , according to our prespecified definitions .
the proportion of patients with a poor outcome who had a benign eeg was very low ( 1% ) , which is an important finding since few markers indicating a good prognosis are available .
however , it is important to recognize that many patients with good outcome lacked a benign eeg due to presence of some malignant eeg features and that the definition of good outcome includes a significant proportion of patients with mild cognitive impairment .
an important limitation of our study is that the local eeg report was available at the neurologic evaluation .
although treatment - refractory postanoxic status epilepticus was the only eeg criterion that justified withdrawal of treatment according to the ttm trial protocol , other eeg features in the local eeg report may have had influence , causing a self - fulfilling prophecy .
importantly , all 4 eeg readers in this study were blinded to outcome and all clinical data .
another limitation is that our eeg investigations are limited to patients who failed to wake up when the active temperature control phase was over .
thus we lack data on spontaneously awakening patients , but we hypothesize that the occurrence of malignant eeg patterns would be highly unlikely in awake patients .
exclusion of early awakening patients with favorable prognosis explains the worse prognosis among patients with eeg recordings in our study . yet this cohort is likely to be representative of comatose patients eligible for an eeg for prognostication after cardiac arrest .
the power of the study is limited by the exclusion of sites that could not export eeg data of reactivity .
this selection of centers allows for homogenous high - quality data and the possibility to confirm findings , apart from reactivity , in the remaining cohort .
we recruited our eeg specialists from different countries and centers without prior collaboration , which would increase the generalizability of our results .
we recognize that all 4 eeg readers are specialists in clinical neurophysiology with 1014 years of experience in reading eegs and that we can not conclude on the transferability of our results to less experienced readers .
all eegs were performed after rewarming and the majority within 24 days after the cardiac arrest and we stress that our results are representative for this time period only .
it has previously been shown that some of our malignant patterns can occur during the early phase after resuscitation and ongoing target temperature management among survivors with good outcome . a highly malignant eeg pattern , defined according to the standardized acns eeg terminology , predicted poor outcome in half of patients who remained in a coma after rewarming without false - positives .
if replicated in a larger cohort , ideally with blinding of the eegs to the treating physician , these patterns are promising candidates to be included in a multimodal prognostication algorithm .
on the other hand , an isolated finding of a single malignant feature could not be used to predict poor outcome .
neither the level of temperature management nor ongoing sedation in clinically used doses significantly affected the prognostic ability of these patterns .
divisions of clinical neurophysiology ( e.w . , i.r . ) , intensive and perioperative care ( h.f . ) , and neurology ( t.c . ) , department of clinical sciences , lund university , sweden ; department of neurology ( a.o.r . ) , chuv and university of lausanne , switzerland ; departments of neurology / clinical neurophysiology ( a .- f.v.r . ) and intensive care medicine ( j. horn ) , academic medical center , university of amsterdam , the netherlands ; neurophysiology center ( t.w.k . ) , roskilde university hospital , roskilde , denmark ; r&d centre skane ( s.u . ) , skane university hospital , lund ; department of anaesthesia and intensive care ( n.n . ) , intensive care unit , helsingborg hospital , sweden ; department of intensive care ( a. . ) , liverpool hospital , sydney , new south wales , australia ; department of clinical sciences ( d.e . ) , lund university , sweden ; department of anesthesiology , pharmacology and intensive care ( y.g . ) , geneva university hospital , switzerland ; departments of cardiology ( c.h . , j.k .
) and cardiothoracic anesthesiology ( m.w . ) , the heart centre , and copenhagen trial unit , centre of clinical intervention research ( j.w . ) , copenhagen university hospital rigshospitalet , denmark ; department of anesthesiology ( j. hovdenes ) , oslo university hospital , rikshospitalet , norway ; department of intensive care , medical center leeuwarden ( m.k . ) , the netherlands ; intensive care unit ( t.p . ) , santa maria degli angeli , pordenone , italy ; department of anesthesiology and intensive care ( p.s . ) , centre hospitalier de luxembourg ; and adult critical care ( m.p.w . ) , university hospital of wales , cardiff , uk .
drafted the manuscript , which was critically revised for intellectual content and finally approved by all coauthors .
supported by the swedish heart and lung association , the skne university hospital foundations , the gyllenstierna - krapperup foundation , the segerfalk foundation , the swedish national health system ( alf ) , the county council of skne , the swedish society of medicine , the koch foundation , the swedish heart - lung foundation , afa insurance , the swedish research council , and the hans - gabriel and alice trolle - wachtmeister foundation , sweden ; the tryg foundation , denmark ; eu programme interreg iv a ; and the swiss national research foundation ( cr32i3_143780 ) ( a.o.r . ) .
a. rossetti receives support by the swiss national research foundation ( cr32i3_143780 ) and received research support from ucb pharma and sage pharmaceuticals .
t. wesenberg kjaer is principal investigator in trials with widex a / s and has received honoraria from eisai and ucb pharma for lectures .
s. ulln reports no disclosures . h. friberg receives support from the swedish national health system ( alf ) , serves as european editor of therapeutic hypothermia and temperature management , is a member of the editorial board of resuscitation , and has received honoraria for lectures from bard medical and natus inc .
d. erlinge has received minor speaker fees or advisory board compensation from zoll , astrazeneca , and lilly .
y. gasche reports no disclosures . c. hassager receives grant support from the interreg iva ks program
; honorarium for lectures from astrazeneca , teva , novartis , and vicare medical ; and travel support to congresses from novartis .
j. wetterslev 's institution , copenhagen trial unit ( ctu ) , received an unrestricted grant from trygfonden in denmark for involvement in the ttm trial .
m. wise was funded 40% wte during the study by a national institute for social care and health research ( nischr ) academic health science collaboration ( ahsc ) clinical research fellowship ; received travel costs from the british thoracic society , intensive care society , scottish intensive care society , and orion ltd . ; received royalties from wiley publishing ; received honorarium for lecturing at educational meetings from fisher & paykel and merck ; serves on the advisory board for kalobius pharmaceuticals , bard ; and is editor of bmj open respiratory research . | objective : to identify reliable predictors of outcome in comatose patients after cardiac arrest using a single routine eeg and standardized interpretation according to the terminology proposed by the american clinical neurophysiology society.methods:in this cohort study , 4 eeg specialists , blinded to outcome , evaluated prospectively recorded eegs in the target temperature management trial ( ttm trial ) that randomized patients to 33c vs 36c .
routine eeg was performed in patients still comatose after rewarming .
eegs were classified into highly malignant ( suppression , suppression with periodic discharges , burst - suppression ) , malignant ( periodic or rhythmic patterns , pathological or nonreactive background ) , and benign eeg ( absence of malignant features ) .
poor outcome was defined as best cerebral performance category score 35 until 180 days.results:eight ttm sites randomized 202 patients .
eegs were recorded in 103 patients at a median 77 hours after cardiac arrest ; 37% had a highly malignant eeg and all had a poor outcome ( specificity 100% , sensitivity 50% ) .
any malignant eeg feature had a low specificity to predict poor prognosis ( 48% ) but if 2 malignant eeg features were present specificity increased to 96% ( p < 0.001 ) . specificity and sensitivity were not significantly affected by targeted temperature or sedation . a benign eeg was found in 1% of the patients with a poor outcome.conclusions:highly malignant eeg after rewarming reliably predicted poor outcome in half of patients without false predictions .
an isolated finding of a single malignant feature did not predict poor outcome whereas a benign eeg was highly predictive of a good outcome . |
daily lifestyle habits , such as physical activity , smoking , alcohol drinking and dietary intake , are associated with the risk of developing impaired glucose tolerance ( igt ) or diabetes mellitus ( dm ) .
recently , sleep duration , one of the daily lifestyle habits , has gained attention with regard to its association with lifestylerelated disease conditions .
a ushaped curvilinear relationship was observed ( < 6 , 6 , 7 , 8 h ) between sleep duration and impaired glucose tolerance .
both a decrease and an increase in sleep duration were associated with an increase in cardiovascular and noncardiovascular mortality . because igt / dm carries a higher risk of cardiovascularrelated mortality , the impact of short sleep duration on glucose regulation suggested a mechanism whereby short sleep duration was associated with an increase in mortality .
furthermore , an association of sleep duration with mental health , cardiovascular disease and metabolic disorders has been reported in other studies .
because previous studies were carried out in usa and european populations where the average body mass index ( bmi ) is higher , a study was needed in a population where obesity is relatively uncommon . in the present study
, we examined the crosssectional relationship between sleep duration , quality and igt , including dm , in a largescale database of healthy workers in japan .
we analyzed baseline data from the highrisk and population strategy for occupational health promotion ( hipopohp ) study . in brief , hipopohp was an interventional survey to establish a methodology for reducing cardiovascular disease ( cvd ) risk factors in the workplace .
researchers followed the data of cvd risk factors , lifestyle habits and consciousness about health based on nutrition , physical activity and smoking for 4 years .
this study was carried out as part of the management of safety and health with the approval of the safety hygiene committee at each company .
accordingly , all employees were enrolled in this study . however , participation was voluntary , and we explained there was no need for participants to answer the required questionnaire if they did not want to .
approval for the study was obtained from the institutional review board of shiga university of medical science for ethical issues ( no .
the present study examined baseline data from 4143 participants ( 3415 men and 728 women ) aged 1969 years ( mean sd , 41.9 9.4 years ) who underwent physical examination , a lifestyle survey and blood chemical examination .
physical and laboratory data were standardized according to the manual of the hipopohp research group . briefly , after 5 min of silent rest measured by a sandglass , blood pressure ( bp ) was measured twice for each participant using an automatic sphygmomanometer ( bp103iii ; omron colin , tokyo , japan ) at each company , and the mean value was recorded . to measure the lipid levels in each participant ,
the company established a contract with a clinical laboratory ; the blood testing was standardized through the us cholesterol reference method laboratory network ( crmln ) .
we calculated lowdensity lipoprotein cholesterol by friedewald s formula when triglyceride did not exceed 300 mg / dl .
the bmi was calculated as weight ( kg ) divided by height squared ( m ) .
sleep duration was investigated using the question , how many hours of sleep do you get on an average weekday per night? in the lifestyle survey
. sleep duration of participants was categorized into four groups : < 6 , 6 to < 7 , 7 to < 8 and 8 h with reference to a precedent study .
sleep quality was investigated using the question how often do you experience difficulty initiating sleep ?
seldom. participants were asked about the type of , and time spent on , physical activities in their spare time for recreation , exercise or sport in the previous month .
physical activity of each participant was converted into metminutes per week (= met level minutes of activity / day days per week ) according to the international physical activity questionnaire ( ipaq ) , and participants were classified into four classes of physical activity as high ( 3000 metminutes per week ) , moderate activity ( < 3000 but 1500 metminutes per week ) , some activity ( < 1500 but 600 metminutes per week ) or sedentary ( < 600 metminutes per week ) .
the frequency of alcohol intake during a typical week and the total alcohol intake on each occasion were determined and used to calculate the alcohol intake per week .
this value was then divided by seven to obtain the average alcohol intake per day .
subjects were asked to estimate their alcohol intake based on gou , a traditional japanese drinking unit corresponding to 23 g of ethanol .
one gou is equivalent to two usa and uk drink units , or 180 ml of sake , and its ethanol content is roughly equivalent to that of a bottle of beer ( 663 ml ) , two single shots of whiskey ( 70 ml ) , a half glass of shochu ( 110 ml ) or 240 ml of wine .
drinkers were defined as those consuming more than 0.3 gou ( 0.6 drinks ) per week ( 1 g of ethanol a day ) .
we defined igt including dm in the present study according to previous studies as follows : fasting blood sugar level 110 mg / dl , or if < 8 h after meals 140 mg / dl , or on medication for diabetes mellitus , or participants diagnosed as having dm . we defined three other metabolic risk factors in the present study as follows : ( i ) high bp
systolic blood pressure ( sbp ) 130 mmhg , or diastolic blood pressure ( dbp ) 85 mmhg , or the use of an antihypertensive drug ; ( ii ) dyslipidemia either serum highdensity lipoproteincholesterol ( hdl ) concentration < 40 mg / dl , or serum triglycerides ( tg ) concentration 150 mg / dl , or on medication for dyslipidemia ; and ( iii ) obesity bmi 25 kg / m .
high bp , instead of usually defined hypertension ( sbp 140 mmhg , or dbp 90 mmhg , or the use of an antihypertensive drug ) was chosen as one of the metabolic risk factors based on the previous studies that showed high normal bp was associated with an increased risk of cardiovascular disease .
the chi squaredtest for nominal variables and oneway anova for continuous variables were carried out to assess whether there were significant differences among the groups stratified by sleep duration . in the logistic regression
, we included sex , age , sleep duration ( < 6 , 6 to < 7 , 7 to < 8 and 8 h ; taking 6 to < 7 h as a reference ) and interaction term ( bodyweight sleep duration ) .
in model 2 , in addition to model 1 covariates , we included ipaq classification , smoking ( non , past or current smoker ;
nonsmoker served as a reference ) , alcohol intake ( ml / day ) and interaction terms ( ipaq male , current smoking male , alcohol intake male ) as covariates . in model 3 , in addition to model 2 covariates and interaction terms , we included high bp , dyslipidemia and obesity as covariates . in model 4 ,
sleep duration of model 3 covariates was replaced by difficulty in sleep initiation ( often , sometimes , seldom ; taking seldom as a reference ) . in model 5 , difficulty in sleep initiation of model 4 covariates was replaced by difficulty in sleep maintenance ( often , sometimes , seldom ; taking seldom as a reference ) .
model 6 included the covariates and interaction terms in model 4 plus sleep duration ( < 6 , 6 to < 7 , 7 to < 8 and 8 h ; taking 6 to < 7 h as a reference ) and interaction term ( difficulty in sleep initiation sleep duration ) .
we also made a sensitivity analysis in model 3 using a different classification of sleep duration as < 6 , 68 , 89 and > 9 h , taking 68 h as a reference .
all statistical tests were twosided and p < 0.05 was considered statistically significant .
all analyses were carried out using sas version 9.1 for windows ( sas institute , cary , nc , usa ) .
we analyzed baseline data from the highrisk and population strategy for occupational health promotion ( hipopohp ) study . in brief , hipopohp was an interventional survey to establish a methodology for reducing cardiovascular disease ( cvd ) risk factors in the workplace .
researchers followed the data of cvd risk factors , lifestyle habits and consciousness about health based on nutrition , physical activity and smoking for 4 years .
this study was carried out as part of the management of safety and health with the approval of the safety hygiene committee at each company .
accordingly , all employees were enrolled in this study . however , participation was voluntary , and we explained there was no need for participants to answer the required questionnaire if they did not want to .
approval for the study was obtained from the institutional review board of shiga university of medical science for ethical issues ( no .
the present study examined baseline data from 4143 participants ( 3415 men and 728 women ) aged 1969 years ( mean sd , 41.9 9.4 years ) who underwent physical examination , a lifestyle survey and blood chemical examination .
physical and laboratory data were standardized according to the manual of the hipopohp research group . briefly , after 5 min of silent rest measured by a sandglass , blood pressure ( bp ) was measured twice for each participant using an automatic sphygmomanometer ( bp103iii ; omron colin , tokyo , japan ) at each company , and the mean value was recorded . to measure the lipid levels in each participant ,
the company established a contract with a clinical laboratory ; the blood testing was standardized through the us cholesterol reference method laboratory network ( crmln ) .
we calculated lowdensity lipoprotein cholesterol by friedewald s formula when triglyceride did not exceed 300 mg / dl .
the bmi was calculated as weight ( kg ) divided by height squared ( m ) .
sleep duration was investigated using the question , how many hours of sleep do you get on an average weekday per night? in the lifestyle survey
. sleep duration of participants was categorized into four groups : < 6 , 6 to < 7 , 7 to < 8 and 8 h with reference to a precedent study .
sleep quality was investigated using the question how often do you experience difficulty initiating sleep ?
seldom. participants were asked about the type of , and time spent on , physical activities in their spare time for recreation , exercise or sport in the previous month .
physical activity of each participant was converted into metminutes per week (= met level minutes of activity / day days per week ) according to the international physical activity questionnaire ( ipaq ) , and participants were classified into four classes of physical activity as high ( 3000 metminutes per week ) , moderate activity ( < 3000 but 1500 metminutes per week ) , some activity ( < 1500 but 600 metminutes per week ) or sedentary ( < 600 metminutes per week ) .
the frequency of alcohol intake during a typical week and the total alcohol intake on each occasion were determined and used to calculate the alcohol intake per week .
this value was then divided by seven to obtain the average alcohol intake per day .
subjects were asked to estimate their alcohol intake based on gou , a traditional japanese drinking unit corresponding to 23 g of ethanol .
one gou is equivalent to two usa and uk drink units , or 180 ml of sake , and its ethanol content is roughly equivalent to that of a bottle of beer ( 663 ml ) , two single shots of whiskey ( 70 ml ) , a half glass of shochu ( 110 ml ) or 240 ml of wine .
drinkers were defined as those consuming more than 0.3 gou ( 0.6 drinks ) per week ( 1 g of ethanol a day ) .
we defined igt including dm in the present study according to previous studies as follows : fasting blood sugar level 110 mg / dl , or if < 8 h after meals 140 mg / dl , or on medication for diabetes mellitus , or participants diagnosed as having dm . we defined three other metabolic risk factors in the present study as follows : ( i ) high bp
systolic blood pressure ( sbp ) 130 mmhg , or diastolic blood pressure ( dbp ) 85 mmhg , or the use of an antihypertensive drug ; ( ii ) dyslipidemia either serum highdensity lipoproteincholesterol ( hdl ) concentration < 40 mg / dl , or serum triglycerides ( tg ) concentration 150 mg / dl , or on medication for dyslipidemia ; and ( iii ) obesity bmi 25 kg / m .
high bp , instead of usually defined hypertension ( sbp 140 mmhg , or dbp 90 mmhg , or the use of an antihypertensive drug ) was chosen as one of the metabolic risk factors based on the previous studies that showed high normal bp was associated with an increased risk of cardiovascular disease .
the chi squaredtest for nominal variables and oneway anova for continuous variables were carried out to assess whether there were significant differences among the groups stratified by sleep duration . in the logistic regression
in model 1 , we included sex , age , sleep duration ( < 6 , 6 to < 7 , 7 to < 8 and 8 h ; taking 6 to < 7 h as a reference ) and interaction term ( bodyweight sleep duration ) . in model 2 , in addition to model
1 covariates , we included ipaq classification , smoking ( non , past or current smoker ; nonsmoker served as a reference ) , alcohol intake ( ml / day ) and interaction terms ( ipaq male , current smoking male , alcohol intake male ) as covariates . in model 3 , in addition to model 2 covariates and interaction terms , we included high bp , dyslipidemia and obesity as covariates . in model 4 ,
sleep duration of model 3 covariates was replaced by difficulty in sleep initiation ( often , sometimes , seldom ; taking seldom as a reference ) . in model 5 ,
difficulty in sleep initiation of model 4 covariates was replaced by difficulty in sleep maintenance ( often , sometimes , seldom ; taking seldom as a reference ) .
model 6 included the covariates and interaction terms in model 4 plus sleep duration ( < 6 , 6 to < 7 , 7 to < 8 and 8 h ; taking 6 to < 7 h as a reference ) and interaction term ( difficulty in sleep initiation sleep duration ) .
we also made a sensitivity analysis in model 3 using a different classification of sleep duration as < 6 , 68 , 89 and > 9 h , taking 68 h as a reference .
all statistical tests were twosided and p < 0.05 was considered statistically significant .
all analyses were carried out using sas version 9.1 for windows ( sas institute , cary , nc , usa ) .
among the 4143 participants ( 3415 men and 728 women ) , those with and without igt / dm accounted for 402 and 3741 , respectively .
there were 642 people ( 11.5% ) in the group of sleep duration < 6 h , 1690 in the group of sleep duration 6 to < 7 h ( 40.8% ) , 1415 in the group of sleep duration 7 to < 8 h ( 34.2% ) and 396 in the group of sleep duration 8 h ( 9.6% ) .
characteristics of participants by group according to sleep duration are shown in tables 1 and 2 .
mean age , sbp , dbp , alcohol intake and percentage of current smokers were higher among the groups with a longer sleep duration .
mean bmi and percentage of obesity were lower among the groups with a longer sleep duration .
there were no differences in mean blood glucose , prevalence of igt / dm , high bp and dyslipidemia among the groups ( table 1 ) .
chi squaredtest for nominal variables and oneway anova for continuous variables were used to examine the difference between the four groups according to sleep duration .
we defined impaired glucose tolerance ( igt)/diabetes mellitus ( dm ) in this study as follows : fasting blood sugar concentration 110 mg / dl , or if < 8 h after meals 140 mg / dl ) , or on medication for diabetes mellitus , or participants diagnosed as having dm .
we defined three other metabolic factors as high blood pressure ( bp ) : systolic blood pressure ( sbp ) 130 mmhg , or diastolic blood pressure ( dbp ) 85 mmhg , or the use of an antihypertensive drug ; dyslipidemia : highdensity lipoprotein < 40 mg / dl , or triglycerides concentration 150 mg / dl , or on medication for dyslipidemia ; obesity : defined as body mass index ( bmi ) 25
ipaq , international physical activity questionnaire classification ; sleep duration , an average of sleeping hours per night on a weekday .
chi squaredtest for nominal variables and oneway anova for continuous variables were used to examine the difference between four groups according to sleep duration .
we defined impaired glucose tolerance ( igt)/diabetes mellitus ( dm ) in this study as follows : fasting blood sugar concentration 110 mg / dl , or if < 8 h after meals 140 mg / dl ) , or on medication for dm , or participants diagnosed as having dm .
we defined three other metabolic factors as high blood pressure : systolic blood pressure 130 mmhg , or diastolic blood pressure 85 mmhg , or the use of an antihypertensive drug ; dyslipidemia : highdensity lipoprotein < 40 mg / dl , or triglycerides ( tg ) concentration 150 mg / dl , or on medication for dyslipidemia ; obesity : defined as body mass index 25 kg / m .
hdlc , serum highdensity lipoprotein cholesterol concentration ; ldlc , serum lowdensity lipoprotein cholesterol concentration tch , serum total cholesterol concentration .
there were fewer participants who had difficulty in sleep initiation or difficulty in sleep maintenance among the groups with a longer sleep duration .
mean glucose , total cholesterol and hdl cholesterol concentrations were not different among the groups .
although mean triglyceride and lowdensity lipoprotein cholesterol concentrations were different among the groups , there were no obvious trends ( table 2 ) .
results of logistic regression analysis on associations between igt / dm and sleep duration and quality adjusted for other lifestyle habits are shown in table 3 . in model 1 ,
independent factors that were associated significantly with igt / dm were age ( or 1.09 , 95% confidence interval [ ci ] 1.081.11 , p < 0.001 ) , being male ( or 2.60 , ci 1.703.99 , p < 0.001 ) , < 6 h sleep ( or 3.97 , ci 2.606.07 , p
< 0.001 ) , 7 to < 8 h sleep ( or 0.37 , ci 0.250.55 , p < 0.001 ) , and 8 h sleep ( or 0.12 , ci 0.060.24 , p < 0.001 ) in comparison with 67 h duration . in model 2 ,
factors that were significantly associated with igt / dm were age ( or 1.09 , ci 1.071.11 , p < 0.001 ) and < 6 h ( or 4.07 , ci 2.636.31 , p < 0.001 ) , 7 to < 8 h sleep ( or 0.38 , ci 0.260.57 , p < 0.001 ) and 8 h sleep ( or 0.12 , ci 0.060.25 , p < 0.001 ) of sleep duration . in model 3 , factors that were significantly associated with igt / dm
were age ( or 1.08 , ci 1.061.09 , p < 0.001 ) , high bp ( or 1.99 , ci 1.572.52 , p < 0.001 ) , obesity ( or 1.36 , ci 1.001.83 , p = 0.049 ) , < 6 h sleep ( or 2.46 , ci 1.494.06 , p < 0.001 ) and 8 h sleep ( or 0.33 , ci 0.130.79 , p = 0.013 ) . contribution of ipaq ( or 1.57 , ci 0.564.38 , p = 0.389 ) , alcohol intake ( or 1.05 , ci 0.971.15 , p = 0.221 ) and current smoking ( or 0.40 , ci 0.121.36 , p = 0.141 ) were not statistically significant .
the interaction terms , current smoking male ( or 3.09 , ci 1.019.42 , p = 0.048 ) and bodyweight sleep duration ( or 1.01 , ci 1.001.01 ,
factors that were significantly associated with igt / dm were age ( or 1.07 , ci 1.061.09 , p
< 0.001 ) , high bp ( or 2.08 , ci 1.642.64 , p < 0.001 ) , obesity ( or 1.77 , ci 1.382.27 , p < 0.001 ) and difficulty in sleep initiation ( or 1.82 , ci 1.242.67 , p = 0.002 , often vs seldom ) . in model 5 , factors that were significantly associated with igt / dm were age ( or 1.07 , ci 1.061.09 , p < 0.001 ) , high bp ( or 2.11 , ci 1.662.67 , p < 0.001 ) and obesity ( or 1.77 , ci 1.382.26 ,
difficulty in sleep maintenance did not contribute significantly ( or 1.27 , ci 0.881.83 , p = 0.211 , often vs seldom ) . in model 6 ,
factors that were significantly associated with igt / dm were age ( or 1.08 , ci 1.071.10 , p < 0.001 ) , high bp ( or 1.94 , ci 1.522.47 , p < 0.001 ) and < 6 h sleep ( or 2.32 , ci 1.184.55 , p = 0.015 ) .
difficulty in sleep initiation did not contribute significantly ( or 1.49 , ci 0.603.70 , p = 0.386 , often vs seldom ) .
the interaction terms , current smoking male ( or = 3.49 , 1.1210.8 , p = 0.031 ) and bodyweight sleep duration ( or 1.01 , ci 1.001.01 , p = 0.004 ) contributed significantly .
results of analysis by logistic regression models on associations between impaired glucose tolerance / diabetes mellitus and lifestyle habits are shown . * * p < 0.01 , * p < 0.05 . model 1 , adjusted for sex , age and sleep duration ( < 6 , 6 to < 7 , 7 to < 8 and 8 h ; taking 6 to < 7 h as a reference ) , and interaction term ( bodyweight sleep duration ) .
model 2 , adjusted for covariates in model 1 plus international physical activity questionnaire ( ipaq ) classification , smoking ( non , past or current smoker ; nonsmoker serves as reference ) , alcohol intake ( ml / day ) and interaction terms ( ipaq male , current smoking male , alcohol intake male , bodyweight sleep duration ) .
model 3 , adjusted for covariates and interaction terms in model 2 plus high blood pressure ( bp ) , dyslipidemia and obesity .
model 4 , adjusted for sex , age , ipaq classification , smoking ( non , past or current smoker ;
nonsmoker serves as reference ) , alcohol intake ( ml / day ) , high bp , dyslipidemia , obesity , difficulty in sleep initiation ( often , sometimes , seldom ; taking seldom as a reference ) and interaction terms ( ipaq male , current smoking male , alcohol intake male , bodyweight sleep duration ) .
model 5 , adjusted for covariates and interaction terms in model 4 covariates , except for difficulty in sleep initiation , which was replaced by difficulty in sleep maintenance ( often , sometimes , seldom ; taking seldom as a reference ) .
model 6 , adjusted for covariates and interaction terms in model 4 plus sleep duration ( < 6 , 6 to < 7 , 7 to < 8 and 8 h ; taking 6 to < 7 h as a reference ) and interaction term ( difficulty in sleep initiation sleep duration ) .
the results of sensitivity analysis in model 3 using a different classification of sleep duration were similar as those using the original classification of sleep duration .
there were 642 people in the group of sleep duration < 6 h , 3105 in the group of sleep duration 6 to < 8 h , 376 in the group of sleep duration 8 to < 9 h and 20 in the group of sleep duration 9
h. sleep duration < 6 h was associated with a higher risk for igt / dm ( or 1.93 , ci 1.243.00 , p
in the present study , we found significant positive associations between short sleep duration ( < 6 h compared with 67 h sleep duration ) and igt / dm .
although 8 h sleep inversely , and difficulty in sleep initiation positively associated with igt / dm , concomitant inclusions of sleep duration and difficulty in sleep initiation covariates in a model resulted in the disappearance of statistical significance of these factors .
several previous studies showed that difficulties in maintaining sleep or short sleep duration were associated with an increased incidence of diabetes . in a study by spiegel et al . , it was suggested that chronic sleep loss , behavioral or sleep disorderrelated , might represent a novel risk factor for weight gain , insulin resistance and type 2 diabetes .
one mechanism by which sleep deprivation might result in increased risk of insulin resistance and diabetes may be either by directly affecting parameters of glucose tolerance or indirectly through a disturbance in appetite regulation , leading to increased food intake and weight gain .
with regard to short sleep duration and metabolic disorders or weight gain , some have argued that in an environment where food is readily available , sleep deprivation might simply represent an increased opportunity to eat , especially if most wake time is spent in sedentary activities , such as watching television .
however , in the epidemiological literature , those studies that attempted to quantify caloric intake found no relationship between sleep duration and dietary consumption .
chronic partial sleep deprivation also clearly leads to feelings of fatigue , and this tiredness might lead to reductions in physical activity . in some previous studies , long sleep duration was associated with increased type 2 diabetes risk .
previous crosssectional studies showed that short and long sleep duration were associated with an increased risk of developing igt or dm , independent of confounding factors .
studied a longitudinal sample of 276 individuals aged 2164 years with follow up for a mean of 6 years , and found that short and long sleeping times were associated with a higher risk of developing type 2 dm / igt .
however , in a study by mallon et al . , long sleep duration was not associated with risk of dm , whereas short sleep duration was significantly associated with it .
although the mechanisms of short sleep duration related to an increased risk of dm are relatively well understood as aforementioned , the mechanisms for long sleep duration for an increased risk of dm are not clear .
it was speculated that in unrecognized conditions , such as sleep apnea syndrome , an increased need for sleep and risk of dm might coexist .
it is also speculated that long sleep duration might be an early symptom of dm . however , none of the aforementioned postulated mechanisms for an association of long sleep duration with dm have been well proven .
the strengths of the present study include being populationbased , largescale and multisite with highly standardized methods . because the study included men and women of a broad range of ages , the findings are likely to be generalizable to middleaged japanese . however , the present study has some limitations .
thus , the causal implications of short sleep duration for igt / dm should be taken cautiously .
second , we did not measure waist circumference ( wc ) . although the measurement of wc is widely advocated as a simple anthropometric marker of health risk , we showed in a population based study that bmi and wc correlated very well in men and women , and that bmi could be used instead of wc in a study when the latter was not available .
fourth , we used nonfasting blood samples and thus we might have misclassified participants with impaired glucose tolerance . however , previous studies by the present authors and others showed that igt / dm identified by the same criteria as in the present study in nonfasting participants was an independent cardiovascular risk factor . in conclusion , the present results showed that a shorter sleep duration ( < 6 h of sleep per night ) was associated with risk of igt / dm independent of other lifestyle habits and metabolic risk factors . | abstractaims / introduction : to examine the crosssectional relationship between sleep duration and impaired glucose tolerance ( igt ) , including diabetes mellitus ( dm ) , we analyzed a largescale healthy workers database in japan.materials and methods : we examined the baseline database of 4143 participants ( 3415 men and 728 women ) aged 1969 years .
sleep duration of participants was categorized into four groups : < 6 , 6 to < 7 , 7 to < 8 and 8 h. the physical activity of each participant was classified according to the international physical activity questionnaire ( ipaq ) .
we defined igt including dm ( igt / dm ) in the present study according to previous studies as follows : fasting blood sugar level 110 mg / dl , or if < 8 h after meals 140 mg / dl , or on medication for diabetes mellitus , or those diagnosed as having dm .
logistic regression was applied to estimate the odds ratio ( or ) to examine the relationship between igt / dm , sleep duration and other related factors.results : the number of participants with igt / dm was 402 ( 9.7% ) .
the factors that significantly associated with igt / dm were age ( or 1.08 , 95% confidence interval [ ci ] 1.071.10 , p < 0.001 ) , high blood pressure ( or 1.94 , ci 1.522.47 , p
< 0.001 ) , and < 6 h of sleep duration in comparison with 6 to < 7 h sleep ( or 2.32 , ci 1.184.55 , p = 0.015 ) .
the associations of difficulty in sleep initiation , ipaq classification , current smoking and alcohol intake with igt / dm were not statistically significant.conclusions : our results showed that shorter sleep duration ( < 6 h of sleep duration per night ) was associated with a risk of igt / dm independent of other lifestyle habits and metabolic risk factors .
( j diabetes invest , doi : 10.1111/j.20401124.2011.00114.x , 2011 ) |
the online version of this article ( doi:10.1007/s12471 - 014 - 0624-y ) contains supplementary material , which is available to authorized users .
pulmonary vein isolation ( pvi ) has become an important treatment modality for atrial fibrillation ( af ) [ 1 , 2 ] although af recurrences can occur .
several pvi techniques have been developed [ 4 , 5 ] in an attempt to increase af - free survival , among which the endoscopic laser balloon ablation system ( eas ) [ 6 , 7 ] .
the eas consists of a flexible , compliant balloon for sustained wall contact and an adjustable laser beam for ablation independent of tissue contact .
the present study aims to report the procedural characteristics and af - free survival after eas pvi of the first 50 patients treated with the eas in the netherlands .
fifty consecutive patients who underwent a primo pvi using the eas in our centre between december 2011 and december 2013 were included in a prospective registry .
the prospective registry has been approved by the institutional review board and all patients consented to their data being registered .
all patients underwent transoesophageal echocardiography to rule out left atrial ( la ) thrombus prior to the procedure .
bridged with low - molecular - weight heparin until the day of ablation , in accordance with local guidelines .
the eas ( cardiofocus , marlborough , ma , usa ) is a balloon - based catheter system .
the eas was manoeuvred to each pulmonary vein ( pv ) ostium under fluoroscopic guidance ( fig . 1 ) .
a ring of atrial myocardium antral to the pv was exposed by varying the eas balloon inflation size .
laser energy was delivered to the exposed ring of atrial tissue . after a full circle
a circular mapping catheter was introduced to assess persistent electrical connection between the pv and the left atrium .
if any existed , the eas was re - introduced to the pv , and additional lesions were applied by the operator .
no adenosine testing was performed . an oesophageal temperature probe ( sensitherm , st jude medical , usa )
was inserted , and energy delivery was instantaneously terminated when the temperature exceeded 39.0 c . during ablation of the right - sided pvs , stimulation of the phrenic nerve ( using 20 ma at 2.9 ms )
was performed , with immediate cessation of energy delivery once capture was diminished or lost.fig .
eas : endoscopic laser balloon ablation system ; pvi : pulmonary vein isolation ; pv : pulmonary vein laser balloon ablation .
eas : endoscopic laser balloon ablation system ; pvi : pulmonary vein isolation ; pv : pulmonary vein
patients visited the outpatient clinic at 3 , 6 , 12 , 18 and 24 months after pvi , including 24-h holter ecg .
secondary endpoints were : acute pvi , procedure time , ablation time and fluoroscopy time .
the safety endpoint was major or minor complications within 30 days of the procedure as described in european guidelines .
continuous variables were expressed as mean with standard deviation in case of normal distribution or median with interquartile range when not normally distributed .
statistical analysis was performed using ibm spss statistics version 20 ( ibm inc . , armonk , ny , usa ) .
mean age was 56 years , 82 % had paroxysmal af . no la thrombi were found during the preoperative transoesophageal echocardiogram .
there were 2 left - sided and 1 right - sided common pvs and there were 2 right - sided accessory pvs.table 1baseline characteristicspatient characteristictotal ( n = 50)gender female ( % ) 28 % age ( years)55.9 ( 10.7)bmi ( kg / m)26.9 ( 3.6)persistent af18 % af duration ( years)7.0 ( 6.5)failed aads ( range)1.4 ( 04)la ventral - dorsal dimension ( mm)41.1 ( 3.9)lvef ( % ) 58.8 ( 3.2)history of hypertension30 % history of diabetes mellitus6 % data are presented as percentages or means their sd or ranges where appropriate ; bmi body mass index ; af atrial fibrillation ; aads antiarrhythmic drugs ; la left atrial ; lvef left ventricular ejection fraction baseline characteristics data are presented as percentages or means their sd or ranges where appropriate ; bmi body mass index ; af atrial fibrillation ; aads antiarrhythmic drugs ; la left atrial ; lvef left ventricular ejection fraction
in 198 out of 199 pvs ( 99.5 % ) , acute pvi was achieved .
one common left - sided pv could not be isolated due to a temperature rise of the oesophagus .
one procedure was converted to radiofrequency catheter ablation after ablation of the right upper pv was complicated by a phrenic nerve palsy , which did not prolong hospital stay and had fully recovered after 6 months .
table 2 displays the procedural characteristics of all patients.table 2procedural characteristicsprocedure time ( min)170 ( 40)ablation time ( min)61 ( 28)fluoroscopy time ( min)36 ( 10)data are presented as mean their sd procedural characteristics data are presented as mean their sd
after a median follow - up of 17.3 ( interquartile range : 12.919.5 ) months , 58 % of patients were free of af after a single eas pvi without the use of antiarrhythmic drugs .
the present study reports the results of the first 50 patients treated with the eas in the netherlands .
moreover , medium - term af - free survival seems to be comparable with other pvi techniques .
the eas combines a compliant balloon design , an endoscope for visualisation of pv antral tissue and a power - adjustable laser beam .
previous reports have shown that acute pvi can be achieved virtually always with the eas , which was also observed in the present study [ 8 , 9 ] . in the present study ,
the complication rate is low ( 2 % ) , which is consistent with previous reports [ 7 , 9 ] .
although pvi is an important treatment modality for af , the medium - term af - free survival is still 6080 % , after pvi using different techniques , such as radiofrequency catheter ablation and cryoballoon ablation . in the present study ,
af - free survival after a single eas pvi attempt was 58 % , which is in line with a previous report , although there are other eas studies reporting a higher af - free survival [ 10 , 11 ] .
potentially , a learning curve may , in part , have affected the af - free survival in the current study .
a randomised trial will provide further evidence of the af - free survival after eas . based on the current literature
, the af - free survival rate after eas pvi seems to be comparable with other techniques .
af recurrences are generally regarded as recurrence of electrical conduction over the pv - la junction [ 13 , 14 ] .
durable lesion sets therefore remain pivotal in improving medium - term af - free survival after pvi .
the medium - term success percentage suggests the lesion sets created with the eas are not persistent .
this was also suggested in another study , which reported that 62 % of the studies patients had 4 isolated pvs 3 months after the initial eas pvi procedure .
future studies should be aimed at identifying factors that are associated with af - free survival after eas pvi .
although the eas consists of a compliant balloon , the catheter - tissue contact may be suboptimal in some patients , limiting the operator s ability to deliver circular , transmural lesion sets which may influence af - free survival .
moreover , in case of insufficient occlusion or ablation near to a blood pool , ablation energy had to be reduced .
one study showed high - energy eas ablation was favourable to low - energy eas ablation in terms of persistent electrical conduction over the pv - la junction and af - free survival after pvi .
although highly speculative , these factors may explain the medium - term af - free survival in the present study .
the eas is a promising technique with a high acute pvi success rate and a low risk of complications .
medium - term af - free survival after eas pvi is comparable with other pvi techniques .
| introductionthe endoscopic laser balloon ablation system ( eas ) is a relatively novel technique to perform pulmonary vein isolation ( pvi ) in the treatment of atrial fibrillation ( af ) .
the present study aimed to report the results of the first 50 patients treated in the netherlands with the eas in terms of procedural characteristics and af - free survival.methodsfifty patients successfully underwent eas pvi .
median follow - up was 17 months .
mean age was 56 years , 82 % had paroxysmal af.results99 % of the pulmonary veins were successfully isolated with the eas .
mean procedure time was 171 min and mean fluoroscopy time was 36 min .
one procedure was complicated by a temporary phrenic nerve palsy ( 2 % ) . during follow - up , 58 % of patients remained free of af without the use of antiarrhythmic drugs.conclusionpvi with eas is associated with a low risk of complications and a medium - term af - free survival comparable with other pvi techniques.electronic supplementary materialthe online version of this article ( doi:10.1007/s12471 - 014 - 0624-y ) contains supplementary material , which is available to authorized users . |
the archetypal animal mitochondrial dna ( mtdna ) is a 15- to 20-kb circular molecule ( boore 1999 ) .
medusozoan cnidarians ( jellyfish and hydroids ) are among the few metazoans whose mtdnas depart significantly from this traditional architecture .
staurozoans , scyphozoans , and most hydrozoans have linear monomeric mitochondrial genomes with lengths of 16 kb ; certain hydra species have mtdnas that are segmented into two 8-kb linear chromosomes ; and all investigated members of the cubozoa ( box jellyfish ) have mitochondrial genomes that are segmented into several linear molecules of 4 kb ( warrior and gall 1985 ; bridge et al .
1992 ; ender and schierwater 2003 ; kayal and lavrov 2008 ; voigt et al .
although nearly complete mtdna data are available from a diversity of medusozoans ( kayal et al .
2011 ) , whole mitochondrial genome sequences , including telomeres , exist for just three : the scyphozoan aurelia aurita and the hydrozoans hydra oligactis and h. magnipapillata ( shao et al . 2006 ; kayal and lavrov 2008 ; voigt et al .
the telomeres from each of these mitochondrial genomes form an inverted repeat ( ir ) , and in h. oligactis and h. magnipapillata , duplicate genes and pseudogenes are found in the subtelomeric regions .
medusozoans , therefore , provide a unique opportunity for understanding the evolution of linear chromosomes , telomeres , and mtdna structural stability . to address all three of these topics , we present the complete mitochondrial genome sequence from the winged box jellyfish alatina moseri ( the first from a cubozoan ) .
this genome has a highly fragmented linear structure and inflated levels of silent - site genetic diversity ; moreover , subtelomeric recombination has changed the actual proteins encoded in the genome , causing genes to initiate or terminate with short regions of subtelomeric genes from other chromosomes .
the a. moseri mitochondrial genome contains 18 unique genes distributed over eight linear - mapping chromosomes , which range from 2.94.6 kb .
the architectural and compositional features of this genome and how they compare with the mtdnas from other medusozoans are shown in figure 1a and b , respectively .
alatina moseri has among the most fragmented mitochondrial genomes observed from animals ( for other examples , see suga et al .
the complete mitochondrial genome assembly for a. moseri supports earlier analyses of this taxon and six other cubozoan species ( carukia barnesi , carybdea marsupialis , carybdea xaymacana , chironex fleckeri , chiropsalmus quadrumanus , and tripedalia cystophora ) , which were indicative of highly subdivided linear mtdna architectures ( ender and schierwater 2003 ; kayal et al .
it also reinforces the hypothesis that within cnidaria , the shift from a single linear mtdna to a fragmented one occurred at least twice : in the cubozoa and within the hydrozoa ( fig .
2011 ) . outside of medusozoans , linear fragmented mtdnas have been observed in various protists , including green algae ( borza et al .
2010 ) , fungi ( valach et al . 2011 ) , and the ichthyosporean amoebidium parasiticum ( burger et al . 2003)one of the closest unicellular relatives of animals ( lang et al .
synteny between the mtdna of alatina moseri and that of aurelia aurita is marked with horizontal black lines , which have labels corresponding to a. moseri chromosome numbers . see figure 2 for details on the a. moseri mtdna pseudogenes .
note : cnidarian mtdna protein - coding genes are translated using the minimally derived genetic code ( uag = tryptophan ) .
the mtdna gene complement of a. moseri closely parallels those from other medusozoans ( fig .
it includes 16 standard mitochondrial genes , representing 13 proteins , 2 ribosomal rnas ( rrnas ) , and 1 transfer rna ( trna ) ( for secondary structure diagrams of the functional rnas , see supplementary fig .
s1 , supplementary material online ) , and two nonstandard ones : polb , which codes for a putative beta dna polymerase , and orf314 , which may have dna - binding properties ( kayal et al .
polb and orf314 are found in other medusozoan mtdnas , except those of hydroidolinan hydrozoans , and are believed to have originated from a mitochondrial plasmid ( shao et al .
both of these genes are located on chromosome 7 , where no other full - length gene is encoded , which is interesting when considering that they are thought to have arisen from a selfish element .
only one type of trna is encoded in the a. moseri mitochondrial genome : trnmcau .
it is present in three identical copies , each located on a different chromosome , and has characteristics that suggest a role in initiation rather than elongation .
although all cnidarians have reduced mitochondrial trna - coding suites , a. moseri is the first medusozoan known to lack an mtdna - encoded trnwuca . in the mtdnas of octocorals ,
the absence of a mitochondrial trnwuca is associated with a complete lack of uga codons . in a. moseri , however , half of the tryptophan residues within protein - coding mtdna are represented by uga codons , implying that either a nucleus - encoded mitochondrial - targeted trnwuca or nonstandard wobble properties of an imported trnwugg is compensating for the lost mitochondrial version of the gene .
a 20-nt sequence that can be folded into a stem - loop structure was found in the telomeric regions immediately adjacent to the coding dna ( supplementary fig .
s2 , supplementary material online ) ; putative stem - loop structures have been identified in the noncoding regions of other medusozoan mtdnas and were proposed to be potential control regions ( kayal et al .
the 16 telomeres of the eight a. moseri mitochondrial chromosomes are identical in sequence and found in opposing orientations so they make up an ir ( fig .
telomeres arranged as irs are a hallmark of linear organelle genomes ( nosek et al .
2004 ) and are found in the three other medusozoan mtdnas for which telomeric data are available ( fig .
however , the subtelomeric regions of the a. moseri mtdna also contain substantial amounts of other repeated sequence ; in this case , fragments of genes found on other mitochondrial chromosomes ( fig .
2 ) . for example , based on our annotations , the beginning of the rnl gene is found within the 5 ends and adjoining intergenic regions of six different protein - coding genes , meaning that the first 15150 nt of these loci are identical and the amino termini of these proteins are presumably translated from a rrna ( for rnl secondary structure diagram , see supplementary fig .
s1 , supplementary material online ) , a statement supported by both the conserved amino acid sequence of these genes and the rnl secondary structure model ( supplementary fig .
s1 , supplementary material online ) , but protein sequencing data will be needed to confirm this .
similarly , the 3 end of cox3 is identical in sequence , but in a different reading frame , to the 3 end of cob , and this same cox3/cob segment and an additional portion of cob are found in the intergenic dna beside cox1 ( fig .
, the 3 end of nad4 contains tracts that show sequence identity to nad1 , trnm , and rnl .
fragmented and sometimes duplicate genes are present in the subtelomeric mtdna of h. oligactis and h. magnipapillata ( fig .
1b ) but to a much lesser extent than in a. moseri ; and a cursory scan of other linear dnas reveals that this is a reoccurring , but poorly understood , feature of organelle telomeres , including those of nucleomorph dnas and mitochondrial plasmids ( table 1 ) ( burger et al .
examples of linear mitochondrial genomes with fragmented or duplicate genes are their termini alignment and architecture of the alatina moseri mitochondrial telomeric and subtelomeric regions .
the alatina moseri mitochondrial genome is divided into eight chromosomes ( chr ) , each of which has two telomeres : left and right ( based on genetic map in fig .
the telomeres ( turquoise ; length = 750 nt ; not to scale ) are identical in sequence and found in opposing orientations so that they make up an ir .
the subtelomeric regions are composed of coding ( dark blue ) and noncoding ( black ) regions and contain fragments of genes ( red ) from neighboring chromosomes .
gray highlighting denotes sequence identity between different subtelomeric regions ; when the sequence identity includes coding dna , these regions are striped .
the inverted architecture of the a. moseri mitochondrial telomeres and partial duplication of adjacent coding and noncoding regions ( fig .
2 ) resemble the expansion of the large ir region of plastid genomes ( plunkett and downie 2000 ) .
one proposed mechanism for plastid genome ir expansion is based on gene conversion ( goulding et al .
, recombination between two irs allows for the branch migration of a holliday junction across the ir boarder into nonidentical sequence ; the branch migration stalls but not before a short stretch of heteroduplex dna is formed .
resolution of the heteroduplex occurs by sequence correction against either of the two strands and can lead to an expansion ( or reduction ) of the ir ( goulding et al .
similarly , in telomerase - lacking cells of the yeast kluyveromyces lactis , it was shown that a nuclear dna marker present at only one telomere can spread to all other nuclear telomeres in the cell via subtelomeric gene conversion ( mceachern and iyer 2001 ) ; it was also shown that these same cells can maintain the lengths of their telomeres through recombination ( mceachern and blackburn 1996 ) , which is one of the primary strategies for the lengthening of telomeres in telomerase - independent systems , including mitochondria ( nosek et al . 2004 ; tomaska and nosek 2009 ) . when the a. moseri telomeres and their abutting loci are aligned ( fig .
2 ) , it is clear how gene conversion could have generated the subtelomeric regions .
subtelomeric gene conversion can also explain why , as a whole , linear dnas with terminal irs tend to have duplicate genes or gene fragments at their ends ( table 1 ) .
moreover , in most systems , gene conversion is biased toward gc ( marais 2003 ) , which may help explain why in a. moseri the sequence that appears to be most frequently involved in conversion events ( rnl ) is also among the more gc - rich genes in the mitochondrial genome .
overall , in the subtelomeric mtdna of a. moseri , a recombination - based mechanism of telomere maintenance appears to have been taken to an extreme so that fragments of genes introduced to a chromosome end by recombination have actually become embedded in the coding regions of preexisting genes .
we measured the mitochondrial genetic diversity between three different isolates of a. moseri : two from hawaii ( collected in the same location and on the same evening ) and one from australia .
their mtdna sequences , however , differed substantially , especially at silent sites ( table 2 ) .
the average number of nucleotide differences per site ( ) between the mtdnas from the two hawaiian isolates was 2.5% , rising to 5% at telomeric positions and 10% at synonymous sites ( synonymous varied from 6.5% ( atp8 ) to 15.5% ( nad6 ) among protein - coding loci ) .
similar amounts of silent - site variation were observed when the almost complete mtdna sequence of the australian specimen was added to the analysis ( synonymous = 7.9% ; telomere = 4.4% ) , meaning that on average the two hawaiian mtdnas were more different from each other than they were from that of the australian isolate . mean diversity at the more functionally constrained sites ( i.e. , amino acid replacement and rrna - coding positions ) was between 0.3% and 1.8% for both the hawaiian and combined data sets
note. n , number of nucleotide sites ( comprises all sites in the nucleotide alignment , including those with indels ) ; s , number of polymorphic ( i.e. , segregating ) sites ; , average number of pairwise nucleotide differences per site ( calculated with dnasp v5.10.01 ; librado and rozas 2009 ) ; and indels : insertion deletion events ( consecutive indels were counted as a single event ) .
two specimens of a. moseri were collected from waikiki , oahu , hi ( i.e. , hawaiian isolates ) and one from osprey reef in the coral sea , queensland , australia .
animal mitochondrial genomes are renowned for having high levels of within - species silent - site diversity ( largely due to high substitution rates ) ( bazin 2006 ; lynch et al .
2006 ; piganeau and eyre - walker 2009 ) , but the a. moseri mtdna is in the upper extreme of what has been documented , making it among the most genetically diverse eukaryotic genomes observed to date
recombination rates often scale positively with genetic diversity and mutation rates ( eyre - walker 1993 ; nachman 2001 ; hellmann et al . 2003 ) , which is significant because sequence data from the a. moseri mitochondrial telomeres and subtelomeres suggest that these regions are recombinogenic , although the high levels of variation found in other parts of the genome are probably associated with a high mutation rate .
an inflated silent can also result from a large effective genetic population size . in this context , it is noteworthy that alatina , unlike most box jellyfish , can be found at considerable ocean depths ( e.g. , morandini 2003 ) and is thought to only come to shallow water when breeding ( e.g. , arneson and cutress 1976 ; yanagihara et al .
moreover , previous genetic diversity analyses of rnl revealed no geographic structure between hawaiian and australian specimens , suggesting that they constitute a single large population ( bentlage et al .
other medusozoans harbor moderate amounts of intra- and interspecific mtdna variation ( cunningham and buss 1993 ; dawson and jacobs 2001 ; schroth et al .
anthozoan cnidarians , however , are one of the few animal lineages that contain very little intraspecific mtdna diversity ( shearer et al . 2002 ; hellberg 2006 ) .
unlike medusozoans and most other animals , octocorallian anthozoans have a mitochondrial - encoded msh1 gene ( a homologue of the bacterial dna repair gene muts ) , which may be linked to their low mtdna substitution rates ( van oppen et al .
similarly , in land plants , msh1 is allied with low rates of mtdna evolution ( davila et al .
2011 ) . moreover , msh1 mutants of arabidopsis thaliana have inflated mitochondrial recombination rates as compared with the wild type , and they also show high levels of asymmetrical genetic exchange , resulting from gene conversion ( davila et al .
it has been hypothesized that within the phylum cnidaria , there was an abrupt shift from slow to fast rates of mtdna evolution in the stem of medusozoa ( hellberg 2006 ) , which may be connected to the loss of an msh1 gene in medusozoans : this gene is absent from all available medusozoan mtdnas and a blast scan of 6 giga bases of a. moseri nuclear dna data failed to uncover a nuclear copy of msh1 .
the potential loss of msh1 in a. moseri , and medusozoans as a whole , could explain both the high levels of genetic diversity and subtelomeric gene conversion in this group .
the a. moseri mitochondrial genome contains 18 unique genes distributed over eight linear - mapping chromosomes , which range from 2.94.6 kb .
the architectural and compositional features of this genome and how they compare with the mtdnas from other medusozoans are shown in figure 1a and b , respectively .
alatina moseri has among the most fragmented mitochondrial genomes observed from animals ( for other examples , see suga et al .
the complete mitochondrial genome assembly for a. moseri supports earlier analyses of this taxon and six other cubozoan species ( carukia barnesi , carybdea marsupialis , carybdea xaymacana , chironex fleckeri , chiropsalmus quadrumanus , and tripedalia cystophora ) , which were indicative of highly subdivided linear mtdna architectures ( ender and schierwater 2003 ; kayal et al .
it also reinforces the hypothesis that within cnidaria , the shift from a single linear mtdna to a fragmented one occurred at least twice : in the cubozoa and within the hydrozoa ( fig .
2011 ) . outside of medusozoans , linear fragmented mtdnas have been observed in various protists , including green algae ( borza et al .
2010 ) , fungi ( valach et al . 2011 ) , and the ichthyosporean amoebidium parasiticum ( burger et al .
2003)one of the closest unicellular relatives of animals ( lang et al . 2002 ) .
synteny between the mtdna of alatina moseri and that of aurelia aurita is marked with horizontal black lines , which have labels corresponding to a. moseri chromosome numbers . see figure 2 for details on the a. moseri mtdna pseudogenes .
note : cnidarian mtdna protein - coding genes are translated using the minimally derived genetic code ( uag = tryptophan ) . the mtdna gene complement of a. moseri closely parallels those from other medusozoans ( fig .
it includes 16 standard mitochondrial genes , representing 13 proteins , 2 ribosomal rnas ( rrnas ) , and 1 transfer rna ( trna ) ( for secondary structure diagrams of the functional rnas , see supplementary fig .
s1 , supplementary material online ) , and two nonstandard ones : polb , which codes for a putative beta dna polymerase , and orf314 , which may have dna - binding properties ( kayal et al .
polb and orf314 are found in other medusozoan mtdnas , except those of hydroidolinan hydrozoans , and are believed to have originated from a mitochondrial plasmid ( shao et al .
both of these genes are located on chromosome 7 , where no other full - length gene is encoded , which is interesting when considering that they are thought to have arisen from a selfish element .
only one type of trna is encoded in the a. moseri mitochondrial genome : trnmcau .
it is present in three identical copies , each located on a different chromosome , and has characteristics that suggest a role in initiation rather than elongation .
although all cnidarians have reduced mitochondrial trna - coding suites , a. moseri is the first medusozoan known to lack an mtdna - encoded trnwuca . in the mtdnas of octocorals ,
the absence of a mitochondrial trnwuca is associated with a complete lack of uga codons . in a. moseri , however , half of the tryptophan residues within protein - coding mtdna are represented by uga codons , implying that either a nucleus - encoded mitochondrial - targeted trnwuca or nonstandard wobble properties of an imported trnwugg is compensating for the lost mitochondrial version of the gene .
a 20-nt sequence that can be folded into a stem - loop structure was found in the telomeric regions immediately adjacent to the coding dna ( supplementary fig .
s2 , supplementary material online ) ; putative stem - loop structures have been identified in the noncoding regions of other medusozoan mtdnas and were proposed to be potential control regions ( kayal et al .
the 16 telomeres of the eight a. moseri mitochondrial chromosomes are identical in sequence and found in opposing orientations so they make up an ir ( fig
telomeres arranged as irs are a hallmark of linear organelle genomes ( nosek et al .
2004 ) and are found in the three other medusozoan mtdnas for which telomeric data are available ( fig .
however , the subtelomeric regions of the a. moseri mtdna also contain substantial amounts of other repeated sequence ; in this case , fragments of genes found on other mitochondrial chromosomes ( fig .
2 ) . for example , based on our annotations , the beginning of the rnl gene is found within the 5 ends and adjoining intergenic regions of six different protein - coding genes , meaning that the first 15150 nt of these loci are identical and the amino termini of these proteins are presumably translated from a rrna ( for rnl secondary structure diagram , see supplementary fig .
s1 , supplementary material online ) , a statement supported by both the conserved amino acid sequence of these genes and the rnl secondary structure model ( supplementary fig .
s1 , supplementary material online ) , but protein sequencing data will be needed to confirm this .
similarly , the 3 end of cox3 is identical in sequence , but in a different reading frame , to the 3 end of cob , and this same cox3/cob segment and an additional portion of cob are found in the intergenic dna beside cox1 ( fig .
, the 3 end of nad4 contains tracts that show sequence identity to nad1 , trnm , and rnl .
fragmented and sometimes duplicate genes are present in the subtelomeric mtdna of h. oligactis and h. magnipapillata ( fig .
1b ) but to a much lesser extent than in a. moseri ; and a cursory scan of other linear dnas reveals that this is a reoccurring , but poorly understood , feature of organelle telomeres , including those of nucleomorph dnas and mitochondrial plasmids ( table 1 ) ( burger et al .
examples of linear mitochondrial genomes with fragmented or duplicate genes are their termini alignment and architecture of the alatina moseri mitochondrial telomeric and subtelomeric regions .
the alatina moseri mitochondrial genome is divided into eight chromosomes ( chr ) , each of which has two telomeres : left and right ( based on genetic map in fig .
the telomeres ( turquoise ; length = 750 nt ; not to scale ) are identical in sequence and found in opposing orientations so that they make up an ir .
the subtelomeric regions are composed of coding ( dark blue ) and noncoding ( black ) regions and contain fragments of genes ( red ) from neighboring chromosomes .
gray highlighting denotes sequence identity between different subtelomeric regions ; when the sequence identity includes coding dna , these regions are striped .
the inverted architecture of the a. moseri mitochondrial telomeres and partial duplication of adjacent coding and noncoding regions ( fig .
2 ) resemble the expansion of the large ir region of plastid genomes ( plunkett and downie 2000 ) .
one proposed mechanism for plastid genome ir expansion is based on gene conversion ( goulding et al .
recombination between two irs allows for the branch migration of a holliday junction across the ir boarder into nonidentical sequence ; the branch migration stalls but not before a short stretch of heteroduplex dna is formed .
resolution of the heteroduplex occurs by sequence correction against either of the two strands and can lead to an expansion ( or reduction ) of the ir ( goulding et al .
similarly , in telomerase - lacking cells of the yeast kluyveromyces lactis , it was shown that a nuclear dna marker present at only one telomere can spread to all other nuclear telomeres in the cell via subtelomeric gene conversion ( mceachern and iyer 2001 ) ; it was also shown that these same cells can maintain the lengths of their telomeres through recombination ( mceachern and blackburn 1996 ) , which is one of the primary strategies for the lengthening of telomeres in telomerase - independent systems , including mitochondria ( nosek et al . 2004 ; tomaska and nosek 2009 ) . when the a. moseri telomeres and their abutting loci are aligned ( fig .
2 ) , it is clear how gene conversion could have generated the subtelomeric regions .
subtelomeric gene conversion can also explain why , as a whole , linear dnas with terminal irs tend to have duplicate genes or gene fragments at their ends ( table 1 ) .
moreover , in most systems , gene conversion is biased toward gc ( marais 2003 ) , which may help explain why in a. moseri the sequence that appears to be most frequently involved in conversion events ( rnl ) is also among the more gc - rich genes in the mitochondrial genome .
overall , in the subtelomeric mtdna of a. moseri , a recombination - based mechanism of telomere maintenance appears to have been taken to an extreme so that fragments of genes introduced to a chromosome end by recombination have actually become embedded in the coding regions of preexisting genes .
we measured the mitochondrial genetic diversity between three different isolates of a. moseri : two from hawaii ( collected in the same location and on the same evening ) and one from australia .
their mtdna sequences , however , differed substantially , especially at silent sites ( table 2 ) .
the average number of nucleotide differences per site ( ) between the mtdnas from the two hawaiian isolates was 2.5% , rising to 5% at telomeric positions and 10% at synonymous sites ( synonymous varied from 6.5% ( atp8 ) to 15.5% ( nad6 ) among protein - coding loci ) .
similar amounts of silent - site variation were observed when the almost complete mtdna sequence of the australian specimen was added to the analysis ( synonymous = 7.9% ; telomere = 4.4% ) , meaning that on average the two hawaiian mtdnas were more different from each other than they were from that of the australian isolate .
mean diversity at the more functionally constrained sites ( i.e. , amino acid replacement and rrna - coding positions ) was between 0.3% and 1.8% for both the hawaiian and combined data sets
mtdna diversity between three isolates of alatina moseri note. n , number of nucleotide sites ( comprises all sites in the nucleotide alignment , including those with indels ) ; s , number of polymorphic ( i.e. , segregating ) sites ; , average number of pairwise nucleotide differences per site ( calculated with dnasp v5.10.01 ; librado and rozas 2009 ) ; and indels : insertion deletion events ( consecutive indels were counted as a single event ) .
two specimens of a. moseri were collected from waikiki , oahu , hi ( i.e. , hawaiian isolates ) and one from osprey reef in the coral sea , queensland , australia .
animal mitochondrial genomes are renowned for having high levels of within - species silent - site diversity ( largely due to high substitution rates ) ( bazin 2006 ; lynch et al .
2006 ; piganeau and eyre - walker 2009 ) , but the a. moseri mtdna is in the upper extreme of what has been documented , making it among the most genetically diverse eukaryotic genomes observed to date
recombination rates often scale positively with genetic diversity and mutation rates ( eyre - walker 1993 ; nachman 2001 ; hellmann et al . 2003 ) , which is significant because sequence data from the a. moseri mitochondrial telomeres and subtelomeres suggest that these regions are recombinogenic , although the high levels of variation found in other parts of the genome are probably associated with a high mutation rate .
an inflated silent can also result from a large effective genetic population size . in this context , it is noteworthy that alatina , unlike most box jellyfish , can be found at considerable ocean depths ( e.g. , morandini 2003 ) and is thought to only come to shallow water when breeding ( e.g. , arneson and cutress 1976 ; yanagihara et al .
moreover , previous genetic diversity analyses of rnl revealed no geographic structure between hawaiian and australian specimens , suggesting that they constitute a single large population ( bentlage et al .
other medusozoans harbor moderate amounts of intra- and interspecific mtdna variation ( cunningham and buss 1993 ; dawson and jacobs 2001 ; schroth et al .
anthozoan cnidarians , however , are one of the few animal lineages that contain very little intraspecific mtdna diversity ( shearer et al . 2002 ; hellberg 2006 ) . unlike medusozoans and most other animals ,
octocorallian anthozoans have a mitochondrial - encoded msh1 gene ( a homologue of the bacterial dna repair gene muts ) , which may be linked to their low mtdna substitution rates ( van oppen et al .
moreover , msh1 mutants of arabidopsis thaliana have inflated mitochondrial recombination rates as compared with the wild type , and they also show high levels of asymmetrical genetic exchange , resulting from gene conversion ( davila et al .
it has been hypothesized that within the phylum cnidaria , there was an abrupt shift from slow to fast rates of mtdna evolution in the stem of medusozoa ( hellberg 2006 ) , which may be connected to the loss of an msh1 gene in medusozoans : this gene is absent from all available medusozoan mtdnas and a blast scan of 6 giga bases of a. moseri nuclear dna data failed to uncover a nuclear copy of msh1 .
the potential loss of msh1 in a. moseri , and medusozoans as a whole , could explain both the high levels of genetic diversity and subtelomeric gene conversion in this group .
specimens of a. moseri ( sometimes called carybdea alata ) were collected from waikiki ( oahu , hi ) as previously described ( bentlage et al .
complete mtdna sequences were generated for two different hawaiian specimens : one using roche 454 ( gs flx titanium ) sequencing ( genbank project srx017292 ) and the other with abi solid sequencing ( genbank project srx063443 ) .
reads corresponding to mtdna were mined from the 454 and solid data sets using blastn ( v2.2.25 + ) ( altschul et al .
mined reads were assembled with geneious pro v5.4.6 ( biomatters ltd , auckland , nz ) , and then extended using the raw 454 or solid data with codoncode aligner v3.7.1.1 ( codoncode corporation , dedham , ma ) .
the data from both sequencing platforms independently gave the same assembly results : eight linear - mapping mtdna chromosomes with 25- to 250-fold coverage .
for both the 454 and solid assemblies , there was an abrupt decrease in sequence coverage at the extreme ends of the contigs , which is consistent with these regions representing the ends of the mitochondrial chromosomes a similar conclusion was made with the assembly of the h. magnipapillata mtdna ( voigt et al .
standard polymerase chain reaction and sanger sequencing of dna isolated from a third specimen of a. moseri ( from osprey reef in the coral sea , queensland , australia ) confirmed the sequence and arrangement of the 454 and solid assemblies .
the mtdna diversity between the different specimens of a. moseri was calculated with dnasp v5.10.01 ( librado and rozas 2009 ) .
supplementary figures s1 and s2 are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) . | animal mitochondrial dnas ( mtdnas ) are typically single circular chromosomes , with the exception of those from medusozoan cnidarians ( jellyfish and hydroids ) , which are linear and sometimes fragmented .
most medusozoans have linear monomeric or linear bipartite mitochondrial genomes , but preliminary data have suggested that box jellyfish ( cubozoans ) have mtdnas that consist of many linear chromosomes . here
, we present the complete mtdna sequence from the winged box jellyfish alatina moseri ( the first from a cubozoan ) .
this genome contains unprecedented levels of fragmentation : 18 unique genes distributed over eight 2.9- to 4.6-kb linear chromosomes .
the telomeres are identical within and between chromosomes , and recombination between subtelomeric sequences has led to many genes initiating or terminating with sequences from other genes ( the most extreme case being 150 nt of a ribosomal rna containing the 5 end of nad2 ) , providing evidence for a gene conversion based model of telomere evolution . the silent - site nucleotide variation within
the a. moseri mtdna is among the highest observed from a eukaryotic genome and may be associated with elevated rates of recombination . |
denvdengue virusesdc - signdendritic cell - specific intercellular adhesion molecule-3-grabbing non - integrinhsp70heat shock protein 70hsp90heat shock protein 90ssrna+single - stranded positive - sense rna virusesmrcamore recent common ancestorutruntranslated regioneenvelope proteinmmembrane proteinns1non - structural protein 1adeantibody - dependent enhancement
dendritic cell - specific intercellular adhesion molecule-3-grabbing non - integrin heat shock protein 70 heat shock protein 90 single - stranded positive - sense rna viruses more recent common ancestor non - structural protein 1 antibody - dependent enhancement
dengue officially causes 50100 million infections and 22 000 deaths per year around the world .
recent cartographic approaches have estimated to be around 390 million infections per year becoming the most important arthropod - borne viral disease in more than 100 tropical and subtropical countries where 2.5 billion people live at risk of infection .
the disease is caused by dengue viruses ( denv ) , which are members of the flavivirus genus ( flaviviridae family ) causing asymptomatic , mild ( dengue with or without warning signs ) , or severe disease ( severe dengue ) , sometimes leading to death .
the infection in humans starts when denv reaches cells of the mononuclear phagocyte lineage by interaction with some of the proposed receptors ( dc - sign , heparan sulfate , hsp70 , hsp90 , etc . ) and the viral particle is internalized by receptor - mediated endocytosis .
subsequently , low ph - dependent membrane fusion and uncoating lead to viral rna release , polyprotein translation and processing , viral rna synthesis by the replicase complex , virus assembly and in the endoplasmic reticulum and golgi , maturation in the golgi , and release of progeny viruses .
there are no specific treatments available for dengue and the development of a vaccine has been limited by there factors : first the huge antigenic and genetic diversity of the virus , the lack of cross - protection immunity among denv serotypes and eventually genotypes , and the host immune interactions that have been associated with disease severity .
though the vaccine studies have shown some promising outcome , the overall studies are not encouraging and needs a lot of research . because of the existence of enormous intra - serotype genetic diversity , the possibility of cross - protection after vaccination is questionable .
a more intense denv genotype surveillance must be conducted in those countries where vaccine candidates are planed .
the results from such surveillance should be available in real - time for helping decision makers about potentially different vaccine composition for administration in the different regions , a novel challenge for vaccine developers attempting a worldwide coverage .
denvs are enveloped single - stranded positive - sense rna viruses ( ssrna ) whose genomes encode for a viral rna - dependent rna polymerase lacking proofreading activity that leads to very high substitution rates , rapid divergence , and the existence of at least four serotypes ( denv-1 to -4 ) with high intra - serotype genetic diversity .
differences in phylogeny - based estimations of denv substitution rates depending of prior assumptions ( strict or relaxed molecular clock , changes in the distribution of variable and invariable sites across the phylogeny , full - length genome or gene - based analysis ) have been reported .
the rates for denv-1 range from 4.55 10 to 9.08 10 substitutions per site per year ( subs / site / year ) ; for denv-2 the rates range from 6.07 10 to 9.84 10 subs / site / year ; for denv-3 the nucleotide substitution rates range from 9.01 10 to 10.40 10 subs / site / year , and estimations for denv-4 range from 6.02 10 to 10.63 10 subs / site / year . based on these rates and the coalescent theory ,
the more recent common ancestor ( mrca ) of the 4 denv serotypes could have existed more than 1000 y ago .
denv-4 was probably the first diverging serotype , followed by denv-2 ( around 350 y ago ) and finally denv-1 ( 125 y ago ) and -3 ( 100 y ago ) .
the 4 denv serotypes were first defined by their antigenic properties as members of the denv serocomplex .
however , the accumulation of genetic diversity during the last centuries has led to inter - serotype genetic distances even higher than those observed among different species within the flavivirus genus .
the spread of the denv serotypes around the world has also allowed the accumulation of intra - serotype genetic variation and the emergence of different monophyletic groups ( genotypes ) in the different geographic regions of the world ( fig . 1 ) .
although genetic diversity has accumulated along the whole viral genome in which the structural and non - structural genes and untranslated regions ( utrs ) are critical in one or more steps of the virus life cycle , the envelope gene has raised more attention because of the role of the envelope ( e ) protein in virus attachment and entry into the cell , as well as membrane fusion and interaction with the immune system .
figure 1.phylogenetic tree of the 4 denv serotypes based on the complete ( 14791485 nt ) envelope gene of 289 viral isolates worldwide .
the intra - serotype genetic diversity has allowed the designation of genotypes within each denv serotype .
phylogenetic tree of the 4 denv serotypes based on the complete ( 14791485 nt ) envelope gene of 289 viral isolates worldwide .
the intra - serotype genetic diversity has allowed the designation of genotypes within each denv serotype .
the e protein of denv is a membrane - anchored glycoprotein of approximately 53 kda that forms homodimers which are organized into rafts , each containing 3 parallel dimers in the mature virus .
the arrangement of these rafts with the viral membrane ( m ) proteins and host - derived lipid membrane leads to formation of the viral coat with icosahedral - like symmetry .
the e protein plays important roles in the life cycle of the denv and in the stimulation of host protective immunity .
the e protein has a major region known as the ectodomain ( soluble fragment containing residues 1394 ) and a minor membrane - anchored insoluble region ( residues 395495 ) .
the soluble fragment contains 3 structural domains ( i , ii , and iii ) , which have been extensively characterized .
domains i and ii function as a molecular hinge for e protein reorganization at low ph .
additionally , domain ii contains the highly conserved fusion peptide responsible for virus - mediated cell - membrane fusion .
domain iii forms an immunoglobulin - like fold containing putative receptor - binding motifs involved in receptor recognition , attachment and virus entry into the host cell .
several studies suggest that immune sera of denv - infected patients contain several antibody populations which target the different antigenic epitopes exposed on both , the virion surface ( e.g. , m and e ) , and nonstructural proteins secreted during viral infection ( e.g. , viral protein ns1 ) .
denv e is the main protein involved in the immunological response and the induction of neutralizing antibodies .
the antigenic epitopes of the e protein recognized by neutralizing antibodies during infection in humans have been mapped in all 3 domains .
antibodies targeting domain i / ii have been correlated with cross - reactive immunity to the 4 denv serotypes and weak neutralization potency , while those targeting domain iii ( an epitope localized on the lateral ridge and other located on the center of the a strand ) have been correlated with a strong neutralizing activity .
denv - specific antibodies in human immune sera are mainly cross - reactive and weakly neutralizing with a very low proportion having strong neutralizing activity against only one serotype .
several in vitro and in vivo studies suggest important differences in the efficacy of antibody - mediated neutralization , due to the inability of certain antibodies to interact with the epitopes exposed by a virus belonging to a different genotype within the same serotype .
although several hypotheses are plausible , it is possible that the low efficacy of a tetravalent vaccine to protect against denv-2 , during a phase 2b clinical trial in thailand could be due to the fact that a different denv-2 genotype circulated during that period of time .
denv envelope gene has genetic variation large enough even to allow performing genotype discrimination and phylogeographic studies .
it is therefore expected that the naturally accumulated variation allow denv to escape a previously acquired immunity to a certain denv genotype especially when a denv strain with different genotype was used for the first challenge . in vitro , it is possible to obtain neutralization escape mutants of denv through selection during serial passages of a wild type virus in the presence of low doses of monoclonal neutralizing antibodies . because accumulation of mutations could lead to antigenic drift , the potential escape of a particular denv genotype from vaccine - elicited antibodies may contribute to disease severity , which occurs by the broadly accepted mechanism of antibody - dependent enhancement ( ade ) .
this hypothesis is based on evidence in vitro and in vivo suggesting that denv infection is enhanced by the administration of denv - immune sera or monoclonal antibodies in cell cultures and monkeys .
additional evidence arises from epidemiological observations that infection in humans with one denv serotype confers long - term protection against that serotype , but not against any other serotype .
cross - reactive antibodies can recognize and attach the virus belonging to a heterologous serotype in a non - neutralizing way .
this cross - reaction could enhance the virus uptake by fc receptor - bearing cells where they can quickly replicate , invade the lymph nodes and cause higher viremia that have been correlated with disease severity . in case
the administered vaccine does not confer protection , another major concern emerges from the fact that the ade could lead to a severe outcome of the disease .
we are therefore encouraged to define the extent of protection against the different serotypes and genotypes and the geographic distribution and continuous surveillance before vaccine approval and subsequent vaccine composition .
the intra - serotype genetic variation of denv in the form of genotypes determined by sequencing was first reported in the early 1990s for denv-1 and -2 .
sequence availability during the last 2 decades allowed the high - resolution genotyping which were named according to their distribution . in spite of intense microevolution of denv ,
the worldwide distribution of the denv genotypes has been stable through the time ( fig .
although viruses belonging to a specific genotype may be reported in places , which are distant from each other , these are frequently considered imported cases and several factors limit these from becoming established .
the denv strains from imported cases where disease establishment has not been demonstrated were excluded from the analysis .
the denv strains from imported cases where disease establishment has not been demonstrated were excluded from the analysis .
the native american genotype of denv-2 co - circulated in several latin - american countries , and was finally replaced by the asian / american genotype during 1990s . also , despite the circulation of denv-3 genotype iii in the americas for a long period , the co / circulation with denv-3 genotype i was recently reported for a short period of time in brazil , colombia , and ecuador .
although genotype co - circulation and replacement are not very common phenomena in the americas , these are frequently observed in several countries in southeast asia and south pacific . because of globalization , commercial relationships , and tourism , no country is exempt of importing and establishing novel denv strains of any genotype .
it is therefore important to maintain active genotype surveillance in all the endemic countries where the denv is transmitted and vaccination is planed in the near future .
major efforts in denv research are currently focused on designing / producing and licensing a tetravalent vaccine .
it is therefore important to understand the role of denv genetic variability in vaccine efficacy .
if the results of vaccine candidates in clinical trials continue showing low efficacy , future vaccine approaches should consider the design of vaccines for regional administration whose antigenic and genetic composition are based on genotype surveillance . | dengue is a major threat for public health in tropical and subtropical countries around the world . in the absence of a licensed vaccine and effective antiviral therapies , control measures
have been based on education activities and vector elimination .
current efforts for developing a vaccine are both promising and troubling . at the advent of the introduction of a tetravalent dengue vaccine , molecular surveillance of the circulating genotypes in different geographical regions
has gained considerable importance .
a growing body of in vitro , preclinical , and clinical phase studies suggest that vaccine conferred protection in a geographical area could depends on the coincidence of the dengue virus genotypes included in the vaccine and those circulating . in this review we present the state - of - the - art in this field , highlighting the need of deeper knowledge on neutralizing immune response for making decisions about future vaccine approval and the potential need for different vaccine composition for regional administration . |
obesity and overweight have both been described as anomalous accumulation of excessive body fat which may be harmful to health .
there is no single cause to explain all cases of obesity and overweight but most studies implicate imbalance in the amounts of calories consumed and those expended .
the disruption of the normal satiety feedback mechanisms , hyperinsulinism , insulin resistance , and genetics are some of the biophysiological causes of obesity and overweight .
some researchers also attribute obesity and overweight to obesogenic environments where people are frequently exposed to and consume savory foods with hidden fats and sugars that can impair metabolism and lead to obesity .
some public health experts also associate the development of obesity and overweight with socioeconomic status , urban lifestyle , family size , physical inactivity , educational status , cultural factors , and poor eating habits [ 46 ] .
persons who spend their leisure inactively such as in prolonged watching of television and playing of video games have been said to be at risk of obesity .
in addition to being pathologically chronic , obesity and overweight have been associated with other comorbidities in both younger and older populations .
heart disease , vision problems , cancer , hepatic impairment , diabetes , and other disease conditions as well as the economic burden involved in managing these conditions have been associated with obesity and overweight [ 8 , 9 ] .
obese and overweight individuals are also stigmatized ; in some societies , people portray a disease stigma towards those suffering from obesity and overweight and see them as immoral , lazy , unclean , and voracious . while the majority of the researches done highlight obesity and overweight as problems of the developed countries , recent studies also show that the third world countries are no exception [ 11 , 12 ] .
reports show that the issue of childhood and adolescence obesity in third world countries requires public health attention [ 12 , 13 ] . during the time of this research ,
this study aimed to determine the prevalence of obesity and overweight among students aged 1020 years in the kumasi metropolis of ghana , to sensitize the public on the emerging trend of childhood and adolescence obesity and overweight in ghana and to provide data for public health professionals and policy planners .
a descriptive cross - sectional study of 500 students aged 10 to 20 years was carried out from may 2010 to june 2010 in the kumasi metropolis , ghana .
simple random sampling was used to select one submetropolis from a total of 10 submetropolises . within the selected submetropolis
, simple random sampling was used to select two junior high schools . in each selected school ,
therefore , students recruited for the study came from diverse ethnic backgrounds ; most of them were ashantis and others belonged to represented minor groups from northern ghana .
parents of the recruited students had little or no formal education and worked as tradesmen and tradeswomen and skilled professionals .
weight was measured without shoes to the nearest 0.1 kg using a single previously standardized portable weighing scale .
height was measured without shoes and recorded to the nearest 0.1 cm with a height rod fixed on a wall .
the body mass index ( bmi ) of each individual was calculated as weight in kilograms divided by height in metres squared .
the bmi classes we adopted are the same as those used by the international obesity task force for the international overweight and obesity cutoffs for children [ 15 , 16 ] .
the statistical package for social scientists ( spss ) software , version 16.0 , chicago , usa , was used to analyze the collected data .
this research was conducted with approval from the heads of the selected junior and senior high schools and from the teachers .
a total of 500 students aged 10 to 20 years took part in the study .
two hundred and ninety ( 58.00% ) males and 210 ( 42.00% ) females were included in the study .
the overall mean age was 15.91 1.78 years ( 16.00 2.00 years for the males and 15.00 2.00 years for the females ) .
table 1 shows a summary of the measured anthropometric parameters per sex of study subjects .
males were significantly heavier and taller than females ( p < 0.05 ) but there were no differences in bmi ( p > 0.05 ) .
the overall prevalence of underweight , normal weight , overweight , and obesity was 7.40% , 79.60% , 12.20% , and 0.80% , respectively . among males and females ,
prevalence of overweight was 6.80% and 5.40% , respectively , while the prevalence of obesity was 0.20% and 0.60% , respectively .
these results are slightly close to those reported in a study done among students in nigeria where there was a high prevalence of overweight and a low prevalence of obesity . comparable to other studies done in ghana and other second world countries
[ 18 , 19 ] , this study showed that the prevalence rate of overweight was higher among female students than male students .
there are however divergent findings in studies done in developed nations [ 20 , 21 ] where prevalence rates of overweight among male study subjects were higher than among females . for this study ,
the difference in the prevalence of obesity and overweight between the genders was insignificant ( p > 0.05 ) ; the apparently higher values for males could be confounded by a greater number of male respondents ( 290 ) as compared to the female respondents ( 210 ) . while challenges to our study design did not allow for understanding reasons for the distribution of obesity and overweight among the genders , other studies report cultural attributions as to why females may be more overweight or obese than their male counterparts .
a study showed that obese females considered putting on weight as a sign of affluence and happiness .
others also believed that being obese afforded the needed strength in their sport and accorded them the necessary respect .
this is supported by undocumented reports from some areas in ghana , where excessive weight gain is considered to be the result of eating good and healthy food .
table 2 summarizes the prevalence of underweight , normal weight , overweight , and obesity according to the gender of the students .
findings from this study must be considered in the light of certain challenges to the study design .
the study depended on anthropometric measurements to establish whether or not the students were of the following categories : underweight , normal weight , overweight , or obesity .
a study conducted in ghana extensively looked at how the various weight statuses were linked to socioeconomic status of subjects and level of education of students ' parents .
another study also found significant associations between smoking and overweight and obesity while other reports reveal positive associations between some psychosocial factors ( e.g. , loneliness and social isolation ) [ 23 , 24 ] .
even though this study did not look at socioeconomic status , smoking , dietary factors , loneliness , behavioural patterns , age of menarche , and order of births of the students and how these may influence their different weight statuses , it is reasonable from other studies [ 17 , 2326 ] to say that a complex interplay of these factors could contribute to our findings in this study . to the best of our knowledge
, the study has provided an insight into the prevalence of obesity and overweight among children and adolescents aged 1020 years in the kumasi metropolis of ghana .
the study revealed a high prevalence of overweight ( 12.20% ) among the students . therefore , there is a need to establish effective prevention and health promotion programmes among the students .
this would enable maintaining healthy weights and avoiding the possible immediate and long - term health complications associated with overweight and obesity . | the aim was to determine the prevalence of obesity and overweight among students in the kumasi metropolis . in a descriptive cross - sectional study ,
500 students aged 10 to 20 years were examined from two junior high schools selected by multistage sampling technique and three randomly selected senior high schools . height and weight were measured in all participants and the body mass index ( bmi ) of each individual was calculated .
body mass index classes were calculated according to the international obesity task force standards . out of the 500 students , 290 ( 58.00% ) were males and 210 ( 42.00% ) were females .
the prevalence of underweight , normal weight , overweight , and obesity was 7.40% , 79.60% , 12.20% , and 0.80% , respectively .
overweight was more prevalent among students than obesity .
there is therefore the need to establish effective public health promotion campaigns among students in order to curtail future implications on health . |
malaria is a major public health problem in tropical and subtropical countries including india and its dynamics vary from place to place . among all anopheline vectors , anopheles culicifacies ( diptera : culicidae ) is a principal malaria vector in rural , periurban and tribal settings ( mishra et al .
culicifacies has a wide distribution that extends from ethiopia , yemen and iran in the west via afghanistan , pakistan , india , bangladesh , myanmar and thailand , to laos and vietnam and combodia in the east ( harrison 1980 , van bortel et al .
it is found in nepal and southern china , and in the south in sri lanka .
culicifacies is a complex of 5 isomorphic types which are designated as species a and b ( green and miles 1980 ) , species c ( subbarao et al . 1983 ) , species d ( subbarao et al .
1989 , vasantha et al . 1991 ) and species e ( kar et al . 1999 ) with varying biological characteristics such as feeding preference , biting activity , and susceptibility to commonly used insecticides in public health programs ( joshi et al .
madhya pradesh is situated in the central part of india with an area of 308 thousand km of which forest cover 76,429 km ( about 25% of the total land area ) .
madhya pradesh ( population 72.6 million ) along with other states like orrisa ( population 42 million ) , jharkhand ( population 33 million ) , and chhattisgarh ( population 25.5 million ) contributes for more than 60% of reported ( confirmed ) malaria cases in india . according to national vector borne diseases control program ( nvbdcp ) epidemiological data for 2010 from predominantly these tribal states with a total population of 173.1 million ( out of a total of the country population i. e. , 1.21 billion ) represent 14.3 % population show persistent malaria transmission with high api ( annual parasite incidence ) , slide positivity rate ( spr ) and very high pf% ( sharma 2012 ) .
madhya pradesh alone account for 6% of the total population of the country but contributes to 8.6% of the total malaria cases .
malaria is complex in madhya pradesh because of vast tracts of forest with tribal settlement ( 20% of state population ) ( singh et al .
2004 , anon 2007 ) . the magnitude of the problem can be accessed from an estimate made in 1987 , that 54 million individuals of various ethnic origins residing in forested area of india and accounting for 8% of the total population contributed 30% of total malaria cases , 60% of total falciparum cases and 50% of malaria deaths in the country ( sharma 1996 ) .
the reasons for such a high diseases prevalence in madhya pradesh is mainly due to locations of the villages in the deep forest and is characterized by rocky undulation interspersed with ravines and foothills .
these streams flow continuously and provide ample breeding sites covered with dense aquatic vegetation for production of number of anophelines particularly an .
therefore malaria control in these areas requires specific approaches and control strategies which includes the proper surveillance for distribution of an .
culicifacies members and their identification ( pattanayak et al . 1994 ) . since sibling species a , b , c , d and e of an .
culicifacies are morphologically indistinguishable at any stage of life and due to practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex , a molecular method using an allele - specific polymerase chain reaction ( as - pcr ) assay targeted to the d3 domain of 28s ribosomal dna was used to distinguish these sibling species ( singh et al .
firstly in d3-pcr the species complex is distinguished in two groups i.e. , a and d in one group and species b , c and e in the second group . in second tier
involves ad - pcr assay which distinguishes species a from species d , whereas the bce - pcr assay distinguishes species b , c and e with each other . with combination of these two tier pcr assays it is possible to identify individual mosquito of the an .
culicifacies species in various districts of madhya pradesh , india . in the earlier reports from this region
species a , b , c and d were identified but species e reported from southern parts of india only was not reported from this area . noticeably in our study we encountered species e from some of the districts in co - habitation with species b which indicates that all the five members of an .
the adult anopheline mosquito species were collected from different districts of madhya pradesh ie , mandla ( dungaria village ) , jabalpur ( barela village ) , chindwara ( chakarpat and chikhla villages ) , hoshangabad ( dhadav and padav villages ) , narsinghpur ( chinki village ) and khandwa ( chighdhalia ) ( fig . 1 and table 1 ) .
these sites were selected on the basis as they represent the tribal belt along the streams of narmada river and also show high incidence of malaria ( singh 2004 , 2006 , 2009 , sharma 2012 ) .
the collections were made during the transmission period i.e. february march and august september in the morning period between 0600 h to 0800 h using mouth aspirator and battery operated torch .
the fed anophelines were captured at various collection sites including human dwellings , cattle sheds , mixed dwelling and random collection sites .
the fed mosquitoes were captured so that f1 generation of these mosquitoes can be utilized for further use after egg lay .
all adult mosquitoes were brought to the lab for their identification by using standard keys ( christopher 1933 , wattal and kalra 1961 , das et al .
each representative sample was pinned as a voucher specimen and kept in laboratory as a reference collection . from these collection
culicifacies female were separated and allowed for egg laying and the adult emerged from them are used for further standardization and identification of mosquito sibling species using allele specific polymerase chain reaction ( as - pcr ) ( goswami et al .
map showing different collection sites in the study list of collection sites and result using as - pcr assay of an .
culicifacies mosquito the dna extraction was done by using method as described in our previous publication ( sharma et al .
each single adult mosquito was homogenized in the micro centrifuge tube by adding 100 l lysis buffer .
the homogenate was immediately kept on ice for 10 minutes and followed by heat treatment at 65 c for 30 minutes .
subsequently , 30 l 5 m potassium acetate was added and immediately transferred to ice for one hour followed by centrifugation at 13,000 rpm for 15 minutes at 10 c . to the supernatant obtained
, a double volume of absolute chilled ethanol was added for precipitation of dna and kept tubes at 20 c for overnight .
after centrifugation at 13,000 rpm for 15 minutes at 10 c , the precipitated dna was washed in 70% ethanol twice .
the dna pallet was allowed to air dry and finally dissolved in 50 l te buffer .
the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al .
another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr .
the sequences for the primers used were given in the table 2 with their annealing temperatures .
culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris .
hcl 10 mm ph 9.0 , kcl 50 mm mg cl2 2 mm , dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase ( mbi fermentas ) and 10ng of genomic dna .
reactions were performed in a ( biorad pcr system icycler ) thermal cycler . the pcr condition consisted of initial denaturation step for 5 min at 95 c followed by 35 cycles of 30 sec at 95 c , 30 sec at 55 c , and 60 sec at 72 c .
a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) .
optimized condition for ad - pcr assay includes 35 cycles of the initial denaturation temperature at 95 c for 40 s , annealing at 50 c for 40 s , and extension 68 c for 40 s , followed by a final extension at 72 c for 10 min .
the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . whereas the condition for bce - pcr assay are similar as described for the ad - pcr assay except for the primer concentration of 25 pmol bcef primer , 12 pmol bcr primer , 25 pmol er primer and 30 pmol cr primer respectively .
the adult anopheline mosquito species were collected from different districts of madhya pradesh ie , mandla ( dungaria village ) , jabalpur ( barela village ) , chindwara ( chakarpat and chikhla villages ) , hoshangabad ( dhadav and padav villages ) , narsinghpur ( chinki village ) and khandwa ( chighdhalia ) ( fig . 1 and table 1 ) .
these sites were selected on the basis as they represent the tribal belt along the streams of narmada river and also show high incidence of malaria ( singh 2004 , 2006 , 2009 , sharma 2012 ) .
the collections were made during the transmission period i.e. february march and august september in the morning period between 0600 h to 0800 h using mouth aspirator and battery operated torch .
the fed anophelines were captured at various collection sites including human dwellings , cattle sheds , mixed dwelling and random collection sites .
the fed mosquitoes were captured so that f1 generation of these mosquitoes can be utilized for further use after egg lay .
all adult mosquitoes were brought to the lab for their identification by using standard keys ( christopher 1933 , wattal and kalra 1961 , das et al .
each representative sample was pinned as a voucher specimen and kept in laboratory as a reference collection . from these collection
culicifacies female were separated and allowed for egg laying and the adult emerged from them are used for further standardization and identification of mosquito sibling species using allele specific polymerase chain reaction ( as - pcr ) ( goswami et al .
map showing different collection sites in the study list of collection sites and result using as - pcr assay of an .
the dna extraction was done by using method as described in our previous publication ( sharma et al .
each single adult mosquito was homogenized in the micro centrifuge tube by adding 100 l lysis buffer .
the homogenate was immediately kept on ice for 10 minutes and followed by heat treatment at 65 c for 30 minutes .
subsequently , 30 l 5 m potassium acetate was added and immediately transferred to ice for one hour followed by centrifugation at 13,000 rpm for 15 minutes at 10 c . to the supernatant obtained
, a double volume of absolute chilled ethanol was added for precipitation of dna and kept tubes at 20 c for overnight .
after centrifugation at 13,000 rpm for 15 minutes at 10 c , the precipitated dna was washed in 70% ethanol twice .
the dna pallet was allowed to air dry and finally dissolved in 50 l te buffer .
the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al .
another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr .
the sequences for the primers used were given in the table 2 with their annealing temperatures .
culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris .
hcl 10 mm ph 9.0 , kcl 50 mm mg cl2 2 mm , dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase ( mbi fermentas ) and 10ng of genomic dna .
reactions were performed in a ( biorad pcr system icycler ) thermal cycler . the pcr condition consisted of initial denaturation step for 5 min at 95 c followed by 35 cycles of 30 sec at 95 c , 30 sec at 55 c , and 60 sec at 72 c .
a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) .
optimized condition for ad - pcr assay includes 35 cycles of the initial denaturation temperature at 95 c for 40 s , annealing at 50 c for 40 s , and extension 68 c for 40 s , followed by a final extension at 72 c for 10 min .
the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . whereas the condition for bce - pcr assay are similar as described for the ad - pcr assay except for the primer concentration of 25 pmol bcef primer , 12 pmol bcr primer , 25 pmol er primer and 30 pmol cr primer respectively .
the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al .
another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr .
the sequences for the primers used were given in the table 2 with their annealing temperatures .
culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris .
hcl 10 mm ph 9.0 , kcl 50 mm mg cl2 2 mm , dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase ( mbi fermentas ) and 10ng of genomic dna .
reactions were performed in a ( biorad pcr system icycler ) thermal cycler . the pcr condition consisted of initial denaturation step for 5 min at 95 c followed by 35 cycles of 30 sec at 95 c , 30 sec at 55 c , and 60 sec at 72 c .
a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) .
optimized condition for ad - pcr assay includes 35 cycles of the initial denaturation temperature at 95 c for 40 s , annealing at 50 c for 40 s , and extension 68 c for 40 s , followed by a final extension at 72 c for 10 min .
the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . whereas the condition for bce - pcr assay are similar as described for the ad - pcr assay except for the primer concentration of 25 pmol bcef primer , 12 pmol bcr primer , 25 pmol er primer and 30 pmol cr primer respectively .
during the collection period a large number of anopheles mosquitoes were collected from the various collection sites ( fig .
1 and table 1 ) and per man per hour ( pmh ) count estimate was also made from each site .
culicifacies during the study period was found to be in the range of 8120 pmh , with a high density during august - september ( 90120 pmh ) and to a low density in february march ( 850 pmh ) . the as - pcr assay using different primers , the a / d specific primer ( aca ) in conjunction with d3b produces 313 bp amplification product and b / c / e - specific primer ( acb ) forms 133 bp product with d3a . additionally , the external primers d3a and d3b form common product in all the samples with 382bp products in species a and d whereas 385bp in species b / c / e serving as positive control ( fig .
2 ) . for further distinguishing the sibling species in a / d and b / c / e individually ,
a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay differentiating sibling species b , c and e from each other . in ad - pcr ,
the sibling species a and d produced the bands of 359 bp for d species and 359 bp and 166 bp for sibling species a. on the other hand in bce - pcr , the products are 248 bp for b , 248 bp and 95 bp for c and 248 and 178 for sibling species e respectively ( fig .
d3-pcr showing the different bands to differentiate a / d and b / c / e sibling species of an .
culicifacies allele specific multiplex ad - bce - pcr showing different bands for each an .
culicifacies collected from different districts from madhya pradesh ie , mandla , jabalpur , chindwara , hoshangabad , narsinghpur and khandwa respectively .
the collected mosquitoes were subjected to two tier pcr assays . from the d3-pcr only narsinghpur and khandwa samples were identified in a / d group whereas all the other districts samples were identified in and identified in b / c / e group ( fig .
results obtained from ad - bce pcr showed the presence of sibling species b from jabalpur , chindwara and hoshangabad , c from hoshangabad only , d from narsinghpur and khandwa and sibling species e from mandla , chindwara and hoshangabad respectively ( fig .
the accurate identification and distribution pattern of anopheline mosquitoes is necessary for planning effective vector control strategies and for a better understanding of their potential role in malaria transmission .
wattal and kalra in 1961 described 32 species of female anophelines in different regions of india .
they divided india in six regions and included madhya pradesh in hyderabad region where about 28 species were described from this region ( wattal and kalra 1961 ) . in our collection
we have also encountered about 11 species of anopheles from the study area which includes an .
but in the study area malaria is mainly transmitted by two efficient vectors i.e. , an .
culicifacies being a major vector of malaria in india is responsible for approximately 65% of total malaria cases ( sharma 1998 ) . in india
culicifacies have been found among these species b was found throughout the country whereas species e was reported only from the southern parts of india .
they may be different in the feeding pattern an important characteristics that influence vectorial capacity .
distinct difference were observed in laboratory studies with reference to insemination rate , fecundity , longevity , oviposition , gonotropic cycle , egg hatching , larval mortality rates and adult emergence time have been observed in different species of an .
attempts to find morphological markers for the members of species complex have not been successful so far except the variation in spermatheca of sibling species a and b ( das 1990 ) but this difference still need to be reconfirmed by other techniques .
the classical technique of cytotaxonomic is difficult and has limited use as this requires semi gravid females only and moreover it requires highly skilled personnel . with the advent of dna based technology we are now able to differentiate members of an .
the dna based technology includes pcr assay which are simple and sensitive at the same time they are applicable to all stages and either sexes of mosquitoes ( goswami et al .
2006 ) . as reported by sharma ( 2012 ) in his review that the epidemiological indices of malaria in madhya pradesh revealed a very dismal picture of malaria .
an international team of experts reported a very high incidence of malaria in pregnancy ( mip ) .
for example in madhya pradesh ( rural ) 183,0001.5 million per year contract malaria in pregnancy , and result in 73,000629,000 lost foetus and 1,500 to 12,600 maternal deaths .
plausible estimate of 220,000 mip cases per year ( 136,000305,000 ) , 95,800 lost fetus ( 56,800147,600 ) and 1,000 maternal deaths ( 6501,600 ) ( diamond et al .
culicifacies mosquito species were collected from different malaria endemic district of madhya pradesh by using as - pcr we were able to identify four species b , c , d and e from these areas .
species e was not reported earlier from these areas as this species is prevalent only in southern peninsular part of india .
species e is highly anthropophagic and possesses high sporozoite rate up to 20% and also known as vector in southern india and srilanka ( kar et al .
we encountered species e from mandla , chindwara and hoshangabad districts respectively which have high disease prevalence and represent the tribal belts ( singh 2004 , 2006 , 2009 , sharma 2012 ) .
noticeably this species e was found sympatric with a non vector species b from chindwara and hoshangabad district , but in jabalpur district only species b and in mandla only species e were identified .
topographically the villages under study from mandla and jabalpur are very close to each other .
although species b is a poor vector of malaria in india but through examination of mitotic y chromosome morphology ( kar et al .
1999 ) , that what was reported as b on the sri lanka island is really a sympatric mixture of b and e ( surendran et al .
moreover these two sibling species in sri lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides ( surendran et al .
it is evident from the literature that species e can not be differentiated from species b because they have homosequential polytene chromosome arrangements .
species e requires mitotic chromosome examination of male progeny and/or vectorial potential needs to be established for distinction from species b ( kar et al .
after screening several enzyme systems , electrophoretic variation at the lactate dehydrogenase ( ldh ) locus was useful .
it could group species a and d in one category and species b and c in another category ( adak et al .
species e showed the same ldhs allele as in species b and c ( kar et al .
e , the paradox that species b females from northern india are not vectors , but homosequential females from rameshwaram island are vectors is resolved .
similar studies are needed to be carried out to see the vectorial capacity and disease transmission in the studied districts of madhya pradesh . as in mainland areas close to rameshwaram , species e were found , populations in other parts of tamilnadu state where species b has been identified also should be examined ( kar et al .
culicifacies populations identified as species b in sri lanka also should be examined immediately for y - chromosome variations and correlated with malaria infection ( surendran et al .
1997 ) has reported acrocentric and submetacentric y - chromosomes within species b but no epidemiological or dissection data is available that indicates that species b is a vector .
thus , there is an urgent need to develop suitable markers that can differentiate species b and e and also to see the other biological characteristics of these two species in such cohabiting areas to conclude that which species is responsible for disease transmission .
moreover new areas should be explored for the presence of species e as it is a potent vector of malaria .
hence there is an urgent need for nationwide surveillance and identification of vector sibling species distribution once again so that modified species pattern in these areas could be established .
apart from confirming their identity , distribution pattern and their differential malaria vector status , it will be important to determine the susceptibility of these sibling species to insecticides in each part of the states/ country as this affects the efficiency of vector control operations in the malaria control programs in india .
in conclusion we can say that as we encountered a new sibling species e of an .
existence of such modified distribution of sibling species may exist in other areas also which necessitate for reconsidering the sibling species distribution in newer area .
knowledge of proper identification and distribution pattern of sibling species may further help us in development of vector control strategies . | background : anopheles culicifacies is an important vector of malaria in southeast asia , contributing to almost 70% of malaria cases in india .
it exists as a complex of five morphologically indistinguishable species a , b , c , d and e with varied geographical distribution patterns . in india
, 8% of the total population of madhya pradesh ( central india ) contributes about 30% of total malaria cases , 60% of total falciparum cases and 50% of malaria deaths .
an .
culicifacies is the major malaria vector in this state .
vector control mainly relies on the proper identification and distribution of vector species exists in a particular area .
the present study was carried out to identify the distribution of an .
culicifacies sibling species in certain endemic district of central india , madhya pradesh.methods:the an .
culicifacies mosquitoes collected from the study districts were identified morphologically .
the genomic dna was isolated from the mosquitoes and subjected to allele specific pcr targeting d3 domain of 28s ribosomal dna.results:the mean prevalence of an .
culicifacies during the study period was in the range of 8120 per man per hour ( pmh ) . from the study areas species
b was identified from jabalpur , chindwara and hoshangabad , species c from hoshangabad only , species d from narsinghpur and khandwa and sibling species e from mandla , chindwara and hoshangabad respectively.conclusion:this is the first report to detect species e from madhya pradesh region which necessitate for reconsideration of species distribution of each an .
culicifacies sibling species that would enable to develop required vector control strategies . |
cancer is a leading cause of death worldwide and accounts for 7.6 million deaths every year .
the first and second most common causes of cancer death in the world are lung cancer ( 1.37 million deaths , annually ) and gastric cancer ( 736000 deaths , annually ) , respectively ( 1 ) . about 70% of all cancer deaths have occurred in low- and middle - income countries .
gastric cancer is the fourth most common cancer in males , while in females it is the fifth most common cancer ( based on statistics published in 2008 ) ( 2 ) .
the incidence rate of gastric cancer in western countries has shown a remarkable decline in the recent years while it is still the most common cancer among males in iran .
based on age - standardized incidence rates ( asr ) the most common cancer in males was stomach ( 10.2 ) , while in females , stomach cancer ( 5.1 ) was the third most common cancer ( 3 ) .
although the incidence of stomach cancer is decreasing , it is still a major health problem and a major cause of cancer death around the world ( 4 ) .
it has been suggested that carcinogenesis of stomach cancer is due to a collection of genetic alterations of many genes such as oncogenes , tumor suppressor , and mismatch repair genes ( 5 ) .
it has been suggested that there are many genes that raise the risk of stomach cancer , because some genes such as mcc , apc , and p53 tumor suppressor genes have been found in a numerous cases of gastric cancers ( 6 ) .
cadherin-1 , also known as cam 120/80 or epithelial cadherin ( e - cadherin ) , is a protein that in humans is encoded by the cdh1 gene , which is a tumor suppressor gene ( 7 ) .
many researchers have reported e - cadherin , a calcium - dependent adhesion molecule that is responsible for cellular binding to adjacent cells , as an important part in the etiology of stomach carcinogenesis ( 8) .
if a second similar allele has been in the e - cadherin gene , then lack of intracellular adhesion causes an increase in intracellular permeability ( 9 ) . a broad range of mutations has been found in gastric cancers ( 10 ) . the frequency of stomach cancer is higher among senile populations , manifesting more at about 70 to 80 years of age . in a study with a large sample ,
worse clinicopathologic characteristics and prognosis for gastric cancer has been suggested among young patients ( 12 ) .
the proto - oncogene myc , located at 8q24.1 , encodes a nuclear phosphoprotein , regulates almost 15% of human genes and is activated in 20% of all tumors ( 13 ) .
activation of unsuitable myc gene , which contributes to the development of human tumors , is functioned through different mechanisms : chromosome translocations , which has already been shown in burkitt lymphoma ; juxtaposition of the promoter region of the immunoglobulin heavy chain gene , which is highly expressed in b cells , because it is next to the myc gene ; excitation of gene transcription , as has been documented in colon cancer cells ; retrovirus insertion next to the myc locus , where through the retroviral regulatory sequences , the myc expression would be activated , and gene amplification and the copy number of myc gene would be excessed , and as a result its expression would be increased between other cells ( 14 ) .
hence , any changes in myc oncogene are usually persuaded by phenomena such as gene amplification , chromosome translocation , point mutations , viral insertion at the myc locus , and resistance of myc protein to ubiquitin - mediated proteolysis and raised translation or transcription by signaling other oncogenic pathways ( 15 ) .
the location of the myc protein is in the nucleus , and acts as a growth promoter and a transcription factor .
transcription factors of bhlh are a big family , which contain myc , and form homo and heterodimers with themselves and with other family members .
these dimeric complexes are related to regulatory sequences called e - boxes ( with cacgtg sequence ) , detected in promoters of target genes , which they regulate ( 16 ) .
one of the necessary ligands for most of biological functions is the max protein , which is used by myc .
although it seems that the myc protein is expressed independently , yet in fact , the myc protein of each cell is complexed with the max protein ( 17 ) . usually , the levels of max are stable within cells , yet the myc levels are strongly affected by mitogenic signals . in this
regard , when normal cells are grown without mitogenic components , the level of myc will fall , while , myc accumulates significantly in the presence of serum mitogens .
this indicates that the levels of myc - max heterodimer are constantly regulated by the current of mitogenic signals , which normal cells receive ( 16 ) .
the myc gene has three exons , and its protein products ( p64 and p67 ) include strongly conserved nuclear phosphoproteins , with high amounts of p64 compared with p67 , called myc-2 and myc-1 , respectively ( 18 ) .
it has been suggested that patients with late stages of gastric cancers or with metastases , show higher levels of myc amplification , and on the contrary , patients at the early stages show lower amplification ( 19 ) .
the amplification of myc and overexpression of its protein are found in 15 to 30% of gastric cancers ( 20 ) .
some authors have suggested that myc overexpression is more frequent than myc gene amplification in stomach adenocarcinoma ( 21 ) . in more than 40% of cases with stomach cancer , overexpression of myc
some researchers have reported on the association between metastasis and higher level of myc expression ( 22 ) .
another report showed that myc mrna levels were also higher in patients with metastasis than in cases with primary lesions ( 23 ) .
yang et al . pointed out that in advanced stomach cancers , myc expression was higher than in early stages ( 24 ) . on the contrary , onoda et al . suggested that myc expression was more frequent in early than in advanced lesions , yet other studies have not proved this difference ( 23 ) .
( 19 ) noticed that myc protein expression elevated gradually as the lesion progressed , such as that found in chronic active gastritis , gastric ulcer , mild non - classic proliferation , severe non - classic proliferation , and early and advanced stomach cancer ( 19 ) these findings proved that myc amplification could be a serious phenomenon of gastric carcinogenesis .
the objective of this study was to find the preferences of cish and ihc in diagnosis and prognosis of gastric cancer among 102 patients with adenocarcinoma .
samples were gathered from 102 paraffin - embedded blocks , and also 50 blocks from adjacent normal stomach mucosa , as negative control samples .
all patients had stomach surgery at the cancer institute hospital , tehran university of medical sciences .
all patients had a negative history of exposure to either chemotherapy or radiotherapy before surgery .
originally , the samples were 105 patients , yet three samples were excluded , because they received a new adjuvant therapy .
the main objective of this study was to investigate the expression of c - myc in patients with gastric cancer .
more than 40% of stomach cancers show abundant expression of myc , as indicated by previous reports .
the sample size was calculated using the following formula : considering that z = 1.96 , p = 40% and accuracy was 10% , 92 samples were sufficient for this study . to ensure a 10% increase ,
patients approval was retained according to local authorities by the ethics committee , tumor bank , cancer institute of iran .
written informed consent was obtained from all participants and the institutional ethics committee approved the study .
the patients were staged using a standard method by tumor - nodes - metastasis ( tnm ) staging ( 26 ) .
chromogenic in situ hybridization ( cish ) was carried out on 3-m - thick formalin - fixed paraffin - embedded tissue sections .
the 3-m - thick sections were cut from paraffin blocks and dewaxed with xylene , and then rehydrated with a series of 70% to 100% ethanol and water . target retrieval and enzyme digestion were achieved using a commercially available tissue pretreatment kit ( no.c-3018 - 40 ; from zytodot germany ) .
on the first day , slides were incubated in pretreatment buffer , in a water bath ( mmert - germany ) to at least 85c for 30 minutes , with a zytodot cish implementation kit ( germany ) .
next , pepsin solution was applied to the tissue followed by incubation for eight minutes at room temperature in a humidity chamber .
ten microliters of zytodot cish probe c - myc were applied to the slides , and the slides were enclosed by a coverslip ( 22 x 22 mm ) and denatured on a hot plate for five minutes at 94 - 95 c .
the slides were then transferred to a humidity chamber and hybridized overnight at 37c . on the second day ,
the coverslip was removed by soaking in standard saline citrate ( ssc ) solution at room temperature for five to ten minutes and washed in ssc buffer for five minutes at 75c .
next , a hybridized probe linked to digoxigenin was detected by mouse anti - digoxigenin antibody followed by polymerized horseradish peroxidase antimouse immunoglobulin .
the interpretation of cish results was performed by the first author ( m.kh ) with a light microscope using a 40 objective ( original magnification , 100 ; figure 1 a - d ) .
a gene copy number of one to two copies per nucleus were scored as no amplification ( figure 1a ) .
a gene copy number of two to four copies per nucleus in at least 50% of cancer cells were considered as low - level amplification ( figure 1b ) .
a gene copy number of four to six copies per nucleus in at least 50% of cancer cells were considered as moderate level amplification ( figure 1c ) .
a gene copy number of more than six copies per nucleus or the presence of clusters in at least 50% of cancer cells was considered as high - level amplification ( figure 1d ) ( 18 , 19 , 27 ) .
non - neoplastic cells in tissues were always considered as in - test quality controls .
chromogenic in situ hybridization were evaluated with leitz microscope images and were captured with a digital nikon camera .
all the slides of patients were confirmed for diagnosis and were checked for cish and ihc by one senior pathologist ( i.j ) .
a , no amplification ; b , low amplification ( 2 - 4 signals ) ; c , moderate amplification ( 4 - 6 signals ) ; d , high amplification ( more than six signals , which looked as large copy gene clusters in the majority of the nuclei of gastric cancer cells ) ( original magnification , 500 ) .
slides were incubated in hydrogen peroxide , and antigen retrieval was carried out by heat treatment in 0.294% wt / vol citrate buffer at ph 6.0 in a domestic microwave oven ( botan - iran ) as follows : 900 w for five minutes and then 600 w for 10 minutes , excluding slides that were dried out .
immunohistochemical staining was performed using the horse - radish peroxidase ( hrp ) method with a monoclonal antibody ( monoclonal rabbit , ready to use , pme 415 aa biocare medical , usa ) , as recommended by the manufacturer .
a universal peroxidase - conjugated secondary antibody kit ( amplistain anti - mouse / rabbit 1-step hrp , germany ) was used for the detection system ( sdt , germany ) .
the slides were visualized with diamino - benzidine - h2o2 ( dab ) and counterstained with hematoxylin ( sdt ) . any nuclear stain with or without cytoplasmic
a myc - positive case was defined as one having 10% or more tumor cells positive for this protein . a , negative ihc in gastric adenocarcinoma
the statistical calculation was carried out using version 18 of the spss software for windows . in order to assess the degree of agreement between cish and ihc test
the variables of patients according to the two types of diffuse and intestinal gastric cancers were analyzed using the chi - square test .
the variables of patients according to c - myc amplification ( cish ) and c - myc expression ( ihc ) with two groups were tested with the mann - whitney test , and variables with more than two groups were tested with kruskal - wallis . for determining the association between cish and ihc , kappa test and the spearman correlation coefficient were used .
commentary of the kappa test was as follows : poor agreement ( less than 0.20 ) , fair agreement ( 0.20 to 0.40 ) , moderate agreement ( 0.40 to 0.60 ) , good agreement ( 0.60 to 0.80 ) , and very good agreement ( 0.80 to 1.00 ) ( 29 ) .
samples were gathered from 102 paraffin - embedded blocks , and also 50 blocks from adjacent normal stomach mucosa , as negative control samples .
all patients had stomach surgery at the cancer institute hospital , tehran university of medical sciences .
all patients had a negative history of exposure to either chemotherapy or radiotherapy before surgery .
originally , the samples were 105 patients , yet three samples were excluded , because they received a new adjuvant therapy .
the main objective of this study was to investigate the expression of c - myc in patients with gastric cancer .
more than 40% of stomach cancers show abundant expression of myc , as indicated by previous reports .
the sample size was calculated using the following formula : considering that z = 1.96 , p = 40% and accuracy was 10% , 92 samples were sufficient for this study . to ensure a 10% increase ,
patients approval was retained according to local authorities by the ethics committee , tumor bank , cancer institute of iran .
written informed consent was obtained from all participants and the institutional ethics committee approved the study .
the patients were staged using a standard method by tumor - nodes - metastasis ( tnm ) staging ( 26 ) .
chromogenic in situ hybridization ( cish ) was carried out on 3-m - thick formalin - fixed paraffin - embedded tissue sections .
the 3-m - thick sections were cut from paraffin blocks and dewaxed with xylene , and then rehydrated with a series of 70% to 100% ethanol and water . target retrieval and enzyme digestion were achieved using a commercially available tissue pretreatment kit ( no.c-3018 - 40 ; from zytodot germany ) .
on the first day , slides were incubated in pretreatment buffer , in a water bath ( mmert - germany ) to at least 85c for 30 minutes , with a zytodot cish implementation kit ( germany ) .
next , pepsin solution was applied to the tissue followed by incubation for eight minutes at room temperature in a humidity chamber .
ten microliters of zytodot cish probe c - myc were applied to the slides , and the slides were enclosed by a coverslip ( 22 x 22 mm ) and denatured on a hot plate for five minutes at 94 - 95 c .
the slides were then transferred to a humidity chamber and hybridized overnight at 37c . on the second day ,
the coverslip was removed by soaking in standard saline citrate ( ssc ) solution at room temperature for five to ten minutes and washed in ssc buffer for five minutes at 75c .
next , a hybridized probe linked to digoxigenin was detected by mouse anti - digoxigenin antibody followed by polymerized horseradish peroxidase antimouse immunoglobulin .
the interpretation of cish results was performed by the first author ( m.kh ) with a light microscope using a 40 objective ( original magnification , 100 ; figure 1 a - d ) .
a gene copy number of one to two copies per nucleus were scored as no amplification ( figure 1a ) .
a gene copy number of two to four copies per nucleus in at least 50% of cancer cells were considered as low - level amplification ( figure 1b ) .
a gene copy number of four to six copies per nucleus in at least 50% of cancer cells were considered as moderate level amplification ( figure 1c ) .
a gene copy number of more than six copies per nucleus or the presence of clusters in at least 50% of cancer cells was considered as high - level amplification ( figure 1d ) ( 18 , 19 , 27 ) .
non - neoplastic cells in tissues were always considered as in - test quality controls .
chromogenic in situ hybridization were evaluated with leitz microscope images and were captured with a digital nikon camera .
all the slides of patients were confirmed for diagnosis and were checked for cish and ihc by one senior pathologist ( i.j ) .
a , no amplification ; b , low amplification ( 2 - 4 signals ) ; c , moderate amplification ( 4 - 6 signals ) ; d , high amplification ( more than six signals , which looked as large copy gene clusters in the majority of the nuclei of gastric cancer cells ) ( original magnification , 500 ) .
slides were incubated in hydrogen peroxide , and antigen retrieval was carried out by heat treatment in 0.294% wt / vol citrate buffer at ph 6.0 in a domestic microwave oven ( botan - iran ) as follows : 900 w for five minutes and then 600 w for 10 minutes , excluding slides that were dried out .
immunohistochemical staining was performed using the horse - radish peroxidase ( hrp ) method with a monoclonal antibody ( monoclonal rabbit , ready to use , pme 415 aa biocare medical , usa ) , as recommended by the manufacturer .
a universal peroxidase - conjugated secondary antibody kit ( amplistain anti - mouse / rabbit 1-step hrp , germany ) was used for the detection system ( sdt , germany ) .
the slides were visualized with diamino - benzidine - h2o2 ( dab ) and counterstained with hematoxylin ( sdt ) . any nuclear stain with or without cytoplasmic staining was considered as positive results , irrespective of intensity ( figure 2 ) .
a myc - positive case was defined as one having 10% or more tumor cells positive for this protein . a , negative ihc in gastric adenocarcinoma ;
the statistical calculation was carried out using version 18 of the spss software for windows . in order to assess the degree of agreement between cish and ihc test , kappa statistics was applied .
the variables of patients according to the two types of diffuse and intestinal gastric cancers were analyzed using the chi - square test .
the variables of patients according to c - myc amplification ( cish ) and c - myc expression ( ihc ) with two groups were tested with the mann - whitney test , and variables with more than two groups were tested with kruskal - wallis . for determining the association between cish and ihc , kappa test and the spearman correlation coefficient were used .
commentary of the kappa test was as follows : poor agreement ( less than 0.20 ) , fair agreement ( 0.20 to 0.40 ) , moderate agreement ( 0.40 to 0.60 ) , good agreement ( 0.60 to 0.80 ) , and very good agreement ( 0.80 to 1.00 ) ( 29 ) .
this investigation included 102 patients from iran with gastric adenocarcinomas . in our samples , 78 patients were males and 24 cases were females , and the average age was 60.62 years old .
the amplification of myc and its protein expression ( cish and ihc tests ) were carried out and analyzed for all patients .
the location of the tumor was the fundus in 35.3% of patients , while the location was the cardia in only 3% .
table 1 shows that some of the variables including gender , location of tumor , necrosis , vascular invasion , perineural invasion , cish , and ihc had significant differences amongst the diffuse type , while gender , location of tumor , vascular invasion , necrosis , cish , and ihc had significant differences amongst the intestinal type .
this means that subgroups of each variable are different in the two types of gastric cancer .
these results also showed that there was no relationship between age , grade and stage , and type of stomach cancer .
our data revealed that both diffuse and intestinal types of gastric cancer occurred significantly more in males than females ( table 1 ) .
the majority of our patients ( 69.6% ) were in grades ii and iii , which was the same as the stages , with 59.8% in stages ii and iii ( table 1 ) .
this study revealed that there was no association between age , gender , tumor location , necrosis , vascular invasion , perineural invasion , stages and type of gastric cancer , and the cish test .
our data showed that there was an indication of some correlation between grades and cish , although the difference was not significant .
our data also showed that cish+ patients ( 43.1% ) were more frequent compared to ihc+ patients ( 14.7% ) .
different amplifications by the cish test are shown in table 2 . according to our cish data
, 58 samples showed no amplification and among 44 samples with cish+ , 24 samples showed low amplification .
kappa : value agreement = 0.284 , percentage of agreement = 28.4% , p value = 0.0001 , statistically significant difference from zero .
immunoreactivity of myc was seen in 15 patients . in 13 patients both myc amplification and myc immunoreactivity
fifty - six samples had no amplification and were ihc negative . also among 44 positive samples for cish , 13 samples had positive signals for ihc and 31 samples had negative signals for ihc .
most of the ihc negative patients had no amplification yet only two ihc positive patients had no amplification .
the fourth group was cish- and ihc- ( 55% ) . amongst our four groups ,
the kappa test result was 0.284 , which indicated fair agreement between cish and ihc ( table 3 ) . to determine the association between cish and ihc testing
the correlation coefficient was -0.365 with a p value of 0.0001 , indicating that these two tests were related . in this investigation 43% of the samples were positive for cish , yet only 14.7% were positive for ihc .
the clinicopathological features are shown in table 1 . the location of the tumor was the fundus in 35.3% of patients , while the location was the cardia in only 3% .
table 1 shows that some of the variables including gender , location of tumor , necrosis , vascular invasion , perineural invasion , cish , and ihc had significant differences amongst the diffuse type , while gender , location of tumor , vascular invasion , necrosis , cish , and ihc had significant differences amongst the intestinal type .
this means that subgroups of each variable are different in the two types of gastric cancer .
these results also showed that there was no relationship between age , grade and stage , and type of stomach cancer .
our data revealed that both diffuse and intestinal types of gastric cancer occurred significantly more in males than females ( table 1 ) .
the majority of our patients ( 69.6% ) were in grades ii and iii , which was the same as the stages , with 59.8% in stages ii and iii ( table 1 ) .
this study revealed that there was no association between age , gender , tumor location , necrosis , vascular invasion , perineural invasion , stages and type of gastric cancer , and the cish test .
our data showed that there was an indication of some correlation between grades and cish , although the difference was not significant .
our data also showed that cish+ patients ( 43.1% ) were more frequent compared to ihc+ patients ( 14.7% ) .
different amplifications by the cish test are shown in table 2 . according to our cish data
, 58 samples showed no amplification and among 44 samples with cish+ , 24 samples showed low amplification .
kappa : value agreement = 0.284 , percentage of agreement = 28.4% , p value = 0.0001 , statistically significant difference from zero .
immunoreactivity of myc was seen in 15 patients . in 13 patients both myc amplification and myc immunoreactivity
fifty - six samples had no amplification and were ihc negative . also among 44 positive samples for cish , 13 samples had positive signals for ihc and 31 samples had negative signals for ihc .
most of the ihc negative patients had no amplification yet only two ihc positive patients had no amplification .
the fourth group was cish- and ihc- ( 55% ) . amongst our four groups , the kappa test result was 0.284 , which indicated fair agreement between cish and ihc ( table 3 ) . to determine the association between cish and ihc testing ,
the correlation coefficient was -0.365 with a p value of 0.0001 , indicating that these two tests were related . in this investigation 43% of the samples were positive for cish , yet only 14.7% were positive for ihc .
our cases included of 78 males and 24 females , with a male : female ratio of nearly 3:1 , and an average age of 60 , 62 .
our data were in agreement with another research , which gave a ratio of 2:1 and the majority of the patients were older than fifty - five ( 30 ) .
another study from iran was also in agreement with our results and mentioned that gastric cancer was the most common cancer in iranian males ( 31 ) . in this study ,
the tumor location was divided into seven subgroups and the majority of our patients had a tumor in the fundus location .
if we divided our patients into two groups of cardia and non - cardia , we would have only 3% cardia and 97% non - cardia .
one research studied gastric cancer among 125 samples , with only two subgroups of cardia and non - cardia , and found that 58.4% of the samples were in the non - cardia subgroup , which is in agreement with our results ( 30 ) . the majority ( 59.9% ) of our patients had the diffuse type , yet some authors reported intestinal type more than the diffuse type ( 30 - 32 ) .
the best explanation for this difference is that their samples were from brazil and japan , both being high - risk areas , yet our data was from tehran , which is considered a low - risk area .
it has been already documented that intestinal type of gastric adenocarcinoma was more frequent than the diffuse type in high - risk countries ( 33 ) .
some authors have reported an association between overexpression of c - myc and over 50% of cancers in humans , and also the effect of this overexpression on invasiveness and worse prognosis in patients .
contribution of myc to tumorigenesis by persuading reinless cellular growth , angiogenesis , proliferation , and genomic instability has been already documented ( 34 ) .
it was suggested that the c - myc constitutive expression owing to amplification , mutation , or chromosome translocation involves the development and progression of various cancers ( 35 ) .
c - myc persuades point mutations , imperfect replication initiation , dna breakage , changes of dna repair , and causes remodeling of the 3d nuclear structure of telomeres and chromosomes , thus leading to topological positions that initiate genomic instability ( 36 ) .
most of the patients in different studies were at the early stage of gastric cancer and from high - risk areas , such as japan and korea .
however , another author showed higher myc amplification in the diffuse - type , than the intestinal - type , which agreed with our results ( 28 ) .
one study of ihc analysis revealed that myc expression was more frequent in the intestinal - type than the diffuse - type ( 37 ) . on the contrary
association of myc over - expression with the proliferative phases of cells has been documented and this expression was shown only in proliferative phases of development , but not in quiescent or terminally differentiated cells ( 38 ) .
this result is similar to the findings of previous results , which revealed that myc expression is down regulated with cell differentiation , and also another investigation , which showed the myc expression was repressed in grade i gastric cancers , whereas there was a significant increase of myc protein expression in grades ii and iii ( 38 ) . in our investigation
however , some studies have shown high levels of myc protein expression at early stages ( 39 ) .
there was no significant difference in the percentage of cells with c - myc gene amplification between early ( pt1 ) and advanced ( pt2 - 4 ) stomach cancers ( 39 ) .
it was suggested that there was no association between c - myc gene amplification and differentiated / undifferentiated carcinomas ( 39 ) . however
, our data showed that there was an indication of some correlation between cish and grades , although the difference was not significant .
the range of myc overexpression was reported from 15.6% to 100% at the early stages of stomach cancer ( 40 ) .
however , in our study only 14.7% showed myc immunoreactivity ( ihc+ ) in both types , and 85.3% had no expression .
the probable reason is that all of our patients were randomly selected from different stages .
the most usual mechanism of deregulation of myc in stomach cancer is myc amplification ( 41 ) .
this mechanism leads to enhanced oncogenic products in quantities that exceed the transcriptional capacity ( 42 ) . in this regard
, we also observed three or more myc gene copies ( dots ) in 43% of gastric tumors among our patients .
therefore , this investigation was not in agreement with previous studies ( 27 , 28 , 43 )
. the probable cause would be that all of our samples were from different stages ( mostly late stage ) , yet in the previous two studies , the samples were only from early stages .
our study also showed that amplification of c - myc in the diffuse type was more frequent than in the intestinal type , which was not in accordance with some previous reports ( 28 , 43 ) . also our study revealed no correlation between age , gender , tumor location , necrosis , vascular invasion , perineural invasion and type of gastric cancer , and cish test
one study from china revealed that any relationship between myc amplification and clinicopathological characteristics ( grade , stage , lymph node metastasis , tumor location ) in gastric cancer is correlated with ethnicity ( 44 ) .
however , due to the fact that our study could not find any relationship between myc amplification and clinicopathological characteristics , it is possible to report that the ethnicity of iranian patients afflicted , might lead to no relationship between these two parameters .
although we observed c - myc amplification ( cish- ) and ihc+ in only two patients , it seems that this may have been an artifact .
overall , 30.3% of our patients showed c - myc amplification ( cish+ ) , yet the protein was not expressed ( ihc- ) ( table 3 ) .
the possible explanation would be that a mechanism might have degraded the protein or the mrna ( 45 ) .
our data showed a correlation between cish and ihc , and the percentage of positive cish and ihc were 43% and 14.7% , respectively .
some authors discussed on the molecular therapeutic targeting of myc and showed that myc inactivation suppresses tumors in animals ( 37 ) .
in other words , specific knockdown of c - myc may decrease the growth and proliferation of gastric cancer cells , therefore , c - myc could be a possible target in gene therapy ( 46 ) . | background : the incidence rate of gastric cancer in western countries has shown a remarkable decline in the recent years while it is still the most common cancer among males in iran .
the proto - oncogene myc , located at 8q24.1 , regulates almost 15% of human genes and is activated in 20% of all tumors .
the amplification of myc and overexpression of its protein product are observed in 15 - 30% of gastric neoplasias.objectives:the objective of this study was to find the preferences of chromogenic in situ hybridization ( cish ) and immunohistochemistry ( ihc ) in diagnosis and prognosis of gastric cancer.patients and methods : we studied 102 samples of gastric cancer in iran and all the patients had undergone primary surgical resection at the cancer institute hospital , tehran university of medical sciences .
the cish and ihc techniques were applied for all our samples .
all of the samples had adenocarcinoma gastric cancer and were selected randomly . also
, the type of study was cross sectional .
the sample size was 100 patients.results:our data revealed that both diffuse and intestinal types of gastric cancer occurred significantly more in males than females .
our results showed that there was an indication of some correlation between grades and cish , although the difference was not significant .
our data also showed that cish positive patients ( 43% ) were more frequent compared to ihc positive patients ( 14.7% ) .
there was a correlation between cish and ihc .
these results revealed that there was a significant difference between grades and ihc .
there was also no statistical difference between cish amplification in diffuse and intestinal types.conclusions:from the results , it could be concluded that for administration of the treatment of stomach cancer , and progress and prognosis of tumor , which is important for patients and clinicians , the cish is a better and more feasible test than ihc , in regards to sensitivity and specificity . |
november 2014 , a total of 20 suspected cases of illness were clinically diagnosed as dengue in wenzhou ( 5 cases ) and wuhan ( 15 cases ) .
the cohort comprised 14 male and 6 female patients 761 ( median 31 ) years of age . of the patients from wuhan ,
11 had recently traveled to guangdong province ; 3 had recently returned from indonesia and 1 from thailand after > 1 year away from china .
similarly , 3 of the patients from wenzhou had traveled recently in fujian , thailand , and surinam . however , there was no evidence of recent travel to endemic regions for the remaining 2 patients , including a 7-year - old boy .
close contacts of these patients denied recent travel to endemic regions , suggesting autochthonous dengue virus transmission in wenzhou .
blood samples from each of the 20 patients were collected on day 1 of hospitalization ( 24 days after onset of fever ) , and were tested for denv igm by a non serotype - specific dengue dual igm- and igg - capture elisa kit ( panbio , windsor , nsw , australia ) .
elisa results showed 16 serum samples were positive for dengue - specific igm and 3 for dengue - specific igg . although the remaining 4 serum samples were negative for dengue - specific igm , we amplified denv sequences from them , as described in the next section
. results of routine microbiologic examinations for bacteria by culture and antigen detection were negative in all cases , as were serologic and genetic tests for hantaviruses , phleboviruses , and rickettsiales bacteria , performed as described ( 10 ) .
the 20 dengue case - patients showed a variety of clinical symptoms ( table ) : high fever ( 100% ) , headache ( 100% ) , dizziness ( 45% ) , myalgia ( 50% ) , nausea and vomiting ( 40% ) , rash ( 40% ) , and petechiae ( 25% ) .
in addition , chills , arthralgia , anorexia , enlarged lymph nodes , cough , and diarrhea were observed in some patients , and most displayed leucopenia ( 60% ) and thrombocytopenia ( 65% ) . however , none showed plasma leakage , severe bleeding , or severe organ involvement .
viral rna in blood samples from individual patients was detected by reverse transcription pcr based on the conserved regions of the e gene ( 11 ) .
consequently , dengue viral rna was recovered in serum samples from 4 travel - associated patients with dengue ( 1 from wenzhou and 3 from wuhan ) within 6 days after onset of disease , but not in the remaining serum samples . by using 24 pairs of primers ,
complete genome sequences were amplified successfully from the serum samples of 4 patients , all of which were characterized as denv-1 ( technical appendix figure ) .
the complete genome of all 4 viruses was 10,703 nt , and the isolates showed very high ( 99.9% ) sequence identity to each other .
using the maximum - likelihood method implemented in phyml v3.0 ( 12 ) , we estimated phylogenetic trees based on the complete e gene or whole genome sequences of the 4 viruses identified in china and reference sequences from genbank ( figure 2 ; technical appendix figure ) .
as expected , the viruses we identified are most closely related to those isolated in guangdong province in 2014 , indicating they are part of the same outbreak , although with independent incursions into wenzhou and wuhan .
the remainder of the phylogenetic trees show a mix of viruses from china and the indian subcontinent ( india , bangladesh , sri lanka ) , indicative of the movement of viruses among these localities , as well as a small number from singapore .
however , because denv sequences were only recovered from 4 patients , our molecular epidemiologic analysis was limited in scope , making extensive viral sampling necessary to reveal detailed transmission routes .
phylogenetic analysis of a subset of dengue virus 1 e gene sequences within genotype iii that are most closely related to those sampled from wenzhou , zhejiang province , and wuhan , hubei province , china , during 2014 .
the viruses identified in this study were designated as the wenzhou - human and wuhan - human sequences , respectively ( genbank accession nos .
dengue has been relatively commonly reported in china , mainly in the southern provinces ( 4,13 ) .
although the sustained transmission of denv is possible in these localities , many cases appear to have resulted from importation from countries in southeast asia ( 8,13,14 ) .
in contrast , denv has been sporadically reported in other regions of china , and those cases have been strongly associated with importation ( 68 ) .
epidemiologic , serologic , and virologic investigations all confirmed the presence of dengue in wenzhou and wuhan , even though dengue has not been reported from either region for several decades .
although 90% of patients had a recent history of travel to dengue - endemic areas within and external to china , 2 patients from wenzhou had no recent travel history to regions in which dengue was endemic , suggesting the occurrence of autochthonous transmission .
although all 4 denvs have been identified in china in recent years , denv-1 appears to be the most common ( 4,5,13 ) and was observed in this study ( figure 2 ) .
these viruses were most closely related to those from guangdong province , where the greatest number of cases were identified during the 2014 epidemic ( figure 1 ) .
the viruses in this study were most closely related to those from the indian subcontinent . although india likely has the highest dengue incidence globally ( 15 ) and is therefore expected to harbor high levels of genetic diversity , the viruses endemic to india were identified > 3 years before those found in china .
hence , although it is possible that the denv-1 viruses in china originated in india and made multiple incursions in recent years , limited sampling in other localities , notably parts of southeast asia , mean that the exact origins of the viruses found in china remain uncertain . finally , sequences recovered during this study from wenzhou and wuhan and from guangdong province in 2014 are very closely related to a virus isolated in guangdong province in 2013 that is likely to be related to the 2014 cluster .
although little is known about this latter virus , it will be critical to determine whether the 2014 epidemic directly arose from local ancestors present in 2013 , rather than being imported .
this and previous studies ( 6,8 ) highlight the increasing risk that denv - infected travelers may pose to public health in china . in humid subtropical regions such as wenzhou and wuhan ,
in which a. albopictus mosquitoes circulate with often poor control measures , imported dengue viruses may infect vector populations during permissive climatic conditions .
comprehensive strategies should be used to prevent the circulation of denv among local aedes mosquitoes .
technical appendix . phylogenetic tree of whole genome sequences of dengue virus 1 isolated in the cities of wenzhou , zhejiang province , and wuhan , hubei province , china , 2014 . | in 2014 , 20 dengue cases were reported in the cities of wenzhou ( 5 cases ) and wuhan ( 15 cases ) , china , where dengue has rarely been reported .
dengue virus 1 was detected in 4 patients .
although most of these cases were likely imported , epidemiologic analysis provided evidence for autochthonous transmission . |
overweight and obesity are serious health problems [ 1 , 2 ] which have increased during the past decades [ 1 , 3 ] in the us , in other
they tend to cause health damages with potentially immense expenditures [ 5 , 6 ] .
an appropriate definition of overweight and obesity is essential to the public health community for designing and monitoring interventions .
thresholds . currently , the body mass index bmi is used for defining overweight and obesity .
the bmi , given in units kg / m , has the dimension of an area density , the average value of which , for normal , healthy subjects , has so far been recommended as 21.7 [ 2 , 6 ] . in the past , also the broca formula was used , a linear mass versus height relationship , which implies a linear density ( kg / m ) , which may be relevant for describing a hair , but not a human body .
the bmi has been recommended for use among all age groups from childhood through adulthood . analyzing the relationship between bmi and adiposity , revicki and israel state
... there exists considerable error associated with the prediction of body fat , using different bmis .
overweight is defined by a bmi between 25.0 and 29.9 and underweight by a bmi below 18.5 .
many physicians and patients find it ( the bmi ) difficult to interpret , a useful body mass relationship , indeed , should have dimensions relevant to the human body , a volume density or pressure .
the bmi has been used for more than three decades by now , but the fraction of overweight and adipose people is still increasing dramatically .
this may be due to the fact that people just do not care . moreover , the bmi formula may be inapplicable for all individuals from birth to old ages .
this is the central issue of this study . in order to clarify these issues ,
these cohorts are ( a ) individuals between 8 years and 76 years of age , mainly adults and ( b ) four age cohorts of children from birth to the age of 14.5 years .
the results indicate on the one hand that the bmi is less accurate than the bsi in individuals older than ~8 years and taller than ~1.3 m. on the other hand , the bmi is more accurate than the bsi for children below the age of ~8 years and shorter than ~1.3 m ( bmi underweight limit 14.4 , overweight limit 17.4 ) .
these bmi values for children are significantly lower than the conventionally used bmi given above .
international standards require that masses are measured in kilogram ( kg ) , lengths or heights in meter ( m ) , and forces or weights in newton ( n ) .
thus , the bmi clearly has the dimension of an area density , which is common , for instance , in characterizing paper . when interpreting the bmi not as body mass index but as body weight index
, its dimension becomes weight / area , which is pressure and that is relevant to the human body .
since the bmi is numerically equal to the bwi , we use the bmi in this paper to be synonymous with bwi , unless stated otherwise .
we also refer to the fact that humans can swim . with air - filled lungs humans can float because the average body density is slightly less than the density of water . without air in the lungs people drown .
the body density varies during breathing by about 2 - 3% . without considering anatomical details ,
the average human body density is ~l000 kg / m . for finding an appropriate relationship between body mass w and body height h
, we apply the model of scaling , the elementary rule of magnification of a three - dimensional body : if the height of a body for example , a sculpture is changed by a factor of x , then also its width and thickness must change by the same factor . because of the nearly constant density of the human body , this immediately leads to the relation w / h = constant , with the dimension kg / m .
if shapes change , as in children developing from babies to adults , the scaling model is no longer valid .
therefore , the bsi obtained in adults and recommended to describe the normal mass - versus - height relationship is expected to mislead when applied to younger children .
the meaning of other exponents ( like 2 in the bmi or 1 for the broca formula ) must be questioned . for these exponents ,
there exists not even a theoretical model but only an arbitrary advance , unless one uses the bwi , as explained above .
because of the complexity of the human body , one should not expect that a simple approach , like with the bsi or the bmi as single parameters ( like 21.7 for the bmi , and 12.36 for the bsi ) is able to describe accurately the relationship between body mass and body height from birth until death .
a useful body mass versus body height relationship should be not only simple but also allow for differences by age , gender , and ethnicity .
this study , thus , presents a critical and numerically careful test of the usefulness of the bmi and the bsi ( models ) in order to determine the relevant parameters and their uncertainties in cohorts of normal weight individuals .
the body height can be measured with a precision of better than 0.2% and the body mass to less than 1% .
this is much better than needed , because the bmi values , for instance , for healthy or normal weight people range between 18.5 and 24.9 , with the average of 21.7 15% .
where scaling does not apply , for instance , for small children , we have used a sufficiently large sample of healthy individuals and measured their heights and weights in order to obtain an adequate data base for our analysis . as people are not equal genetically or in their environmental situations and as considerable differences exist regarding their bodily proportions , one expects a cloud of points in the mass versus height diagram ( figure 1 ) . the task here is then to determine how the bmi and bsi approaches mathematically fit the individual data points . for that purpose
, we define the bmi value for each individual as ami = wi / hi and the corresponding bsi values as asi = wi / hi
. then we apply a least squares fit to all data points , both for ami and asi .
we use masses and body heights of individuals ( tables 1 , 2 , and 3 ) for comparing bmi with bsi . for such tasks ,
decades ago investigators restricted their analyses to average heights and weights because of the ease of calculation .
flegal importantly noted the difference between younger and older men and women , as was also emphasized by shreeve and pierson . in view of the natural changes of normal bodies with growing and ageing
( healthy young subjects usually have less body fat than healthy older subjects ) , it seems appropriate to follow flegal .
we propose to extend this approach to observe the effects of age and gender , of ethnic , genetic , and other distinctions on the bsi and bmi values .
the emphasis of this paper is to clarify the applicability of the bmi and bsi and thus to provide a solid bases for these studies .
in a first step , we have taken data of 20 healthy and normal weight people listed in table 1 , all ethnically caucasions .
the two dashed lines depict the 18.5 kg / m and 24.9 kg / m bmi curves , the lower and upper limits for normal as determined by others [ 2 , 6 ] .
for the 20 healthy people , data points relate individual masses in terms of weights and heights , as listed in table 1 .
the crosses identify people from 8 to 32 years , open circles people between 36 and 57 years , and full circles people from 58 to 76 years .
the scatter also explains the difficulty of finding a correct relationship between body height and body mass .
the crosses , open circles , and full circles even provide evidence for an age dependence of the human weight in support of flegal and shreeve and pierson .
moreover , people who have masses in agreement with the bsi appear bmi - overweight if they are tall and bmi - underweight if they are short .
this results from the mathematical differences in slopes of the bmi and bsi curves . to find out whether and where bsi or bmi is superior for classifying the relationship between observed weights and heights of a group of individuals , we have used the individual values of the 20 healthy people listed in table 1 to calculate each individual bmi and bsi . as introduced above
, we express the individual bmi by the symbol ami = wi/(hi ) and the individual bsi by the symbol asi = wi/(hi ) , with the subscript i specifying the individual . with standard error analyses ,
the advantage of the standard error analysis is that it provides the standard deviations ( uncertainties ) (as ) and (am ) of as and am , and also the mean uncertainties (asi ) and (ami ) for the scatter of the individual data points .
we list only the numerical values , omitting the dimensions , and obtain :
( 1)as=12.36 , (as)=0.30 , (asi)=1.4 ,
( 2)am=21.48 , (am)=0.74 , (ami)=3.3 .
(as ) is 2.4% of as , and (am ) = 3.4% of am .
for am we obtained 21.48 , which is only 1% and thus insignificantly smaller than the average bmi value of 21.7 [ 2 , 6 ] .
this shows that our sample average is in very good agreement with the bmi average , determined by others [ 2 , 6 ] .
even our small sample indicates that the bsi approach yields standard deviations which are a factor of ~1.4 smaller than the bmi approach .
the scattering of the data clouds ( (asi ) and (ami ) ) is 11% ( = 1.4/12.36 ) and 15% ( = 3.3/21.48 ) , respectively .
the latter corresponds extremely well with the distance of 15% of the bmi curves 18.5 and 24.9 from the mean at 21.7 .
however , the 11% suggest a more narrow range , for the bsi ( 15%/11% = 1.4 ) .
thus , the bsi model fits our data set ( table 1 ) better than the bmi .
one may ask whether a sample size of only n = 20 individuals is acceptable for assessing bmi and bsi .
(as ) is proportional to n. therefore , a factor 10 improvement in precision would have required a hundred times larger sample size , in our case 2000 .
answer 2 is that the scattering of the individual data , (asi ) , is practically independent of the sample size .
therefore , a sample size of 20 is sufficient for discriminating between bmi and bsi .
the data in figure 1 already show an age dependence . selecting the five youngest people in this group ( symbol y ) , we find
( 3)as , y=11.06 , (as , y)=0.39 , (as , yi)=0.9 .
the difference between as , y = 11.06 and as = 12.36 , as given above , is 1.3 and significant .
we find correspondingly for the 9 seniors ( symbol a ) :
( 4)as , a=13.05 , (as , a)=0.38 , (as , ai)=1.1 .
here the difference between as , a ( 4 ) and as , y ( 3 ) is ~2.0 and amounts to 3.7 standard deviations .
we also compute the analogous values for the bmi and obtain for the five younger people :
( 5)am , y=17.87 , (am , y)=1.4 , (am , yi)=3.2 .
it is worth noting that am , y is lower than the bmi value of 18.5 , the limit to bmi - underweight . for the 9 seniors , we find correspondingly
( 6)am , a=23.24 , (am , a)=0.79 , (am , ai)=2.4 .
the difference between am , a ( 6 ) and am , y ( 5 ) , that is , between 23.24 and 17.87 is ~5.4 .
thus , also the bmi approach shows clearly an age dependence . for a more detailed study of the body mass relationships
, we have analysed in addition data of 79 children , 42 girls ( table 2 ) and 37 boys ( table 3 ) .
these data have been determined by one of us ( sz ) during routine preventive medical checkups of healthy children with normal growth not selected for their weights , most of which appeared normal .
figure 2 shows the data points for the girls ( x ) and boys ( ) .
independently shown are the two conventional bmi curves of 18.5 for underweight and 24.9 for overweight , and the bsi curve of 12.36 . for the analysis , we have split the data of the total cohort into 4 age cohorts of both the girls and boys , with data listed in tables 2 and 3 , and obtained the following results :
cohort 1age group from birth1.0 year
( 7)girls : am=15.1 , (am)=0.4 , (ami)=1.4,as=24.0 , (as)=0.9 , (asi)=3.3.boys : am=16.9 , (am)=0.5 , (ami)=1.8,as=26.0 , (as)=0.9 , (asi)=3.0 .
age group from birth1.0 year
( 7)girls : am=15.1 , (am)=0.4 , (ami)=1.4,as=24.0 , (as)=0.9 , (asi)=3.3.boys : am=16.9 , (am)=0.5 , (ami)=1.8,as=26.0 , (as)=0.9 , (asi)=3.0 .
cohort 2age group above 1 year3.0 years
( 8)girls : am=15.6 , (am)=0.4 , (ami)=0.8,as=17.3 , (as)=1.0 , (asi)=2.2.boys : am=16.1 , (am)=0.5 , (ami)=1.3,as=17.2 , (as)=0.8 , (asi)=2.1 .
age group above 1 year3.0 years
( 8)girls : am=15.6 , (am)=0.4 , (ami)=0.8,as=17.3 , (as)=1.0 , (asi)=2.2.boys : am=16.1 , (am)=0.5 , (ami)=1.3,as=17.2 , (as)=0.8 , (asi)=2.1 .
am=16.1 , (am)=0.4 , (ami)=1.0,as=15.2 , (as)=0.6 , (asi)=1.6.boys : am=15.3 , (am)=0.6 , (ami)=1.4,as=14.5 , (as)=0.7 , (asi)=1.9 .
age group 3.1 years7.1 years
( 9)girls : am=16.1 , (am)=0.4 , (ami)=1.0,as=15.2 , (as)=0.6 , (asi)=1.6.boys : am=15.3 , (am)=0.6 , (ami)=1.4,as=14.5 , (as)=0.7 , (asi)=1.9 .
cohort 4age group 8 years14.5 years
( 10)girls : am=19.6 , (am)=1.0 , (ami)=3.7,as=12.9 , (as)=0.8 , (asi)=2.9.boys : am=18.1 , (am)=0.9 , (ami)=2.7,as=12.3 , (as)=0.5 , (asi)=1.3 .
age group 8 years14.5 years
( 10)girls : am=19.6 , (am)=1.0 , (ami)=3.7,as=12.9 , (as)=0.8 , (asi)=2.9.boys : am=18.1 , (am)=0.9 , (ami)=2.7,as=12.3 , (as)=0.5 , (asi)=1.3 . the bmi analysis for children in the youngest three age cohorts ( cohort 13 )
yields am values which are consistent with a single number of ~15.9 both for girls and boys .
as the children become older ( cohort 4 ) , the am for girls rises to 19.6 and for boys to 18.1 .
these am values are still significantly below the conventional average bmi value of 21.7 . however , regarding the bsi analysis in children of the first cohort , up to one year , the as was found to be 24.0 for girls and 26.0 for boys , which is much higher than the bsi for adults ( 12.36 ) . as the children grow older ( see cohorts 2 ) , the as value decreases to ~17.3 , being about equal for girls and boys . in the cohort 3 , children with ages between ~3 to ~7 years
however , the as in children of cohort 4 , ageing between ~8 and 14.5 years , is 12.9 for the girls and 12.3 for the boys .
these results agree with the bsi value of 12.36 that has been found for healthy weight adults .
this shows that the bsi based on scaling ( magnification ) underestimates the normal weights of small children .
the reason for this is easily explained by the differences in the body proportions typically for these children compared to those of adults .
the results of our analysis , separately for girls and boys , clearly show that for children older than ~8 years or taller than ~1.3 m , the bsi is preferable to the bmi for classifying normal and abnormal weights of children .
the ratios of s/m and si/mi , as given above , are on the average 1/1.6 , which implies lower ranges of uncertainties with bsi and thus its higher precision .
for the children of cohort 4 , that is , 25 girls and boys older than ~8 years and taller than ~1.3 m , we obtain for the bsi :
( 11)as=12.70 , (as)=0.47 , (asi)=2.3 .
the as - value , 12.70 , differs from 12.36 ( adult bsi ) by 0.34 , which is only 60 per cent of the combined errors of the bsi values of 12.70 and 12.36 .
this means that the as value of all 25 children older than ~8 years and taller than ~1.3 m agrees well with the reference bsi value of 12.36 for adults and is again indicative of the higher precision of the bsi compared with the bmi in this age group . however , for the individual cohorts 13 , children younger than ~8 years or shorter than ~1.3 , we have listed above the am values , which are close to ~15.9 . the standard error analysis of the whole group of 54 girls and boys ( cohorts 13 ) yields for the bmi :
( 12)am=15.9 , (am)=0.20 , (ami)=1.5 .
the am - value of 15.9 , being the average for the cohorts 13 with a total of 54 children , differs from the conventionally used bmi average of 21.7 .
this difference of 5.8 corresponds with 29 standard deviations of (am ) = 0.20 ( see above ) , the most significant discrepancy between the bmi here found for children and the conventionally used bmi .
the above equation ( 12 ) with am of 15.9 and (ami ) of 1.5 implies over- and underweight limits of 17.4 and 14.4 , respectively , and relative to 15.9 , a precision of 9.4% , in contrast to the conventional bmi which has a precision of ~15% .
note that over- and underweight limits are defined here as am (ami ) .
therefore , healthy short people would be lighter than what the conventional bmi suggests .
this agrees with what we find for people older than 8 years using the bsi , as discussed above . as the bmi has been recommended for use among all age groups from childhood through adulthood and as our children are
normal , the bmi values , in particular the bmi- underweight value of 18.5 , should be revised and not be applied for children below ~8 years of age . for children older than 8 years
, the bmi should be replaced by the bsi . the results , in summary ,
the conventional bmi appears less precise than the bsi for adults and individuals older than 8 years and should accordingly be replaced .
the bmi applies well for children younger than 8 years or shorter than 1.3 m provided it 's average is reduced from the conventional value of 21.7 to 15.9 with underweight and overweight limits being 14.4 and 17.4 , respectively .
applying standard data analysis on our first sample of 20 , mainly adult healthy individuals , we have observed that the bsi body mass equation yields parameters that are a factor of 1.4 more precise than those for the bmi .
the bmi analysis leads to an average body mass index ( 21.48 ) for our sample in excellent agreement with the bmi average of 21.7 .
our analysis of the data ( tables 2 and 3 ) on children demonstrates that the overwhelming part of them is bmi underweight .
this casts serious doubt on the usefulness of the bmi - underweight limit if applied to children .
for very young children , caution should be exercised when using the bsi because the body shape of these children can not be reproduced by scaling shapes of adults down to these children . for children older than ~8 years and taller than ~1.3 m
, the bsi values for girls and boys are in good agreement with 12.36 , the bsi value of the sample cohort with data listed in table 1 .
our analysis of 54 children with ages less than ~8 years or shorter than ~1.3 m shows that the bmi is the adequate body mass relationship with an average bmi value of 15.9 , albeit different from the corresponding bmi for adults .
moreover , the number 15.9 is less than the bmi - underweight limit . the analysis of the children data yields a bmi - underweight limit of 14.4 . as a first reasonable estimate
, we define underweight if the bmi value of a child is more than (ami ) = 1.5 less than 15.9 , which means : below bmi = 14.4 .
we observe that 9 of our 79 children , 5 girls and 4 boys , are in the category of underweight , ~ one sixth of them are in agreement with expectation for a normal statistical distribution .
similarly , we define overweight if the individual bmi value is more than (ami ) , above 15.9
interestingly , this would imply that we should consider children already as overweight if they are even 1.1 units below the standard bmi - underweight limit of 18.5 .
this obvious and serious discrepancy needs correction perhaps by revising the bmi recommendation in order to differentiate normal values for short or very young children from values for older , taller children plus adults .
there exist slide - rule - type circular plastic discs exforge , where body height and body weight can be matched by rotating one disc relative to the other , and then the bmi value can be read off . in a little window ,
the study of danish school children with 7 through 13 years of age by baker et al . has shown that higher bmi during childhood is associated with an increased risk of coronary heart disease in adulthood .
the very large sample size in the danish study ensures that these data are of high statistical relevance .
table 1 of the publication by baker et al . shows fourteen bmi values between 15.4 and 18.6 that are , with a single exception ( 18.6 ) lower than 18.5 , below which people are judged underweight , if one adopts the recommendation for use among all age groups from childhood through adulthood .
the bmi values of our children ( cohorts 13 ) are fully consistent with the data given by baker et al . in table 1 of their paper . furthermore , we find that the weights and heights of our children from 814.5 years can be well described by the bsi model that fits the data of adults better than the bmi does .
our study provides both bmi and bsi fits for children from birth up to an age of ~8 years .
shorter children may appear of normal or even subnormal weight when using the bmi as recommended so far , but they actually may be overweight as we have discussed above on the basis of our bmi analysis . using the bmi with the underweight limit of 18.5 as the only reference may influence parents of children with normal weight ( according to our study ) to feed them until they have normal weight with bmi = 21.7 , but actually then they are overweight .
an essential question is which bmi / bsi values signal a significant risk of a given age group not only for cardiovascular diseases in adulthood but also of other diseases associated with overweight .
this paper provides the answer . obviously , the most appropriate tools needed to be applied , bmi and bsi , at the appropriate ages and heights for correct prognostic values . the bsi formula is identical with that known as rohrer 's ponderal index ( pi ) [ 1618 ] used to evaluate perinatal problems in new - born infants or near - term foetuses who are small for gestational age , or who have intrauterine growth retardation .
the pi has been shown to be better than birth weight for prediction of short - term problems such as neonatal asphyxia , acidosis , hypoglycaemia , and hypothermia .
other studies [ 19 , 20 ] have used the pi to predict long - term comorbidities such as microalbuminuria , insulin resistance , hypertension , and other cardiovascular diseases . obviously , the bmi value for children , as shown above , supersedes the precision of the pi . the individually measured bmis , interpreted as body weight indices , are proportional to the pressure in all weight - bearing joints . when people with identical heights are compared , their joint pressures are proportional to their bmis , or simply to their masses or weights .
individuals with the same bsi have pressures in their weight - bearing joints proportional to their heights .
therefore , the shorter person who is lighter for a given bsi has an advantage .
the studies of bhargava et al . suggest that it would be valuable to measure the bmi for sufficiently many people , males and females , from birth on , preferably annually and in various regions all over the world .
the present study shows that the bsi is more precise than the bmi for people older than ~8 years or taller than ~1.3 m. our corresponding average bsi value is 12.5 with a limit to underweight around 11.1 and the limit to overweight around 13.9 . for children with ages less than ~8 years or shorter than ~1.3 m
, we find the bmi to be superior to the bsi , if the conventional bmi value of 21.7 is replaced by 15.9 as average value , bringing a reduction by 27 percent . for improving the am- and as - values and finding better underweight and overweight limits for bmi and bsi we strongly recommend a worldwide study of many subgroups for which individual
we propose that such studies evaluate the bmi and the bsi metric for achieving a higher predictive power for genetic and other variations in different world regions , consequential ranges of metabolic differences , and possible environmental effects .
such a global investigation could provide the most useful information about normal metabolism , the epidemiology of adiposity under the multiple and interacting influences of nutrition , exercise , race , and other measurable factors
. it would be interesting to include the bsi together with the bmi in the bode index .
for this future work , we therefore propose to study large samples , of healthy people in age categories , irrespective of height , separately for males and females .
additionally , one should simultaneously consider racial / ethnic and other distinctions and measure their weights and heights ; also the blood pressure appears of interest in this context . if scaling would apply also to human vascular system
, one could directly show the proportionality between bsi and blood pressure using the hagen - poiseuille - law .
overall , in doing so one may easily have to deal with 300 or more subgroups
preferably more than 2000 individuals , the fit will allow to find the mean numbers asj , the numerical bsi values for each group j. similarly numerical values should be obtained for the bmis .
as the fits also will yield the standard errors of the asj- and amj - values , the changes with age for each gender can be described quantitatively .
the results of such future studies should allow setting narrower and more accurate definitions for healthy weight than those presently achieved by the exclusive use of the bmi . with the bsi values , it should be possible to quantify overweight or adiposity more precisely than with the bmi alone , in people older than ~8 years or taller than 1.3 m. based on these a - values taken only for healthy weight , one should then consider all people , healthy and ill , assign each person to the relevant subgroup
j , and record , in addition to the weight and height of each person , clinical data including information about food intake in order to link a person 's body mass data with his or her clinical status .
gives healthy weights calculated with the bsi formula w = bsi h = 12.5 kg / m h for ages above ~8 years and heights above ~1.3 m , and with the bmi formula w = bmi h = 15.9 kg / m h for ages less than 8 years and heights below 1.3 m. for heights between the tabulated values hi and hi+1 , interpolation between the weights wi and wi+1 is sufficiently precise . in the future
in conclusion we find that the bsi with a mean of ~12.5 is a dimensionally correct body mass relationship for people older than ~8 years or taller than ~1.3 m and has a higher precision than the bmi .
if understood as body weight index ( bwi ) with the dimension of weight / area , the bmi expresses pressure and is relevant to the human body .
we recommend the bmi with a mean of 15.9 for children less than ~8 years and/or shorter than ~1.3 m ; this category includes babies .
it appears urgent to study the intrinsic causes of overweight and obesity with the most accurate tools .
the challenge is of great consequence to both the affected individual and society as a whole with burdens of large costs , both in money and public health efforts . | validation of body - mass relationships requires a careful statistical analysis of data of normal weight individuals .
bmi
( ratio between body mass and square of body height ) and bsi values ( ratio between mass and cube of body height ) have been calculated
for 99 persons with ages between 1 day and 76 years .
these bmi or bsi values have been used for least squares fits yielding mean bmi
or bsi values , their variances ( providing precision ) , and average deviations of individual bmi / bsi values from the bmi / bsi means .
the latter allows limits to over- and underweight .
for adults we found mean values of bsi of 12.36 and confirmed 21.7 for the mean
bmi ; but the bsi was 1.4 times more precise than the bmi .
for children shorter than 1.3 m and younger than 8 years we
found the bmi average of 15.9 and over-/underweight limits of 17.4/14.4 being significantly smaller than and incompatible with
the recommended bmi values . |
esophagorespiratory fistula ( erf ) is an uncommon condition , despite of the anatomical proximity of these structures .
malignancy of esophagus , lung , or mediastinum is recognized as the most common cause .
causes of erf development include direct tumor invasion and subsequent perforation or after radiation , laser therapy , chemotherapy , or pre - existing stents.1 benign erf is rare and may be due to trauma or infection.2 however , there have been few reports describing benign erf and its treatments in south korea . here
we report a case of nonmalignant esophagopleural fistula successfully treated by covered self - expanding metallic stents .
a 73-year - old man was admitted to chungnam national university hospital complaining right chest discomfort .
he had 20 pack - years smoking history but had neither an operation history nor a medical history such as diabetes , hypertension , pulmonary tuberculosis , or hepatitis .
physical examination was unremarkable except for diminished respiratory sounds in the whole right lung field .
the results of laboratory studies showed a white blood cell ( wbc ) count of 8,500/mm ; hemoglobin of 12.5 g / dl ; and platelet count of 164,000/mm .
the serum biochemistry presented na of 140 meq / l ; k 4.3 meq / l ; cl 107 meq / l ; aspartate aminotransferase 19.5 iu / l ; alanine aminotransferase 12.4
iu / l ; total bilirubin 0.9 mg / dl ; and lactate dehydrogenase ( ldh ) 246 iu / l ( all within normal ranges ) with hypoalbuminemia ( 2.8 g / dl ) and elevated serum creatinine level ( 1.65 mg / dl ) . chest x - ray revealed right pleural effusion ( fig .
pleural fluid analysis revealed ph of 7.0 ; red blood cell 3,200/mm ; wbc 4,800/mm ; total protein 4.0 g / dl ; and ldh 2,246
iu / l compatible with exudative parapneumonic effusion , for which an antibiotic therapy was initiated with chest tube catheter insertion ( fig .
stent insertion was considered as the first treatment due to the large fistula and pleuritis . a 14-cm long , 18-mm diameter covered choo stent (
three months after the stent insertion , the stent was easily removed by pulling removal snare with an alligator forceps ( fig .
erf is used to describe all fistulas located between the esophagus and the airway tree ; however , esophagotracheal and esophagobronchial fistulas mostly account for the remaining erf cases ( 3% to 11% ) of esophagopulmonary fistulas.3,4 accordingly , there have been very few previous reports concerning benign esophagopleural fistula and its treatment .
recent reports5 regarding malignant esophagopulmonary fistula other than esophagotracheal and esophagobronchial fistulas showed technically successful stent placement outcome in 14 patients and complete fistula sealing in 12 patients ( 86% ) .
known causes of acquired benign erf include trauma and infection.6 - 8 the former lesions may be due to the increase in fistula caused by surgical procedures , blunt chest trauma , and prolonged ventilator assistance .
other reported infectious causes include syphilis , mycotic disease , and crohn 's disease . in our case , the cause of the esophagopleural fistula was unclear .
also a specific infectious agent was not isolated at the time of diagnosis and treatment .
leaks of the esophagus are associated with a high mortality rate and need to be treated as soon as possible .
therapeutic options include surgical repair or resection or conservative management with antibiotic therapy and cessation of oral intake .
endoscopic techniques to overcome this issue have been reported with obliterating agents , such as fibrin glue or cyan acrylic glue,9 - 11 endoscopic clip application,12,13 and endoscopic suturing device.14,15 recently several types of covered and uncovered stents have been used in the treatment of erfs .
self - expanding metal stents ( semss ) have been considered a safe and effective treatment for malignant dysphagia stricture , and fistula in inoperable patients.16 primarily sems has been used to palliate malignant state but are less commonly used in benign state due to concerns about their nonremovability and high complication rate .
recent technical improvement overcame these limitations . to evaluate fistula closure and stent patency or migration , repeat esophagogram at 1 week , then every 1 to 2 months after the procedure is suggested . in our case , the cause of the esophagopleural fistula was unclear .
therefore , it appears that implantation of membrane - covered metal stents is an effective alternative for the treatment of esophagopleural fistula . | the most common cause of esophagorespiratory fistulas ( erfs ) is associated with malignancy .
the use of self - expandable metal stents is effective for the treatment of malignant erfs , but benign erf is rare , which is why its optimal treatment is not defined yet . there have been few reports describing benign esophagopleural fistula and its treatments in south korea . here ,
we report a rare case of spontaneous esophagopleural fistula , which was successfully treated by endoscopic placement of a membrane covered metal stent . |
sjgren 's syndrome is a slowly progressing autoimmune disease characterized by lymphocytic infiltration of the exocrine glands , mainly the lacrimal and salivary glands , resulting in impaired secretory function .
simultaneously , systemic features of cutaneous , respiratory , renal , hepatic , neurologic , and vascular nature often occur .
the syndrome can present either alone ( as primary sjgren 's syndrome ) or in the context of underlying connective tissue disease ( as secondary sjgren 's syndrome ) .
renal involvement is well - recognized extra glandular manifestation of primary sjgren 's syndrome ( pss ) .
most common manifestations are related to tubular dysfunction resulting from chronic interstitial nephritis and can manifest as distal renal tubular acidosis ( rta ) , proximal rta , tubular proteinuria , and nephrogenic diabetes insipidus .
hypokalemic paralysis rarely occurs as the first manifestation of a renal tubule disorder due to pss .
we herein present two cases who presented to us with hypokalemic paralysis , secondary to pss .
a 19-year - old unmarried lady presented to our hospital for evaluation of two episodes of generalized weakness .
both the episodes were acute in onset , with weakness of all four limbs and neck muscles , and occurred during recovery from febrile illness .
meq / l ) on both the occasions and complete recovery occurred in a span of 48 h on receiving intravenous and oral potassium .
there was no history of fever , joint pain , skin rash , photosensitivity , or parotid swelling .
there is no family history of a similar illness . at presentation to us , she had normal general physical and neurological examination .
laboratory parameter of patients a diagnosis of distal rta was made in view of an alkaline urinary ph ( > 5.5 ) in the setting of metabolic acidosis .
she was evaluated for etiology of distal rta , which revealed positive rheumatoid factor ( rf ) and antinuclear antibody ( ana ) , while serum c3 level was normal and anti - dsdna was negative .
schirmer 's test was positive in both eyes and her serum anti - ro and anti - la antibody levels were unequivocally elevated .
she was discharged on oral potassium and alkali supplementation . a 22-year - old married lady presented for evaluation of an episode of acute onset flaccid quadriparesis without any sensory , bladder , or bowel involvement .
the episode occurred 15 days before she presented to us . at the time of episode
she had been taking oral potassium supplementation regularly since then and did not develop any further episodes of weakness .
there was no history of fever , joint pain , skin rash , photosensitivity , or parotid swelling .
there is no family history of a similar illness . her general and systemic examination including neurological examination was normal .
bicarbonate loading test ruled out an associated proximal tubular dysfunction . on working up for the etiology of distal rta
, she was found to have elevated titer of rf and ana , her serum c3 level was normal , and anti - dsdna was negative .
a 19-year - old unmarried lady presented to our hospital for evaluation of two episodes of generalized weakness .
both the episodes were acute in onset , with weakness of all four limbs and neck muscles , and occurred during recovery from febrile illness .
meq / l ) on both the occasions and complete recovery occurred in a span of 48 h on receiving intravenous and oral potassium .
there was no history of fever , joint pain , skin rash , photosensitivity , or parotid swelling .
there is no family history of a similar illness . at presentation to us , she had normal general physical and neurological examination .
laboratory parameter of patients a diagnosis of distal rta was made in view of an alkaline urinary ph ( > 5.5 ) in the setting of metabolic acidosis .
she was evaluated for etiology of distal rta , which revealed positive rheumatoid factor ( rf ) and antinuclear antibody ( ana ) , while serum c3 level was normal and anti - dsdna was negative .
schirmer 's test was positive in both eyes and her serum anti - ro and anti - la antibody levels were unequivocally elevated .
a 22-year - old married lady presented for evaluation of an episode of acute onset flaccid quadriparesis without any sensory , bladder , or bowel involvement .
the episode occurred 15 days before she presented to us . at the time of episode
she had been taking oral potassium supplementation regularly since then and did not develop any further episodes of weakness .
there was no history of fever , joint pain , skin rash , photosensitivity , or parotid swelling .
bicarbonate loading test ruled out an associated proximal tubular dysfunction . on working up for the etiology of distal rta
, she was found to have elevated titer of rf and ana , her serum c3 level was normal , and anti - dsdna was negative .
pss is a disease of exocrine glands presenting with manifestations related to dry eyes and dry mouth .
nonexocrine organ systems may also be involved , including skin , lung , gastrointestinal tract , central and peripheral nervous system , muscular skeletal apparatus , and the kidney .
the reported rate of renal involvement in pss in literature is variable ranging from 4.2% to 50% .
the spectrum of renal disease includes interstitial nephritis , which can manifest as distal rta , proximal rta , tubular proteinuria , nephrogenic diabetes insipidus , glomerular diseases , or renal failure .
the most common manifestations are related to tubular dysfunction which results from chronic interstitial nephritis .
. the mechanisms of distal rta - induced hypokalemia include decreased distal tubular na delivery , secondary hyperaldosteronism , defective h - k atpase , and bicarbonaturia .
although hypokalemia is frequent sequel of rta , a severe symptomatic decrease in serum potassium concentration has been described in a few cases only .
subsequently , approximately 25 more cases has been reported.[821 ] here , we present a report of two cases that presented to us with hypokalemic paralysis secondary to distal rta , which on further work up , was found secondary to pss . both of our patients did not have any evidence of proximal tubular dysfunction . in conclusion ,
the kidney involvement in pss can uncommonly present as hypokalemic paralysis in the absence of significant sicca symptoms or may precede sicca symptoms .
sjgren 's syndrome should be instigated in any patient presenting with hypokalemic paralysis from rta , even in the absence of the sicca syndrome . | primary sjgren 's syndrome ( pss ) is a chronic autoimmune disease characterized by a progressive lymphocytic infiltration of the exocrine glands with varying degrees of systemic involvement .
overt or latent renal tubular acidosis ( rta ) , caused by tubulointerstitial nephropathy , is a common extraglandular manifestation of pss .
hypokalemic paralysis is a well known , albeit rare complication of severe distal rta from any cause .
cases of pss manifesting for the first time as hypokalemic paralysis caused by distal rta have been rarely reported .
we herein present our experience of two cases , who presented to us for evaluation of hypokalemic paralysis and on work up found evidence of distal rta , which on further work up found to be secondary to pss .
a high index of suspicion for pss should be kept in all patients with hypokalemic paralysis . |
a 59-year - old
female receiving alemtuzumab for chronic lymphocytic leukemia was admitted to a
local hospital near her home in eastern kentucky in october 2007 for altered
mental status of two weeks duration .
admission laboratory values included a white blood count of 5 900/mm ,
hemoglobin of 10.4 g / dl , platelets of 120 000/mm , but absence of
eosinophilia .
there was evidence of
hypoalbuminemia with serum albumin of 2.4 g / dl .
chest roentgenogram revealed right upper lobe infiltrate , and
computerized tomography of the head showed only maxillary and ethmoid
sinusitis .
however , bloody cerebrospinal
fluid ( csf ) was found with a nontraumatic lumbar puncture ( lp ) with 200 000
red cells / mm , 172 white cells / mm , and protein of 750 mg / dl .
csf studies including herpes simplex virus ( hsv ) polymerase chain reaction ( pcr ) , cryptococcal
neoformans antigen , and bacterial culture were negative . despite broad - spectrum antibiotics , the
patient encephalopathy and pulmonary infiltrates did not improve , and
she was
transferred to a tertiary hospital in lexington , ky , usa . neurologic findings at the time of transfer included a glasgow coma score of five , with no localization to pain .
sputum analysis on
admission revealed 1 + white blood cells , gram - positive cocci , budding yeast , and strongyloides stercoralis was
identified in the filariform stage by gram 's stain .
oral ivermectin was started at 15 grams as a single daily dose ( ~147 mcg / kg ) via nasogastric tube [ 15 , 16 ] .
the following day , the ivermectin dose was
increased to 30 grams per day ( 293 mcg / kg ) for three weeks to account for the
patient large body habitus ( 102 kg ) and questionable ileus .
lps 6 days apart revealed 9 00 and 6688 red
cells / mm , respectively , and xanthochromic csf from the second
lp .
csf studies , performed as described elsewhere , for cytomegalovirus ( cmv ) pcr , hsv pcr , epstein - barr virus ( ebv ) , jc virus ( jcv ) pcr ,
cryptococcal antigen , fungal culture , acid - fast stain and culture , and vdrl were
all negative .
stool specimens
demonstrated rhabditiform larvae , eggs , and adult s. stercoralis ,
with filariform larvae in the sputum for one week
after initiation of ivermectin .
the stronglyoides antibody was
0.13 iv ( index value ) which was negative by laboratory reference values ( < 1.49
considered negative ) .
lack of neurologic
improvement and persistence of s.
stercoralis from the sputum led to concern for malabsorption of oral
ivermectin .
discussion with the food and
drug administration ( fda ) for permission to use parenteral veterinary
ivermectin led to the measurement of plasma and csf ivermectin concentrations
in collaboration with the university of iowa by high - performance liquid
chromatography .
the ivermectin
plasma and csf concentrations were 49.3 ng / ml and 0.14 ng / ml , respectively , on
day 10 of oral ivermectin therapy .
repeat microscopy of sputum after three weeks of therapy did not reveal
organisms . despite 22 days of oral
therapy , the patient showed no sign of neurologic recovery . after discussion and permission from the
surrogate decision maker ,
this report describes an immunocompromised patient with disseminated
strongyloides presenting to a community
hospital with two weeks of altered mental status . on transfer to a tertiary
hospital , repeat
lp confirmed evidence of hemorrhagic csf with persistent
elevation in csf red blood cells and xanthrochromia .
s. stercoralis organisms were observed
on sputum gram stain , which was subsequently confirmed in stool and additional
sputum samples . despite immediate
institution of ivermectin after transfer to the tertiary care center , the
patient did not improve neurologically and expired after being transitioned to
comfort care .
the patient may have died
from subarachnoid hemorrhage and advanced encephalitis secondary to
disseminated strongyloidiasis .
the hemorrhagic csf and
xanthochromia were likely secondary to capillary rupture or local inflammation
from the host immune response .
previous neuropathology has demonstrated the
presence of the strongyloides larvae within capillaries ,
meninges , and brain tissue in a case of disseminated strongyloidiasis
after administration of corticosteroids . as in this patient ,
amajor
difficulty in disseminated strongyloidiasis is overcoming the delay in
diagnosis both because of delayed presentation for medical evaluation and the
vague symptoms that accompany disseminated disease .
however , pulmonary and
gastrointestinal abnormalities with an infiltrate and ileus in an
immunocompromised host should
warrant consideration of the diagnosis .
eosinophilia may be mild and nonspecific and because of low parasite
load and irregular larval output .
microscopy of a single stool specimen not surprisingly fails to detect
larvae up to 70% of the time .
strongyloides antibody may show
cross - reactivity with other helminth infections including filariasis ,
ascaris lumbricoides , and acute
schistosomiasis [ 21 , 22 ] .
while serology
has a negative predictive value of 95% , our patient had a negative antibody
test for strongyloides despite
microscopic evidence of pulmonary and gastrointestinal diseases .
it is possible that false - negative serology
in our patient was related to immunosuppression from either her hematologic
malignancy or chemotherapy .
while delay in
diagnosis may have contributed to the poor
outcome , inadequate central nervous system penetration of ivermectin or
ivermectin neurotoxicity may have played a role .
bowel obstruction and ileus are
commonly described in hyperinfection syndrome , and intestinal inflammation , and
bowel edema from penetration of strongyloides may
impair absorption . in this situation the
absorption of oral medications is unclear , and determination of ivermectin
concentrations appears useful to assess adequacy of oral treatment .
ivermectin is highly bound to
serum albumin , and lower levels of albumin can increase clearance of unbound drug
as well as additional effects of contribution to tissue edema which can slow
absorption and increase the volume of distribution . increased clearance or volume of distribution may interfere with
achievement of effective levels in the central nervous system .
we were able to
achieve a relatively high ivermectin plasma level in our patient , with a daily
dose of 293 g / kg .
the plasma
concentrations in our patient were similar to those observed in healthy
volunteers following multiple oral doses of 60 mg given on days 1 , 4 , and 7 .
because the concentrations were similar to those observed in healthy
volunteers after multiple oral doses , we concluded that there was adequate intestinal
absorption and we did not further pursue use of parenteral ivermectin . despite the higher
oral ivermectin dose and achievement of plasma concentrations that were higher
than circulating ivermectin plasma levels in previous patients ,
therefore , establishing cns penetration by
measuring adequate csf concentrations may be important in the prediction of
outcome . at steadystate ,
the csf
concentration in our patient suggested less than 1% csf penetration of orally
administered ivermectin .
after five daily subcutaneous ivermectin
doses of 200 g / kg , there were no detectable levels in the csf using the same
assay methodology used in this study .
the
plasma levels measured after the initial dose and the seventh daily dose were
5.8 ng / ml and 12.1 ng / ml , respectively , and at 14 days steady - state plasma
concentrations
were 11.417.4 ng / ml .
lastly ,
it seems unlikely that neurotoxic side - effects of ivermectin contributed to the
poor outcome of the patient .
adverse
neurologic effects include mydriasis , ataxia , tremors , emesis , lethargy , and
coma [ 13 , 2426 ] . however , our patient was comatose prior to and after three
weeks of therapy .
moreover , healthy volunteers have tolerated single doses of
2 000 g / kg , and doses up to 1 091 g / kg given three times at 72-hour intervals
without evidence of toxicity .
when orally
administered ivermectin therapy fails to produce a clinical response , alternate
methods of therapy should be considered .
subcutaneous ivermectin has been used
in numerous cases to overcome poor oral absorption [ 9 , 14 , 25 , 27 , 28 ] .
while no
parenteral antihelminthic medications are fda approved for use in humans , permission may be obtained on
an individual basis to administer these agents by alternative routes with
evidence of problems of enteral absorption .
the mortality in disseminated
strongyloidiasis is high , likely from progression of disease prior to
diagnosis .
ivermectin remains the drug
of choice and is typically given orally at a dose of 200 g / kg per day for two
weeks or until resolution of symptoms .
because of possible impaired absorption , hypoalbuminemia , or extensive
central nervous system disease , serum concentrations may help discriminate when
either higher oral doses or parenteral ivermectin is necessary . two case reports as well as ours showed
microscopic evidence of strongyloides eradication with serum concentrations ranging from 11.4 to 49.6 ( ng / dl ) .
we were able to measure a csf ivermectin
concentration of 0.14 ng / dl , which suggests a less than 1% central nervous
system penetrance .
because of extensive mortality ,
early diagnosis and treatment are paramount in disseminated strongyloidiasis . | strongyloides stercoralis affects over 100 million people worldwide .
those people most susceptible to infection are those with an immunocompromising condition , such as cancer or human immunodeficiency virus ( hiv ) .
local disease may spread throughout the body of the host , causing a condition termed disseminated strongyloidiasis .
standard treatment for strongyloides stercoralis infection is oral ivermectin .
we describe a patient with chronic lymphocytic leukemia diagnosed with disseminated strongyloidiasis two weeks after initial presentation .
after repeated dosing of oral ivermectin with no clinical response , serum and cerebral spinal fluid ( csf ) concentrations of ivermectin were measured to assess absorption .
the peak serum concentration of 49.3 ng / ml correlated with a csf concentration of 0.14 ng / ml . despite these concentrations , the patient eventually succumbed to multi - system organ failure .
we discuss the reasons for treatment failure and explore the utility of measuring ivermectin concentrations . |
the study was conducted at specialized deaddiction services provided at a tertiary care hospital in south india .
the participants for the study were recruited from an inpatient setting . to be included in the study ,
the patients were required to be older than 18 years of age , fulfilling the diagnostic criteria for substance dependence as per the international classification of the diseases , 10 revision ( icd-10 ) . to be included in the study ,
participants with comorbid psychiatric disorders , currently in withdrawal state clinically , and those who were not cooperative for interview were excluded from the study .
the instrument development was through a multistage process of item generation , selection and scaling , field testing , and refinement procedures .
item generation was done from the detailed , qualitative interviews with 48 persons seeking treatment for substance use disorders admitted to the center over a 3-month period .
interviews were done , after ensuring persons were free of withdrawal symptoms and from sedative effects of drugs for detoxification by a qualified psychiatrist .
the phrases used by the patients during the qualitative interviews were considered while framing the items for the questionnaire .
forty - five items were selected for the initial scale and a five - point likert scale was used with frequency options ranging from always to never .
the expert 's opinions on clarity , comprehensiveness , item relevancy , and ease of use were sought .
items that received a score of < 4 from > 20% of experts were revised or deleted .
we recruited 168 patients admitted in the ward and among them five patients refused to be part of the study , six patients were not able to read the questionnaire , and seven patients had comorbid psychiatric conditions .
participants who were informed about the questionnaire and its purpose and taken consent for their participation .
the scale was administered after 1 week when the patient was clinically free from withdrawal symptoms and the sedative medications as per the qualified psychiatrist . among 150 patients ,
50 completed the questionnaire twice over 15 day 's interval to provide data for test retest reliability .
the mean age of the sample was 35.6 years ( standard deviation [ sd ] 8.6 years ) , with a mean of 12 years ( sd 2.7 years ) education .
the occupation of the sample include 19 ( 13% ) were unemployed , 6 ( 4% ) students , 13 ( 9% ) farmers , 28 ( 19% ) unskilled , 26 ( 18% ) semiskilled , 21 ( 14% ) skilled , 10 ( 7% ) clerical , 22 ( 15% ) , and 5 ( 3% ) were professionals with 58% of them were working in unorganized sectors .
most participants had received a diagnosis of alcohol - dependence syndrome as per the icd-10 [ table 1 ] . diagnostic breakup of the participants the scale was sent to six experts in the field of addiction and rehabilitation .
they gave their opinion on the adequacy of the content , clarity , item relevancy , and ease of use by rating the item on a five - point likert scale ranging from strongly disagree to strongly agree .
the mean age of the expert consensus group was 48 years ( sd 10.5 years ) , ranging from 33 to 63 years and their mean experience in the field of psychiatry and rehabilitation was 20.5 8.6 years .
items regarding employers were revised to accommodate students as four of the six experts felt the need to revise those items .
two items on the family expectations on work performance and its influence on substance use were added to the instrument to make it a 47-item one . test
retest reliability of responses to the statements was examined using the cohen 's kappa statistic .
the cohen 's kappa coefficients ranged from 0.247 up to 0.776 [ table 2 ] .
the items with a weighted kappa coefficient below 0.4 were removed and other 45 items were retained .
kappa coefficient values for corresponding items the 45 items of the scale were subjected to a factor analysis and subsequent orthogonal ( varimax ) rotation to understand the factor structure .
this yielded five factors based on the observation of the scree plot of eigenvalues ; values were 9.5 , 2.6 , 2.4 , 2.1 , and 1.8 for factors 15 ; the sixth factor had an eigenvalue of 1.5 and thus this and subsequent factors were not considered further . after rotation ,
items with loadings < 0.3 on any of the first five factors were not retained .
internal consistency of the final scale ( and subscales ) was estimated using cronbach 's alpha .
the mean age of the sample was 35.6 years ( standard deviation [ sd ] 8.6 years ) , with a mean of 12 years ( sd 2.7 years ) education .
the occupation of the sample include 19 ( 13% ) were unemployed , 6 ( 4% ) students , 13 ( 9% ) farmers , 28 ( 19% ) unskilled , 26 ( 18% ) semiskilled , 21 ( 14% ) skilled , 10 ( 7% ) clerical , 22 ( 15% ) , and 5 ( 3% ) were professionals with 58% of them were working in unorganized sectors .
most participants had received a diagnosis of alcohol - dependence syndrome as per the icd-10 [ table 1 ] .
they gave their opinion on the adequacy of the content , clarity , item relevancy , and ease of use by rating the item on a five - point likert scale ranging from strongly disagree to strongly agree .
the mean age of the expert consensus group was 48 years ( sd 10.5 years ) , ranging from 33 to 63 years and their mean experience in the field of psychiatry and rehabilitation was 20.5 8.6 years . items regarding employers were revised to accommodate students as four of the six experts felt the need to revise those items .
two items on the family expectations on work performance and its influence on substance use were added to the instrument to make it a 47-item one .
test retest reliability of responses to the statements was examined using the cohen 's kappa statistic .
the cohen 's kappa coefficients ranged from 0.247 up to 0.776 [ table 2 ] .
the items with a weighted kappa coefficient below 0.4 were removed and other 45 items were retained .
the 45 items of the scale were subjected to a factor analysis and subsequent orthogonal ( varimax ) rotation to understand the factor structure .
this yielded five factors based on the observation of the scree plot of eigenvalues ; values were 9.5 , 2.6 , 2.4 , 2.1 , and 1.8 for factors 15 ; the sixth factor had an eigenvalue of 1.5 and thus this and subsequent factors were not considered further . after rotation , items with loadings < 0.3 on any of the first five factors were not retained .
internal consistency of the final scale ( and subscales ) was estimated using cronbach 's alpha .
the present study aimed at developing a brief standardized vocational assessment tool to measure the difficulties faced by the persons with substance use disorders at workplace .
the goal of the vocational assessment therapy in mental health is to develop skills and obtain the supports needed for independent , interdependent , productive living , improved participation in daily life , and better quality of life . in substance use disorders
, a direct relation exists between the viable employment and recovery and also it acts as a reinforcing factor in recovering persons to maintain his or her sobriety .
the research aimed at developing brief scale so that the vocational difficulties perceived by the substance use disorder patients documented and appropriate rehabilitation can be done to keep them in viable employment .
the tool developed in the research was developed through a multistaged process of item generation , selection and scaling , field testing , and refinement procedures .
all possible items were pooled and measurements of reliability , consistency , and the factor analysis were used to understand the final dimensions .
the sociodemographic and clinical profile of the sample was a representation of the sample from the center .
the vocational assessment tool developed aims to understand the occupation in terms of the personal factors , work factors , environmental factors , substance - related factors , and time - related factors . even though the items pooled were from the qualitative interviews with the substance users , all the items were either the contributing factors or the consequence of the substance use , and their mention in the literature is unquestionable .
the first factor contained 11 items which mainly concerned with the individual decision - making capabilities , satisfaction , attention , and confidence at the workplace .
these items are found in literature as the factors having an impact on the performance at workplace . the second factor involved 12 items that mainly concerned with the perception of the person regarding problems at workplace , warning at workplace , family expectations , promotion at the workplace , and underpaid at workplace .
these items are related to interpersonal relation at workplace and family expectations on work performance .
literature supports the influence of items such as family influence and pressure from others on the work performance .
the third factor involved 11 items which were concerned with the perception of the individual regarding the problem with anger , anxiety , restless , stress at normal situations , interests , and insecurity .
these items are mainly concerned with individual behaviors , one 's interest , and attitudes and thus were grouped as a personal factor .
these items are concerned with interests , values , and vulnerabilities such as anger , anxiety referring to the process by which one choose , experiments , and interprets occupational behaviors .
the fourth factor involved six items on the effect of the substance on the person regarding health complications , withdrawal symptoms , criticism , isolation at the workplace , and less money due to substance use .
these items were grouped as substance - related factors , and these have an effect on the work performance due to substance abuse .
the fifth factor involved five items that mainly concerned with the problem with the timings and punctuality at the workplace .
the overall content of the scale addressed all of the commonly concerned items in vocational assessment literature such as behaviors , attitudes , personal characteristics , communication skills , and lifestyle conditions that can contribute to the individual 's success in occupation .
item on personal development and additional responsibilities scored 0.247 and 0.277 , respectively , on kappa coefficient for test retest reliability may have been due to the ambiguity and ambivalence attached to these items .
other items of the scale kappa coefficient value ranged from 0.4 to 0.776 which is moderate to substantial agreement over the time .
internal consistency of the final version of the scale with cronbach 's alpha value of 0.91 reveals excellent reliability .
the scale is a brief instrument which will take around 1015 min for administration and it will be helpful to know the perceived difficulties of persons with substance use disorders .
the content of this scale arose directly from earlier qualitative data obtained from the detailed interview with the cases admitted to a substance use treatment facility .
the questionnaire was administered when participants were sober , free from withdrawal symptoms and was not on sedating medications to get a more accurate perception from them .
the tool is subjective in the study followed a convenient sampling method which might have led to sample bias , with those who chose to participate having different perceptions than those who did not .
further evaluation in larger groups of participants with this instrument may reveal a better picture of vocational difficulties perceived by them . nevertheless , it has promise as a vocational self - evaluation tool in patients with substance use disorders to assist occupational therapist to document vocational problems and develop rehabilitation packages for patients .
the content of this scale arose directly from earlier qualitative data obtained from the detailed interview with the cases admitted to a substance use treatment facility .
the questionnaire was administered when participants were sober , free from withdrawal symptoms and was not on sedating medications to get a more accurate perception from them .
the tool is subjective in the study followed a convenient sampling method which might have led to sample bias , with those who chose to participate having different perceptions than those who did not .
further evaluation in larger groups of participants with this instrument may reveal a better picture of vocational difficulties perceived by them .
nevertheless , it has promise as a vocational self - evaluation tool in patients with substance use disorders to assist occupational therapist to document vocational problems and develop rehabilitation packages for patients .
| background : work - related problems are a serious concern among persons with substance use but due to lack of a standardized tool to measure it ; these problems are neither systematically assessed nor appropriately addressed .
most existing measures of work performance cater to the needs of the workplace rather than focusing on the workers ' perception of the difficulties at work.aim:to develop a standardized instrument to measure work - related problems in persons with substance use disorders.methods:qualitative data obtained from interviews with substance users were used to develop a scale .
the refined list of the scale was circulated among an expert panel for content validation .
the modified scale was administered to 150 cases , and 50 cases completed the scale twice at the interval of 2 weeks for test retest reliability.results:items with a test retest reliability kappa coefficient of 0.4 or greater were retained and subjected to factor analysis .
the final 45-item scale has a five - factor structure .
the value of cronbach 's alpha of the final version of the scale was 0.91.conclusions:this self - report questionnaire , which can be completed in 10 min , may help us in making a baseline assessment of the work - related impairment among persons with substance use and the impact of substance use on work . |
a cyst is a pathological epithelial cell - lined cavity that is filled with fluid or soft material .
they are frequently found as intra - bony lesions of the jaw , which can expand so drastically that sometimes the facial appearance can change .
although removal of the cyst is necessary in these cases , enucleation of these lesions can bring forth a risk of infection , fracture of the jaw , or nerve injury if the cyst is large enough to push on important anatomic structures . to avoid those complications , marsupialization or decompression
is recommended to reduce the size of the cyst , hence making it safe to enucleate the lesion .
the benefit of marsupialization or decompression includes maintenance of pulp vitality , preservation of the inferior alveolar nerve or maxillary sinus , prevention of fracture of the jaw , and low risk of recurrence1,2 .
many previous studies have also suggested that marsupialization or decompression can be considered a good treatment option in large odontogenic cyst cases3,4,5 .
in particular , some studies have reported good post - op results in radicular cysts , dentigerous cysts ( dcs ) , and keratocystic odontogenic tumors ( ktocs)6,7,8 .
ameloblastoma is the most frequent odontogenic cyst in the jaw , and shows high recurrence and invasiveness9 . among the four subtypes of ameloblastoma , unicystic ameloblastoma
ua , with its cyst - like character , is considered to be a good indication for marsupialization or decompression5,11
. however , there have been few studies that have focused on postoperative success based on the ameloblastoma 's size , or other contributing factors affecting the velocity of its shrinkage .
this study is devised to evaluate the effectiveness of the decompression of cystic lesions of the mandible , especially , dc , kcots and ua using a panoramic view . furthermore , to extract more meaningful clinical insights , we investigated the correlation between decompression and several host factors , such as age and the initial size of the cyst .
thirty - two patients with cystic lesions were chosen . among them , 13 were dcs ( 12 males and 1 female ; mean age 40.8 years ) , 14 were ktoc cases ( 10 males and 4 females ; mean age 27.9 years ) , and 5 were ua cases ( 1 male and 4 females ; mean age 18.6 years ) .
the subjects were selected from patients who visited the seoul national university dental hospital from 2000 to 2012 . among the mandibular cyst cases , only the cases in which the center of the lesion was localized to the posterior part of the premolar were selected .
decompression was performed under local anesthesia , making a hole through the mucosa and the submucosal bony surface in the vicinity of the lesion . after setting a tube through the hole ,
a biopsy was done in all cases to confirm that the lesion was indeed an odontogenic cyst .
the patients were followed - up monthly . because cystic lesions are relatively radiopaque , these looked darker than the jawbone in the panoramic view .
a change of light and darkness in the panoramic view was remarkable around the boundary of the cystic lesion . by investigating the contrast of light and darkness in the panoramic view
tracing of the defined boundary was performed using a computer program ( sketchup version 8.0 for windows ; trimble navigation ltd . ,
1 ) before and after decompression , the size of the cystic lesion was measured .
panoramic radiography was performed using panoramic units ( orthopantomograph opr 100 ; instrumentarium co. , forchheim , finland ) operated at 70 kvp and 10 ma using photostimulable phosphor plates .
the absolute and relative velocities of shrinkage were calculated by measuring the change of size before , during , and after decompression . here , the absolute velocity of shrinkage was defined as follows : absolute velocity of shrinkage ( mm / mo)=(size of decreasing area after decompression [ mm])/(duration of decompression [ mo ] ) the relative velocity of shrinkage was defined as follows : relative velocity of shrinkage ( % /mo)=(percentage of decreasing area after decompression [ % ] ) /(duration of decompression [ mo ] ) with the aid of statistics , we studied whether the absolute or relative velocity of shrinkage had a meaningful correlation with the host factors , such as the age of the patient and the initial size of the lesion .
data were analyzed with pasw statistics version 18.0 software ( ibm co. , armonk , ny , usa ) . a kolmogorov- smirnov test , shapiro - wilk test , kruskal - wallis test , spearman 's rank correlation analysis , pearson 's correlation analysis , and simple regression analysis were used to evaluate the change of the cystic lesion within each parameter . a p - value less than 0.05 was determined to be significant .
thirty - two patients with cystic lesions were chosen . among them , 13 were dcs ( 12 males and 1 female ; mean age 40.8 years ) , 14 were ktoc cases ( 10 males and 4 females ; mean age 27.9 years ) , and 5 were ua cases ( 1 male and 4 females ; mean age 18.6 years ) .
the subjects were selected from patients who visited the seoul national university dental hospital from 2000 to 2012 . among the mandibular cyst cases , only the cases in which the center of the lesion was localized to the posterior part of the premolar were selected .
decompression was performed under local anesthesia , making a hole through the mucosa and the submucosal bony surface in the vicinity of the lesion . after setting a tube through the hole ,
a biopsy was done in all cases to confirm that the lesion was indeed an odontogenic cyst .
because cystic lesions are relatively radiopaque , these looked darker than the jawbone in the panoramic view .
a change of light and darkness in the panoramic view was remarkable around the boundary of the cystic lesion . by investigating the contrast of light and darkness in the panoramic view
tracing of the defined boundary was performed using a computer program ( sketchup version 8.0 for windows ; trimble navigation ltd . ,
1 ) before and after decompression , the size of the cystic lesion was measured .
panoramic radiography was performed using panoramic units ( orthopantomograph opr 100 ; instrumentarium co. , forchheim , finland ) operated at 70 kvp and 10 ma using photostimulable phosphor plates .
the absolute and relative velocities of shrinkage were calculated by measuring the change of size before , during , and after decompression . here ,
the absolute velocity of shrinkage was defined as follows : absolute velocity of shrinkage ( mm / mo)=(size of decreasing area after decompression [ mm])/(duration of decompression [ mo ] ) the relative velocity of shrinkage was defined as follows : relative velocity of shrinkage ( % /mo)=(percentage of decreasing area after decompression [ % ] ) /(duration of decompression [ mo ] ) with the aid of statistics , we studied whether the absolute or relative velocity of shrinkage had a meaningful correlation with the host factors , such as the age of the patient and the initial size of the lesion .
data were analyzed with pasw statistics version 18.0 software ( ibm co. , armonk , ny , usa ) . a kolmogorov- smirnov test , shapiro - wilk test , kruskal - wallis test
, spearman 's rank correlation analysis , pearson 's correlation analysis , and simple regression analysis were used to evaluate the change of the cystic lesion within each parameter . a p - value less than 0.05 was determined to be significant .
in the dc group , the initial mean size of the lesion and the standard deviation ( sd ) in the panoramic view were 1,474.3 mm and 930.7 mm .
the decompression lasted for 6.9 months and a 39.4% reduction was obtained during the procedure .
the relative velocity of shrinkage ( meansd ) was 6.12.9%/mo and the absolute velocity of shrinkage was 81.140.0 mm / mo .
in the ktoc group , the initial mean size of the lesion ( mean sd ) was 2,356.31,135.7 mm and the decompression was performed for 7.4 months .
the relative velocity of shrinkage ( meansd ) was 4.72.9%/mo and the absolute velocity of shrinkage was 106.081.9 mm / mo .
in the ua group , the initial mean size of the lesion ( meansd ) was 2,115.4888.9 mm in the panoramic view .
the relative velocity of shrinkage ( meansd ) was 4.65.6%/mo and the absolute velocity of shrinkage ( meansd ) was 115.8112.9 mm / mo.(table 1 ) no statistically significant inter - group difference was found in the absolute or relative velocities of shrinkage.(fig .
2 ) some of the host factors seemed to contribute to the process of healing after decompression .
the age of the patient showed a statistically significant correlation with the relative velocity of shrinkage in the ua group only ( p=0.04 , r=0.46).(fig .
a ) in the ktoc group , the absolute velocity of shrinkage was significantly associated with the age of the patient ( p=0.04 , r=0.45).(fig .
b ) when analyzing the initial size of the lesion , all three kinds of cystic lesions showed no correlation between the initial size of the lesion and the relative velocity of shrinkage . however , in the dc and ktoc groups , absolute velocity of shrinkage was larger when the initial size of the lesion was larger ( dc : p=0.02 , r=0.65 ; ktoc : p=0.02 , r=0.56).(fig .
computed tomography ( ct ) is one of the best tools to evaluate the size of an intra - bony lesion of the jaw . unlike the panoramic image , which reproduces the actual anatomy on a two - dimensional plane , ct data provides three - dimensional images with precision . in some previous studies ,
researchers have tried to use ct as a tool to measure the size of the cyst but the procedure had various limitations such as high cost and the large amount of radiation associated with ct scans .
resorting to panoramic view instead of ct , we referred to yoshiura and kubota 's report12,13 that says that the area of the cyst measured on the panoramic image has a linear relationship with the volume of the lesion on ct when the lesion exists in the posterior part of the mandible .
they argue that this is because odontogenic cysts rarely expand bucco - lingually in the posterior part of the premolar region12,13 . with the aid of this report
, we used a panoramic view to study the size of the cysts located in the posterior part of the mandible .
marsupialization is a surgical procedure in which the supra - wall of the cyst is removed and the internal wall of the cyst is sutured with the oral mucosa so that part of the lining epithelium can be transitioned into the oral epithelium . with decompression
, we expect to reduce the size of the lesion by inserting a rubber tube or a stent through a small hole into the cyst14 .
both of these procedures disrupt the continuity of the cystic wall , and the suturing of the internal tissue of the cyst with the oral mucosa in marsupialization is challenging to the operator .
moreover , post - operative deformation or surface depression is less likely with decompression compared to with marsupialization ; hence prosthetic reconstruction is easier in decompression cases15,16 .
, some studies have reported that the tissue left after decompression could develop into a more aggressive lesion17,18,19,20 .
because of this , periodic follow - ups and radiographic imaging should be conducted after decompression , and we followed this principle in this research .
marker et al.21 recommended that the decompression procedure should be performed for over a year , and reported that the longer the decompression period lasted ( to the extent that the drain be removed spontaneously ) , the better the prognosis was .
their alleged criterion of success was a 50% to 60% decrease in the cystic volume . according to the recommendation of marker et al.21 , the duration of decompression in our study was too short .
the duration after decompression was 6.9 months for the dc group , 7.4 months for the ktoc group , and 13.4 months for the ua group .
however , in spite of the short duration of decompression , we at least fulfilled the minimum and most crucial goal : the size of the lesion was reduced enough to be enucleated without damaging the adjacent tissue .
considering the fact that most of the shrinkage observed by miyawaki et al.22 was accomplished during the first 3 months after the decompression , it is reasonable to conclude that a considerable amount of shrinkage had already been attained during the follow - up period in this study . in terms of the effect of the cyst type on the velocity of shrinkage ,
gao et al.23 reported that decompression was more effective with radicular cysts than with ktocs or ua .
kubota et al.6 also obtained similar results : the velocity of shrinkage after decompression was faster in radicular cysts than in ktocs or dcs .
in contrast , kim and lee15 reported that dcs shrank more dramatically after decompression compared to ktocs or radicular cysts .
however , anavi et al.1 reported that very few statistically significant inter - group differences were found in the velocity of shrinkage among radicular cysts , dcs , and ktocs . in this study , there were also no intergroup differences in the velocity of shrinkage .
previous studies have noted that shrinkage is faster when the initial size of the cyst is larger24,25 . although anavi et al.1 reported that the relative velocity of shrinkage was faster in the initially small - sized cases , his result can be interpreted in different ways given that the absolute velocity of shrinkage could give opposite results . in this study , however , ua showed little correlation between the initial size of the lesion and its shrinkage velocity .
the reason for this is not clear but may involve the histologic diversity of ua cases . among ackermann 's classification of ua ( the unilocular luminal type , the intraluminal with solid growth inside the lumen of the cystic lesion type , and the intraluminal growth with mural invasion within adjacent tissues type)26
the inter - type difference in behavior and recurrence seems to impose different characteristics on its response to decompression .
there is little consensus about whether the velocity of shrinkage is affected by the age of the patient .
kubota et al.6 said that there seemed to be little correlation between the age of the patient and the relative velocity of shrinkage .
a similar result was obtained for the ua group by gao et al.23 . however , anavi et al.1 reported that the period that was required to decrease the size of the cyst to a certain level was shorter when the patient was under 18 years old .
kim and lee15 also revealed that the relative velocity of shrinkage was faster when the patient was younger , especially in the dc cases . in this study , the relative velocity of shrinkage was higher when the patients were younger in the ua group . in the ktoc cases ,
although active osteogenic metabolism can partly explain these results , more focused research is required to verify the effect of decompression on cystic pathophysiology .
among the dc , ktoc and ua groups , there was little inter - group difference in shrinkage velocity . in the dc and the ktoc groups , the absolute velocity of shrinkage was faster for a larger initial size of the lesion ( dc : p=0.02 , r=0.65 ; kcot : p=0.02 , r=0.56 ) .
also , in the ktoc and ua groups , the absolute velocity of shrinkage was faster for younger patients ( kcot : p=0.04 , r=0.45 ; ua : p=0.04 , r=0.46 ) . | objectivesthe aim of this study was to verify the clinical effectiveness of decompression in decreasing the size of a cyst .
in addition to the different types of cysts , we tried to reveal what effect host factors , such as the initial size of the lesion and the age of the patient , have on the velocity of cyst shrinkage.materials and methodswith the aid of a panoramic view , we measured the size of the cysts before and after decompression in 13 dentigerous cysts ( dcs ) , 14 keratocystic odontogenic tumors ( ktocs ) , and 5 unicystic ameloblastoma ( ua ) cases .
the velocity of shrinkage in the three cystic groups was calculated .
relationships between the age of the patient , the initial size of the cyst , and the shrinkage velocity were investigated.resultsthe three types of cysts showed no inter - type differences in their velocity of shrinkage .
however , there was a statistically meaningful relationship between the initial size of the lesion and the absolute velocity of shrinkage in the dc group ( p=0.02 , r=0.65 ) and the ktoc group ( p=0.02 , r=0.56 ) .
there was also a significant relationship between the age of the patient and the absolute velocity of shrinkage in the ktoc group ( p=0.04 , r=0.45 ) and the ua group ( p=0.04 , r=0.46).conclusionthere was no difference in the decrease in size due to decompression among the different types of cysts .
however , the age of the patient and the initial size of the lesion showed a significant relationship with the velocity of shrinkage . |
fracture of dual columns of the acetabulum is the most complicated type of acetabular fracture .
posterior - anterior column fixation is considered to be important for joint stability and early mobilization after surgery .
ochs et al . investigated screw placement into the anterior column with ct analysis for 260 cadaveric bony hemi - pelvis specimens .
chen et al . analyzed 164 cases of three - dimensional ct pelvic data and concluded that there is a sex difference in screw placement into fractured anterior columns .
few studies have focused on a fixation model involving fractured posterior - anterior columns of the acetabulum .
thus , we designed a plate - screw fixation model for fractured columns of the acetabulum ( figure 1 ; patent no .
this model is divided into 3 parts : the reconstructive plate , which acts to fix the anterior column fracture ; the square plate , which is used to support the fracture of the square area ; and the third part is a lag screw for the posterior column , which is the key point of our design because the entry point of the lag screw determines the implant of the guide plate as well as the direction of the screw holes .
we first used the modified stoppa approach for measuring the reference point , which can improve the entry point and angle of the lag screw , thus providing an anatomic basis for accurate placement of the screw .
after cadaveric research , the comprehensive studies on the placement of the model and entry point of the lag screw of the acetabulum were measured by computed tomography ( ct ) images in healthy adults .
the ethics board review of south medical university ( guangzhou , china ) approved the study after a thorough investigation and verification .
nineteen recently embalmed adult bony hemi - pelvis specimens were obtained from the anatomy department of south medical university .
all of the specimens had complete integrity and there were no deformities or fractures based on visual inspection and an x - ray study .
the surgical field of vision was marked by the modified stoppa approach in the hemi - pelves ( figure 2 ) .
the screw was inserted in a retrograde motion on the minimum of the ischial tuberosity ( figure 3 ) .
a screw , 3.5 mm in length , was inserted parallel to the posterior line of the ischiadic ramus .
the distance between the entry point and the vertical line of the arcus marginalis ( op ) , and distance between the apex - q of the greater sciatic notch and the vertical line of the arcus marginalis ( pi ) were measured ( figure 4 ) . after projecting the line of the screw on an -square , the angle of the screw placement direction and the -square ( )
the angle of the screw placement direction and the arcus marginalis ( ) were measured ( figure 4 ) .
the distance between the entry point and the front line of the sacroiliac joint may exhibit a sex difference , thus the distance between the ischiadic spine and the posterior inferior iliac spine was also measured . according to the data obtained from the cadaveric study
, we selected 50 healthy adults ( 30 males and 20 females ; age range , 25~69 years ; mean age , 46.4 years ) in this study .
the data was collected by ct scan ( 16-slice multidetector scanner [ slice thickness , 1.25 mm ] ; ge , [ fairfield , connecticut ] , usa ; ) .
after data screening , the specimens that included deformities , fractures , tumors , osteoporosis , or incomplete scanning were excluded .
format and imported to mimics 10.01 ( materialise , [ leuven ] , belgium ) for measurement .
the threshold value was set with bone ct , and three - dimensional reconstruction was performed by region growth .
the screw hole had a 2.5 mm radius , with a supplementary hole 1 cm from the screw hole .
the 3-dimensional reconstruction images of the hemi - pelvis specimens were rotated laterally and the square area was fully exposed .
a line was drawn along the posterior border of the ischiadic ramus and extended to cross the arcus marginalis at point i. anterior to this point was the placement area of the guide module , which was 3 cm in length and 1.5 cm in width .
a 6.5-mm screw was placed in the posterior column of the acetabulum from the iliac fossa to the tuberosity of the ischium .
the entry point of the posterior column was 15.27 mm from the anterior aspect of the sacroiliac joint and 13.62 mm from the arcus marginalis .
thus , the entry point of the posterior column was guaranteed in the placement area of the guide module .
a 3-mm radius cylinder was virtually placed in the hemi - pelvis using mimics - cad .
then , both ends of the cylinder were extended and crossed out of the iliac fossa and tuberosity of the ischium .
the distance between the out - point , o , and the arcus marginalis , op , as well as the length of the screw hole and posterior border of the guide module , pi , were measured ( figure 5 ) .
all the hemi - pelvis specimens were placed with screws virtually and the entry points were all guaranteed in the placement area of the guide module ( figure 6 ) . because the placement area of the guide module , the fossa iliaca was parallel to the undersurface of the guide module ; the relative position between the cylinder and the placement area was the direction of the locking screw .
the relative position can be determined by and . these 2 angles were measured using the rp slice module of mimics ( figure 7 ) .
the corresponding data were analyzed using spss ( statistical program for social sciences , version 20.0 ; spss , inc . ,
the ethics board review of south medical university ( guangzhou , china ) approved the study after a thorough investigation and verification .
nineteen recently embalmed adult bony hemi - pelvis specimens were obtained from the anatomy department of south medical university .
all of the specimens had complete integrity and there were no deformities or fractures based on visual inspection and an x - ray study .
the surgical field of vision was marked by the modified stoppa approach in the hemi - pelves ( figure 2 ) .
the screw was inserted in a retrograde motion on the minimum of the ischial tuberosity ( figure 3 ) .
a screw , 3.5 mm in length , was inserted parallel to the posterior line of the ischiadic ramus .
the distance between the entry point and the vertical line of the arcus marginalis ( op ) , and distance between the apex - q of the greater sciatic notch and the vertical line of the arcus marginalis ( pi ) were measured ( figure 4 ) . after projecting the line of the screw on an -square , the angle of the screw placement direction and the -square ( )
the angle of the screw placement direction and the arcus marginalis ( ) were measured ( figure 4 ) .
the distance between the entry point and the front line of the sacroiliac joint may exhibit a sex difference , thus the distance between the ischiadic spine and the posterior inferior iliac spine was also measured .
according to the data obtained from the cadaveric study , we selected 50 healthy adults ( 30 males and 20 females ; age range , 25~69 years ; mean age , 46.4 years ) in this study .
the data was collected by ct scan ( 16-slice multidetector scanner [ slice thickness , 1.25 mm ] ; ge , [ fairfield , connecticut ] , usa ; ) .
after data screening , the specimens that included deformities , fractures , tumors , osteoporosis , or incomplete scanning were excluded .
format and imported to mimics 10.01 ( materialise , [ leuven ] , belgium ) for measurement .
the threshold value was set with bone ct , and three - dimensional reconstruction was performed by region growth .
the screw hole had a 2.5 mm radius , with a supplementary hole 1 cm from the screw hole .
the 3-dimensional reconstruction images of the hemi - pelvis specimens were rotated laterally and the square area was fully exposed .
a line was drawn along the posterior border of the ischiadic ramus and extended to cross the arcus marginalis at point i. anterior to this point was the placement area of the guide module , which was 3 cm in length and 1.5 cm in width .
a 6.5-mm screw was placed in the posterior column of the acetabulum from the iliac fossa to the tuberosity of the ischium .
the entry point of the posterior column was 15.27 mm from the anterior aspect of the sacroiliac joint and 13.62 mm from the arcus marginalis .
thus , the entry point of the posterior column was guaranteed in the placement area of the guide module .
a 3-mm radius cylinder was virtually placed in the hemi - pelvis using mimics - cad .
then , both ends of the cylinder were extended and crossed out of the iliac fossa and tuberosity of the ischium .
the distance between the out - point , o , and the arcus marginalis , op , as well as the length of the screw hole and posterior border of the guide module , pi , were measured ( figure 5 ) .
all the hemi - pelvis specimens were placed with screws virtually and the entry points were all guaranteed in the placement area of the guide module ( figure 6 ) .
because the placement area of the guide module , the fossa iliaca was parallel to the undersurface of the guide module ; the relative position between the cylinder and the placement area was the direction of the locking screw .
the relative position can be determined by and . these 2 angles were measured using the rp slice module of mimics ( figure 7 ) .
the corresponding data were analyzed using spss ( statistical program for social sciences , version 20.0 ; spss , inc . ,
the data for cadaveric specimens are recorded in table 1 and figure 8 . the distance between the out - point , o , and
the arcus marginalis , op , was 0.960.32 cm in females and 0.920.16 cm in males ( p>0.05 ) .
the length of the screw hole and posterior border of the guide module , pi , was 0.980.28 cm in females and 0.750.23 cm in males ( p>0.05 ) .
for the width of the entry point and the anterior border of the sacroiliac joint , the value was 2.430.66 cm in females and 1.870.26 cm in males ( p<0.05 ) .
the distance between the ischiadic spine and posterior inferior iliac spine was 5.740.46 cm in females and 5.160.29 cm in males ( p<0.05 ) .
the angle of the screw and the arcus marginalis , , was 59.686.28 in females and 56.753.22 in males ( p>0.05 ) .
the angle of the screw and square , , was 41.272.76 in females and 34.312.78 in males ( p<0.05 ) .
the length of pi was 1.080.22 cm in females and 0.850.27 cm in males ( mean , 0.980.13 cm ; range , 0.67~1.56 cm ; p>0.05 ) , and the length of op ( the entry point and arcus marginalis ) was 1.060.29 cm in females and 1.120.24 cm in males ( mean , 1.090.26 cm ; range , 0.70~1.59 ; p>0.05 ) .
the angle of the screw and the arcus marginalis ( ) was 55.334.00 in females and 55 .
the angle of the screw and the plate ( ) was 39.212.45 in females and 35.582.31 in males ( p<0.05 ) .
the width of the entry point and the anterior border of the sacroiliac joint was 2.210.46 cm in females and 1.980.12 cm in males ( p=0.51 ) .
the distance between the ischiadic spine and posterior inferior iliac spine was 5.490.61 cm in females and 4.860.54 cm in males p<0.001 ) .
a comparison of the corresponding data was made between specimens and healthy adults , and there was no significant difference based on sex and side ( p>0.05 ) .
the data for cadaveric specimens are recorded in table 1 and figure 8 . the distance between the out - point , o , and
the arcus marginalis , op , was 0.960.32 cm in females and 0.920.16 cm in males ( p>0.05 ) .
the length of the screw hole and posterior border of the guide module , pi , was 0.980.28 cm in females and 0.750.23 cm in males ( p>0.05 ) .
for the width of the entry point and the anterior border of the sacroiliac joint , the value was 2.430.66 cm in females and 1.870.26 cm in males ( p<0.05 ) .
the distance between the ischiadic spine and posterior inferior iliac spine was 5.740.46 cm in females and 5.160.29 cm in males ( p<0.05 ) .
the angle of the screw and the arcus marginalis , , was 59.686.28 in females and 56.753.22 in males ( p>0.05 ) .
the angle of the screw and square , , was 41.272.76 in females and 34.312.78 in males ( p<0.05 ) .
the length of pi was 1.080.22 cm in females and 0.850.27 cm in males ( mean , 0.980.13 cm ; range , 0.67~1.56 cm ; p>0.05 ) , and the length of op ( the entry point and arcus marginalis ) was 1.060.29 cm in females and 1.120.24 cm in males ( mean , 1.090.26 cm ; range , 0.70~1.59 ; p>0.05 ) .
the angle of the screw and the arcus marginalis ( ) was 55.334.00 in females and 55 .
the angle of the screw and the plate ( ) was 39.212.45 in females and 35.582.31 in males ( p<0.05 ) .
the width of the entry point and the anterior border of the sacroiliac joint was 2.210.46 cm in females and 1.980.12 cm in males ( p=0.51 ) .
the distance between the ischiadic spine and posterior inferior iliac spine was 5.490.61 cm in females and 4.860.54 cm in males p<0.001 ) .
a comparison of the corresponding data was made between specimens and healthy adults , and there was no significant difference based on sex and side ( p>0.05 ) .
acetabular fractures , particularly those involving the anterior and posterior columns , remain technically challenging for orthopedic surgeons .
dual column fixation is considered to be important for joint stability and early mobilization in surgery .
we designed a plate - screw fixation model for both fractured columns of the acetabulum . in this model ,
an excellent anatomic understanding of the hemi - pelvis can be very useful in determining the screw insertion into the hip joint .
thus , an anatomic study was conducted regarding screw fixation in the posterior column of the acetabulum because the placement of the screw determines fixation of the plate - screw model .
the angle of screw insertion in the posterior column was improved using a modified stoppa approach , which can expose an area of 1519 mm from the iliac fossa to the arcus marginalis .
this area , as well as the distal end of the square , was considered as the penetration plate . to determine the insertion angle ,
the included angle of square , , and screw , , was measured and the angle of the screw and arcus marginalis , , was measured for assistance .
there was no significant sex difference in , while was significantly different . during insertion
, the deviation of the screw should be near 34 in males and 41 in females .
the data maintained from the cadaveric specimens can support designation of the plate - screw . to assist the design and clinical application of the guide module
, the measured parameters were revised with reference to the data obtained from the cadaveric specimens .
the reference section of the entry point , the anterior border of the sacroiliac joint , was changed to the intersection point of the arcus marginalis and the extension line of posterior border of the ischiadic ramus .
the reason for this revision indicates a significant sex difference by defining the anterior border of the sacroiliac joint as a reference section because the greater sciatic notch is wider in females than males .
accordingly , the distance between the anterior border of the sacroiliac joint and the entry point is also wider in females .
if the reference section is defined as the intersection point of the arcus marginalis and the extension line of the posterior border of the ischiadic ramus , there is no sex difference .
this was approved in ct reconstruction in our study and the distance was approximately 1 cm .
the intersection point was closer to the fracture line and was determined by touching the greater sciatic notch in the anterior incision .
second , the absolute angle of the horizontal and sagittal planes was revised as the angle of the screw and the placement square , as well as the major axis of the placement square .
the horizontal and sagittal planes were difficult to define because of the posture change of the patients .
moreover , the ct data may have error when the different postures of patients were adjusted .
therefore , the relative angle may help with the design and manufacture of the guide module .
as our research demonstrated promising results , we consider that the design may be valuable for safer fixation of bilateral acetabular fractures in peri - operative planning .
the major limitation in this study was that we only included a small number of cases based on the design in our study .
we will continue to apply this method in larger numbers of healthy adults with dual acetabular fractures .
the more comprehensive research on a larger number of cases will be beneficial to confirm the validity and efficiency of this design in clinical practice .
in this study , cadaveric specimens and healthy adults were selected to study the entry point and angle of the lag screw .
the data of measured parameters obtained in this study , which are consistent , provided an anatomic basis for the designation of the guide module ; therefore , accurate and safe placement of the lag screw into the posterior column of the acetabular fracture is possible . | backgroundthe aim of this study was to provide valid data for a plate - screw fixation model for fractured posterior - anterior columns of the acetabulum.material/methodsnineteen cadaveric bony hemi - pelvis specimens were obtained and 50 healthy adults were enrolled . the modified stoppa approach and computed tomography ( ct ) imaging were used to collect the measured parameter data of the module.resultsthe measured parameter data were as follows : op , 0.960.32 cm in females and 0.920.16 cm in males ( p>0.05 ) ; pi , 0.980.28 cm in females , and 0.750.23 cm in males ( p>0.05 ) ; , 59.686.28 in females and 56.753.22 in males ( p>0.05 ) ; and , 41.272.76 in females and 34.312.78 in males ( p<0.05 ) .
the corresponding ct image data were as follows : pi , 1.080.22 cm in females and 0.850.27 cm in males ( p>0.05 ) ; op , 1.060.29 cm in females and 1.120.24 cm in males ( p>0.05 ) ; , 55.334.00 in females and 55 .
503.43 in males ( p>0.05 ) ; and was 39.212.45in females and 35.582.31in males ( p<0.05 ) .
no significant difference with respect to sex and side existed between specimens and healthy adults ( p>0.05).conclusionsthe measured parameter data obtained in healthy adults and cadaveric specimens provided an anatomic basis for the designation of the guide module , and thus confirmed the accuracy and safety of screw placement in fractured columns of the acetabulum . |
on march 6 , 2015 , we conducted an aerial survey covering a 24-km radius around the pope county facility and identified 100 resident mallards ( anas platyrhynchos ) and 21 trumpeter swans ( cygnus buccinator ) . during march 912 , 2015 , we collected 148 representative waterfowl fecal samples , pooled in groups of up to 3 , to determine whether wild birds were actively shedding hpaiv .
we did not detect hpaiv , although 2 pooled samples contained lpaiv ( detailed methods in the technical appendix ) . in march 2015 , we chose 5 counties with infected facilities ( kandiyohi , lac qui parle , meeker , nobles , and stearns ) and 5 waterfowl production areas ( technical appendix ) where facilities were uninfected ( figure 1 ) to test for a spatial difference in hpaiv shedding . within these areas , we compiled a list of wetlands and lakes and scouted those areas for waterfowl activity and sampled feces . for each area ,
we collected 20 samples from a given spatiotemporal point to obtain representation within a target area .
minnesota collection sites for waterfowl feces sampled for highly pathogenic avian influenza virus ( hpaiv ) in spring 2015 ( n = 3,139 ) .
although hpaiv was confirmed in a nicollet county poultry facility on may 5 , 2015 , our sampling occurred during april 22april 27 , 2015 , and we consider this a control area ( control no .
we solicited agency staff and the public to report any deceased wild birds or live birds exhibiting neurologic signs consistent with hpaiv infection , including raptors , wild turkeys , and groups of > 5 dead birds from which we obtained samples .
we refer to these as morbidity and mortality samples , and our collection efforts targeted birds that had died < 24 h previously . in april 2015 , which coincided with the peak rates of infection in minnesota facilities (
8) , we collected 2,991 waterfowl fecal samples and pooled them into 1,027 brain - heart infusion media vials ; 1,591 samples ( 548 pooled ) were obtained from counties with infected facilities , and 1,400 samples ( 479 pooled ) were collected from waterfowl production areas without facilities ( figure 1 ) .
although hpaiv was not detected in these samples , 30 pooled samples ( representing 85 individual birds ) tested positive for lpaiv .
apparent lpaiv fecal prevalence was 0.012 ( 95% ci 0.0070.018 ) in counties with infected poultry , 0.008 ( 95% ci 0.0040.014 ) in counties without infection , and 0.010 ( 95% ci 0.0070.014 ) in the combined study area . given that hpaiv was not detected and that we could not sample every individual bird in the waterfowl population , if hpaiv were present , there was a 95% probability that fecal prevalence was between 0 and 0.181% in areas with infection and 0 and 0.224% in areas without infection . through june 4 , 2015 (
last confirmed positive facility ) , we collected and tested 104 morbidity and mortality samples ( table ) and detected a single hpaiv - positive bird , a cooper s hawk ( accipiter cooperii ) from yellow medicine county ( 20 km from an infected facility ) ; this infection was confirmed on april 29 , 2015 ( figure 2 ) .
we suspect that this woodland predator and opportunistic scavenger was exposed to hpaiv through a food item .
although not discovered as part of minnesota department of natural resources surveillance , 3 black - capped chickadees ( poecile atricapillus ) were found in an urban neighborhood exhibiting neurologic signs and submitted to the university of minnesota veterinary diagnostic laboratory by the minnesota wildlife rehabilitation center in june 2015 ; in 1 bird there was weak detection of eurasian h5 rna , but no virus was recovered and no sequence could be obtained directly from the sample ( 7 ) .
all 3 birds demonstrated multifocal encephalitis , which was likely the cause for the neurologic signs ( a. armien , pers .
wild bird morbidity and mortality samples ( n = 104 ) screened for highly pathogenic avian influenza virus ( hpaiv ) in minnesota through june 4 , 2015 .
a cooper s hawk was confirmed to be hpaiv positive in yellow medicine county on april 29 , 2015 , whereas weak titers of eurasian h5 rna were detected in a sampled black - capped chickadee from ramsey county collected in june 2015 .
morbidity and mortality samples yielded the only hpaiv detected in our surveillance of minnesota wild birds , despite the relatively small number of samples .
this sample type has proven valuable for hpaiv detection in wild birds in other states ; 32% of hpaiv detections nationwide and 90% of hpaiv detections within the mississippi flyway were derived from this source during december 2014june 2015 ( 7 ) .
evolving hpaiv strains can elicit clinical signs and death in young immunologically naive ducks ( 10 ) , and targeted sampling of waterfowl postbreeding areas for dead or neurologically impaired hatch - year birds might prove useful for future hpaiv surveillance ( 11 ) .
careful thought has been given to the design of surveillance programs for avian influenza ( 12 ) .
the study objectives , coupled with the methodologic limitations of available approaches , drive the sampling tool ultimately applied .
although opportunistic sampling ( e.g. , morbidity and mortality surveillance ) is accessible to most agencies , it is not suited for formal population - level inferences . for estimating aiv shedding prevalence , swab sampling of oropharyngeal and cloacal cavities in live birds or the trachea and cloaca in recently deceased birds is optimal because aiv replicates and sheds through the digestive tract ( 13 ) and the upper respiratory system ( 14 ) . for investigating exposure history , sampling blood from live or recently dead birds for serologic testing
would be more appropriate , although timing , location , and mechanism of exposure can not be determined .
the outbreak s speed required a quickly deployable method to collect adequate sample sizes and implement spatial design elements that would allow a meaningful comparison between known areas with infection and areas of the state apparently without infection .
modeling has shown that aiv maintenance in wild bird populations is mediated by environmental transmission ( 15 ) , and the detection of lpaiv in waterfowl fecal samples supports that conclusion .
no hpaiv was detected in waterfowl feces , although there was a 95% probability of apparent fecal prevalence throughout the study area of 0 to 0.1% .
thus , we conclude that during the 2015 hpaiv ( h5n2 ) outbreak in minnesota poultry , hpaiv contamination in wild waterfowl feces was not widespread .
detailed description of methods used to detect highly pathogenic avian influenza virus in wild birds , minnesota , usa , 2015 . | in 2015 , a major outbreak of highly pathogenic avian influenza virus ( hpaiv ) infection devastated poultry facilities in minnesota , usa .
to understand the potential role of wild birds , we tested 3,139 waterfowl fecal samples and 104 sick and dead birds during march 9june 4 , 2015 .
hpaiv was isolated from a cooper s hawk but not from waterfowl fecal samples . |
type 2 diabetes mellitus ( t2 dm ) has continued to increase worldwide , reaching a figure of about 370 million in 2011 , with 4.6 million associated deaths per year.1 the standard therapy for this epidemic disease includes diet , exercise , use of oral hypoglycemic drugs , and/or subcutaneous insulin injections.2,3 several multicenter trials have demonstrated that different pharmacological agents can successfully lower blood glucose and reduce the risk of developing microvascular and macrovascular diabetic complications . however , the large number of limitations and unwanted side effects that still exist limit their use in clinical practice.4,5 consequently , a small , albeit significant , proportion of diabetic patients are also advised to resort to complementary and alternative medical therapies . among the effective herbal derivatives ,
berberine has aroused great interest for its glucose - lowering69 and lipid - lowering activity.10,11 berberine has been demonstrated to be effective in diabetic patients , for whom it has been shown to significantly decrease fasting and postprandial blood glucose and glycosylated hemoglobin ( hba1c ) levels . its effect is similar to that of metformin,12 although it acts with partly different mechanisms , ie , by increasing insulin receptor expression.13 berberine regulates glucose metabolism through multiple mechanisms of action : 1 ) stimulation of glucose uptake by glucose transporter type 4 ( glut-4 ) upregulation ; 2 ) activation of 5 adenosine monophosphate ( amp)-activated protein kinase ( ampk ) , as a consequence of the inhibition of mitochondrial function ; 3 ) suppression of adipogenesis , by inhibiting peroxisome proliferator - activated receptor gamma ( ppar ) and c - enhancer - binding protein alpha ( c / ebp ) function ; 4 ) stimulation of glucagon - like peptide-1 ( glp-1 ) release from ileal cells ; 5 ) suppression of human protein tyrosine phosphatase 1b ( h - ptp 1b ) ; 6 ) stimulation of the pancreatic g protein - coupled receptor 40 ( gpr40 ) ; and 7 ) reduction of intestinal glucose absorption , by inhibiting -glucosidase activity.14 with regard to the lipid profile , berberine upregulates low - density lipoprotein ( ldl)-receptor ( ldl - r ) expression independent of sterol regulatory element - binding proteins but dependent on extracellular signal - regulated kinase ( erk ) and c - jun n - terminal kinase ( jnk ) activation , which results in total cholesterol ( c ) and ldl - c reduction ( by about 30% and 25% , respectively ) .
this upregulation occurs through a posttranscriptional mechanism that stabilizes messenger ribonucleic acid ( mrna ) , making berberine a cholesterol - lowering drug endowed with a different mechanism of action from that of statins.15 along with its cholesterol - lowering properties , berberine also reduces triglycerides by about 35% .
these actions on the lipid profile have been observed in both animals and humans.15,16 berberine , an isoquinoline alkaloid of the protoberberine type found in a variety of plants , has been used in indian and chinese medicines for many decades .
it is found in hydrastis canadensis ( goldenseal ) , coptis chinensis ( coptis or goldthread ) , berberis aquifolium ( oregon grape ) , berberis vulgaris ( barberry ) , and berberis aristata ( tree turmeric).17 in spite of its functions as a glucose - and lipid - lowering agent , berberine remains rather defective in terms of its oral bioavailability.18 in humans , this appears to be due to a p - glycoprotein ( p - gp)-mediated gut extrusion process19 and a massive biliary excretion.20 the amount of berberine capable of crossing enterocytes seems to be reduced by about 90% by p - gp , and this suggests that either the use of a potential p - gp inhibitor21 or a chemical modification of berberine that would allow it to overcome p - gp antagonism22 might enhance its poor oral bioavailability , thus increasing its clinical effectiveness . among the potential p - gp inhibitors ,
silymarin ( derived from silybum marianum ) , a herbal drug traditionally used as a liver protectant , could be considered a good candidate , owing to its very poor oral bioavailability and very high safety profile.23 we therefore decided to test the clinical role played by silymarin when added to berberine in the treatment of glycemic and lipid alterations in patients with t2 dm .
this 4-month , single - blind , randomized , controlled clinical trial was conducted in the setting of routine clinical practice , in accordance with the principles stated in the declaration of helsinki and consistent with good clinical practice , as defined by the international conference on harmonization and in accordance with the ethical principles underlying european union directive 2001/20/ec and the united states code of federal regulations , title 21 , part 50 ( 21cfr50).24 the protocol and subject consent and privacy forms were approved by the local review board before the study began .
it was carried out in a single center in italy between october 2012 and may 2013 .
suitable patients , identified from the review of case notes and/or computerized clinic registers , were contacted by the investigators while at the center or by telephone .
all patients provided their written informed consent to participate in this study after a full explanation of the study had been given .
the inclusion criteria were : 1 ) providing informed consent , with a signed and returned privacy agreement ; 2 ) history of continuous ( for 3 months ) suboptimal glycemic control ( hba1c ranging between 7.0% and 9.0% ) ; 3 ) age between 25 and 75 years ; and 4 ) a negative pregnancy test , for female patients .
the exclusion criteria were : 1 ) refusal to sign the informed consent or privacy agreement ; 2 ) moderate - to - severe liver disorders , including serum alanine amino - transferase and aspartate amino - transaminase greater than threefold the upper limit of normality and/or abnormal renal function ( serum creatinine greater than 115 mol / l ) ; 3 ) severe heart dysfunction ( new york heart association25 class phase iii or higher ) ; 4 ) history of acute diabetic complications , including diabetic ketoacidosis or hyperosmolar hyperglycemic nonketotic coma ; 5 ) psychiatric disease or severe infection ; 6 ) pregnancy or planned pregnancy ; 7 ) fasting plasma glucose with a value of 200 mg / dl or higher ; and 8) insulin therapy and use of p - gp antagonists .
all participants were instructed to follow their usual hypocaloric , low - glycemic index diet throughout the study . the controlled - energy diet (
a daily caloric deficit of about 600 kcal ) was based on the national cholesterol education program adult treatment panel ( ncep - atp ) iii recommendations26 and contained 50% of calories from carbohydrates , 30% from fat ( < 7% saturated fat , up to 10% polyunsaturated fat , and up to 20% monounsaturated fat ) , and 20% from proteins , with a maximum cholesterol content of 300 mg / day and 35 g / day of fiber .
the participants were also encouraged to maintain their usual standard physical activity ( riding a stationary bike for 20 to 30 minutes , three to four times a week or brisk walking for 30-minute sessions , three to four times a week ) .
all the enrolled subjects were randomized by an independent investigator , using a computer - generated random number table , to either of two groups : one receiving berberis aristata and one receiving berberis aristata plus silybum marianum tablets .
all the patients of the berberis aristata group received a galenic preparation containing a standardized extract corresponding to 500 mg of pure berberine per tablet .
all the patients of the berberis aristata group took 1 tablet on an empty stomach twice a day ( before breakfast and dinner ) for the whole length of the study ( 120 days ) .
all the berberis aristata tablets were prepared by the same pharmacist and herbal specialist authorized to manufacture galenic preparations according to a medical prescription .
all the patients of the berberis aristata / silybum marianum group received the add - on oral therapy in a nutraceutical combination , in tablet form ( berberol ; pharmextracta srl , pontenure , italy ) , containing 588 mg / tablet berberis aristata extract ( titrated in 85% berberine ) plus 105 mg / tablet silybum marianum extract ( titrated in > 60% flavanolignans ) .
the two active ingredients of the product were provided by siit srl ( berberis aristata extract ) and indena ( silybum marianum extract ) , both of milan , italy .
the product , in agreement with italian law , was registered with the ministry of health , in 2010 ( registration number : e10 40753y ) , as a food supplement , with both active ingredients ( berberis aristata and silybum marianum standardized extracts ) belonging to the list of accepted botanical nutraceuticals , and with all excipients of food grade .
like the patients of the berberis aristata group , the berberol patients consumed 1 tablet on an empty stomach twice a day ( before breakfast and dinner ) for the whole length of the study ( 120 days ) .
all participants of both groups were instructed to record the onset of any adverse events in a personal daily document , with the specific description of their symptoms ( including severity , duration , and possible cause - effect relationship with drug administration ) , the number of missed tablets , and any changes in diet , physical exercise , or weight . the glycemic control of the participants of both groups was suboptimal despite a prescribed diet , physical exercise , and/or hypoglycemic drugs . on enrollment , among the patients in the berberis aristata group ,
five were only treated with diet and without any antidiabetic drug , nine were on metformin monotherapy , two were on sulphonylurea monotherapy , and 15 were on oral combination therapy ( eleven with metformin and sulphonylureas , two with metformin plus dipeptidyl peptidase-4 [ dpp-4 ] inhibitors , one patient with metformin plus pioglitazone , and one patient with metformin plus sulphonylurea and pioglitazone ) .
sixteen patients in the berberis aristata group were on statin monotherapy , three were on a combination therapy ( two with a statin plus ezetimibe and one with statin plus omega-3 oil ) , and one patient was taking a fibrate .
on enrollment , among the patients in the fixed combination group ( berberis aristata plus silybum marianum ) , four were treated with diet without any antidiabetic drug , four were on metformin monotherapy , one was on sulphonylurea monotherapy , and 23 were on oral combination therapy ( of which four were treated with metformin plus sulphonylureas , five with metformin plus dpp-4 inhibitors , one with metformin plus pioglitazone , one with sulphonylurea plus dpp-4 inhibitor , four with metformin plus sulphonylurea and pioglitazone , four with metformin plus sulphonylurea and dpp-4 inhibitors , one with metformin plus pioglitazone and dpp-4 inhibitor , and three with metformin plus sulphonylurea , pioglitazone and acarbose ) .
twenty - three patients in the berberis aristata plus silybum marianum group were on statin monotherapy , and one patient was taking a fibrate .
, all patients underwent an initial screening assessment that included medical history , physical examination , vital signs ( blood pressure and heart rate ) , a 12-lead electrocardiogram , measurement of height and body weight , calculation of body mass index ( bmi ) , abdominal circumference ( waistline , wl ) , assessment of fasting blood glucose ( fg ) , total cholesterol ( tc ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , and triglycerides ( tg ) .
after 120 treatment days the following parameters were evaluated : weight , bmi , wl , hba1c , fg , tc , ldl - c , hdl - c , tg , aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) . in all participants ,
the bmi was calculated , by the investigators , as the weight in kilograms divided by the square of the height in meters .
the wl was measured midway between the lateral lower rib margin and the iliac crest , and its reduction was determined with a gulick anthropometric spring - loaded tape measure ( model 5829 ; bell medical services , neptune , nj , usa ) . all plasmatic variables were determined after a 12-hour overnight fast .
we used the plasma obtained by adding 1 mg / ml na - ethylenediaminetetraacetic acid ( edta ) ( sigma - aldrich , st louis , mo , usa ) and centrifuged at 3000 rpm for 15 minutes at 4c .
the blood samples were drawn by laboratory technicians , and the assays performed by the biologist in charge of the laboratory .
treatment tolerability was assessed through interviews with the patients by the investigators and the comparison of clinical and laboratory values with baseline levels .
safety monitoring included physical examination , vital sign assessment , weight , electrocardiogram , and adverse event recording . according to the two variables affecting the results treatment ( berberis aristata versus berberol ) and time ( before versus after )
the statistical analysis used was the between - within subject design analysis of variance ( anova ) and analysis of covariance ( ancova ) .
a multiple comparison test ( tukey honestly significant difference [ hsd])27 was used to analyze the possible differences between the average values during the observation period .
the level was set at 0.05 , and values were considered significant at p<0.05 .
ncss 8 statistical analysis and graphics ( ncss , llc , kaysville , ut , usa ) and jmp 10 ( sas institute , inc , cary , nc , usa ) were the two software used for analysis .
this 4-month , single - blind , randomized , controlled clinical trial was conducted in the setting of routine clinical practice , in accordance with the principles stated in the declaration of helsinki and consistent with good clinical practice , as defined by the international conference on harmonization and in accordance with the ethical principles underlying european union directive 2001/20/ec and the united states code of federal regulations , title 21 , part 50 ( 21cfr50).24 the protocol and subject consent and privacy forms were approved by the local review board before the study began .
it was carried out in a single center in italy between october 2012 and may 2013 .
suitable patients , identified from the review of case notes and/or computerized clinic registers , were contacted by the investigators while at the center or by telephone .
all patients provided their written informed consent to participate in this study after a full explanation of the study had been given .
the inclusion criteria were : 1 ) providing informed consent , with a signed and returned privacy agreement ; 2 ) history of continuous ( for 3 months ) suboptimal glycemic control ( hba1c ranging between 7.0% and 9.0% ) ; 3 ) age between 25 and 75 years ; and 4 ) a negative pregnancy test , for female patients .
the exclusion criteria were : 1 ) refusal to sign the informed consent or privacy agreement ; 2 ) moderate - to - severe liver disorders , including serum alanine amino - transferase and aspartate amino - transaminase greater than threefold the upper limit of normality and/or abnormal renal function ( serum creatinine greater than 115 mol / l ) ; 3 ) severe heart dysfunction ( new york heart association25 class phase iii or higher ) ; 4 ) history of acute diabetic complications , including diabetic ketoacidosis or hyperosmolar hyperglycemic nonketotic coma ; 5 ) psychiatric disease or severe infection ; 6 ) pregnancy or planned pregnancy ; 7 ) fasting plasma glucose with a value of 200 mg / dl or higher ; and 8) insulin therapy and use of p - gp antagonists .
all participants were instructed to follow their usual hypocaloric , low - glycemic index diet throughout the study . the controlled - energy diet ( a daily caloric deficit of about 600 kcal ) was based on the national cholesterol education program adult treatment panel ( ncep - atp ) iii recommendations26 and contained 50% of calories from carbohydrates , 30% from fat ( < 7% saturated fat , up to 10% polyunsaturated fat , and up to 20% monounsaturated fat ) , and 20% from proteins , with a maximum cholesterol content of 300 mg / day and 35 g / day of fiber .
the participants were also encouraged to maintain their usual standard physical activity ( riding a stationary bike for 20 to 30 minutes , three to four times a week or brisk walking for 30-minute sessions , three to four times a week ) .
all the enrolled subjects were randomized by an independent investigator , using a computer - generated random number table , to either of two groups : one receiving berberis aristata and one receiving berberis aristata plus silybum marianum tablets .
all the patients of the berberis aristata group received a galenic preparation containing a standardized extract corresponding to 500 mg of pure berberine per tablet .
all the patients of the berberis aristata group took 1 tablet on an empty stomach twice a day ( before breakfast and dinner ) for the whole length of the study ( 120 days ) .
all the berberis aristata tablets were prepared by the same pharmacist and herbal specialist authorized to manufacture galenic preparations according to a medical prescription .
all the patients of the berberis aristata / silybum marianum group received the add - on oral therapy in a nutraceutical combination , in tablet form ( berberol ; pharmextracta srl , pontenure , italy ) , containing 588 mg / tablet berberis aristata extract ( titrated in 85% berberine ) plus 105 mg / tablet silybum marianum extract ( titrated in > 60% flavanolignans ) .
the two active ingredients of the product were provided by siit srl ( berberis aristata extract ) and indena ( silybum marianum extract ) , both of milan , italy .
the product , in agreement with italian law , was registered with the ministry of health , in 2010 ( registration number : e10 40753y ) , as a food supplement , with both active ingredients ( berberis aristata and silybum marianum standardized extracts ) belonging to the list of accepted botanical nutraceuticals , and with all excipients of food grade .
like the patients of the berberis aristata group , the berberol patients consumed 1 tablet on an empty stomach twice a day ( before breakfast and dinner ) for the whole length of the study ( 120 days ) .
all participants of both groups were instructed to record the onset of any adverse events in a personal daily document , with the specific description of their symptoms ( including severity , duration , and possible cause - effect relationship with drug administration ) , the number of missed tablets , and any changes in diet , physical exercise , or weight .
the glycemic control of the participants of both groups was suboptimal despite a prescribed diet , physical exercise , and/or hypoglycemic drugs . on enrollment , among the patients in the berberis aristata group ,
five were only treated with diet and without any antidiabetic drug , nine were on metformin monotherapy , two were on sulphonylurea monotherapy , and 15 were on oral combination therapy ( eleven with metformin and sulphonylureas , two with metformin plus dipeptidyl peptidase-4 [ dpp-4 ] inhibitors , one patient with metformin plus pioglitazone , and one patient with metformin plus sulphonylurea and pioglitazone ) .
sixteen patients in the berberis aristata group were on statin monotherapy , three were on a combination therapy ( two with a statin plus ezetimibe and one with statin plus omega-3 oil ) , and one patient was taking a fibrate .
eleven participants were not taking any hypolipidemic treatment . on enrollment , among the patients in the fixed combination group ( berberis aristata plus silybum marianum ) ,
four were treated with diet without any antidiabetic drug , four were on metformin monotherapy , one was on sulphonylurea monotherapy , and 23 were on oral combination therapy ( of which four were treated with metformin plus sulphonylureas , five with metformin plus dpp-4 inhibitors , one with metformin plus pioglitazone , one with sulphonylurea plus dpp-4 inhibitor , four with metformin plus sulphonylurea and pioglitazone , four with metformin plus sulphonylurea and dpp-4 inhibitors , one with metformin plus pioglitazone and dpp-4 inhibitor , and three with metformin plus sulphonylurea , pioglitazone and acarbose ) .
twenty - three patients in the berberis aristata plus silybum marianum group were on statin monotherapy , and one patient was taking a fibrate .
before starting the study , all patients underwent an initial screening assessment that included medical history , physical examination , vital signs ( blood pressure and heart rate ) , a 12-lead electrocardiogram , measurement of height and body weight , calculation of body mass index ( bmi ) , abdominal circumference ( waistline , wl ) , assessment of fasting blood glucose ( fg ) , total cholesterol ( tc ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , and triglycerides ( tg ) .
after 120 treatment days the following parameters were evaluated : weight , bmi , wl , hba1c , fg , tc , ldl - c , hdl - c , tg , aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) . in all participants , the bmi was calculated , by the investigators , as the weight in kilograms divided by the square of the height in meters . the wl was measured midway between the lateral lower rib margin and the iliac crest , and its reduction was determined with a gulick anthropometric spring - loaded tape measure ( model 5829 ; bell medical services , neptune , nj , usa ) .
we used the plasma obtained by adding 1 mg / ml na - ethylenediaminetetraacetic acid ( edta ) ( sigma - aldrich , st louis , mo , usa ) and centrifuged at 3000 rpm for 15 minutes at 4c .
the blood samples were drawn by laboratory technicians , and the assays performed by the biologist in charge of the laboratory .
treatment tolerability was assessed through interviews with the patients by the investigators and the comparison of clinical and laboratory values with baseline levels .
safety monitoring included physical examination , vital sign assessment , weight , electrocardiogram , and adverse event recording .
according to the two variables affecting the results treatment ( berberis aristata versus berberol ) and time ( before versus after ) the statistical analysis used was the between - within subject design analysis of variance ( anova ) and analysis of covariance ( ancova ) . a multiple comparison test
( tukey honestly significant difference [ hsd])27 was used to analyze the possible differences between the average values during the observation period .
the level was set at 0.05 , and values were considered significant at p<0.05 .
ncss 8 statistical analysis and graphics ( ncss , llc , kaysville , ut , usa ) and jmp 10 ( sas institute , inc , cary , nc , usa ) were the two software used for analysis .
a total of 69 patients were enrolled in the trial . of these , 63 completed the study .
a total of 31 were randomized to receive berberis aristata , and 32 were randomized to receive berberol . during the study , six patients ( four males and two females ) did not complete the study , and the reasons for premature withdrawal included protocol violation , failure to follow up , or noncompliance .
specifically , four patients belonged to the berberis aristata group and two patients to the berberol group .
as mentioned in the materials and methods section , once the enrolment and randomization procedures had been completed , the individuals enrolled in the study started their oral , 120-day treatment with either berberis aristata or berberol .
= 0 ( before in table 2 ) , the baseline parameters did not show any statistically significant difference between groups .
both treatments did not modify weight , wl , or bmi but significantly reduced , respectively for berberis aristata and berberol , fasting glycemia ( 19.05% and 18.13% ) and hba1c ( 7.18% and 12.35% ) , with the latter difference being statistically significant for berberol . with regard to the lipid profile , both berberis aristata and berberol significantly reduced tc and tg , whereas only berberol significantly decreased ldl ( 16.92% ) , with no significant differences between treatments .
ast and alt were equally reduced in both groups . in terms of safety , no patients reported any serious adverse events . in both groups ,
none of the patients experienced any musculoskeletal disorders , such as myopathy , or showed clinical signs of liver toxicity .
several studies , mostly performed in the chinese population , have reported the effects of berberine on the lipidic and glycemic profile,10,11 but very few have reported on the effects of berberine in caucasians.2830 at a careful reading , these studies reveal a number of slight differences in the effects of berberine in the chinese and caucasians , which probably reflect a genotype / ethnic difference .
one of the most important differences is surely efficacy , which seems to be more apparent in the chinese than in caucasian subjects .
this fact has prompted many formulators to develop compounds of berberine and other herbal / natural ingredients , with the most widely exploited combination being that of berberine with monascus purpureus .
however , these formulations do not consider the low standardization profile of the monascus raw material or the patient risk arising from the possible presence of mycotoxic contaminants , such as citrinin.3134 nevertheless , the association of berberine and monascus offers real advantages in terms of its capacity to reduce tc , ldl , and tg.35 this is likely due to the berberine downmodulation of proprotein convertase subtilisin / kexin type 9 ( pcsk9 ) , a protein reducing the cholesterol - lowering properties of statins.36 this effect of berberine may be of great importance in the context of hypercholesterolemia but is useless when administered to hyperglycemic patients .
berberine is , anyway , rather defective in terms of oral bioavailability , and this appears to be mainly due to the p - gp - mediated gut extrusion process .
the results obtained by shan et al,22 using the berberine analog imb - y53 , suggest that overcoming the cellular efflux and in particular , the efflux linked to the p - gp function improves berberine bioavailability and its hypoglycemic effect .
chemical modifications of the berberine skeleton and the use of oral bioavailability enhancers coadministered with berberine may represent possible improvements . among these , silymarin ,
also known as milk thistle extract , is a herbal drug that has been known for years as a liver protectant . in the standardized form ,
titrated in flavanolignans , silymarin also suffers from poor oral bioavailability.37,38 in addition , silymarin has been proposed as an enterocyte p - gp antagonist,23 and thus , its use could prove useful to improve berberine efficacy .
on this basis , we developed a preparation , berberol , which contains both berberis aristata ( titrated in 85% berberine ) and silybum marianum ( containing silymarin ) .
an early pilot study conducted in statin - intolerant caucasians39 had demonstrated that the addition of 210 mg silymarin to 1,000 mg berberine , as sole therapy , exerted extremely effective results on the lipid profile ( 30% in tc ) , which had often been described in literature as obtainable only through the administration of 1,500 mg.810 we thus decided to verify , in a larger sample enrolled on the base of a t2 dm diagnosis , whether the addition of 210 mg silymarin to 1,000 mg berberine could improve upon the antihyperglycemic effect of berberine alone , administered at the same dose of 1,000 mg / day . although the administration of berberis aristata ( berberine [ 1,000 mg / day ] ) as add - on therapy did not result in statistically significant reductions of the main anthropometric measures ( weight , bmi , and wl )
, it caused statistically significant reductions of a few important glycemic ( fg and hba1c ) and lipidic ( tc and tg ) parameters , while reducing the ldl value to a considerable , albeit not statistically significant , extent .
similarly , the administration of berberol did not affect the anthropometric parameters but reduced the fg and hba1c values , as well as tc , ldl , and tg , in a statistically significant manner .
the comparison of the results obtained from treatment with berberol ( berberis aristata and silybum marianum ) against those obtained from the administration of berberis aristata alone indicates that hba1c is the parameter that most benefits from the presence of silymarin .
indeed , glycosylated hemoglobin reduced by 7.2% approximately following the administration of 1,000 mg berberine and by about 12.3% following the administration of 1,000 mg berberine and 210 mg silymarin . in the comparison between the two treatments ,
this result is the only parameter for which the difference was found to be statistically significant . in the light of this evidence
, it can be stated that in patients with t2 dm , the administration of berberol ( berberis aristata and silybum marianum ) can improve the glycemic status in a statistically significant manner in comparison with the administration of berberis aristata alone .
this result is not so clear from the point of view of the lipidic profile .
although ldl significantly reduced only in the berberol arm , the comparison between the groups did not show any statistically significant difference .
this may be explained by variations in the study populations , treatments , and enrollment criteria . as a matter of fact ,
the patients enrolled in the study were either normocholesterolemic ( and thus , not receiving any pharmacological therapy ) or hypercholesterolemic and thus , treated with statins . in either case ,
even if the individuals were not really targeted patients , they were rather well controlled , with average tc values below 180 mg / dl and average ldl values below 98 mg / dl on enrollment .
perhaps a study designed to enroll individuals with a markedly unbalanced lipidic profile , ie , untreated patients or with residual hyperlipidemia , would have highlighted the additional effect of silymarin when added to berberine .
first , the study was limited by the small size of the two samples , which makes it impossible to draw final conclusions .
second , the study was not conducted in a double - blind manner , and the individuals could have been influenced by the fact that they knew they were being treated with a galenic preparation ( berberis aristata extract titrated in berberine ) or a food supplement registered with the ministry of health the common perception of a galenic is often different from that of a food supplement .
third , we lack clear evidence such as could be provided with for example , the caco-2 cell culture method that the results obtained were due to improved enterocyte crossing caused by the presence of silymarin and were not due to another unexpected type of synergism between berberine and silymarin . because of this
, the next step will be the validation of the fixed mixture berberis aristata / silybum marianum in a caco-2 cell model .
the test will also enable a better definition of the most beneficial ratio in terms of dosages . as a matter of fact
, the doses of the active ingredients of berberol have so far , been established on the basis of the limitation arising from the fact that in italy , silybum marianum has also been registered as an over - the - counter ( otc ) drug . as an otc medication
, the product is used clinically at 420 mg doses . on the other hand ,
berberis aristata has never been registered as either a prescription or otc drug and is marketed in italy only as food supplement , at 500 mg doses ( calculated as pure berberine ) .
consequently , the combination of 105 mg pure silymarin with 500 mg berberine was chosen to make it impossible to exceed the set dose of 420 mg silymarin even with the ( improbable ) administration of 2,000 mg pure berberine ( 4 tablets ) .
on the basis of the study results , it may be confirmed that use of a fixed ( 210 mg ) amount of silymarin ( obtained from silybum marianum ) added to 1,000 mg berberine ( obtained from berberis aristata ) and administered as add - on therapy in the form of a nutritional supplement , in tablets ( berberol ) , is effective in improving lipidic and glycemic profiles in diabetes.40 it may be also concluded that berberol seems to be equally safe and tolerated and more effective than tablets containing 1,000 mg berberine only in improving hba1c in patients with a diagnosis of t2 dm .
berberine and above all , berberol could represent a good treatment option before initiating insulin therapy in diabetic patients with suboptimal glycemic control . | backgroundberberine is an isoquinoline alkaloid widely used to improve the glucidic and lipidic profiles of patients with hypercholesterolemia , metabolic syndrome , and type 2 diabetes .
the limitation of berberine seems to be its poor oral bioavailability , which is affected by the presence , in enterocytes , of p - glycoprotein an active adenosine triphosphate ( atp)-consuming efflux protein that extrudes berberine into the intestinal lumen , thus limiting its absorption . according to some authors , silymarin , derived from silybum marianum ,
could be considered a p - glycoprotein antagonist.aimthe study aimed to evaluate the role played by a possible p - glycoprotein antagonist ( silymarin ) , when added to a product containing berberis aristata extract , in terms of benefits to patients with type 2 diabetes.methodsthe study enrolled 69 patients with type 2 diabetes in suboptimal glycemic control who were treated with diet , hypoglycemic drugs , and in cases of concomitant alterations of the lipid profile , hypolipidemic agents .
the patients received an add - on therapy consisting of either a standardized extract of berberis aristata ( titrated in 85% berberine ) corresponding to 1,000 mg / day of berberine , or berberol , a fixed combination containing the same standardized extract of berberis aristata plus a standardized extract of silybum marianum ( titrated as > 60% in silymarin ) , for a total intake of 1,000 mg / day of berberine and 210 mg / day of silymarin.resultsboth treatments similarly improved fasting glucose , total cholesterol , low - density lipoprotein ( ldl ) cholesterol , triglyceride , and liver enzyme levels , whereas glycosylated hemoglobin ( hba1c ) values were reduced to a greater extent by the fixed combination.conclusionthe association of berberine and silymarin demonstrated to be more effective than berberine alone in reducing hba1c , when administered at the same dose and in the form of standardized extracts in type 2 diabetic patients . |
drug delivery strategies commonly use
endocytosis to access the
cell interior ; however , the potential for disease to affect endocytic
behavior remains relatively unexplored , hindering the design of robust
therapeutics .
endocytosis encompasses several energy - dependent and
highly regulated processes through which cells internalize substances
and objects from their surroundings in membrane - bound vesicles , permitting
essential functions such as nutrient uptake , signal transmission ,
plasmalemma recycling , and pathogen defense .
many endocytic
pathways have been described , each distinguished by its molecular
players , vesicle size , and cargo .
perhaps
the most studied and most widespread mechanism in eukaryotic cells
is clathrin - mediated endocytosis ( cme ) , recognizable via electron
microscopy by the distinct lattice of clathrin proteins that enclose
newly formed vesicles .
clathrin - coated vesicle formation begins at plasma
membrane invagination sites called pits and ultimately involves as
many as 30 proteins ( clathrin , adaptor protein-2 , epsin , dynamin ,
and others ) in a multistep process of recruitment , budding , and vesicle
release into the cytosol .
as clathrin - coated vesicles
form , adaptor protein-2 and other specific adaptor proteins recruit
transmembrane receptors into the fledgling vesicle by binding to their
cytosolic domains and the clathrin lattice simultaneously .
receptors of transferrin , insulin , low - density lipoprotein , growth
factors , and numerous other ligands have been shown to internalize
in this fashion .
receptor recruitment mechanisms vary , and internalization
can be independent of ligand attachment ( constitutive ) or specific
to the conformation of a ligand receptor complex . as a result , the clathrin pathway provides flexibility
for drug design , enabling continuous uptake of compounds in the fluid
phase and selective uptake of drugs conjugated to antibodies , peptides ,
and natural ligands that target receptors in clathrin - coated vesicles .
lysosomal storage diseases offer an example
where cme is key to
intracellular drug delivery .
they include around 50 different inherited
disorders where a dysfunctional lysosomal protein ( hydrolase , transmembrane
protein , chaperone , etc . )
patients
present with varying , but often devastating symptoms including abnormal
tissue development , peripheral organ enlargement , seizures , and neurodegeneration .
although treatment options remain limited , intravenous
infusion of recombinant enzymes has proven therapeutic in the clinics
for a few lysosomal storage diseases ( gaucher , fabry , pompe , and mucopolysaccharidosis
types i , ii , and vi ) .
known as enzyme replacement therapy ,
this treatment takes advantage of exposed mannose-6-phosphate residues
on lysosomal hydrolases , which target cell surface mannose-6-phosphate
receptors and internalize in clathrin - coated vesicles . upon trafficking to endosomal compartments , the enzyme
receptor
complex dissociates , the receptor recycles to the cell surface , and
the enzyme is delivered to the lysosome .
several obstacles have hindered enzyme replacement therapy ,
including
enzyme clearance from the bloodstream , immune reactions , and difficulty
traversing cellular barriers .
traffic
jams , including altered transport of endocytic vesicles , impaired
recycling of membrane proteins to the cell surface , cytoskeletal rearrangement ,
altered signaling , and diminished biosynthesis .
lipidoses in particular ( lysosomal
storage diseases characterized by lipid accumulation ) have been associated
with abnormal trafficking of plasma membrane materials ( e.g. , sphingolipids
and the mannose-6-phosphate receptor ) to subcellular compartments .
these findings offer indirect evidence of endocytic deficits in
these diseases , potentially reducing the efficiency of current therapies ;
yet , the internalization of other receptors amenable for drug targeting
warrants further investigation .
we have previously reported
lysosomal enzyme delivery ( acid sphingomyelinase ,
-galactosidase , and -glucosidase ; deficient in type
a b niemann pick , fabry , and pompe diseases , respectively )
by targeting intercellular adhesion molecule-1 ( icam-1 ) .
this receptor is highly expressed in inflammatory conditions , including
lysosomal storage disease , and mediates a nonclassical endocytic mechanism
independent of clathrin , caveolin , and other known pathways .
icam-1 engagement induces nano- and micrometer - scale vesicle formation ,
permitting targeting and internalization of nanocarriers of a variety
of dimensions .
cell culture and mouse
models have demonstrated that polymer nanocarriers targeted to icam-1
( anti - icam ncs ) markedly increase accumulation of enzymes in lysosomes
and relieve lysosomal storage .
importantly , anti - icam nc uptake seems to surpass that of otherwise
equivalent nanocarriers targeted to the transferrin receptor , a receptor
associated with clathrin - coated vesicles .
furthermore , these nanocarriers readily internalize even when classical
endocytic pathways are pharmacologically inhibited , suggesting a useful
treatment modality for pathological endocytic dysfunction . here
we have examined clathrin - mediated endocytic activity
in wild - type
fibroblasts and fibroblasts from patients with type a niemann pick
disease ( npd ) . in these cells , the absence of acid sphingomyelinase
( asm ) leads to marked accumulation of sphingomyelin in intracellular
compartments , which has been linked to aberrant mannose-6-phosphate
receptor internalization .
whether this represents
an isolated endocytic deficit or a symptom of broader dysfunction
remains an open question .
we therefore examined the uptake of other
clathrin - associated ligands , the formation of clathrin - coated vesicles ,
and the advantage of targeting icam-1 as an alternative enzyme delivery
strategy .
the murine
monoclonal antibody
against human icam-1 ( anti - icam ) was r6.5 from the american type culture
collection , as in our previous studies .
alexa fluor 594 transferrin , bodipy - fl c12sphingomyelin ,
10000 mw texas red dextran , and fluorescent secondary antibodies were
from molecular probes ( eugene , or ) .
anti - transferrin and anti - clathrin
heavy chain were from calbiochem ( la jolla , ca ) .
green fluoresbrite
100 nm polystyrene beads were from polysciences ( warrington , pa ) .
edward schuchman ( department
of genetics and genomics sciences , mount sinai school of medicine ,
new york , ny ) . unless otherwise noted ,
all other reagents were from sigma - aldrich ( st .
wild - type and asm - deficient skin fibroblasts
from type a npd patients ( homozygous for the r496l mutation ) were
kindly provided by dr .
cells were seeded on glass
coverslips and cultured in dulbecco s modified eagle medium
( gibco brl , grand island , ny ) supplemented with 10% fetal bovine serum ,
2 mm glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin .
cells were incubated at 37 c , 5% co2 , and 95% relative
humidity and , where indicated , stimulated with 10 ng / ml tumor necrosis
factor- ( tnf ) overnight prior to the assay to upregulate
icam-1 expression .
model polymer
nanocarriers were prepared by coating 100 nm diameter
polystyrene beads by surface absorption with a mix of anti - icam and
asm ( anti - icam / asm ncs ) at a 50:50 mass ratio as described .
unbound molecules were separated by centrifugation
at 13000 g for 3 min and removed .
coated carriers
were resuspended in a solution of phosphate buffered saline supplemented
with 1% bovine serum albumin and then sonicated at low power to separate
aggregates .
this protocol has been shown to produce carriers with
active asm capable of degrading accumulated lysosomal substrates . where indicated ,
either anti - icam or the enzyme
cargo were labeled with i , and the amount of radiolabeled
antibody or enzyme per carrier was determined with a gamma counter .
final carrier diameter ( 190 7 nm ) and polydispersity index
( 0.16 0.02 ) were determined by dynamic light scattering as
described previously .
enzyme loading
efficiency was 80% with a content of 230 24 asm molecules and
135 17 anti - icam molecules per carrier .
lysosomal enzymes have
been shown to remain steadily attached to these nanocarrier formulations ,
with only minimal release by mechanical stress ( 10% release
by pipetting , centrifugation , or sonication ) , storage ( < 5% release
after 3 days in saline at 4 c ) , or physiological fluid ( 10%
release after 5 h in serum at 37 c ) . while these prototype carriers are not intended for clinical use ,
they are a valid model with coating efficacy , targeting , and intracellular
transport comparable to anti - icam ncs made of poly(lactic - co - glycolic acid ) , a material approved by the us food and
drug administration .
wild - type and npd fibroblasts
were incubated with medium containing
200 g / ml alexa fluor-594 transferrin for 1 h at 37 c
to measure uptake by cme .
cells were
then washed and fixed with 2% paraformaldehyde at room temperature .
to distinguish the surface - bound fraction of transferrin , fixed cells
were stained with goat anti - transferrin then fluorescein isothiocyanate
( fitc)-labeled rat anti - goat igg .
this protocol renders double - labeled
surface transferrin ( green + red ) vs single - labeled internal transferrin .
alternatively , to measure the colocalization of clathrin and transferrin ,
fixed cells were permeabilized with 0.2% triton x-100 and stained
with mouse anti - human clathrin heavy chain , then fitc goat anti - mouse
igg .
vesicle formation was confirmed by transmission and scanning electron
microscopy ( tem and sem ) in cells treated with transferrin for 30
min . for tem or sem ,
pits were defined as invaginations of
the plasmalemma 50 nm in diameter with a visible electron - dense
protein coat under the cytosolic leaflet of the plasmalemma in the
case of tem , or an electron - light halo surrounding the opening of
the invagination for sem .
semiquantitve analysis was only obtained
from sem , where each frame encompassed 5 pits ( 50
pits analyzed per condition ) .
fluorescent samples were observed
with an eclipse te2000-u microscope , using 60 planapo objectives ,
and filters optimized for texas red and fitc fluorescence ( nikon ,
melville , ny ) .
color channels were imaged separately with an orca - iccd
camera ( hamamatsu , bridgewater , nj ) , merged , and analyzed with imagepro
3.0 software ( media cybernetics , silver spring , md ) .
as described
previously , a custom imagepro macro was used to quantify all surface
( yellow ) vs internalized ( green ) material .
wild - type
and npd fibroblasts were pretreated for 30 min at 37 c with
control media or media supplemented with one or a combination of the
following pharmacological inhibitors : 50 m monodansylcadaverine
( mdc ; inhibits clathrin endocytosis ) , 1 g / ml filipin ( inhibits
caveolar endocytosis ) , and 3 mm amiloride ( inhibits cam endocytosis ) .
cells were then incubated for 1 h with inhibitor - supplemented
media containing 1 mg / ml texas red dextran ( a nondegradable fluid
phase marker for endocytosis ) .
cells were fixed and the number of
fluorescent dextran - filled compartments were quantified by fluorescence
microscopy using an algorithm that quantifies fluorescent objects
whose intensity is above a threshold background level .
following overnight
treatment with tnf to mimic inflammation in many lysosomal
storage diseases , npd fibroblasts were
incubated at 37 c with a similar concentration of free i - asm or i - asm on anti - icam ncs for 1 h ( 2.3
g / ml ) .
cells were then washed to remove unbound asm and scraped
from the well in a lysis buffer ( 2% triton x-100 ) .
the mass of asm
present in cell lysates was determined in a gamma counter as described .
asm activity was measured with an amplexred
sphingomyelinase assay kit ( molecular probes , eugene , or ) .
tnf-stimulated
npd fibroblasts were incubated with free asm and anti - icam / asm ncs
for 1 h at 37 c , washed , and lysed as described above .
asm in
the cell lysate hydrolyzes sphingomyelin in the assay kit , ultimately
reacting to form fluorescent resorufin ( excitation and emission maxima
of 571 and 585 nm , respectively ) .
fluorescence was measured using
an f2500 fluorescent spectrophotometer ( hitachi , schaumburg , il ) .
to determine intracellular asm activity , surface - bound asm was removed
with an acid glycine wash prior to collecting cell lysates , a technique
that has been shown to detach many types of ligands ( including antibody
conjugates ) bound to their respective cell - surface receptors . to verify
asm activity within
the lysosome
, fibroblasts were incubated overnight with tnf
and fluorescent bodipy - fl - c12-sphingomyelin , which integrates
with cellular sphingomyelin stores and fluoresces red ( 620 nm ) at
high concentrations and green ( 530 nm ) at physiological concentrations .
cells were incubated for 1 h with free asm or
anti - icam / asm ncs , then washed and fixed 3 h later , permitting degradation
of intracellular sphingomyelin stores .
the clathrin - mediated
uptake of glycosylated enzymes via the mannose-6-phosphate receptor
represents the basis of current lysosomal enzyme replacement therapies ,
yet the diminished uptake of this receptor in type a b niemann pick
disease poses an obstacle for therapeutic efficiency .
internalization parameters ( binding efficiency , uptake
kinetics , intracellular routing , etc . ) vary widely between ligands ,
even those utilizing the same endocytic pathway .
hence , we examined the uptake of fluorescent transferrin ,
a ligand with a well - described clathrin - mediated internalization pathway .
wild - type fibroblasts internalized transferrin
within widespread intracellular compartments within 1 h , unlike npd
fibroblasts ( figure 1a ) .
we counted less than
half as much internalized transferrin in npd cells as wild - type cells
( 32 4 vs 88 28 ) ; however , there was a comparable amount
of transferrin bound to the surface of the plasma membrane in both
cell types ( figure 1b ) .
these findings suggest
that the transferrin receptor may still be available on the cell surface
for binding , but npd fibroblasts less readily internalize the ligand
( a ) fluorescence microscopy images of wild - type ( wt ) and npd fibroblasts
incubated with transferrin ( green ) for 1 h at 37 c .
unbound
transferrin was washed away , cells were fixed , and surface transferrin
was immunostained ( red + green = yellow ; arrowheads ) to distinguish
from the internalized ligand ( green ; arrows ) . dashed lines = cell
borders .
data are the mean sem .
* comparison with wt cells ( p < 0.05 by student s t test ) .
tem of wild - type and npd fibroblasts exposed to transferrin revealed
far fewer membrane invaginations and clathrin - coated pits on the surface
of npd cells ( figure 2a ) . in parallel , sem
enabled visualization of the cell surface and quantification of endocytic
invaginations ( figure 2b ) .
in addition to dramatic
bulges underneath the cell surface ( asterisks , presumably engorged
storage compartments ) , npd fibroblasts exhibited smaller pit diameters
( 107 8 vs 140 6 nm in wild - type cells ) and fewer pits
over 100 nm ( 6 3 vs 18 5 pits per m ; figure 2c ) , verifying diminished cme capability .
wt and npd fibroblasts were incubated with transferrin for 30 min
at 37 c , washed , and fixed .
( a ) nascent clathrin vesicles and
membrane invaginations were imaged by transmission electron microscopy
( tem ; arrowheads point to only some , not all , examples ) .
( b ) endocytic pits on the cell surface ( arrowheads show
only some , not all , examples ) and bulges beneath the membrane ( asterisks )
were imaged by scanning electron microscopy ( sem ) .
scale bar = 1 m .
( c ) the number and size of the pits in ( b ) were quantified .
data are
the mean sem . * comparison with wt cells ( p < 0.05 by student s t test ) .
clathrin recruitment to ligand binding
sites represents a key step in clathrin - coated vesicle formation ,
so we examined the intracellular distribution of clathrin heavy chain .
without transferrin added to the cell media , clathrin occupied a central
area within the wild - type cell body ( possibly associating with the
golgi and assisting with transport through the biosynthetic route ) ,
but a perinuclear area within the npd cells , potentially indicating
sequestration around lysosomes ( figure 3a ) .
with the addition of transferrin to the cell media , the distribution
broadened in wild - type cells ( figure 3b ) , where
it largely colocalized with transferrin ( 63% 6% ; figure 3c ) . on the contrary
, clathrin remained largely within
3 m of the nucleus in npd cells ( 85% 4% vs 36%
11% perinuclear localization in wild - type cells ) , resulting in low
colocalization with transferrin ( 17% 4% ) .
( a ) fluorescence microscopy images of wt and npd fibroblasts showing
the distribution of clathrin heavy chain in the absence of ligands
added in the cell media .
( b ) cells were incubated with transferrin
for 1 h at 37 c , washed , fixed , and clathrin heavy chain was
immunostained red .
bottom panels show colocalization ( yellow ) of clathrin ( red ) and transferrin
( green ) in the same cells .
( c ) quantification
of the percent of bound transferrin that colocalized with clathrin ,
and the percent of clathrin distributed within 3 m
of the cell nucleus ( perinuclear ) .
data are the mean sem . * comparison
with wt cells ( p < 0.05 by student s t test ) . diminished
clathrin recruitment to ligand
binding sites suggests
a mechanism for diminished cme , which can potentially affect not only
ligand internalization but also constitutive pinocytosis .
we examined
the uptake of texas red dextran ( a nondegradable fluid phase marker
for endocytosis ) . by fluorescence microscopy
, we observed a more widespread
distribution of dextran - filled endocytic vesicles throughout the cytosol
in npd fibroblasts compared to the wild - type ( figure 4a ) , consistent with aberrant intracellular trafficking observed
in some lipidoses .
indeed , we observed only 60%
as much dextran uptake in npd fibroblasts as wild - type cells , indicative
of diminished endocytosis overall ( figure 4b ) . to evaluate whether the reduction was due to impaired cme as
observed above , we treated cells with mdc , an inhibitor of clathrin - mediated
uptake . in wild - type cells ,
mdc decreased pinocytosis of dextran by
48% , indicating that this pathway accounts for dextran uptake .
contrarily ,
applying mdc to npd cells had little effect , confirming that the disease
disrupted the clathrin pathway .
( a ) fluorescence
microscopy images of wt and npd fibroblasts incubated for 1 h at 37
c in media containing texas red dextran .
the number of dextran - positive vesicles
was quantified by fluorescence microscopy in cells treated with or
without ( b ) 50 m monodansylcadaverine ( mdc , an inhibitor of
clathrin endocytosis ) and ( c ) a cocktail of 1 g / ml flipin ( inhibits
caveolar endocytosis ) and 3 mm amiloride ( inhibits macropinocytosis
and cam - mediated endocytosis ) .
# comparison
between control and inhibitor within each cell type group ( p < 0.05 by student s t test ) .
next we evaluated whether cme
can compensate for uptake when other
pathways are inhibited .
wild - type cells treated with a cocktail of
filipin and amiloride ( inhibitors of the caveolar , and cam + macropinocytosis
pathways ) maintained basal levels of bulk uptake ( 115 11 vesicles ,
figure 4c ) .
others have reported compensation
between clathrin / caveolar pathways in response to inhibition , which
may be the case here . however , such compensation
was not observed in npd cells treated with the same inhibitor cocktail ,
further validating that cme is impaired in these cells .
taken together ,
these data demonstrate that not only cme of ligands , but also clathrin - mediated
pinocytosis is affected in npd fibroblasts .
because several
enzyme replacement therapies depend on cme via the mannose-6-phosphate
receptor , deficient receptor - mediated
and pinocytic cme in npd cells may affect the efficacy of this strategy ,
calling for other therapeutic approaches .
we have previously reported
asm delivery to npd fibroblasts using nanocarriers targeted to icam-1
on the cell surface , inducing endocytosis and lysosomal delivery via
a well - studied pathway distinct from clathrin , caveolin , and other
known pathways .
here we compared the uptake of asm carried by anti - icam ncs to the
uptake of free asm .
over the course of 1 h , npd fibroblasts internalized
greater amounts of enzyme ( > 10 fold ) when presented on anti - icam
ncs
( figure 5a ) . as a result
, npd cells treated
with anti - icam / asm ncs recovered more basal asm activity than those
treated with free enzyme ( 30% vs 3% of the asm activity
of wild - type cells ; figure 5b ) .
furthermore ,
activity of anti - icam / asm ncs was localized to the intracellular space
( 57% intracellular enzyme activity ) , whereas most free asm
activity was localized to the cell surface ( 10% intracellular
enzyme activity ) , as evidenced after washing the cells with acid glycine
( figure 5c ) .
therefore , alternative targeting
strategies , such as targeting the cam pathway , offer a means to bypass
dysfunctional clathrin endocytosis and deliver enzymes to intracellular
targets .
( a ) tnf-stimulated
npd fibroblasts were incubated for 1 h at 37 c with free i - asm or anti - icam / i - asm ncs .
the amount of
cell - associated asm was determined in a gamma counter from the cell
lysates .
( b ) additionally , asm activity was measured with a sphingomyelinase
kit ( molecular probes ) to detect the conversion of sphingomyelin into
fluorescent resorufin in cell lysates .
( c ) intracellular enzyme activity after removing
the surface - bound asm fraction with an acid glycine solution .
( a c ) data are the
mean sem . * comparison with cells treated with free asm ( p < 0.05 by student s t test ) . given the enhanced intracellular asm delivery
by anti - icam ncs , we tested their potential to attenuate lysosomal
storage , the central goal of enzyme replacement therapy .
npd and wild - type
fibroblasts were incubated overnight with fluorescent sphingomyelin
to permit visualization of lipid stores , and npd fibroblasts were
treated with free or carrier - bound asm as before ( figure 6a ) .
untreated npd fibroblasts accumulated 5-fold
more sphingomyelin in compartments around the nucleus than wild - type
cells ( figure 6b ) .
treatment with anti - icam / asm
ncs reduced 89% of storage , whereas treatment with the same
amount of enzyme free in solution reduced only 35% of storage ,
consistent with impaired cme . reduction of sphingomyelin accumulation in npd
fibroblasts by asm
delivery .
tnf - stimulated wt and npd fibroblasts were incubated overnight
with fluorescent bodipy - fl - c12-sphingomyelin to permit
visualization of lipid accumulation .
( a ) fluorescence microscopy images
of cells receiving no treatment , free asm , or anti - icam / asm ncs for
1 h at 37 c .
cells were washed and fixed 3 h later to permit
sphingomyelin degradation . dashed lines
comparison with untreated npd control ( p <
0.05 by student s t test ) .
finally , the reduction of lysosomal storage by
anti - icam ncs correlated
with restoration of cme .
npd fibroblasts treated with anti - icam / asm
ncs demonstrated a more widespread distribution of clathrin heavy
chain and a 4-fold improvement of clathrin transferrin colocalization ,
reaching levels comparable to that of the wild - type cells ( figure 7c ) .
furthermore , treated cells exhibited recovery
of transferrin uptake , from 36% to 71% of the wild - type
levels ( figure 7a ) , suggesting a relationship
between lysosomal storage and endocytic efficiency .
recovery of clathrin
distribution , recruitment , and endocytosis
in npd fibroblasts treated with anti - icam / asm ncs .
( a ) untreated npd
fibroblasts vs npd fibroblasts treated with anti - icam / asm ncs were
incubated with fluorescent transferrin as in figure 1 .
( b ) fluorescence microscopy
images of untreated and treated npd fibroblasts incubated for 1 h
at 37 c with transferrin , and then immunostained for clathrin
heavy chain .
bottom
panels show the colocalization ( yellow ) of clathrin ( red ) and transferrin
( green ) in the same cells .
( c ) quantification of the percent of bound transferrin
that colocalized with clathrin , and the percent of clathrin distributed
within 3 m of the cell nucleus .
( a , c ) data are the
mean sem . * comparison with untreated npd cells ( p < 0.05 by student s t test ) .
as one of the predominant
endocytic mechanisms in most mammalian
cells , cme represents a valuable target for intracellular drug delivery ,
either through passive association with the fluid phase of clathrin - coated
vesicles or active targeting of the cargo receptors that regulate
this pathway . in the case of enzyme replacement
therapy for lysosomal storage disorders , where therapeutic recombinant
enzymes target the mannose-6-phosphate receptor , clathrin endocytic
activity is key to efficient enzyme uptake . using a patient fibroblast model ,
we have examined the effect of
type a npd on cme , the implications for enzyme delivery , and the potential
role for icam-1-targeted nanocarriers to enhance enzyme delivery by
bypassing the clathrin pathway .
the lysosomal storage phenotype
at the cellular level appears to
correlate with diminished uptake of clathrin - associated cargo .
we
observed diminished internalization of fluorescent transferrin in
asm - deficient fibroblasts as compared to the wild - type ( figure 1 ) .
the quantity of transferrin associated with the
cell surface remained the same , suggesting that the ligand receptor
interactions may remain intact at the cell surface , while the internalization
machinery or the recycling kinetics may be impaired .
several groups
have also observed impaired receptor mobilization in cells with lysosomal
storage disorders .
for example , atypical mannose-6-phosphate receptor
internalization and trafficking has been observed in aveolar macrophages
from asm - knockout mice , in fibroblasts with i - cell disease , and in
fibroblasts with pompe disease .
interestingly , previous studies have also described either enhancement
or reduction of mannose-6-phosphate receptor expression in different
lysosomal storage disorders , while our work did not identify changes in this parameter with regard
to the transferrin receptor . because the mannose-6-phosphate receptor
is directly involved in transporting lysosomal enzymes in the biosynthetic
route , it is possible that the receptor
accumulates at this stage as an effect of lysosomal storage disease ,
thereby altering expression on the cell surface and explaining differences
with our study .
it is also possible that our experimental set up may
not detect a reduction of transferrin receptor expression on the cell
surface because we do not directly evaluate this , but rather experiments
involve concomitant endocytosis , which may in itself modify the amount
of receptor exposed at the plasmalemma .
importantly , transferrin is
often an advantageous targeting moiety that can be chemically conjugated
to drugs or nanoscale drug carriers , and the mannose-6-phosphate receptor
is also a key target for current enzyme replacement therapies . yet ,
results shown here indicate that these may not represent the most
efficient targeting schemes for lysosomal storage diseases .
we examined endocytosis
in these cells and found that dysfunction
was not limited to specific receptors but also affected clathrin - mediated
pinocytosis , posing significant implications to current therapies .
upon examination with tem , npd fibroblasts exposed to transferrin
formed fewer clathrin pits ( observable by the characteristic cytosolic
lattices ) and exhibited generally less invaginations and furrows in
their membrane ( figure 2 ) .
these observations
correlate well with the smaller size and number of endocytic pits
observed by sem ( figure 2 ) and with the diminished
clathrin - mediated uptake of extracellular fluid ( figure 4 ) . given
established diameters for traditional pinocytic vesicles
formed by macropinocytic , clathrin- , and caveolin - mediated pathways
( > 1 m , 120 nm , 60 nm , respectively ) , the
pits
we observed were most consistent with cme in size . aberrant pit formation in npd cells is a relevant parameter
for therapeutic design , restricting the amount of material and the
size of objects these cells internalize .
for example , diminished fluid
phase uptake may impair substrate reduction therapy , a treatment strategy
where small molecules remove excess storage or inhibit metabolite
biosynthesis in order to attenuate storage . and
while smaller pit sizes may still accommodate entry of small
molecules , macromolecules and drug conjugates larger than 100 nm in
diameter ( such as enzyme - loaded nanoparticles or certain viral gene
delivery vectors ) may be excluded .
diminished pit formation
can be explained by poor trafficking of
endocytic machinery ( e.g. , clathrin ) to the pit site .
the diseased
cells exhibited perinuclear clathrin localization that was poorly
recruited to transferrin binding sites ( figure 3b ) .
pits recruit clathrin directly from the cytosol , and cytosolic diffusion may be impaired due to the storage
phenotype .
for example , clathrin coats assemble on endosomes / lysosomes
and the trans - golgi network during the formation of transport vesicles
and on autophagosomes during lysosome reformation
. aberrant membrane fusion due to elevated cholesterol ( a feature
of several lipidoses , including type a b npd ) has been linked to vesicle and autophagosome accumulation .
the delayed turnover of these
intermediate compartments may lead to sequestration of a fraction
of the clathrin pool , consistent with the observed perinuclear clathrin
localization ( figure 3 ) and limiting clathrin
availability at the plasma membrane .
impaired cme in npd cells
may be responsible for poor internalization
of free asm .
inefficient enzyme uptake has long been an obstacle to
enzyme replacement therapy ; the aforementioned aberrant uptake of
mannose-6-phosphate receptors has been linked to poor internalization
of asm in a murine npd model and -glucosidase in pompe fibroblasts .
it is worth noting , however ,
that this is not a universal finding .
renal tubular cells with metachromatic
leukodystrophy ( a lysosomal storage disease characterized by sulfatide
accumulation ) showed a 2-fold increase in enzyme uptake via this receptor
in one study .
this is a key aspect to
consider : given that different cell types and tissues in the body
are differently affected by npd ( as for other lysosomal storage disorders ) ,
it is likely that the manner and severity of endocytic dysfunction
will vary ( e.g. , neurons are severely affected in type a npd , vs fibroblasts
used here as an available model ) .
therefore , a thorough and comparative
investigation of endocytic patterns is necessary in other cells types .
furthermore , systematic examination of these patterns in different
lysosomal storage diseases may help identify efficient routes of internalization .
for example , the present study demonstrated the utility of nanocarriers
that target the cam pathway ( a clathrin- and caveolin- independent
pathway ) , offering a well - studied means to bypass aberrant cme and
more efficiently deliver enzyme cargo .
therapeutic effect only requires 1020% of
wild - type enzyme activity within the lysosome , which was achieved with anti - icam / asm ncs after only 1
h. the nanocarriers more efficiently reduced sphingomyelin storage
( figure 6 ) , and the reduction of the storage
phenotype led to a recovery of clathrin - mediated endocytosis ( figure 7 ) .
the restorative effect could be due to relief
of the intracellular traffic jam , permitting increased cytosolic diffusion
of endocytic machinery and active transport of trafficking compartments
along opened cytoskeletal pathways .
alternatively , several lipidoses
( such as npd ) are characterized by increased cholesterol storage ,
which
has been implicated in aberrant suppression of lipid synthesis , switching
between lipid biosynthetic and degredative pathways , and abnormal
lipid trafficking between the membrane and intracellular compartments .
atypical membrane composition
resulting from these disrupted processes may play a role in endocytic
dysfunction and could be revived upon normalizing stored lipids to
a basal level .
it would be interesting to test if a similar restoration
of cme could be observed in npd cells following substrate reduction
therapy , using compounds that have been demonstrated to reduce lipid
accumulation , such as cyclodextrin or miglustat .
for drugs , drug conjugates , and drug carriers that
target endocytosis
for cellular uptake , the relationship between disease and endocytic
behavior represents a crucial and often overlooked design parameter .
for example , this work examined for the first time a broader relationship
between lysosomal storage and cme .
impairment of clathrin - associated
ligand uptake in asm - deficient fibroblasts poses a challenge to current
enzyme replacement therapies dependent on this pathway , as well as
novel therapies that may seek to use clathrin - targeting agents . targeting
alternate mechanisms of endocytosis , such as the cam pathway ,
may
improve therapeutic delivery to desired intracellular compartments ,
whether for small molecule , gene delivery , or enzyme replacement platforms .
recognizing
the relative activity of different endocytic pathways
in diseased cells may help identify appropriate drug targets , and
whether by ligand - mediated or pinocytic uptake , improve the internalization
efficiency of drug delivery systems overall .
additionally , by analogy
to endocytic defects observed in lysosomal storage diseases , it becomes
vital to examine whether accumulation within endolysosomal compartments
of foreign materials employed in drug delivery could impart similar
endocytic alterations .
because most drug carrier strategies converge
at the endolysosomal route , this knowledge will be key in designing
strategies for intracellular delivery built of materials whose degradation
does not cause secondary endocytic defects . | drugs
often use endocytosis to achieve intracellular delivery ,
either by passive uptake from the extracellular fluid or by active
targeting of cell surface features such as endocytic receptors .
an
example is enzyme replacement therapy , a clinically practiced treatment
for several lysosomal storage diseases where glycosylated recombinant
enzymes naturally target the mannose-6-phosphate receptor and are
internalized by clathrin mediated endocytosis ( cme ) .
however , lysosomal
substrate accumulation , a hallmark of these diseases , has been indirectly
linked to aberrant endocytic activity .
these effects are poorly understood ,
creating an obstacle to therapeutic efficiency .
here we explored endocytic
activity in fibroblasts from patients with type a niemann pick
disease , a lysosomal storage disease characterized by acid sphingomyelinase
( asm ) deficiency .
the uptake of fluid phase markers and clathrin - associated
ligands , formation of endocytic structures , and recruitment of intracellular
clathrin to ligand binding sites were all altered , demonstrating aberrant
cme in these cells .
model polymer nanocarriers targeted to intercellular
adhesion molecule-1 ( icam-1 ) , which are internalized by a clathrin - independent
route , enhanced the intracellular delivery of recombinant asm more
than 10-fold compared to free enzyme .
this strategy reduced substrate
accumulation and restored clathrin endocytic activity to wild - type
levels .
there appears to be a relationship between lysosomal storage
and diminished cme , and bypassing this pathway by targeting icam-1
may enhance future therapies for lysosomal storage diseases . |
the elderly are at risk for nutritional disorders and malnutrition , including undernutrition and nutrient deficiencies and imbalances .
several age - related physical , clinical , economic , and social factors can compromise nutritional status ; furthermore , changes in the body composition of elderly people and sedentary lifestyles reduce energy requirements and daily energy intakes .
the highly intercorrelated nature of the consumption of dietary components makes it difficult to evaluate specific unconfounded effects through observational studies.1
2
eating patterns have been considered risk factors for various metabolic and circulatory changes that cause several symptoms including dizziness , especially among elderly individuals.3
4
dizziness is a change in balance characterized by the illusion of movement of the individual or the environment that surrounds them .
rotational dizziness is called vertigo.5 this symptom is highly prevalent worldwide , affecting 2% of young adults , 30% at 65 years , and up to 50% in the elderly over 85 years.6
especially in the elderly , a lack of regular physical activity , low level of physical fitness , and nutritional disorders are risk factors for several metabolic and circulatory changes that cause various symptoms , such as dizziness and even benign paroxysmal positional vertigo ( bppv).1
7
current evidence shows that good dietary habits in the elderly can improve their quality of life .
the prevalence of malnutrition is increasing in this population and is associated with declines in functional status , impaired muscle function , decreased bone mass , immune dysfunction , anemia , reduced cognitive function , poor wound healing , delayed recovery from surgery , higher hospital readmission rates , and mortality.8
malnutrition is defined as a state in which a deficiency , excess , or imbalance of energy , protein , and other nutrients causes adverse effects on body form , function , and clinical outcome.9
there is evidence to suggest that dietary habits such as low sodium can alter inner ear fluid homeostasis and auditory function .
the experiments indicate that the endolymph compartment has a built - in mechanism for maintaining a low - sodium concentration while keeping ionic balance with the surrounding perilymph and serum.10
the labyrinth system , which depends on a continuous supply of energy , is influenced by the circulating levels of glucose and hormones , which depend on the generation of energy by adenosine triphosphate .
data in the literature estimate that the occurrence of glucose metabolic disorder is between 42 and 80% in patients with tinnitus and dizziness , whereas 2.5 to 15% of the population presents asymptomatic hypoglycemia or some affection of glucose tolerance curves . in brazil
, glucose metabolic disorder has already been considered the most frequent cause of labyrinthic metabolic dysfunctions.11
12
vertigo is the most common type of dizziness and bppv is the most common cause of vertigo in adults .
it is considered the most common cause of dizziness in the elderly , and 30% of people had the condition at least once.13
14
in the united states , bppv has an estimated prevalence of 2.4% in the general adult population , and although this disorder affects people throughout life , it tends to affect individuals aged 50 to 70 years and therefore affects payroll taxes and the health.15
based on the considerations presented , this study aimed to investigate the possible association between the presence of bppv with the practice of food habits in the elderly .
this cross - sectional study was approved by the human research ethics committee.16 it is part of a broader investigation , the eelo project ( from portuguese : studies on aging and longevity ) , which has been conducted in londrina since 2009 .
the city of londrina ( 500,000 inhabitants ) is situated in the north region of paran state , brazil .
the city has a population of 43,610 elderly people enrolled in the 38 primary care units in the urban city area .
the sample was a randomly stratified set , considering the gender and the five regions of the city ( 15% from the central region , 27% from the northern region , 23% from the southern region , 19% from the eastern region , and 16% from the western region ) .
the study included individuals aged 60 years and over , of both genders , who were living independently and classified at level 3 or 4 as proposed by spidurso.17 this classification evaluates the independence level of the elderly , with level 1 indicating a lack of self - mobility and level 5 indicating athletes .
elderly who had any illness or limitation that would prevent the testing , such as physical or mental disabilities , were excluded from the sample .
bppv was found in 117 , and 53 of them had recurrent bppv confirmed by the questionnaire .
the dietary information was collected by means of the dietary 24-hour recall methods.18 the interviews were conducted on three different days : one day on the weekend and two in the middle of the week . with the aid of photo album with pictures of portion sizes and foods ,
the interviews took place with notation of the food consumed in the order of dialed meals .
the types of food , the quantities consumed , and how they were prepared were recorded .
the quantities of these foods were reported in household measures and converted into grams or milliliters .
dietary data were processed and analyzed with the nutritional evaluation software avanutri online.19 the analysis of dietary intake of protein , carbohydrate , lipid , fiber , and cholesterol took into account the recommended dietary reference intakes.20
the presence of vertigo was established through questions about vertigo ( attacks , symptoms , and familiar history of vertigo ) , and the diagnosis of bppv among study participants with vertigo was established with the dix - hallpike maneuver and answers on the questionnaire about vertigo.21
the chi - square test was performed , and p 0.05 was considered statistically significant .
the significance of food habit variables and the variables carbohydrates , polyunsaturated fat , monounsaturated fat , saturated fat , lipids , protein , and fiber were all considered .
based on a sample of 487 subjects , 117 had bppv and 370 did not .
we did not observe a significant association ( p = 0.3064 ) between food habits and bppv in the total population ( odds ratio = 1.3017 ; table 1 ) . abbreviation : arr , absolute risk reduction ; bppv , benign paroxysmal positional vertigo ; nnh , number needed to harm .
note : odds ratio = 1.3017 ; confidence interval 0.82722.0483 ; arr = 5.41% ; nnh = 19 ;
corr = 1.303 ( p = 0.3064 ) . of the 117 people who had bppv , 102 ( 87.17% ) had inadequate carbohydrate intake and 15 ( 12.82% ) did not . and of the 370 people without bppv , 330 ( 89.18% ) had inadequate carbohydrate intake and 40 ( 10.81% ) had normal intake .
the mann - whitney test was used for statistical analysis between inadequate carbohydrates and bppv and gave a value of u = 19,351.50 and p = 0.0419 , a statistically significant difference ( see table 2 ) .
note : u = 19351.50 ( p = 0.0419 ) . of the 117 people who had bppv , 82 ( 70.08% )
had polyunsaturated fat intake and 35 ( 29.91% ) did not . of the 370 people without bppv , 289 ( 78.10% )
had polyunsaturated fat intake and 81 ( 21.89% ) did not . for statistical analysis between polyunsaturated fat and bppv
, we performed the mann - whitney test , which gave a value of u = 18470.00 and p = 0.0084 , a statistically significant difference ( see table 3 ) . abbreviation : bppv , benign paroxysmal positional vertigo .
we did not observe a significant association between inadequate protein intake ( p = 0.78 ) , inadequate intake of saturated fats ( p = 0.97 ) , inadequate intake of lipids ( p = 0.43 ) , and inadequate intake of monounsaturated fats ( p = 0.79 ) , but there was important significance between bppv and inadequate fiber intake ( p = 0.03 ; table 4 ) . abbreviation : bppv , benign paroxysmal positional vertigo .
in this study , we observed the association of bppv with a diet of inadequate carbohydrate intake , rich in polyunsaturated fatty acids , and insufficient fiber intake .
the elderly often have reduced appetite and energy expenditure , which can occur along with a decline of biological and physiological functions , reduction of lean body mass , and changes in cytokine and hormonal levels .
other disturbances include changes in fluid electrolyte regulation , delayed gastric emptying , and diminished senses of smell and taste .
in addition , pathologic changes of aging such as chronic diseases and psychological illness can lead to bad nutrition in the elderly .
nutritional assessment is important to identify , along with treating patients at risk.8
vertigo , tinnitus , and hearing loss are common complaints among the elderly in industrial countries .
numerous agents are known to incite vertigo , tinnitus , and hearing loss , such as hyperinsulinemia and hyperlipidemia . according to the study of kamierczak and doroszewska,22 who assessed the occurrence of hyperinsulinemia and hyperlipidemia in patients suffering from vertigo , tinnitus , or hearing loss of unknown origin
however , the authors concluded that disturbances of metabolism by glucose , such as diabetes mellitus and hyperinsulinemia , may be responsible for inner ear diseases , whereas the disturbance of lipid metabolism remains vague .
micronutrient insufficiency and high saturated fat intake have been associated with chronic diseases.23 the study also reported that disturbances of glucose may be responsible for inner ear diseases.23 we also detected an excess of carbohydrates ; these nutrients are interconvertible , raising each other through degradation of its components : 85% excess carbohydrate becomes fats ( lipids ) in the individual by the liver , increasing the lipids , cholesterol , and triglycerides in the blood , which may cause accumulation of sodium and potassium in the inner ear .
huffman et al confirmed our data showing that a lack of fiber leaves the human body unprotected , without the minimum number of regulators provided by a good diet.23 fiber has an effect on lipid metabolism ( propionate ) and glucose ( acetate , propionate , and butyrate ) , delaying absorption of glucose and starch hydrolysis , helping to maintain electrolyte balance of blood capillaries and also assisting the vestibule - cochlear apparatus health .
british dietary recommendations are to decrease total fat intake to less than 30% of daily energy consumption and saturated fat to less than 10%
. the energy and fat intake seems to be reduced on the diet rich in polyunsaturated fatty acids .
insulin sensitivity and plasma low - density lipoprotein cholesterol concentrations are improved with a diet rich in polyunsaturated fatty acids compared with the diet rich in saturated fatty acids.24 however , other literature has reported that the elderly interviewed in large urban centers of brazil have not joined the new trend of developed countries ; consumption of traditional foods were replaced by processed foods of easy preparation causing dyslipidemia and excess of sodium , damaging the natural physiology chemical level in the inner ear among other comorbidities.25
in this study , we detected a diet high in polyunsaturated fatty acids ( table 3 ) .
reviewing the literature , we could observe that polyunsaturated fatty acids , when processed ( hydrogenated ) , can be transformed into trans fats , which are harmful to health .
however , we can not analyze this information by itself ; an individual who consumes excess fat ( even good fat ) and carbohydrates can still eat a low concentration of dietary fiber , which is likely to have dyslipidemia affects in the ear .
even though not much data regarding disorders of lipid metabolism exist in the literature , the ingestion of high amounts of polyunsaturated fats and trans fat could be related in part to lipid metabolism disorders.26
mantello et al wrote that the changes in glucose metabolism are the main metabolic changes that lead to vestibulocochlear disorders.27 as the labyrinthine structures possess an intense metabolic activity , glucose is necessary for energy production and for maintaining proper concentrations of sodium and potassium in the endolymph.27 some habits , such as alcohol , tobacco , sugar , salt , saturated fats , and caffeine , in addition to physical inactivity , should be banned from the lives of patients with vertigo because they can exacerbate symptoms of cochleovestibular and make vestibular compensation even slower.28 we suggest that the same procedures are taken in cases of bppv , intensely studied here , for the same reasons cited above . through these results ,
we emphasize the importance of a multidisciplinary care team , expanding the procedures and results to prevention and treatment and minimizing episodes of recurrent bppv in the elderly .
the association between bppv with inadequate carbohydrate intake and a diet rich in polyunsaturated fatty acids and inadequate fiber intake has been observed .
these data represent an important tool for better understanding the overall health and comorbidities of the elderly , assisting with the reasoning and awareness of a necessary change in their lifestyle ; through guidelines and nutritional treatments , alongside multidisciplinary care , the team can help to decrease hearing symptoms of bppv .
this study showed the importance of further studies associating the relationship between bppv and the types of food habits .
further research is needed to develop a prevention and rehabilitation of bppv with associate food habits . | introduction poor diet habits and inadequate intake of nutrients are a concern in the elderly .
nutritional education with guidance may improve the results of the treatment of vertigo .
objective evaluate the presence of benign paroxysmal positional vertigo ( bppv ) associated with feeding habits .
methods cross - sectional study with elderly people living independently .
we evaluated nutritional habits through the method of dietary 24-hour recall and manipulation of dix - hallpike .
results based on a sample of 487 individuals , 117 had bppv . among the 117 elderly patients with bppv , 37 ( 31.62% ) had inadequate feeding .
from those 370 individuals without bppv , 97 ( 26.21% ) had inappropriate feeding .
no significant association between nutritional habits and bppv in the total population was observed ( p = 0.3064 ) .
however , there was significant relation between bppv and inadequate carbohydrate intake ( p = 0.0419 ) and insufficient fiber intake ( p = 0.03 ) , and the diet of these subjects was rich in polyunsaturated fatty acids ( p = 0.0084 ) .
conclusion these data correlate with the dyslipidemia and hypertriglyceridemia status , making it extremely important to reduce the intake of fats and carbohydrates and increase the fiber intake to stabilize triglycerides and thus minimize harmful effects on the inner ear .
food readjustment is suggested in patients with bppv , along with the work of a multidisciplinary team to improve the quality of the elderly . |
injuries to the extrahepatic biliary system in blunt abdominal trauma are uncommon [ 17 ] .
extrahepatic hepatic ductal injuries ( ehdis ) occur even less frequently [ 18 ] . because of their rarity and the frequently insidious onset of symptoms ,
ehdis are commonly missed during the initial trauma evaluation , and debate continues regarding the best way to diagnose and treat them
[ 2 , 9 ] .
diagnostic tools useful in ehdis include computed tomography ( ct ) , abdominal ultrasound ( aus ) , nuclear imaging ( hida ) , percutaneous transhepatic cholangiography ( ptc ) , and endoscopic retrograde cholangiopancreatography ( ercp ) .
traditional management options in ehdi include primary repair with or without a t - tube , biliary - enteric anastomosis , ductal ligation , stenting , and drainage .
simple drainage and biliary decompression is often the most appropriate treatment option in unstable patients [ 2 , 3 ] .
more recently , ercp has allowed trauma surgeons to diagnose and potentially treat ehdis via stenting and/or papillotomy , even in the face of previous abdominal surgical procedures [ 4 , 10 , 11 ] .
a comprehensive review of 53 cases of ehdis reported in the english - language literature was conducted , focusing on the evolving role or ercp in diagnosis and treatment of these injuries .
we also report an illustrative case of blunt traumatic injury involving the extrahepatic portion of the left hepatic duct
( lhd ) and the confluence of the lhd and the right hepatic duct ( rhd ) .
diagnostic and treatment algorithms that incorporate ercp are presented in order to help systematize and simplify the management of ehdis .
a 26-year old motorcycle rider was struck on his right side by a mid - sized passenger car traveling at approximately 30 miles per hour .
he was hemodynamically unstable upon arrival to the hospital ( systolic blood pressure 60 mmhg , heart rate 120/min ) .
the patient remained hypotensive despite aggressive fluid resuscitation and was promptly taken to the operating room .
he was found to have a large stellate laceration of the liver involving medial segments of the right lobe .
a jackson - pratt drain was left in the left upper quadrant ( luq ) . after a damage control dressing
was placed , the patient was taken to interventional radiology where several branches of the right hepatic artery were embolized .
the patient 's early hospital course was uneventful and his abdomen was definitively
closed on postoperative day two .
however , he subsequently began draining
increasing amounts of bile from his abdominal drain , associated with concurrent
rise in serum bilirubin . computed tomography ( ct ) of the abdomen demonstrated a
large fluid collection in the upper abdomen ( figure 1 ) .
a percutaneous
drain was placed into this collection and drained approximately 500 ml of bile .
due to
continued drainage of several hundred milliliters of bile per day , an
ercp was obtained .
this demonstrated a proximal transection of the extrahepatic
portion of the lhd as well as a leak at the confluence of lhd and rhd
( figure 2(a ) ) .
stenting across the transected lhd was attempted but the guidewire could not be passed across the injury .
a sphincterotomy was performed and the
common bile duct ( cbd ) was stented in order to decompress the biliary tree . over the next several days ,
the drainage markedly decreased and the patient was discharged to home with drains in place .
a repeat ercp four months after patient 's initial injury showed
filling of both the rhd and the lhd ( figure 2(b ) ) .
his liver function
tests ( lfts ) at the time were within normal limits .
he is now four years out from his original trauma , has normal lfts , and a recent abdominal sonogram showed normal cbd size .
the first case of bile duct rupture due to blunt abdominal trauma was reported by wainwright in 1799 .
. the frequencies of injuries to the biliary tree , in decreasing order , are those of gallbladder , common bile duct ( cbd ) , hepatic
ducts ( hd ) and junction of left hepatic duct ( lhd ) , and right hepatic duct ( rhd )
[ 1 , 14 ] .
we will first discuss the demographics , anatomy , and pathophysiology of ehdis , followed by a description of some traditional treatment methods and a detailed discussion of the emerging role of ercp in the treatment of ehdis , along with diagnostic and treatment algorithms that incorporate ercp .
extrahepatic hepatic ductal injuries ( ehdis ) occur predominantly in men , and the male - to - female ratio increases with patient age [ 2 , 12 , 15 ] .
approximately 50% of ehdis
are automobile - related , with the remaining half due to crush injuries ,
motorcycle crashes , sports / recreational injuries , and falls .
the rarity of ehdis combined with over 50% frequency of severe associated injuries contributes
to an average diagnostic delay of about 2 weeks [ 12 , 16 ] .
, the relative fixation of the proximal hepatic ducts to the liver can lead to a
shearing force , inducing intraductal hypertension and tearing , as seen with
high - speed deceleration [ 14 , 17 ] .
another mechanism involves compression of the biliary system and gallbladder against the vertebral column and ductal blowout , which may be seen when the gallbladder rapidly empties into a short cystic duct [ 2 , 8 , 16 ] .
ischemic necrosis of the ducts has also been proposed , perhaps
accounting for delayed injuries .
a combination of mechanisms is
likely involved in each individual case . abdominal ultrasound ( aus ) and
percutaneous evacuation of bile can help confirm the diagnosis . in cases of persistent bile drainage
, scintigraphy may be useful , although it is poor in pinpointing the site of injury [ 6 , 19 ] .
more recently , ercp has emerged as a valuable adjunct in treatment of ehdi , and can be both diagnostic and therapeutic [ 12 , 21 , 22 , 23 ] .
one third of ehdis are missed at initial laparotomy or investigation , and another 2% are
not recognized on repeat surgery [ 8 , 12 ] . over
50% of patients with ehdis who do not undergo immediate trauma laparotomy typically have a diagnostic delay of more than 1 day , which can result in significant morbidity and mortality [ 2 , 8 ] .
sterile bile causes minimal peritoneal reaction , with vague abdominal pain and distention , nausea , vomiting , and jaundice [ 8 , 24 ] .
liver injury is the most common associated injury ( 55% of patients ) , followed by extremity ( 19% ) , pelvic fractures ( 17% ) , and splenic and gallbladder injury .
pneumothorax , rib fractures , and head injury are less frequent . others report combined injuries involving the duodenum , stomach , colon , pancreas , and non - ehdi biliary duct injuries
( 5% ) [ 12 , 25 ] .
mortality was reported in 3.8% to 12.7% cases of ehdi , with blunt injuries being
associated with higher mortality than penetrating injuries [ 1 , 2 , 12 , 25 , 26 ] .
ehdis are associated with long hospitalizations ( average of 40 days ) and many potential complications .
early complications include wound infection , abscess , and biliary fistula
[ 8 , 27 , 28 ] .
late complications include ductal stricture with or without cholangitis and posttraumatic hepatic atrophy [ 25 ,
29 , 30 ] .
when the diagnosis of ehdis occurs during an emergent celiotomy , the
primary focus should be on patient stabilization , hemostasis , temporizing stenting , ligation , and t - tube placement should be considered
[ 1 , 8 , 31 ] . at the very least , the injured duct should be tagged and the area drained , with definitive repair performed later .
long - term stenting across injured hepatic ducts may be considered , even without suture repair [ 17 , 32 ] .
choledochoenterostomy and hepaticoenterostomy have been used for major ductal injuries , including
complete transections [ 1 , 15 , 33 ] .
when performing biliary reconstruction ,
the size of the duct and viability of its blood supply have to be considered ,
and end - to - end ductal repair should be avoided in complete injury due to the
risk of stricture formation [ 8 , 16 , 25 ] .
bilioenteric anastomoses produce good long - term outcomes in 8590% of cases [ 3538 ] .
long - term anastomotic stenting ( 69 months ) has been supported by some authors [ 25 , 36 ] , while others stent for a shorter period of time or
not at all [ 35 , 39 ] .
anastomotic stents decompress the biliary tree , allow postoperative radiographic followup , and there may be a correlation between outcome and the time stented as anastomotic catheters may limit the contraction of collagen and stricturing .
opponents of stenting argue that stents contribute to complications ( stent dislodgment , occlusion by biliary debris ,
and cholangitis ) [ 25 , 40 ] .
while many biliary strictures appear in the first 2 postoperative years , it may take up to 5 years for 80% of strictures to occur , with approximately 20% of failures after that period , suggesting that a
long - term followup of 710 years may be optimal [ 25 , 35 , 41 ] .
tacking of the
roux - en - y jejunal loop marked with metallic clips to the abdominal wall can
help facilitate future biliary tree access .
management of ehdis depends on the patient 's overall clinical status , associated injuries ,
and the location and extent of the injury .
patients who are hemodynamically
stable on initial presentation and do not require immediate surgical
intervention can safely undergo nonoperative management of bile duct injuries an attractive therapeutic alternative [ 42 ,
43 ] .
this is further supported by the use of ercp in treatment of iatrogenic
extrahepatic bile duct injuries , which is well described and accepted [ 12 , 22 ] .
more recently , ercp has emerged as a valuable adjunct in treatment of ehdi , and has
been used to define the anatomy of the injury as well as to provide definitive
therapy [ 12 ,
2123 , 43 ] .
in fact , a total of 19 cases in this review involved ercp utilization in either diagnostic or therapeutic capacity ( table 1 ) .
the safety and efficacy of ercp has been advocated in increasing number of publications , with excellent
( > 90% ) ductal visualization success and low
( < 10% ) morbidity [ 7 , 43 ] .
endoscopic retrograde cholangiopancreatography has been successfully utilized in treatment
of hepatic ductal injuries both as a primary treatment modality and as an
adjunct to surgery , with some of the patients having previously undergone at
least one laparotomy .
indeed , it may be that ercp is the optimal choice
for treatment of bile duct injuries regardless of whether the patient underwent
recent surgery .
one might speculate that performing a potentially therapeutic
ercp for ehdis in the setting of a recent laparotomy may actually constitute the
safest initial approach , given the possibility of postoperative adhesions and
the risk of bile duct devascularization due to surgical dissection .
in fact , at
least one reported death was due to massive hepatic bleeding encountered during
an operative attempt at repair of rhd stricture .
the usefulness of ercp in such setting is exemplified by the current
case , where the diagnosis of ehdi was not made until after the initial trauma
laparotomy , and reoperation to restore biliary continuity would have been very
difficult and risky .
not only did ercp confirm the diagnosis of ehdis and
facilitated definitive treatment of the injury , but also indirectly pointed to
the potential cause of surgical failure if operative management was attempted
small transected lhd that could not be traversed with the guidewire . because surgical repair of small extrahepatic
bile ducts can be exceedingly difficult [ 62 ,
63 ] , a topic beyond the scope of this discussion
, ercp may
be the preferred treatment method in this scenario as well . in majority of reported cases , including the current report , ercp - facilitated ductal
stenting was performed [ 8 ,
10 , 16 ,
43 ] . in fact , ercp with sphincterotomy and
drainage avoids surgery in 7090% of iatrogenic ductal injuries by reducing the
biliary intraductal pressure gradient .
percutaneous drainage of any bile collections should be performed as well , with prophylactic drainage suggested by some even in the absence of an active bile leak . in the current case ,
while a ct scan revealed a fluid collection , it failed to fully delineate the anatomic injury .
much like in other reports of both traumatic and nontraumatic bile duct injuries , ercp was used to define the anatomy of the injury and to treat it definitively with biliary decompression and stenting
[ 12 , 2123 ] .
it is likely that endoscopic stenting provides similar effects to operative anastomotic stenting via biliary decompression and by potentially decreasing stricturing
through limiting collagen contraction .
failures of
endoscopic therapy are rare , and have been associated with leaks from noncommunicating or
anatomically excluded ductal injuries .
a recent review of ehdis with an average followup period of 26 months
reported an increasing use of ercp in both diagnostic and therapeutic
capacities .
among patients treated primarily with ercp , 9/19 had followup studies [ 4 , 8 , 11 , 12 , 16 , 43 , 61 ] . among these patients ,
8/9 showed resolution of biliary leak and no evidence of biliary stricture [ 4 , 8 , 11 , 16 , 43 , 61 ] and
1/9 showed nonvisualization of the previously injured lhd .
as more long - term followup data confirm good clinical results , the ercp
will likely take the dominant position as the initial treatment of choice for ehdis .
complication rates associated with ercp use in the setting of bile duct injuries are low
( < 10% ) . reported post - ercp complications include
pancreatitis and persistent hyperamylasemia [ 10 , 14 ] .
in addition , stent migration or clogging may occur . in adult patients , stent migration has been noted in upto 5% of patients .
stent clogging is more common , with upto 30% incidence
within 3 months of stent placement .
there is also a low risk of infection and bleeding related to percutaneous catheter drainage of ehdi - associated bile
collections .
ductal stenosis at the site of injury is an important late complication of ercp and stenting .
it has been postulated that prolonged
stenting ( up to 12 months ) , sometimes with multiple stents , may provide both
treatment and prevent further stricturing .
after a literature search was conducted , table 1 was
constructed to summarize all known cases of ehdis from 1952 to 2006 .
based on our case as well as the
literature search , proposed diagnostic ( figure 4 ) and treatment
( figure 5 ) algorithms were designed in order to systematize clinical
decision - making in the setting of ehdis .
because the clinical presentation is often insidious , ehdis are frequently missed on the initial clinical evaluation .
availability of ercp presented
trauma surgeons with a new diagnostic and therapeutic alternative . with good short - term results of ercp
being well established , a growing body of data is demonstrating equally good results on long - term followup .
we recommend the use of diagnostic and treatment algorithms to standardize care , decrease diagnostic delay , and potentially improve outcomes . | extrahepatic hepatic ductal injuries ( ehdis ) due to blunt abdominal trauma are rare . given the rarity of these injuries and the insidious onset of symptoms , ehdi are commonly missed during the initial trauma evaluation , making their diagnosis difficult and frequently delayed .
diagnostic modalities useful in the setting of ehdi include computed tomography ( ct ) , abdominal ultrasonography ( aus ) , nuclear imaging ( hida scan ) , and cholangiography .
traditional options in management of ehdi include primary ductal repair with or without a t - tube , biliary - enteric anastomosis , ductal ligation , stenting , and drainage .
simple drainage and biliary decompression is often the most appropriate treatment in unstable patients .
more recently , endoscopic retrograde cholangiopancreatography ( ercp ) allowed for diagnosis and potential treatment of these injuries via stenting and/or papillotomy .
our review of 53 cases of ehdi reported in the english - language literature has focused on the evolving role of ercp in diagnosis and treatment of these injuries .
diagnostic and treatment algorithms incorporating ercp have been designed to help systematize and simplify the management of ehdi .
an illustrative case is reported of blunt traumatic injury involving both the extrahepatic portion of the left hepatic duct and its confluence with the right hepatic duct .
this injury was successfully diagnosed and treated using ercp . |
diabetes mellitus ( dm ) is a chronic progressive metabolic disorder due to absolute ( type 1 ) or relative ( type 2 ) deficiency of insulin hormone . worldwide ,
366 million people were estimated to have diabetes mellitus in the year 2011 , and numbers are predicted to double by 2030 [ 24 ] .
dm has caused approximately 4.6 million deaths in the age group of 2079 years in a ten - year period from 2001 to 2011 , accounting for approximately 8.2% of mortality [ 5 , 6 ] .
almost 80% of deaths related to diabetes occur in low- and middle - income developing countries .
the incidence of type 2 diabetes ( t2d ) with an early onset associated with complications has risen in recent years in oman compared to other middle east countries [ 810 ] .
the impact of t2d may limit function and quality of life among men and women .
individuals with t2d need a disciplined balance between the demands of self - care and preferred lifestyles .
type 2 diabetes is developing into an international public health problem , with a significant increase in the middle east region [ 6 , 11 ] . in oman ,
the prevalence of t2d escalated from 11.6% ( 2000 ) to 15% ( 2005 ) and rose to 16.1% ( 2010 ) , with rising prevalence among all age groups [ 12 , 13 ] .
these figures are expected to double by 2030 due to the life threatening long term complications [ 14 , 15 ] and substantial impact on health and well - being [ 1618 ] . a significant number of omani men and women lack knowledge , skills , and information on self - care management while coping with t2d .
hence , an exploration of personal and clinical factors to improve self - care behaviors among omani men and women with t2d is important in assisting them in managing their health .
health related quality of life ( hrqol ) is a multidimensional construct with bearing on a person 's physical , cognitive , social , emotional , psychological , role , and spiritual status [ 19 , 20 ] .
hrqol is an acceptable outcome or efficacy of self - care among adults with t2d ( figure 1 ) .
the literature on perceptions of living with t2d is extensive and has been shown to correlate with quality of life ( qol ) [ 21 , 22 ] .
several studies show that adults with t2d rate their qol lower than the general population [ 2326 ] as compared to those with type 1 diabetes ( t1d ) [ 27 , 28 ] .
women with t2d have been found to have a lower quality of life than men [ 2931 ] , and those with a longer duration of t2d had poor qol .
a self - care management model leads to better glycemic control and qol , while those with poor glycemic control were found to have low qol [ 36 , 37 ] .
no studies focusing on the predictors of qol and health status among omani men and women have been reported .
hence , the purpose of this study was to examine the predictors of qol and health state and to examine comparisons among omani men and women .
a cross - sectional descriptive study was conducted among adults with t2d in the outpatient diabetes clinic in a selected public hospital .
a sampling framework list of the accessible population with known type 2 diabetes ( n = 2000 ) was obtained from the diabetes clinic in a selected public tertiary hospital in oman .
a simple random sampling using random number tables was used to recruit omani adults who were screened in this diabetes clinic .
the inclusion criteria were adults above 18 years diagnosed with t2d for two years who were able to understand , communicate , and converse in arabic or english language and were not currently pregnant .
the exclusion criteria were adults with known diagnosis of t1d , unknown t2d , cognitive / neurological impairment , mental / physical disability , or critical or advanced complications .
sample size was estimated with the gpower software at a power of 0.95 with an effect size of 0.15 using 10 predictors ( independent variables ) , an alpha of 0.05 , and standard deviation of 1% on two - tailed testing [ 38 , 39 ] . to obtain a power of 0.95 and assuming a 30% incompletion rate , a total sample size of 330
a review of the literature was conducted to select standardized instruments to measure the identified concepts of hrqol and health / self - care activities .
the sociodemographic and clinical - related information was primarily gathered by a demographic and clinical baseline tool which included age , gender , schooling ( educational level ) , duration of diabetes , diabetes education , knowledge and management , ability to manage , activities of daily living , and medication .
health related quality of life was evaluated with the euroqol ( eq-5d ) [ 40 , 41 ] .
the eq-5d-5l consists of five dimensions ( mobility , self - care , usual activities , pain / discomfort , and anxiety / depression ) .
each dimension had five levels : no problems , slight problems , moderate problems , severe problems , and extreme problems .
the eq visual analog scale ( vas ) then recorded the respondent 's self - rated health on a 20 centimeter ( 10-point interval ) vertical vas with endpoints categorized as
the best health you can imagine and the worst health you can imagine .
worst imaginable health state was recorded as 0 at the bottom of the scale , and best imaginable health state was achieved as 100 at the top .
both the 5-item index score and the vas score were then converted into a value score between 0 ( worst health state ) and 1 ( best health state ) [ 24 , 40 ] .
self - care activities ( sca ) were evaluated with the revised summary of diabetes self - care activities scale ( sdsca ) to assess aspects of the diabetes regimen and evaluate the dietary management skills of the participants .
the sdsca scale is a self - reporting measure of the frequency of performing 13 diabetes self - care tasks and consisted of six subscales of the diabetes self - management ( dsm ) behaviors : diet , exercise , blood glucose testing , medication taking , foot care , and smoking behavior over the prior seven days .
interitem correlations had a range of r = 0.200.76 ( mean = 0.47 ) for four sdsca subscales and 6-month test - retest reliability had a range of r = 0.000.58 ( mean = 0.40 ) .
the glycosylated or glycated hemoglobin ( hba1c ) value was classified into good glycemic control if the hba1c values are less than or equal to ( ) 7% and poor glycemic control if hba1c values are greater than ( > ) 7% .
glycemic control of 7.0% is endorsed as a treatment goal [ 17 , 43 ] .
body mass index ( bmi ) in weight in kilograms / square of height in meters ( kg / m ) was categorized as underweight if 18.5 kg / m , normal if 18.524.9 kg / m , overweight if 2529.9 kg / m , and obese if 30 kg / m . weight and height were measured by a portable digital scale and a portable stadiometer .
the eq-5d , sdsca , and demographic and clinical characteristics instruments were translated to arabic and back - translated and checked with monolingual testing .
the linguistic validation of the arabic version of the tools was found to be adequate .
the final instruments used in this study were administered to 30 omani adults with t2d twice in a 2-week interval .
intraclass correlation coefficient was intended to evaluate the test - retest reliability for the subdimensions of the eq-5d ( 0.75 and 0.91 ) .
interitem correlations had a range of r = 0.750.86 for four sdsca subscales and item - to - total correlations had a range of 0.770.91 for the sdsca . good evidence for internal consistency was shown using cronbach 's alpha for the sdsca which demonstrated = 0.87 , which was considered acceptable .
data were collected using eq-5d and sdsca standardized questionnaires after the pilot study between january and june 2010 among 330 adults with t2d in the diabetes clinic .
study participants were provided with an explanation of the study , and informed consent was obtained .
ethical approval was provided by the ethics and research committee , college of nursing at sultan qaboos university . informed written and verbal consent was acquired from each participant who met the inclusion criteria through a written letter .
informed consent and the completed questionnaires were stored and kept in locked cabinets . during the study , 30 selected adults dropped from the study . a 91% ( n = 300 )
descriptive and inferential statistics were calculated using the spss statistical package version 21.0 ( spss inc . ,
chicago , il , usa ) . a normality test and multicollinearity checks were performed .
the determinants of qol were assessed with anova and multivariate generalized linear model ( glm)/manova [ 4548 ] .
the highest percent of omani men was between 5059 years ( 40.6% ) as compared to 21.7% among the women ( table 1 ) .
the highest frequency of adherence to self - care activities was an average of three days / week among men ( 61.5% ) and women ( 60.5% ) .
a higher percentage of the men ( 62.2% ) had poor hba1c values as compared to women ( 46.5% ) .
for men , 30.1% reported that t2d mostly prevented their activities of daily living as compared to 26.8% reported among the women .
nearly half percentage of the women ( 53.5% ) and men ( 50.3% ) had moderate ability to manage diabetes positively .
more women ( 79.6% ) were on prescribed oral hypoglycemic agents as compared to men ( 70.6% ) ; while the remaining participants were on insulin .
women had slightly higher mean qol scores ( p < 0.05 ) for age , schooling , prevention of activities of daily living , ability to manage positively , and knowledge of diabetes and its management as compared to men ( table 2 ) .
women had higher mean health state scores for age and prevention of activities of daily living and knowledge of diabetes and its management as compared to men .
schooling and ability to manage positively were highly significant predictors of qol among women ( p < 0.05 ) as compared to men .
age , prevention of activities of daily living and knowledge / management of diabetes were significant predictors of health state among women as compared to men .
ability to manage diabetes positively was a significant predictor of health state among men as compared to women .
schooling and ability to manage diabetes positively were significant with qol among women ; while age , prevention of activities of daily living , and knowledge of diabetes and management were significant with health state among women as compared to men ( table 2 ) .
ability to manage diabetes positively was significant with health state among men compared to women .
women had higher mean qol scores for duration of diabetes , diabetes education , and medication as compared to men ( table 3 ) .
positively higher perception on health state scores was found with duration of diabetes , sca , diabetes education , medication , bmi , and hba1c among women .
sca and medication were highly significant predictors of health state among women ; while bmi was highly significant among men .
sca and medication were significant with health state among women ; while bmi was significant with health state among men ( table 3 ) . in this study ,
manova models were used with all determinants emerging from the anova tests as predictors of qol and health state ( tables 25 ) .
these personal and clinical characteristics interact with the specific domains valued as important in life , which explains the significant differences in qol and health state among omani men and women . a further glm technique was useful to explore the relationship between qol and health state , interdependent variables with the predictors ( like age and duration of diabetes ) as seen in tables 4 and 5 .
the combined effect of predictors on qol and health state using wilks 's lambda multivariate tests ( table 4 ) shows duration of diabetes , prevention of activities of daily living , and ability to manage positively were significant predictors of qol and health state among men as compared to women .
the test of overall model significance ( table 5 ) showed the model is important for each dependent variable ( qol and health state ) .
manova models were used with all independent variables in the anova tests as predictors of qol and health state ( table 5 ) .
manova results are explained with the test of overall model significance and the test of overall individual effects of predictors . among women with t2d ,
30.6% of the variance in the total qol and 14% of the variance in health state could be explained by personal and clinical characteristics ( table 5 ) , while 35.7% of the variance in the total qol and 23% of the variance in health state was explained by personal and clinical characteristics among men .
these r values indicated a supportive relationship among the predictors of qol and health state .
qol and health state scores were strongly correlated with the age , diabetes duration , and prior diabetes education .
hence , personal and clinical characteristics had a significant positive effect on qol and health state supporting the self - care diabetes management model .
apply an f test of significance to the relation of each covariate ( age , diabetes duration , sca , diabetes education , ability to manage positively , and bmi ) in relation to each of the dependent variables ( qol and health state ) .
age and diabetes education were significant predictors of qol , and sca was a significant predictor of health state among women as compared to men .
duration of diabetes , diabetes education , ability to manage diabetes positively , and bmi were significant predictors of health state among men as compared to women .
higher schooling , increased ability to manage diabetes , higher age , moderate level of prevention of activities of daily living , higher knowledge of diabetes and management , higher sca , and use of medication among women influenced their qol and health state as compared to men .
this state contributed to an increased qol and health among women as they overcome challenges in coping with t2d .
results of the study were congruent with previous studies [ 49 , 50 ] that show better self - care leads to improved qol . in this study , men with t2d had low qol which was consistent with a previous study . in middle aged women ,
perceived diabetes did not prevent their activities of living , and they showed above average knowledge and management of diabetes .
a strong effect was found for interactions between females and qol due to higher schooling and ability to manage diabetes positively ; while higher age , prevention of adl , and knowledge / management of diabetes were significant with health state among women .
better physical activity among women contributes to higher qol and had better understanding of their diabetes .
these findings were consistent with other studies that show younger age , education , longer duration of dm , fasting glucose levels , strong knowledge , and positive attitude [ 5759 ] had significantly explained higher qol scores .
specific elements like ability to manage positively and bmi influenced qol and health state among men as compared to women . in turn , low qol affects hba1c .
some studies show that men can experience more restrictions in daily life than women due to unexplained physical and emotional problems [ 57 , 60 ] .
an important finding is an impact of higher ability to manage diabetes positively and prevention of prevents activities of daily living that significantly predicted qol ; while bmi significantly predicted health state among men .
the effects were stronger for those with high school and diploma level education and longer duration of diabetes , prevention of activities of daily living , ability to manage positively , and body mass index which were significant determinants among men .
some men have more self - confidence in their ability to manage diabetes and are less likely to be depressed or anxious .
hence , good knowledge and a positive attitude are predictors of adherence to self - care and promote qol .
men with higher educational levels , strong knowledge , and positive attitude had a higher probability of attaining greater qol scores .
age , psychological perception , sca , hba1c , and lower levels of physical activity were significantly associated with higher qol among men .
the most striking difference was that women had higher qol scores and health state for higher age and low bmi and adhered better to oral medications and sca .
this finding reflects the inclination of women with higher education to participate in their own self - care .
age , duration of diabetes , diabetes education , and ability to manage diabetes had higher qol and health state among men .
hence , chronicity of t2d has a differential impact on qol and health state among women and men .
similar studies show high bmi is a strong predictor of decreased qol [ 57 , 64 ] and lower bmi was associated with higher qol .
insulin and higher bmi were associated with lower qol [ 33 , 61 , 63 ] .
qol and health state scores were lower in women compared with men and lower with longer duration of t2d . hba1c and qol have a significant association in previous studies [ 66 , 67 ] .
higher age , income , and education had better qol among women [ 56 , 61 , 67 , 68 ] .
men and women who have good health have significant health states with t2d [ 52 , 69 ] .
the study limitations are interactive effects of psychological and clinical predictors that may be relevant for comprehensive understanding of the impact on the domains of health - related qol among men and women .
the amount of variance influenced by the personal and clinical factors and explained in the glm is useful in understanding how hrqol influences omani men and women .
poor glycemic control increases the risk of developing long term complications of t2d , which causes poor health state and qol .
maintaining hba1c within a desirable range is an indicator of good glycemic control and was a contributor to better qol . the assessment of qol and health state is a key component of the self - care management model ( figure 1 ) .
this assessment is culturally specific and may assist in early identification to allow for appropriate self - care among individuals with t2d who are at risk for decreased qol .
this study gives useful information to help design appropriate culturally specific interventions related to various aspects of qol .
the scm model approach indicates that adults need to use their self - care behaviors for goal attainment and to take control of t2d , thus enhancing hrqol .
this study provides important qol evidence that may help the diabetes nurse educator ( dne ) to identify adults who are at risk of low qol and develop interventions for healthy lifestyle behaviors based on personal needs , clinical characteristics , and health state .
the dne can educate assist in motivating the patient with t2d to control blood glucose levels , have an annual screening examination , report any changes in health immediately , and engage in rigorous scm .
an effective scm model empowers men and women in proactively managing t2d and finding ways to overcome the problems with mobility , usual care , self - care , anxiety , and pain .
scm interventions by the dne should be tailored to the individual taking into account personal needs and motivation to change as well as clinical factors that influence better qol ( figure 1 ) .
the nurse as an educator has an opportunity to positively influence outcomes ( qol and health state ) by using effective behavioral skills and a collaborative health care approach . | the aim of this study was to explore predictors of health related quality of life ( hrqol ) among men and women with type 2 diabetes .
this cross - sectional descriptive study consisted of a random sample of 300 adults with type 2 diabetes in a selected public hospital .
euro - qol and revised summary of diabetes self - care activities scales were used to collect data between january and june 2010 .
schooling and ability to manage positively were highly significant predictors of quality of life ( qol ) among women as compared to men .
age , prevention of activities of daily living and knowledge / management of diabetes were significant predictors of health state among women as compared to men .
findings demonstrate that 30.6% ( versus 35.7% ) of the variance in the total qol and 14% ( versus 23% ) of the variance in health state could be explained by personal and clinical characteristics among women and men , respectively .
the study underlines the importance for nurse educators to assess hrqol among men and women and to develop effective self - care management strategies based on personal and clinical characteristics . |
all subjects were evaluated during a 2010 family forum in chicago , as part of our monogenic diabetes registry ( http://monogenicdiabetes.uchicago.edu ) ( 11 ) . all parents provided informed written consent as approved by the university of chicago institutional review board .
the beery - buktenica developmental test of visual - motor integration ( vmi ) presents drawings of geometric forms in order of increasing difficulty to be copied with paper and pencil for subjects > 2 years of age .
the vmi is often administered to evaluate visual - motor and visual - perceptual deficits .
it has a coefficient of 0.82 , and its validity has been established ( 1214 ) .
nonparametric analysis was performed using the kruskal - wallis anova test ( with value h ) for group comparisons , as well as spearman correlations .
all subjects were evaluated during a 2010 family forum in chicago , as part of our monogenic diabetes registry ( http://monogenicdiabetes.uchicago.edu ) ( 11 ) . all parents provided informed written consent as approved by the university of chicago institutional review board .
the beery - buktenica developmental test of visual - motor integration ( vmi ) presents drawings of geometric forms in order of increasing difficulty to be copied with paper and pencil for subjects > 2 years of age .
the vmi is often administered to evaluate visual - motor and visual - perceptual deficits .
it has a coefficient of 0.82 , and its validity has been established ( 1214 ) .
nonparametric analysis was performed using the kruskal - wallis anova test ( with value h ) for group comparisons , as well as spearman correlations .
eight subjects had the r201h mutation characterized by isolated diabetes without neurodevelopmental concerns , eight had v59 m or v59a ( v59m / a ) mutations associated with the intermediate dend syndrome , and three had mutations that have an inconsistently reported neurodevelopmental phenotype ( one each with r201c , y330c , and e322k ) ( table 1 ) .
overall , no group differences were found in age ( kruskal - wallis anova group comparison h [ 2 , n = 19 ] = 1.9 ; p = 0.39 ) , sex distribution ( = 0.79 ; p = 0.68 ) , age of diabetes diagnosis ( h [ 2 , n = 19 ] = 2.1 ; p = 0.35 ) , or age of treatment initiation ( h [ 2 , n = 19 ] = 2.2 ; p = 0.33 ) among the three groups . clinical characteristics and results of beery - buktenica vmi testing in patients with monogenic neonatal diabetes caused by r201h ( n = 8) , v59 m or v59a ( n = 8) , or other ( n = 3 ) mutations in kcnj11 the three groups differed significantly ( h [ 2 , n = 19 ] = 12.78787 ; p = 0.0017 ) from each other in graphomotor constructional abilities necessary for accurate copying of the vmi geometric figures ( table 1 and supplementary fig .
namely , in children with r201h , scores fell within the normal range ( median = 107 , lower quartile = 93 , and upper quartile = 118.5 ) , whereas children with v59m / a scored low to very low ( median = 49 , lower quartile = 49 , upper quartile = 74.5 ) , and scores of children with other mutations were intermediate ( median = 89 , lower quartile = 65 , and upper quartile = 91 ) .
age at treatment initiation was significantly inversely correlated with vmi scores only in the v59m / a group ( spearman correlation = 0.79 , p < 0.05 ) ( supplementary table 1 ) .
all three children with v59m / a who had transitioned to sulfonylureas before their first birthday had standard scores > 70 , whereas the five children whose treatment started later scored < 50 ( greater than three sds below the mean ) .
to our knowledge , our study represents the largest cohort of rare kcnj11 neonatal diabetic patients undergoing an identical neurodevelopmental assessment at one time . using a well - validated , age - standardized measure
, we show that those with intermediate dend - associated v59m / a mutations have significant impairment of eye - hand coordination , whereas those with the r201h mutation not associated with neurodevelopmental concerns performed in the normal range .
early treatment with sulfonylureas was associated with better vmi scores in the v59m / a group ; however , this observation must be confirmed in larger numbers of patients with more comprehensive longitudinal assessments .
why mutations such as r201h do not cause a similar level of impairment as seen in v59m / a is uncertain , especially since the diabetes caused by these mutations appears to be equivalent .
given that a few of the r201h and other mutation case subjects exhibited low normal scores , it may be that the vmi is not sensitive enough to quantify mild subclinical difficulty .
other factors could contribute to the neurodevelopmental concerns in these patients , including diabetic ketoacidosis at diagnosis during a very young age critical for brain development , as well as longer - term metabolic control characterized by prolonged hyperglycemia and sometimes frequent episodes of severe hypoglycemia , which has been associated with a spectrum of visual - spatial , memory , attention , and executive dysfunctions ( 15 ) .
the current study and previous reports of improved cognitive and motor symptoms after the change in treatment suggest the benefit of sulfonylurea blockade of activated glucose - responsive channels that have an unknown function in the brain ( 810 ) .
this implies that sulfonylureas cross the blood - brain barrier ; however , the degree to which they do may be a critically important factor and warrants further study .
the three v59m / a subjects who started sulfonylurea therapy before 1 year of age had better vmi scores than the five subjects who did not ; however , it remains to be determined whether there is a definitive critical age for treatment .
this finding raises hope for prevention of at least some of the neurodevelopmental disability in those who start sulfonylureas at an early age .
this bolsters consideration of an empirical trial of sulfonylureas in newly diagnosed neonatal diabetic patients before results of genetic testing are available , given that atp - sensitive potassium channel mutations cause almost 50% of cases .
however , the risk / benefit of sulfonylureas should be carefully considered and should in no way supplant mandatory genetic testing . notably , vmi scores in the three v59m / a children treated early were still low and developmental challenges are likely to persist , as is also likely in rare severe dend cases not assessed in this study .
efforts should include comprehensive specialty assessment and support of optimal progress and continued collection of data on greater numbers of patients tracking long - term neurodevelopmental outcome . in this regard
| objectiveto assess performance on an age - standardized neuromotor coordination task among sulfonylurea - treated kcnj11-related neonatal diabetic patients.research design and methodsnineteen children carrying kcnj11 mutations associated with isolated diabetes ( r201h ; n = 8) , diabetes with neurodevelopmental impairment ( v59 m or v59a [ v59m / a ] ; n = 8) , or diabetes not consistently associated with neurodevelopmental disability ( y330c , e322k , or r201c ; n = 3 ) were studied using the age - standardized beery - buktenica developmental test of visual - motor integration ( vmi).resultsalthough r201h subjects tested in the normal range ( median standard score = 107 ) , children with v59m / a mutations had significantly lower than expected vmi standard scores ( median = 49 ) .
the scores for all three groups were significantly different from each other ( p = 0.0017 ) .
the age of sulfonylurea initiation was inversely correlated with vmi scores in the v59m / a group ( p < 0.05).conclusionsneurodevelopmental disability in kcnj11-related diabetes includes visuomotor problems that may be ameliorated by early sulfonylurea treatment .
comprehensive longitudinal assessment on larger samples will be imperative . |
diagnosis of fronto - temporal dementia ( ftd ) requires beside brain - imagining a careful evaluation of genetic and neuropathological , cognitive processes disorders , and behaviour problems .
the complexity of the symptoms observed after frontal lobe damage creates the necessity for a very careful neuropsychological examination .
gustafson and nielsen already in the last century proposed a way of evaluating the behaviour of patients with frontal dementia enabling a differentiation of pick s and alzheimer disease .
the manchester group evaluated also the symptoms of ftd in retrospective studies in order to compare the clinical diagnosis with post mortem examination .
this made possible a differential diagnosis of dementia and of alzheimer type ( dat ) .
lopez et al . stated more symptoms of depression , anxiety , irritability , mood lability , disinhibition , inertia as well as social withdrawal in ftd patients in comparison to dat patients , in whom paranoic symptoms were more often observed .
gregory and hodges conducted a review of psychotic symptoms in ftd patients with at least 5 basic diagnostic features . in a half of the patients a diagnosis of ftd already established , 1/3 had been diagnosed with schizophrenia , psychosis , depression with obsessive - compulsive traits , alcohol addiction , and psychogenic memory disorders .
the course of illness in ftd is similar to dat and it includes three stages . the nature of the stages including disorders of cognitive , emotional processes as well as disorders of behaviour were described in many works .
lebert applied an inventory of symptoms to discriminate ftd , alzheimer disease , and vascular dementia .
the inventory took into account the following disorders : lack of self - control , egocentric behaviours , miller et al . performed a retrospective evaluation of occurrence and the lack of symptoms of the lund - manchester scale in 30 patients with ftd
the following differences between patients with ftd and were noted : stereotyped behaviours and perseveration , logopenia with preserved spatial orientation .
kertesz et al . developed a frontal behavioural inventory ( fbinv ) in order to delineate specific behaviours , which would make possible a reliable diagnosis of frontal dementia .
the inventory may be used both for the purpose of an introductory evaluation and to perform retrospective diagnosis .
the items of the inventory were selected from the elementary diagnostic of the lund - manchester group and out of the most common syndromes of ftd observed in the patients examined by kertesz et .
the test was administered to 12 ftd patients , 16 dat patients at the early stage of illness , and 11 patients with dementia caused by depression diagnosed by a psychiatrist and the beck depression test during a pilot study .
the depth of dementia was defined on the basis of scores gained on the mattis dementia rating scale ( mdrs ) .
ftd patients were selected taking into account clear cut behavioural symptoms , yet several developed logopenia at the later state of illness , and a typical motor neuron disorder ( mnd ) was stated in one of them .
pick disease was confirmed in 3 patients post - mortem , while in one cortico - basal symptoms were noted , while in two tau - negative ubiquitin inclusions as well as clinical symptoms of mnd were stated .
results of the pilot study revealed that patients with frontal lobe lesions obtained much higher scores in the fbinventory than did the two control groups , something which was confirmed with anova .
no significant differences between the scores of patients with alzheimer dementia and dementia following depression were noted .
patients with alzheimer dementia were older , than patients with ftd and depression with one exception all ftd patients were 65 years old at the onset of illness ( which is a common time for falling ill in such cases ) .
an analysis of the mean scores gained at fbinv revealed that the most common behavior disorders were : lack of insight , thinking rigidity , concreteness , personal neglect , inappropriate remarks due to disinhibition .
a significant difference , however , was stated between the scores of ftd patients , and the control groups .
the results made it possible to define a cutting point , and to operationalize the behavioural diagnosis of ftd .
the point consists of 27 out of 72 points , which can be gained in fbinv , and includes all the ftd patients .
symptoms of depression were confirmed by vegetative symptoms as well as by scores recorded on the beck depression inventory .
kertesz notes , that the cutting points of fbinv may enable a grouping of the patients in order to perform a further examination .
there are many inventories of behaviour evaluation that aim at screening deviant behaviours in geriatric , psychiatric or dementive patients [ 1522 ] . geriatric scales frequently combine cognition , behaviour , and everyday activities in order to measure global functional deterioration but they do not discriminate particular behavioural syndromes .
a direct evaluation of behaviour of ftd patients takes into account items measuring motor or cognitive perseverations , verbal inclusions , disinhibition , aspontaneity , and utilization behaviours .
there were attempts to make the evaluation of symptoms of frontal lobe injuries more formal with the use of executive interviews .
logopenia appears at the later stages of ftd , and is sometimes connected with verbal and oral apraxia .
frequent hesitations in speech , prozodic disorders , the leaving out of initial consonants , and stuttering was also noted .
yet the scores of items evaluating those disorders were as a rule lower as they appear at a later stage of the illness in ftd patients .
those symptoms used to be called a primary progressive aphasia ( ppa ) . at the later phases of ppa personality disorders are quite common .
the new version consists of a series of 24 items enabling an evaluation of social behaviour disorders as well as motor behaviours and the syndrome of alien hand .
they create a lot of anxiety in family members who are reluctant to speak about them .
the author believe that the inventory discussed may be of use in the case of ppa if administered along with specific language tests .
the aim of our research was to show the diagnostic value of fbinv [ 1214 , in distinguishing ftd from such syndromes as vad or depression as well as in evaluating the efficacy of neurotherapy .
the examination included 112 patients , 68 men and 46 women , who were diagnosed and treated at the reintegrative - training centre of the foundation for brain damaged persons in krakw , and in clinic for developmental psychiatry , psychotic disorders and the old age psychiatry at the medical university of gdask , suffering from various types of dementia including depression .
the group consisted of 41 patients with alzheimer dementia including 25 men and 16 women ; the mean age was 69.84 ( sd 9.7 ) , 14 patients with vascular dementia including 9 men and 5 women ; the mean age was 65.73 ( sd 8.4 ) , 18 ftd patients including 11 men and 9 women ; with a mean age of 56.41 ( sd 9.8 ) , 27 dd patients including 16 men and 11 women ; mean age 56.18 ( sd 12.3 ) , 12 ppa patients including 7 men and 5 women ; mean age 61.36 ( sd 6.9 ) .
the following tasks were applied : neuropsychological tests [ wechsler inteligence scale - revised wais - r ; wechsler memory scale - revisd wms - r ; western aphasia battery revised
wab - r ; lines evaluation test , face recognition test , screening with mmse to evaluate the depth and progression of dementia and with the polish version of the boston naming test ( bntvpl ) which aimed at discrimination of language and behavioural variants of fronto - temporal dementia , frontal behaviour inventory ( fbinv ) adapted for polish .
the final version of the fbinv consists of questions that include both correct as well as incorrect , negative aspects of behaviour out of which the carer selects those he thinks describe the patient best .
the particular questions can be put in other words if the carer has problems with understanding them .
they are : negative behaviors , such as apathy , aspontaneity , indifference , thinking rigidity , concreteness , personal neglect , distractibility , inattention , loss of insight , logopenia , verbal apraxia , and alien hand syndrome [ the last 3 items were included to evaluate the specific motor and linguistic behaviours that might occur in ftd ] .
positive behaviours , connected with disinhibition , such as perseveration , irritability , excessive jocularity , unpredictability , irresponsibility , inappropriateness , impulsivity , restlessness , aggression , hyperorality , hypersexuality , utilization behaviour , and incontinence .
the inventory was given to a patient caregiver , and the examiner provided explanations of the meaning of particular questions in the case of such a need .
the inventory was given to a patient caregiver , and the examiner provided explanations of the meaning of particular questions in the case of such a need .
the scores gained by the examined patients with ftd did not differ in as far as sex is concerned , therefore , further analysis will be conducted for both men and women together in each group .
the screening of cognitive functions taking into account intellectual abilities , memory , visual functions , language functions , and general features of dementia in individual groups is presented in table 2 .
an analysis of wais - r scores shows that the lowest global i.q . was noted in patients with alzheimer dementia , fvftd patients , and ppa patients .
yet , it may be observed that the distribution of results in nonverbal i.q . and verbal i.q .
thus , patients with alzheimer dementia gained significantly lower scores in nonverbal i.q . when compared with their verbal i.q . on the other hand ,
is most probably connected with the different profile of disorders in alzheimer dementia , in which visual - spatial deficits connected with a performance part of wais - r are more pronounced , while language disorders dominate in ppa and ftd patients ( five patients with semantic dementia sd were included in that group , and this might have influenced the results ) .
wms - iii test revealed disorders of logical memory in all examined patients , yet the depth of those disorders is different .
most severely impaired were dat patients , less ppa and vad patients , and the least dd patients .
it should be noted that dat patients , as might be expected , exhibit more clear cut impairments in visual memory than other groups .
in fact they practically do not accomplish any delay tasks whatsoever . on the other hand , ftd and ppa patients score much worse in auditory memory than in the visual memory . on reproduction with delay
the most pronounced disorders of visual - spatial functions occurred as was expected for dat patients both in the benton test as well as in the face recognition test , the disorders were less pronounced in vad patients .
ftd and ppa patients revealed non - significant disorders , while dd patients stayed within a norm .
it can be noticed that ftd and ppa patients tend to abandon the tasks , which may be the cause of a lower scoring .
visual - spatial dysfunctions may be also connected with deficits in memory selectivity , as the patients spare much less time to individual tasks .
western aphasia battery ( wab ) showed the occurrence of aphasic disorder in all examined patients with the exception of patients with depression .
as expected most pronounced disorders were observed in ppa patients with aphasia quotient = 64.3 , slightly lower in ad patients with aq=87.3 , and in ftd patients with aq=85.1 .
slightly surprising are the profiles of the cortical quotients as they show the reverse tendency .
the worst scores were gained by dat patients with a cortical quotient = 68.6 .
it was probably due to the fact that picture recognition scores are included in the cq , and as we know this is frequently impaired in alzheimer dementia .
comparison of the scores of the examined groups in the mmse test did not reveal statistically significant differences among the examined groups .
the lowest mean , however , was gained by patients with dat , and the lowest patients with depression and ppa , which was in agreement with expectations .
the fbinv scores of patients with various types of dementia are presented in table 3 . it may be observed that the highest scores ( much above the cutting point ) were obtained by ftd patients .
the scores of ppa patients were higher than in the control groups , but not as high as in the case of patients with ftd .
it confirms the observations of other authors that the so called frontal type occurs within vad which must be connected with dissociation of the frontal subcortical structures taking place in vad .
it was stated that disinhibition symptoms , which prevail over apathy and withdrawal , are helpful in distinguishing ftd patients from vad or dd patients .
frequency of the occurrence of individual frontal lobe symptoms in 18 patients with bvftd , correlated with the data gathered from the structured interview is shown in table 4 .
this will enable the reader to better see the spectrum of behaviour disorders that appear in bvftd patients .
in our study bvftd patients were given a half - a - year neuropsychotherapy aimed at a reduction of frontal symptoms in accordance with a program elaborated by pchalska .
the results of the study are shown in table 5 , and in figure 1a , b. it may be observed that rehabilitation resulted in an improvement of some deviant behaviours .
thus , a significant decrease of indifference and emotional flatness as well as of apathy was observed .
yet , some disorders , mainly the behaviours connected with disinhibition did increase along with the progression of the illness despite neurotherapy .
behaviours resulting from difficulties in passing from an idea to action as well as apathy also increased . those were apraxia of speech , concreteness , disorganization , and inattention . as might have been expected alien hand symptoms as well as incontinence also increased .
mean scores of patients with bvftd in i i ii examination in the first 12 traits of the inventory , i.e. negative behaviours resulting from difficulty in passing from an idea to action as well as apathy is shown in figure 1a .
mean scores of bvftd patients in i and ii examination in the other 12 trait of the inventory , which measure positive behaviours connected with disinhibition are shown in figure 1b . to sum up
, it is worth pointing out that the above results are in agreement with the results obtained in the studies of other authors that , however , were performed much later than our study .
the screening of cognitive functions taking into account intellectual abilities , memory , visual functions , language functions , and general features of dementia in individual groups is presented in table 2 .
an analysis of wais - r scores shows that the lowest global i.q . was noted in patients with alzheimer dementia , fvftd patients , and ppa patients .
yet , it may be observed that the distribution of results in nonverbal i.q . and verbal i.q .
thus , patients with alzheimer dementia gained significantly lower scores in nonverbal i.q . when compared with their verbal i.q . on the other hand ,
is most probably connected with the different profile of disorders in alzheimer dementia , in which visual - spatial deficits connected with a performance part of wais - r are more pronounced , while language disorders dominate in ppa and ftd patients ( five patients with semantic dementia sd were included in that group , and this might have influenced the results ) .
wms - iii test revealed disorders of logical memory in all examined patients , yet the depth of those disorders is different .
most severely impaired were dat patients , less ppa and vad patients , and the least dd patients .
it should be noted that dat patients , as might be expected , exhibit more clear cut impairments in visual memory than other groups .
in fact they practically do not accomplish any delay tasks whatsoever . on the other hand , ftd and ppa patients score much worse in auditory memory than in the visual memory . on reproduction with delay
the most pronounced disorders of visual - spatial functions occurred as was expected for dat patients both in the benton test as well as in the face recognition test , the disorders were less pronounced in vad patients .
ftd and ppa patients revealed non - significant disorders , while dd patients stayed within a norm .
it can be noticed that ftd and ppa patients tend to abandon the tasks , which may be the cause of a lower scoring .
visual - spatial dysfunctions may be also connected with deficits in memory selectivity , as the patients spare much less time to individual tasks .
western aphasia battery ( wab ) showed the occurrence of aphasic disorder in all examined patients with the exception of patients with depression .
as expected most pronounced disorders were observed in ppa patients with aphasia quotient = 64.3 , slightly lower in ad patients with aq=87.3 , and in ftd patients with aq=85.1 .
slightly surprising are the profiles of the cortical quotients as they show the reverse tendency .
the worst scores were gained by dat patients with a cortical quotient = 68.6 .
it was probably due to the fact that picture recognition scores are included in the cq , and as we know this is frequently impaired in alzheimer dementia .
comparison of the scores of the examined groups in the mmse test did not reveal statistically significant differences among the examined groups .
the lowest mean , however , was gained by patients with dat , and the lowest patients with depression and ppa , which was in agreement with expectations .
the fbinv scores of patients with various types of dementia are presented in table 3 . it may be observed that the highest scores ( much above the cutting point ) were obtained by ftd patients .
the scores of ppa patients were higher than in the control groups , but not as high as in the case of patients with ftd .
it confirms the observations of other authors that the so called frontal type occurs within vad which must be connected with dissociation of the frontal subcortical structures taking place in vad .
it was stated that disinhibition symptoms , which prevail over apathy and withdrawal , are helpful in distinguishing ftd patients from vad or dd patients .
frequency of the occurrence of individual frontal lobe symptoms in 18 patients with bvftd , correlated with the data gathered from the structured interview is shown in table 4 .
this will enable the reader to better see the spectrum of behaviour disorders that appear in bvftd patients .
in our study bvftd patients were given a half - a - year neuropsychotherapy aimed at a reduction of frontal symptoms in accordance with a program elaborated by pchalska .
the results of the study are shown in table 5 , and in figure 1a , b. it may be observed that rehabilitation resulted in an improvement of some deviant behaviours .
thus , a significant decrease of indifference and emotional flatness as well as of apathy was observed . yet , some disorders , mainly the behaviours connected with disinhibition did increase along with the progression of the illness despite neurotherapy .
behaviours resulting from difficulties in passing from an idea to action as well as apathy also increased .
those were apraxia of speech , concreteness , disorganization , and inattention . as might have been expected alien hand symptoms as well as incontinence also increased .
mean scores of patients with bvftd in i i ii examination in the first 12 traits of the inventory , i.e. negative behaviours resulting from difficulty in passing from an idea to action as well as apathy is shown in figure 1a .
mean scores of bvftd patients in i and ii examination in the other 12 trait of the inventory , which measure positive behaviours connected with disinhibition are shown in figure 1b . to sum up
, it is worth pointing out that the above results are in agreement with the results obtained in the studies of other authors that , however , were performed much later than our study .
in the course of diseases following degeneration of the brain a number of behaviour disorders and other psychopathological symptoms occur , beside the classical cognitive deficits .
such symptoms as psychotic disorders , restlessness , mood disorders , sleep disorders , journeying and many others , which accompany degenerative processes are called behavioural and psychological symptoms . in table 6 ,
a differential analysis of ftd and dat was presented on the basis of both our own research and other studies [ 33,3843 ] .
it can be noticed that executive functions disorders are more pronounced than memory disorders in fronto - temporal dementia , while the reverse pattern of disorders occurs in alzheimer dementia .
it is worth pointing out , however , that the intensity of those symptoms as well as the clinical picture varies in accordance with the degree of degenerative changes , and with so called cognitive reserve .
the behavioural manifestation of the process depends both upon neurobiological factors , such as the localization of changes , as well as the dynamics of the process of dementia .
both negative behaviours connected with passing from thought to action and apathy as well as positive behaviours following disinhibition were noted .
the results confirm the usefulness of fbinv for description and the distinguishing of bvftd from other types of dementia as well as an evaluation of neurotherapy results .
it should stressed here that classic descriptions of the frontal syndrome pointed to the appearance of a number of early behavioural symptoms before clinical disorders of memory .
behaviours and symptoms most frequent in ftd as observed in our study ( table 3 ) are in agreement with the typical clinical picture of that disease .
the possibility of noting differences and similarities among the examined groups was increased by including groups of patients with a decrease in cognitive functions following various degenerative processes .
biochemical aspects were taken into account by the inclusion of one group with taupathy ( ftd ) and amyloidopathy ( alzheimer disease ) , forms conditioned by the primary vascular process , and functional disorders of cognitive functions following depression .
the neuroanatomic discrimination is reflected in our results . since speech disorders are quite frequent in ftd semantic dementia as well as primary progressive aphasia treated as a clinical variant of ftd , this is distinguished by some authors alongside with the temporal and frontal form .
such symptoms as : stereotyped utterances , echolalia , perseveration , mutism , semantic aphasia , semantic deficit , and spontaneous speech reduction are considered to belong to the prodromal symptoms of ftd .
it seems questionable whether processes at the basis of progressive aphasia and ftd are identical or close to each other .
those nosographic doubts follow from the fact that the level of behavioural disorders registered in fbinv was significantly different in both groups .
it should be also borne in mind that a clinical picture caused by vascular factors varies to a considerable degree , and thus does not fit a classic description of dementia .
it is true that diagnostic criteria contrast vascular dementia with primary degenerative diseases of the brain .
yet , the border line may be not clear , since vascular factors are present also in primary degenerative processes such as alzheimer disease for example .
such vascular changes were observed in about 1/3 of alzheimer patients . some of the vascular degenerations ( amyloid angiopathy , leukoencephalopathy ) are observed in almost all the examined cases of alzheimer disease .
it is generally accepted that behavioural disorders are more frequent in vascular dementias than in alzheimer disease , at least at the early stages of the illness .
this concerns mainly disorders of affect something that was also confirmed in the present study . a similar level of deviations was noted in the study of persons with dat and with depression , which may suggest some connections between those diseases . it might seem surprising if we realize that two different disorders are being compared there : one following the slow degeneration of brain tissue , and the other that passes sometimes even without treatment . yet
, the connection between alzheimer disease , or dementia in its broader sense , and depression have much in common .
the more frequent occurrence of mood disorders ( particularly of a depressive type ) in dat has already been mentioned . yet
, the present study makes it necessary to point out that depressive disorders are connected with a decrease of cognitive ability , which sometimes remind one of dementia due to their degree .
quite often depression appears at the early phases of dementia , even in the preclinical stages . and
hence , episodes of depression are treated as a significant factor pointing to the possibility of the occurrence of dementia , and of the alzheimer type in particular .
those complex relations may be , at least in part , explained on a neurobiological basis .
degenerative changes in alzheimer disease start in the hippocampus and olfactory cortex , and then expand to the frontal , temporal , and parietal associative cortex .
moreover , the loss of subcortical neurons in the basal nucleus and bluish nucleus occurs quite often . in the case of the relation between alzheimer
dementia and depression the basic role seems to be played by the damage of the hippocampus , which leads to impairment of the mechanisms of control of the corticosteroids level . in dat both
an increase in the level of basic cortisol , and a lack of decrease of its level after the application of deksametason was observed [ 6163 ] .
the damage to the hippocampus causing an increase in the crh level leads to an excessive activity of the hypothalamic - pituitary - adrenal pivot , which is often observed in depression too .
disruptions of the control of the corticosteroids level in depressions may also be connected with disorders of intellectual functions . for our studies
it is of importance that a hippocampus lesion occurs at the early or even preclinical stages of dat .
not only biological mechanisms indicating the connection of depression with alzheimer disease are taken under consideration .
some studies point to a significant loss of neurons in the bluish nucleus in alzheimer patients , in whom depression was diagnosed at the same time .
noted also a more marked degeneration of neurons in the bluish nucleus in patients with alzheimer dementia , in whom depression was stated before the clinical development of dementia .
we should also take into account some other factors , while discussing the possible neurobiological mechanisms of creating psychological and behavioural symptoms of the diseases examined here .
it is worth pointing out that the pathogenesis of psychological and behavioural symptoms is very complex .
it is possible that morphological degenerations observed in dementias predestine some behaviours , which , however , are determined by premorbid personality traits as well as environmental factors .
, aggressive behaviours appear more often in periods preceding faster progression . at this present stage of the analysis of the results an evaluation of the process dynamics
better knowledge of the psychological picture , and of behavioural symptoms in particular is of great practical value .
it should be borne in mind that dementive processes are proceeded by an signalling period , during which behaviour disorders may dominate over cognitive disorders , which at that time are difficult to distinguish during a clinical examination . especially in the case of ftd
, the symptoms of dementia may be neglected for a long time . in consequence ,
such persons are often given the wrong diagnosis , which often results in a delay of proper treatment .
naturally , the limbic structures , which play an essential role in basic drives and their affective correlates , may not be damaged at the early stage of ftd .
the lesions may be noted after cutting of the ways leading from the frontal lobes to the limbic system or if that system is destroyed as a consequence of neurodegenerative changes .
ablation of the frontal part of the temporal lobe connected with the bilateral ablation of the amygdalia body in animals is followed by dramatic changes in sexual and aggressive behaviours .
klver - bucy syndrome , including hypersexuality , lack of fear reaction and visual agnosia , show how dramatic the results of lesions of the limbic structures for emotional - motivational functions can be .
they exhibit various deviant social behaviors , such as disinhibition , verbal or physical aggression , and executive functions disorders connected with apathy .
dysfunctions observed in those patients remind one of the behaviours of animals with lesions disrupting the activity of the limbic system .
microgenetic theory of a symptom developed on the basis of an evolutionary , four - dimensional model of brain work may be of help in understanding those disorders .
damage to the limbic system or of one of its connections with other brain structure including the frontal lobes ( figure 2 ) disrupts the normal activity of the brain , and due to that basic limbic - cortical dysfunction many patients with dementia exhibit difficulties with adult or
behaviour disorders are often the first symptom of an illness , which may enable putting an early by a well trained evaluator .
the microgenetic model of brain work makes us aware that in fronto - temporal dementia the whole of the brain systems become disrupted and not only specific cognitive functions as in the case of local brain injuries .
the function of those systems are at first disorganized only in part , but then undergo global disruption , after the destabilization reaches a level , which causes a loss of integrity .
it needs to be stressed that even in the case of the linguistic variant of ftd ( lvftd ) , neither the course nor the nature of the disorders observed , speech and language disorders , recalls aphasia .
there is a difference in reaction of the dynamic system that receives one strong and localized blow as is the case in a brain stroke ; and of the system , which loses its energy and undergoes entropy as in the case of dementia . in such case
the changes are vertical : the upper levels of complex brain processes lose energy , become disorganized , and finally disintegrate , in contradistinction to the horizontal effects of focal lesions , when one part of the system is damaged , and others remain untouched .
as cortical dementia traits grow , which occurs both in alzheimer disease as well as in ftd , the process of neurodegeneration runs counter to microgenesis , that is : from the upper areas to the lower ( deeper ) areas of the brain .
the deeper the neurodegenerative changes in the brain are , the less specific will be the symptoms . and
after the illness assaults the lowest level ( brain stem and midbrain ) , basic life functions will also undergo entropy : will lose energy and disintegrate , which results in decease .
therefore , at the final stage of any neurogenerative dementive disease differences in the clinical picture , which might be clear at the very beginning , become difficult to distinguish . in other words ,
each dementia : starts with an upset of one s own identity , due to the arising disturbances , runs in a way specific for the basic pathomechanism of a given type of dementia , with time its specific traits are lost and each ends in a similar way .
naturally , the limbic structures , which play an essential role in basic drives and their affective correlates , may not be damaged at the early stage of ftd .
the lesions may be noted after cutting of the ways leading from the frontal lobes to the limbic system or if that system is destroyed as a consequence of neurodegenerative changes .
ablation of the frontal part of the temporal lobe connected with the bilateral ablation of the amygdalia body in animals is followed by dramatic changes in sexual and aggressive behaviours .
klver - bucy syndrome , including hypersexuality , lack of fear reaction and visual agnosia , show how dramatic the results of lesions of the limbic structures for emotional - motivational functions can be .
they exhibit various deviant social behaviors , such as disinhibition , verbal or physical aggression , and executive functions disorders connected with apathy .
dysfunctions observed in those patients remind one of the behaviours of animals with lesions disrupting the activity of the limbic system .
microgenetic theory of a symptom developed on the basis of an evolutionary , four - dimensional model of brain work may be of help in understanding those disorders .
damage to the limbic system or of one of its connections with other brain structure including the frontal lobes ( figure 2 ) disrupts the normal activity of the brain , and due to that basic limbic - cortical dysfunction many patients with dementia exhibit difficulties with adult or
behaviour disorders are often the first symptom of an illness , which may enable putting an early by a well trained evaluator .
the microgenetic model of brain work makes us aware that in fronto - temporal dementia the whole of the brain systems become disrupted and not only specific cognitive functions as in the case of local brain injuries .
the function of those systems are at first disorganized only in part , but then undergo global disruption , after the destabilization reaches a level , which causes a loss of integrity .
it needs to be stressed that even in the case of the linguistic variant of ftd ( lvftd ) , neither the course nor the nature of the disorders observed , speech and language disorders , recalls aphasia .
there is a difference in reaction of the dynamic system that receives one strong and localized blow as is the case in a brain stroke ; and of the system , which loses its energy and undergoes entropy as in the case of dementia . in such case
the changes are vertical : the upper levels of complex brain processes lose energy , become disorganized , and finally disintegrate , in contradistinction to the horizontal effects of focal lesions , when one part of the system is damaged , and others remain untouched .
as cortical dementia traits grow , which occurs both in alzheimer disease as well as in ftd , the process of neurodegeneration runs counter to microgenesis , that is : from the upper areas to the lower ( deeper ) areas of the brain .
the deeper the neurodegenerative changes in the brain are , the less specific will be the symptoms . and
after the illness assaults the lowest level ( brain stem and midbrain ) , basic life functions will also undergo entropy : will lose energy and disintegrate , which results in decease .
therefore , at the final stage of any neurogenerative dementive disease differences in the clinical picture , which might be clear at the very beginning , become difficult to distinguish . in other words ,
each dementia : starts with an upset of one s own identity , due to the arising disturbances , runs in a way specific for the basic pathomechanism of a given type of dementia , with time its specific traits are lost and each ends in a similar way .
to sum up , it should be stressed that the scores of the present study enable differentiation due to the specific behavioural profiles of the various dementive syndromes and are of significance for the course of therapy . at the same time
they enable evaluation of the efficacy of neurotherapy , and hence they make possible a raising in the quality of life of persons with various types of dementia .
the results obtained in the present study confirmed the diagnostic value of fbinv in the differential diagnosis of various types of ftd and in the evaluation of neurotherapy efficacy . | summarybackgroundthe aim of the paper is the differential diagnosis of various types of fronto - temporal dementia ( ftd ) , with the focus on its behavioural variant ( bvftd).material / methodmaterial and method .
screening was done in order to assess the depth of dementia with the short version of mmse , while evaluation of various variants of ftd was performed with the use of such neuropsychological tests as newcomb and chicago fluency tests , wechsler memory scale - iii ( wms - iii ) , western aphasia battery ( wab - r ) , and the boston naming test ( bnt ) .
behaviour was evaluated with a polish version of the frontal behavioral inventory ( fbinv ) .
the inventory consists of 24 questions which enable an evaluation of social behaviour disorders .
the study included 112 patients - 68 men and 46 women treated in the reintegrative -training centre of the foundation for persons with brain dysfunctions in krakw and in the clinic for developmental psychiatry , psychotic disorders and old age psychiatry , of the medical university at gdask , who were suffering from various types of dementia.resultsit was found that ftd patients scored the highest , while the vad patients scored somewhat lower in the fbinv . at the same time the scores obtained by ppa patients were higher in comparison to the control groups , but not as high as in the case of patients with ftd . in the process of
the neurotherapy of ftd patients we found a reduction of the behavioral disturbances , despite the progression of the illness.conclusionsthe results obtained in the present study confirmed the diagnostic value of fbinv in the differential diagnosis of various types of ftd and in the evaluation of neurotherapy efficacy . |
arterial baroreflex is the main mechanism for the regulation of the cardiovascular system , and sinoaortic denervation ( sad ) is a widely used tool to study its function.1 it is well established that chronic sad increases blood pressure variability ( bpv ) without increasing mean blood pressure.2 previous studies have demonstrated that bpv,3 baroreflex sensitivity,4 and the renin angiotensin system ( ras)5 were independent determinants in hypertensive end - organ damage in rats .
in addition to the classic circulating ras , there is growing evidence supporting the existence of a local functioning cardiac ras.6 all components of the ras ( renin , angiotensinogen , angiotensin - converting enzyme ( ace ) , and angiotensin ii receptors ) are identified in the heart at the mrna and protein levels,7 and this system is activated in experimental left ventricular hypertrophy ( lvh ) induced by hemodynamic overload.8 the hypertension - induced lvh initially serves as an adaptive ventricular response to pressure overload ; nevertheless , it is a major independent risk factor associated with increased cardiovascular morbidity and mortality.9 the aim of this study was to evaluate ventricular hypertrophy parameters induced by baroreflex impairment and the contribution of the local cardiac angiotensins ( ang ) i , ii and 17 in this process in normotensive ( wistar ) and hypertensive ( shr ) rats .
experiments were performed on male wistar and shr rats aged 16 weeks,10 housed in cages with free access to water and food , and maintained in a room with a constant temperature ( 23c ) on a 12-hour light dark cycle .
all surgical procedures and protocols used were in accordance with the guidelines for ethical care of the experimental animals and were approved by the institutional animals care and use committee ( cep 1016/08 ) .
rats were randomized into four groups : wistar control ( wc ) , n = 9 , wistar denervated ( wd ) , n = 7 , shr control ( shrc ) n = 8 , shr denervated ( shrd ) n = 8 .
sad was performed under anesthesia with ketamine , 80 mg / kg ( parke - davis , brazil ) and xylazine , 12 mg / kg ( bayer , brazil ) . a 3-cm midline incision was made , and sternocleidomastoid muscles were reflected laterally , exposing the neurovascular sheath . the common carotid arteries and
the vagal trunk were isolated , and the aortic depressor fibers either traveling with the sympathetic nerve or as an isolated aortic nerve were cut .
the third contingent of aortic baroreceptor fibers traveling with the inferior laryngeal nerve was interrupted by resection of the superior laryngeal nerve after the carotid bifurcation was exposed extensively for carotid stripping . to complete the procedure ,
the sinus nerve , all carotid branches and the carotid body were resected.11 the mortality rate was 35% higher in the animals submitted to sad than in their respective controls .
ten weeks after sad , animals were anesthetized with ketamine , 80 mg / kg ( parke - davis , brazil ) and xylazine , 12 mg / kg ( bayer , brazil ) , and femoral artery and vein catheters were inserted for direct measurements of blood pressure ( bp ) , heart rate ( hr ) and drug administration .
twenty - four hours after catheterization , the arterial catheter was connected to a transducer ( blood pressure xdcr , kent scientific , litchfield , ct ) , and bp signals were recorded in conscious , freely moving rats , over a 30-min period by a microcomputer equipped with an analog - to - digital converter board ( windaq , 2000 hz sampling frequency , dataq instruments , inc , akron , oh ) .
the recorded data were analyzed on a beat - to - beat basis to quantify changes in mean bp and hr .
increasing doses of phenylephrine ( 0.2532.0 g / kg ) and sodium nitroprusside ( 0.051.6 g / kg ) were given as sequential bolus injections ( 0.1 ml ) to produce pressure responses ranging from 5 to 30 mmhg .
a 3- to 5-min interval between doses was necessary for bp to return to baseline .
peak increases or decreases in mean bp after vasoactive drugs injection and the corresponding peak reflex changes in hr were recorded for each dose of the drug .
baroreflex sensitivity was evaluated by a mean index that associated changes in hr to the changes in mean bp , thus allowing for a separate analysis of gain for reflex bradycardia and reflex tachycardia.12 the mean index was expressed as bpm / mmhg .
lvh was evaluated in animals at 16 weeks of age by ventricular hypertrophy index ( calculated by lv / body mass index ( mg / g ) ) , echocardiography and lv collagen volume fraction .
transthoracic echocardiography was performed in all the groups , by double - blind observers with the use of a sequoia 512 ( acuson corporation , mountain view , ca ) , which offers a 1013 mhz multifrequency linear transducer .
images were obtained with the transducer placed on each animal 's shaved chest ( lateral recumbence ) . to obtain a more distinctive image display
, a transmission gel was used between the transducer and the animal 's chest ( general imaging gel , atl , reedsville , pa ) .
animals were scanned from below , at a 2-cm depth with focus optimized at 1 cm .
rats were anesthetized with a combination of ketamine , 80 mg / kg ( parke - davis ) and xylazine , 12 mg / kg ( bayer ) i.p .
wall thickness and left ventricular ( lv ) dimensions were obtained from a short - axis view at the level of the papillary muscles .
lv mass was calculated by using the following formula , assuming a spherical lv geometry and validated in rats : lv mass = 1047 [ ( lvd + pwd + ivsd)3 lvd3 ] , where 1047 is the specific gravity of muscle , lvd is lv end - diastolic diameter , pwd is end - diastolic posterior wall thickness and ivsd is end - diastolic interventricular septum thickness .
in addition , another morphological index was evaluated , the relative wall thickness ( rwt ) , which is expressed by 2 pwd / lvd .
it represents the relation between the lv cavity in diastole and the lv posterior wall .
lv fractional shortening was calculated as ( lvd lvs)/lvd 100 , where lvs is lv end - systolic diameter .
two - dimensional guided pulsed - wave doppler recordings of lv inflow were obtained from the apical four - chamber view .
maximal early diastolic peak velocity ( e ) and late peak velocity ( a ) were derived from mitral inflow .
the lv outflow tract velocity was measured just below the aortic valve , from an apical five - chamber view .
the velocity of circumferential fiber shortening ( vcf ) was measured following the formula ( lvd lvs)/(lvd et ) , where et is the ejection time .
the sample volume was then placed between the mitral valve and lv outflow tract so that the aortic valve closure line and the onset of mitral flow could be clearly identified .
the isovolumic relaxation time ( ivrt ) was measured from aortic valve closure to the onset of mitral flow .
global cardiac function was evaluated by using the myocardial performance index ( mpi ) , which is the ratio of total time spent in isovolumic activity ( isovolumic contraction time and isovolumic relaxation time ) to the et .
these doppler time intervals were measured from the mitral inflow and lv outflow time intervals . interval a , from the cessation to onset of mitral inflow is equal to the sum of the isovolumic contraction time , et and isovolumic relaxation time .
the mpi was calculated with the formula ( a b)/b . at the end of the experimental protocol
, a transverse incision below the diaphragm and bilateral thoracotomy incisions were performed in the sodium pentobarbital ( 40 mg / kg i.p . ) anesthetized rats , in order to expose the heart .
the animal 's heart was arrested in diastole by perfusion with a nacl 0.9% plus 14 mm kcl solution ( pressure equal to a 13 cm water column ) , followed by 4% tamponed formalin for tissue fixation .
hearts were excised , trimmed , weighed and immersed in paraformol 4% in pbs for 24 h. thereafter , the heart was transected perpendicular to the long axis and processed and embedded in paraffinso that histological sections could be performed .
lv sections of 3 m were stained with picrosirius red for collagen quantification.13 computerized image acquisition ( nikon optiphot ) and analysis were used to access collagen volume fraction in lv of all studied animals using the digital image analyzer program image pro plus 6.0 ( media cybernetics , usa).14 tissue was homogenized as described by oliveira et al.15 in 100 mmol / l phosphate buffer ( pb ) , ph 7.2 , containing 340 mmol / l sucrose , 300 mmol / l nacl ( 1 g tissue : 10 ml buffer ) and inhibitor pmsf(100 mmol / l ) . the homogenates were centrifuged at 3.000 rpm , at 40c for 10 min and the supernatant was frozen at 70c until experimental use .
angiotensins i , ii , and 17 were quantified in tissue extracts using reverse - phase high - performance liquid chromatography coupled with ultraviolet ( 214 nm ) detection ( hplc - uv ) .
peptide separation was accomplished using a 4.6 250 mm , 7-m aquapore od 300 column ( applied biosciences , foster city , ca ) , equilibrated with 0.1% phosphoric acid in 5% acetonitrile .
the peptides were initially separated by isocratic elution for 5 min , followed by a linear gradient from 5% to 35% acetonitrile in 0.1% phosphoric acid for 20 min at a flow rate of 1.5 ml / min .
the left ventriclewas pre - homogenized in 8 ml of 0.1 m sodium phosphate buffer containing 0.34 m sucrose and 0.3 m nacl ( ph 7.2 ) .
extraction of angiotensins i , ii and 17 was achieved using sep - pak - c18 column chromatography ( millipore , milford , ma ) .
the column was activated with the following steps : 5 ml of methanol , 5 ml of tetrahydrofuran , 5 ml of hexane , 5 ml of methanol , and 10 ml of h2o ( milliq water ) .
the samples were then loaded onto the column and eluted by the following steps : 10 ml of h2o , 5 ml of 4% acetic acid , and 5 ml of ethanol / acetic acid / h2o ( 9046 , v / v ) .
the peptides were eluted in the last phase and were evaporated to dryness in a speedvac sc110 sample concentrator ( savant instruments , holbrook , ny ) .
sample extracts were reconstituted with 500 l of 0.1% phosphoric acid in 5% acetonitrile , after which they were filtered and injected onto the analytical column of the hplc system .
retention time was used to identify peaks of interest , previously determined by the elution of peptide standards .
calculations were based on peak area , and angiotensins i , ii and 17 concentrations were expressed as nmol / g of tissue .
experiments were performed on male wistar and shr rats aged 16 weeks,10 housed in cages with free access to water and food , and maintained in a room with a constant temperature ( 23c ) on a 12-hour light dark cycle .
all surgical procedures and protocols used were in accordance with the guidelines for ethical care of the experimental animals and were approved by the institutional animals care and use committee ( cep 1016/08 ) .
rats were randomized into four groups : wistar control ( wc ) , n = 9 , wistar denervated ( wd ) , n = 7 , shr control ( shrc ) n = 8 , shr denervated ( shrd ) n = 8 .
sad was performed under anesthesia with ketamine , 80 mg / kg ( parke - davis , brazil ) and xylazine , 12 mg / kg ( bayer , brazil ) . a 3-cm midline incision was made , and sternocleidomastoid muscles were reflected laterally , exposing the neurovascular sheath . the common carotid arteries and the vagal trunk were isolated , and the aortic depressor fibers either traveling with the sympathetic nerve or as an isolated aortic nerve were cut .
the third contingent of aortic baroreceptor fibers traveling with the inferior laryngeal nerve was interrupted by resection of the superior laryngeal nerve after the carotid bifurcation was exposed extensively for carotid stripping . to complete the procedure ,
the sinus nerve , all carotid branches and the carotid body were resected.11 the mortality rate was 35% higher in the animals submitted to sad than in their respective controls .
ten weeks after sad , animals were anesthetized with ketamine , 80 mg / kg ( parke - davis , brazil ) and xylazine , 12 mg / kg ( bayer , brazil ) , and femoral artery and vein catheters were inserted for direct measurements of blood pressure ( bp ) , heart rate ( hr ) and drug administration .
twenty - four hours after catheterization , the arterial catheter was connected to a transducer ( blood pressure xdcr , kent scientific , litchfield , ct ) , and bp signals were recorded in conscious , freely moving rats , over a 30-min period by a microcomputer equipped with an analog - to - digital converter board ( windaq , 2000 hz sampling frequency , dataq instruments , inc , akron , oh ) .
the recorded data were analyzed on a beat - to - beat basis to quantify changes in mean bp and hr .
increasing doses of phenylephrine ( 0.2532.0 g / kg ) and sodium nitroprusside ( 0.051.6 g / kg ) were given as sequential bolus injections ( 0.1 ml ) to produce pressure responses ranging from 5 to 30 mmhg .
a 3- to 5-min interval between doses was necessary for bp to return to baseline .
peak increases or decreases in mean bp after vasoactive drugs injection and the corresponding peak reflex changes in hr were recorded for each dose of the drug .
baroreflex sensitivity was evaluated by a mean index that associated changes in hr to the changes in mean bp , thus allowing for a separate analysis of gain for reflex bradycardia and reflex tachycardia.12 the mean index was expressed as bpm / mmhg .
lvh was evaluated in animals at 16 weeks of age by ventricular hypertrophy index ( calculated by lv / body mass index ( mg / g ) ) , echocardiography and lv collagen volume fraction .
transthoracic echocardiography was performed in all the groups , by double - blind observers with the use of a sequoia 512 ( acuson corporation , mountain view , ca ) , which offers a 1013 mhz multifrequency linear transducer .
images were obtained with the transducer placed on each animal 's shaved chest ( lateral recumbence ) . to obtain a more distinctive image display ,
a transmission gel was used between the transducer and the animal 's chest ( general imaging gel , atl , reedsville , pa ) .
animals were scanned from below , at a 2-cm depth with focus optimized at 1 cm .
/ kg ( parke - davis ) and xylazine , 12 mg / kg ( bayer ) i.p .
wall thickness and left ventricular ( lv ) dimensions were obtained from a short - axis view at the level of the papillary muscles .
lv mass was calculated by using the following formula , assuming a spherical lv geometry and validated in rats : lv mass = 1047 [ ( lvd + pwd + ivsd)3 lvd3 ] , where 1047 is the specific gravity of muscle , lvd is lv end - diastolic diameter , pwd is end - diastolic posterior wall thickness and ivsd is end - diastolic interventricular septum thickness . in addition , another morphological index was evaluated , the relative wall thickness ( rwt ) , which is expressed by 2 pwd / lvd .
it represents the relation between the lv cavity in diastole and the lv posterior wall .
lv fractional shortening was calculated as ( lvd lvs)/lvd 100 , where lvs is lv end - systolic diameter .
two - dimensional guided pulsed - wave doppler recordings of lv inflow were obtained from the apical four - chamber view .
maximal early diastolic peak velocity ( e ) and late peak velocity ( a ) were derived from mitral inflow .
the lv outflow tract velocity was measured just below the aortic valve , from an apical five - chamber view .
the velocity of circumferential fiber shortening ( vcf ) was measured following the formula ( lvd lvs)/(lvd et ) , where et is the ejection time .
the sample volume was then placed between the mitral valve and lv outflow tract so that the aortic valve closure line and the onset of mitral flow could be clearly identified .
the isovolumic relaxation time ( ivrt ) was measured from aortic valve closure to the onset of mitral flow .
global cardiac function was evaluated by using the myocardial performance index ( mpi ) , which is the ratio of total time spent in isovolumic activity ( isovolumic contraction time and isovolumic relaxation time ) to the et .
these doppler time intervals were measured from the mitral inflow and lv outflow time intervals . interval a , from the cessation to onset of mitral inflow is equal to the sum of the isovolumic contraction time , et and isovolumic relaxation time .
at the end of the experimental protocol , a transverse incision below the diaphragm and bilateral thoracotomy incisions were performed in the sodium pentobarbital ( 40 mg / kg i.p . ) anesthetized rats , in order to expose the heart .
the animal 's heart was arrested in diastole by perfusion with a nacl 0.9% plus 14 mm kcl solution ( pressure equal to a 13 cm water column ) , followed by 4% tamponed formalin for tissue fixation .
hearts were excised , trimmed , weighed and immersed in paraformol 4% in pbs for 24 h. thereafter , the heart was transected perpendicular to the long axis and processed and embedded in paraffinso that histological sections could be performed .
lv sections of 3 m were stained with picrosirius red for collagen quantification.13 computerized image acquisition ( nikon optiphot ) and analysis were used to access collagen volume fraction in lv of all studied animals using the digital image analyzer program image pro plus 6.0 ( media cybernetics , usa).14
tissue was homogenized as described by oliveira et al.15 in 100 mmol / l phosphate buffer ( pb ) , ph 7.2 , containing 340 mmol / l sucrose , 300 mmol / l nacl ( 1 g tissue : 10 ml buffer ) and inhibitor pmsf(100 mmol / l ) . the homogenates were centrifuged at 3.000 rpm , at 40c for 10 min and the supernatant was frozen at 70c until experimental use .
angiotensins i , ii , and 17 were quantified in tissue extracts using reverse - phase high - performance liquid chromatography coupled with ultraviolet ( 214 nm ) detection ( hplc - uv ) .
peptide separation was accomplished using a 4.6 250 mm , 7-m aquapore od 300 column ( applied biosciences , foster city , ca ) , equilibrated with 0.1% phosphoric acid in 5% acetonitrile .
the peptides were initially separated by isocratic elution for 5 min , followed by a linear gradient from 5% to 35% acetonitrile in 0.1% phosphoric acid for 20 min at a flow rate of 1.5 ml / min .
the left ventriclewas pre - homogenized in 8 ml of 0.1 m sodium phosphate buffer containing 0.34 m sucrose and 0.3 m nacl ( ph 7.2 ) .
angiotensin iii ( 320 ng ) was added to each sample as an internal standard .
extraction of angiotensins i , ii and 17 was achieved using sep - pak - c18 column chromatography ( millipore , milford , ma ) .
the column was activated with the following steps : 5 ml of methanol , 5 ml of tetrahydrofuran , 5 ml of hexane , 5 ml of methanol , and 10 ml of h2o ( milliq water ) .
the samples were then loaded onto the column and eluted by the following steps : 10 ml of h2o , 5 ml of 4% acetic acid , and 5 ml of ethanol / acetic acid / h2o ( 9046 , v / v ) .
the peptides were eluted in the last phase and were evaporated to dryness in a speedvac sc110 sample concentrator ( savant instruments , holbrook , ny ) .
sample extracts were reconstituted with 500 l of 0.1% phosphoric acid in 5% acetonitrile , after which they were filtered and injected onto the analytical column of the hplc system .
retention time was used to identify peaks of interest , previously determined by the elution of peptide standards .
calculations were based on peak area , and angiotensins i , ii and 17 concentrations were expressed as nmol / g of tissue .
two - way analysis of variance ( anova ) followed by the student newman keuls post hoc test was used .
hypertensive groups ( shrc and shrd ) showed higher values in mean blood pressure than the normotensive groups ( wc and wd ) ( 155 2.2 ; 162 7.3 vs 100 1.8 ; 100 2.8 mmhg , respectively ) .
tachycardia and bradycardia indexes in wd group were 0.44 0.08 and 0.63 0.05 bpm / mmhg , respectively ; in the shrd group , the tachycardia index was 0.23 0.03 bpm / mmhg and bradycardia index was 0.67 0.09 bpm / mmhg .
the bpv ( expressed by standard deviation ) was increased in the groups submitted to sad compared with their respective controls , and the shrd group presented increased values of bpv compared with the other groups ( fig .
animals of the wd , shrc and shrd groups presented higher ventricular hypertrophy index than wc , and sad associated with hypertension induced a higher increase in left ventricle mass in the shrd group ( fig .
the size of left ventricle cavity in diastole ( lvdia ) was increased in the animals of the hypertensive groups ( shrc and shrd ) .
the interventricular septum ( ivs ) and left ventricle posterior wall ( lvpw ) were increased in wd and shr groups . the relative wall thickness ( rwt ) and left ventricle mass ( lvm ) were increased in the wd , shrc and shrd groups , and the shr groups presented increased values of lvm compared with wd group .
isovolumic relation time ( ivrt ) was increased in the wd , shrc , shrd groups .
the e / a ratio was lower in the wd group than the wc group ; and animals of the shr groups presented increased values compared with the wd group .
only the animals of the wd group presented increased values of ejection fraction ( ef ) and velocity of circumferential shortening ( vcf ) compared with the wc group ( table 1 ) . left ventricular fibrosis , accessed by collagen volume fraction evaluation ( fig .
3a , b ) , showed that hypertensive rats ( shr and shrd ) presented increased percentage of collagen compared with normotensive groups .
interestingly , denervated rats ( wd and shrd ) had sharply increased collagen content compared with baroreflex intact rats . fig .
4 shows the concentration of the ang i , ang ii and ang 17 in the left ventricle of different groups .
ang ii concentration was increased in the shr groups compared with the wc group , whereas the ang 17 concentration was decreased in the shrd group compared with the wistar groups .
chronic sad ( 10 weeks ) did not increase the bp and hr of normotensive and hypertensive groups .
additionally , sad also increased the bpv in wistar groups , as the variability became the same as the shr animals without denervation .
the most consistent cardiovascular alteration produced by acute or chronic sad is the huge increase in bpv .
previous studies have described similar high variability in dogs,16 cats17 and rats.18 usually , the bpv in sad animals is characterized by an increase in the bp standard mean , obtained through the moment - to - moment records of bp signals.18 the increased bpv may be due to the absence of straight beat - to - beat synchronization between bp and sympathetic nerve activity normally provided by baroreceptors.2 the ventricular hypertrophy index was increased in shrd compared with shrc , and in the shrc compared with wc , which suggests that hypertension may play a role in the development of the lvh .
this also seems to indicate that hypertension associated with the bpv and baroreflex sensitivity damage results in a higher lvh .
a similar behavior was observed for the collagen volume fraction , an important marker of pathological lvh .
the collagen network may help to maintain cardiac muscle structural organization , may facilitate tensile strength transmission from cardiomyocytes to lv , and may contribute towards myocardial relaxation .
equally important , the collagen network is responsible for myocardial passive stiffness and for ventricular volume.13 despite these beneficial aspects of the collagen network , pressure overload to the heart may cause excessive accumulation of collagen fibers among cardiac cells due to mechanisms yet to be fully understood .
this process , known as fibrosis , considerably increases cardiac tissue stiffness , impairing cardiac function.19 reparative and reactive fibrosis may occur in different situations . in hypertension ,
the reactive fibrosis to increased pressure overload seems to be linked to myocyte apoptosis.20 in addition , since increased bpv is associated with enhanced end - organ damage4 , the amount of collagen in the heart of denervated rats is consequently augmented . in this study , we showed that baroreflex impairment may mediate these stimuli regardless of pressure overload , as denervated rats presented increased collagen volume fraction , and when sad was associated with hypertension this increase became considerably more evident .
both hypertension and high bpv are involved in cardiovascular damage . a study carried out by miao
et al.21 showed that in the shr ( hypertension accompanied by an elevated bpv ) the lvh is more severe than in animals with chronic sad ( only elevated bpv , without hypertension ) , corroborating our findings of a positive correlation between left ventricle mass and bpv ( r = 0.56 ) .
this correlation has also been described by van vliet et al.22 in animals submitted to carotid denervation , sad and modified sad ( preservation of the carotid chemoreceptors ) .
our results for left ventricle angiotensins show that shr groups presented increased ang ii levels compared with wc .
ang ii is the key effector of ras and a vasoconstrictor acting in the cardiovascular system.23 li et al.24 have shown that the gene expressions of all components of ras were elevated in the heart and aorta of the shr compared with wistar kyoto rats .
this significant increase lends supports to the hypothesis that activation of local tissue ras may contribute to cardiac hypertrophy and arterial remodeling .
ras plays a central role in the development of hypertension and the progression of end - organ damage.5 our results showed a positive correlation between the cardiac hypertrophy index and ang ii levels ( r = 0.41 ) , thus corroborating other data in that this peptide is closely related to the development of the cardiac hypertrophy.25 li et al.24 have suggested that the lvh in shr was associated with ace and ace2 gene expression cardiac ras , while aortic hypertrophy was associated with angiotensinogen and ace2 gene expression of aortic ras . this seems to support the postulation that , since there is no correlation between the plasma ang ii concentration and organ damage parameters , it is indeed the tissue ras which may exert a detrimental effect on cardiovascular function26 rather than the circulating ras .
miao et al.27 have shown that plasma and tissue ang ii concentrations remain unaltered after chronic sad ( 2 , 10 and 16 weeks ) . accordingly
, our study could not find any significant differences between animals submitted to sad compared with each control group , although a qualitative tendency may be observed in the wd group compared with the control .
on the other hand , shan et al.28 have observed an increase in the ang ii levels in the cardiac and kidney tissues of rats submitted to chronic sad , and these ang ii levels were more elevated when these animals were submitted to chronic stress , suggesting that elevated bpv and ang ii liberation may be related to end - organ damage development induced by baroreflex dysfunction . in transgenic animals
the overexpression of the angiotensinogen gene in the cardiac myocytes resulted in increased cardiac ang ii concentration and right and left ventricular hypertrophy , unaccompanied by either hypertension or increased circulating levels of ang ii.29 these data strongly suggest that ang ii may induce cardiac hypertrophy regardless of its hemodynamic effects . in our data ,
a reduced ang 17 concentration in the shrd group was observed compared with normotensive groups .
campbell et al.30 found similar results when comparing shr ( 20 weeks ) with donryu rats as a control group .
previous studies have shown that ang 17 partially prevented the pressoric response elicited by ang ii in sad rats but not in sham - operated animals.31 it is well established that ang 17 can act as a physiological antagonist of ang ii by means of the activation of masreceptors , which in turn blocks the vasoconstrictor effect of ang ii.32 different researchers have shown that opposite effects of ang ii are enhanced in animal models of hypertension.33 benter et al.34 have demonstrated that ang 17 attenuates ang ii vasoconstriction in shr but not in normotensive animals .
other studies ( hcht et al.31 ) have also demonstrated that ang 17 blocked the pressor response to ang ii in hypertensive shr and aortic coarcted rats , but not in normotensive control rats .
the enhanced effect of ang 17 in situations of ang ii overactivity may be related to an increased sensitivity to ang 17 . in our data ,
the qualitative and quantitative reduction of the ang 17 levels were accompanied by the significant increase in the ang ii levels .
this reduction of ang 17 may be related to the increase in its sensitivity in the attempt to tone down the effects generated by ang ii overactivity .
our results suggest that not only bp variability and reduced baroreflex sensitivity may contribute to the development of left ventricular hypertrophy as previously suggested ; our findings indicate that elevated levels of ang ii and reduced concentration of ang 17 may also play a role in this process .
these data indicate that baroreflex dysfunction associated with changes of ras may be predictive factors of lvh and cardiac failure .
| objective : the aim of this study was to evaluate the role of angiotensin i , ii and 17 on left ventricular hypertrophy of wistar and spontaneously hypertensive rats submitted to sinoaortic denervation.methods:ten weeks after sinoaortic denervation , hemodynamic and morphofunctional parameters were analyzed , and the left ventricle was dissected for biochemical analyses.results:hypertensive groups ( controls and denervated ) showed an increase on mean blood pressure compared with normotensive ones ( controls and denervated ) .
blood pressure variability was higher in denervated groups than in their respective controls .
left ventricular mass and collagen content were increased in the normotensive denervated and in both spontaneously hypertensive groups compared with wistar controls .
both hypertensive groups presented a higher concentration of angiotensin ii than wistar controls , whereas angiotensin 17 concentration was decreased in the hypertensive denervated group in relation to the wistar groups .
there was no difference in angiotensin i concentration among groups.conclusion:our results suggest that not only blood pressure variability and reduced baroreflex sensitivity but also elevated levels of angiotensin ii and a reduced concentration of angiotensin 17 may contribute to the development of left ventricular hypertrophy .
these data indicate that baroreflex dysfunction associated with changes in the renin angiotensin system may be predictive factors of left ventricular hypertrophy and cardiac failure . |
increased expression of genes necessary for detecting and repairing dna damage can result when cells are exposed to certain genotoxic compounds [ 13 ] .
these same treatments can also induce differential expression of genes in the ubiquitin- ( ub- ) mediated proteolytic pathway [ 4 , 5 ] , suggesting interplay between dna repair and protein degradation [ 69 ] .
mechanisms to transiently stabilize or reduce the abundance of repair proteins following detection and/or removal of dna damage could provide a means for regulating these processes .
defects in ub - mediated protein degradation have been linked to breast cancer , angelman syndrome , von hippel - lindau disease , and altered responses to clinical anesthetics , while defects in dna damage repair are associated with human disorders such as xeroderma pigmentosum , cockayne syndrome , and trichothiodystrophy .
this faithful transmission of genetic material is critical to cell survival , while proper functioning of dna repair processes ensures that genomic integrity is maintained .
ubiquitination results in the modification of protein function , thereby controlling cellular processes such as cell cycle progression , differentiation , and stress responses [ 15 , 16 ] .
this 8.5 kda protein is first activated by ubiquitin - activating enzymes ( e1 ) before being transferred to ubiquitin - conjugating enzymes ( e2 ) and ubiquitin protein ligases ( e3 ) .
it is ultimately attached to lysine residues in target proteins , and multiubiquitin chains form through the activity of ubiquitin chain assembly factors ( e4 ) .
the anaphase promoting complex / cyclosome ( apc / c ) is one of several multisubunit e3 ubiquitin ligases that control progression through the cell cycle [ 18 , 19 ] .
in addition to e1 and e2 enzymes , apc / c - mediated ubiquitination depends on the activator proteins , cdc20 or cdh1 [ 2022 ] , with the former regulating the metaphase to anaphase transition through the degradation of securin .
not surprisingly , apc / c is a target of the spindle checkpoint , not allowing the degradation of securin until proper attachment and alignment of all kinetochores to the spindle are completed .
clarke et al . have reported that in budding yeast cdc20 is capable of acting independently of the apc / c , suggesting an alternative mechanism to its ability to regulate mitotic exit . in metazoans , the apc / c is also active in postmitotic differentiated cells , implying that it has assumed functions in nonproliferating cells as well .
nucleotide excision repair ( ner ) is a highly conserved mechanism that detects and removes bulky lesions from dna following chemical treatment or uv irradiation [ 2628 ] .
compromising ner activity has pleiotropic effects , leading to increased mutation frequency and risk of carcinogenesis [ 29 , 30 ] .
bulky adducts repaired by the ner include n - acetyl-2-aminofluorene ( aaf ) adducts , cyclobutane pyrimidine dimers ( cpds ) , ( 6 - 4 ) photoproducts , and cisplatin intrastrand crosslinks [ 3133 ] .
global genome ner ( ggr ) , responsible for the repair of untranscribed regions of the genome , can be divided into five distinct steps : damage recognition , incision , excision , repair synthesis , and dna ligation .
the expression of a number of ner proteins is upregulated as a result of dna damage ; however , once the dna damage is repaired and the proteins are no longer needed , they are degraded to restore basal levels [ 1 , 34 ] . multiple interactions between members of the ner , ubiquitin - associated enzymes , and proteasomal subunits have been revealed by both genetic and biochemical approaches [ 57 , 9 , 35 , 36 ] .
this would indicate that one means of regulating the repair process is through ub - mediated degradation of ner proteins once they are no longer needed .
many of the more than 30 ner proteins are essential to the repair processes ; however , a subset is considered accessory , and deletion of these results in a less severe sensitivity to dna damaging agents in comparison to the essential components , which have near 0% survival with uv treatment [ 3739 ] .
rad4 and rad23 are two such accessory members , interacting strongly with one another to control the damage recognition step of ner [ 40 , 41 ] .
rad4 , which transiently accumulates following uv irradiation , is stabilized by rad23 and this interaction stimulates binding of the rad4/rad23 complex to the damaged dna [ 5 , 43 ] .
ng et al . have concluded that the primary function of rad23 in ner is the stabilization of rad4 , although diminished rad4 stability is not the primary cause of deficient ner in rad23 mutant cells [ 9 , 44 ] .
to examine the interplay between cdc20 and ner , we utilized yeast strains deleted for rad4 and harboring the conditional cdc20 - 1 mutation .
we report here that the diminished capacity of cdc20 results in extreme uv sensitivity in ner - compromised mutants of s. cerevisiae , specifically those harboring rad4 .
all deletion strains used in this study carry a gene deletion linked to a kanamycin - resistance marker kanmx that confers resistance to the antibiotic geneticin ( g418 ) and were obtained from the mississippi functional genomic network ( mfgn ) core facility ( university of southern mississippi , hattiesburg , ms ) or open biosystems ( now thermo fisher scientific ) . a cdc20 - 1 mutant strain containing the clonat resistance marker was generated using a one - step gene replacement .
double mutants harboring various gene deletions and cdc20 - 1 were also generated by one - step gene replacement , making the single and double mutants isogenic with respect to one another .
all other double mutants were generated by traditional crosses and verified by temperature sensitivity and dual resistance to g418 and clonat .
a plasmid containing cdc20 - 1 was constructed by amplifying the mutant allele from strain 405 - 1 - 1 ( gift from d. burke ) , which included 400 bp upstream of the start codon and 500 bp downstream of the stop codon .
the clonat resistance gene was excised from p4339 ( obtained from mfgn ) and blunted .
the fragment was ligated at a stui site 150 bp downstream of the stop codon of cdc20 - 1 .
dna sequencing was performed to verify the presence of the cdc20 - 1 mutation and the resistance marker .
the resulting recombinant plasmid was linearized with spei to be used for one - step gene replacement .
plasmids designed to overexpress cdc20 were generated using the pyes.2 vector ( invitrogen ) , which carries a ura3 marker , 2 ori , and multiple cloning site downstream of the gal1 inducible promoter .
plasmids harboring cdc20 or cdc20 - 1 were constructed by ligating a 2.0 kb hindiii fragment into pyes.2 at the hindiii site and orientation was verified through restriction enzyme analysis .
all other overexpression plasmids were obtained from open biosystems , in which the genes of interest were subcloned behind the gal1 inducible promoter in the bg1805 plasmid vector .
yeast strains were incubated at 22c for 1216 hours in yeast - peptone - dextrose ( ypd ) or the appropriate selective medium .
cultures were normalized to an a660 of 1.0 , and a 1 : 10 serial dilution was performed in sterile water . from each dilution , 5 l was spotted onto ypd agar containing methyl methanesulfonate ( mms ) ( 0.05% ) , hydroxyurea ( 100 mm ) , phleomycin ( 5 g / ml ) , benomyl ( 12 g / ml ) , and 4-nitroquinoline ( 4-nqo ) ( 0.2 , 0.5 , and 1.0 g / ml ) or exposed to 10 or 25 j / m of uv radiation in a stratalinker model 1800 ( stratagene ) . for those strains harboring genes to be overexpressed , transformants were assayed on synthetic complete dextrose media lacking uracil and containing either 2% glucose or 3% galactose/0.5% glucose .
quantification of survival was determined by plating a known number of cells on selective media , followed by uv irradiation ( 10 and 25 j / m ) and incubation at 22c for 3 - 4 days .
uv survival was calculated by dividing the number of surviving colonies following treatment by the number of colonies on untreated plates .
briefly , 0.5 ml of an overnight culture was used to inoculate 4.5 ml of fresh selective media .
after 810 hours at 22c , cells were treated with 0.2 , 0.5 , or 1.0 g / ml of 4-nqo or uv irradiated at 10 , 25 , or 50 j / m .
cells were returned to 22c for another 4 hours prior to performing the -gal assay .
four hours was found to be optimal for induction of the rnr3:lacz fusion protein in studies by jia and xiao .
activities were determined , and fold induction was calculated as a ratio of -galactosidase activity in the cultures with and without treatment .
the pyes.2 vector ( invitrogen ) was digested with pvuii , cutting the plasmid outside of the ura3 marker .
linearized and uncut plasmids of a known concentration were transformed in parallel into multiple strains and plated on scd lacking uracil .
following 3 - 4 days of incubation at 22c to allow for sufficient colony growth , colonies were counted .
the results are the average of at least three independent experiments , and standard error of the mean was determined .
a strain carrying rad4 with a c - terminal gfp fusion was obtained from the mfgn core facility .
the cdc20 - 1 allele was introduced into this strain via a traditional cross , and gene replacement was verified by pcr , resistance to clonat , and temperature sensitivity at 37c .
cultures were grown at 22c in scd lacking histidine with or without clonat for 1216 hours and subsequently uv irradiated at 25 j / m and 100 j / m .
an outgrowth of 4 hours was allowed before cells were briefly centrifuged and washed once with water .
samples were examined and photographed on a zeiss imager m1 axio fluorescence microscope paired with a photometrics coolsnap hqcamera .
uv exposures were done in triplicate , and greater than 150 cells were counted and analyzed for each treatment .
the rad4/cdc20 - 1 double mutant was initially identified as displaying a synthetic sick phenotype in a synthetic genome analysis using a query strain harboring the cdc20 - 1 temperature sensitive allele ( figure 1(a ) ) .
the uv sensitivity of the rad4 strain was increased approximately 10-fold by the point mutation in cdc20 - 1 ( figure 1(b ) ) .
when cdc20 under the control of the gal1 promoter was reintroduced into the double mutant strain and then irradiated , uv resistance comparable to that seen in rad4 strains was restored ( figure 1(c ) ) .
this was not observed when cdc20 - 1 was transformed into the double mutant . when cdc20 - 1 was introduced into the rad4 strain survival at 10 j / mwas less than that caused when cdc20 was overexpressed in the same strain , indicating a dominant negative phenotype of the cdc20 - 1 mutant allele .
the sensitivities of neither the single nor double mutant to hydroxyurea , mms , phleomycin , and benomyl were significantly different as compared to wild - type or the cdc20 - 1 strains alone ( figure 1(d ) ) .
it has been reported that inactivation of certain dna repair pathways in s. cerevisiae may diminish or enhance gene expression in response to dna damage . in this assay ,
plasmid with rnr3 fused to lacz was transformed into rad4/cdc20 - 1 , rad4 , cdc20 - 1 , and wild - type strains .
rnr3 is the large subunit of the ribonucleotide reductase complex , which catalyzes dntp synthesis and is subject to both dna replication and repair checkpoints .
we examined the response of rad4 and rad4/cdc20 - 1 strains to both uv radiation and 4-nqo .
deletion of rad4 enhanced rnr3-lacz induction by 4-nqo 3.5- and 2.5-fold at concentrations of 0.2 and 0.5 g / ml ( figure 2(a ) ) .
uv radiation at energy levels of 10 and 25 j / m enhanced this induction by 2- and 7-fold , respectively ( figure 2(b ) ) . with the introduction of cdc20 - 1 into the rad4 background , however , the fold increase was diminished and not different than either wild - type or cdc20 - 1 strains alone .
these data are not consistent with the values for survivability following uv exposure and indicate an altered capacity for dna repair that is not related to cell survival in strains harboring both rad4 and cdc20 - 1 simultaneously . to determine whether introduction of cdc20 - 1 affected the cell 's ability to repair double strand breaks , a plasmid end - joining assay was performed [ 47 , 48 ] .
briefly , strains of s. cerevisiae were transformed with a linearized plasmid ( pyes.2 ) containing the ura3 marker . to normalize for differences in transformation efficiencies between strains , an uncut version of the same plasmid
since the plasmid must be recircularized in order to revert the ura phenotype of the selected strains , the number of transformants obtained with the linearized plasmid normalized to the number obtained with the uncut plasmid provides a quantitation of the dsb - repair ability of the yeast strains . as shown in figure 3
, rad4 strains were able to repair dsbs , although not as efficiently as wild type .
the double mutant , however , had significantly diminished ability to repair these defects as compared with the single deletion alone .
these results indicate that introduction of cdc20 - 1 affects the cells ability to repair damaged dna in an ner - defective strain . to determine whether cdc20 - 1 may have an effect on expression or stability of rad4 we utilized a strain of yeast harboring a c - terminal gfp tag on rad4 , with or without a chromosomal copy of cdc20 - 1 .
liquid cultures were irradiated at 25 and 100 j / m and examined four hours after exposure .
fluorescence was observed in strains carrying the tagged copy of rad4 in an otherwise wild - type background , regardless of treatment or time of outgrowth . when cdc20 - 1 was present , however , fluorescence was diminished following uv irradiation such that around 60% of the cells had visible fluorescence ( figure 4 ) .
since strains with cdc20 - 1 were capable of survival on solid media at these energy levels , we do not attribute decreased fluorescence to cell death .
here we demonstrate that rad4 and cdc20 - 1 exhibit a synthetic growth defect upon uv exposure in saccharomyces cerevisiae .
we examined strains carrying a deletion of rad4 and the conditional cdc20 - 1 allele and found that the two mutations in combination resulted in extreme uv sensitivity .
overexpression of cdc20 in rad4/cdc20 - 1 restored the slight uv resistance observed in the single mutant rad4 .
microscopic analyses indicate that in the presence of cdc20 - 1 , rad4-gfp fluorescence is significantly diminished relative to wild type after a four outgrowth following uv irradiation . when introduced into a ner - defective strain ( rad4 ) , survival following uv irradiation
our data demonstrate that this uv sensitivity may be the result of a reduced capability to repair double strand breaks , as the rad4/cdc20 - 1 strain had a diminished capacity to anneal dsbs in a plasmid end - joining assay .
we propose that cdc20 may act to indirectly modulate the nucleotide excision repair pathway by way of its role in protein degradation . in a report by gillette et al .
a novel cullin - based e3 ubiquitin ligase ( ecs ligase ) was identified that ubiquitinates rad4 following uv exposure .
if cdc20 has a role in regulation of ecs ligase activity , the ubiquitination of rad4 could be affected indirectly ; however , cdc20 may also directly regulate its ubiquitination through the apc / c .
rad4 contains a putative cyclin destruction box ( d box ) in its amino terminus , although its expression does not exhibit cell cycle periodicity .
d box motifs contain two conserved residues ( rxxl ) and several other more moderately conserved residues .
they are a highly conserved sequence common to substrates of the apc / c [ 4952 ] .
previous studies have shown that rad4 is stable , with a half - life of approximately 4 hours .
upon uv exposure , these levels decrease as the protein is proteolyzed by the 26s - proteasome .
the ubiquitination , not the stabilization , of rad4 in response to uv irradiation via the ecs ligase regulates ner in part . without an examination of rad4 ubiquitination in cdc20 - 1 mutants , however , it is impossible for us to state that this is indeed the case , although microscopic examinations lend proof to this argument .
despite the strong genetic evidence that cdc20 affects the ability of saccharomyces cerevisiae to carry out ner in the absence of rad4 we have yet to demonstrate biochemically how cdc20 acts in this pathway .
there are a number of ner proteins that contain putative d boxes and many other candidate proteins that may be targeted for ubiquitination via the apc / c or acted upon by cdc20 alone .
we do not exclude the possibility that unidentified protein is a member of the chromatin remodeling complex as rad4/rad23 complex is known to have a role in maintenance of heterochromatin structure [ 53 , 54 ] . in addition , recent studies have shown that rad4 can bind to the proteasome without affecting overall protein degradation ability of the complex , and studies have yet to elucidate mediators of this direct binding .
further research is necessary to dissect this interaction and to understand the roles of each component .
this study is the first , however , to present evidence that cdc20 may play a role in the degradation of proteins involved in nucleotide excision repair . | regulation of dna repair can be achieved through ubiquitin - mediated degradation of transiently induced proteins . in saccharomyces cerevisiae , rad4 is involved in damage recognition during nucleotide excision repair ( ner ) and , in conjunction with rad23 , recruits other proteins to the site of damage .
we identified a synthetic interaction upon uv exposure between rad4 and cdc20 , a protein that modulates the activity of the anaphase promoting complex ( apc / c ) , a multisubunit e3 ubiquitin ligase complex .
the moderately uv sensitive rad4 strain became highly sensitive when cdc20 - 1 was present , and was rescued by overexpression of cdc20 .
the double mutant is also deficient in elicting rnr3-lacz transcription upon exposure to uv irradiation or 4-nqo compared with the rad4 single mutant .
we demonstrate that the rad4/cdc20 - 1 double mutant is defective in double strand break repair by way of a plasmid end - joining assay , indicating that rad4 acts to ensure that damaged dna is repaired via a cdc20-mediated mechanism .
this study is the first to present evidence that cdc20 may play a role in the degradation of proteins involved in nucleotide excision repair . |
a ruptured infected aortic aneurysm with staphylococcal sepsis is rare yet potentially life - threatening condition .
although ruptured infected aneurysm is usually treated surgically , it has still high operative mortality and morbidity , especially high surgical risk . despite recent improvement in endovascular treatment of abdominal aortic aneurysm and antibiotic therapy , a ruptured infected aortic aneurysm coexisting with sepsis is still a lethal condition .
we report one case that in a highly risk patient endovascular approach with prolonged antibiotic therapy was one of options for successful results .
a 81-year - old female with history of hypertension was admitted to the hospital with abdominal pain , anorexia , and mild fever for two months ' duration .
she was admitted to the gastroenterology for gastritis two months ago and at that time she was diagnosed as infectious colitis and her blood culture yielded staphylococcus aureus , which was treated with intravenous administration with ceftriaxone . the computed tomography ( ct ) of her abdomen at that time showed a normal abdominal aorta ( fig .
the patient looked unwell , and had a temperature of 37.7 , pulse rate of 72 beats per minute , respiration rate of 20 per minute , and blood pressure of 150/70 mmhg .
a ct image of abdominal aorta demonstrated irregular aneurysmal dilatation ( maximum 6 cm ) of the abdominal aorta at the l3-l4 level , along with out - pouching area and soft tissue masses that showed heterogeneous enhancement . enhancing and nonenhancing elements were seen within the dilated aortic lumen , along with a crescent - shaped low - signal area outside the aorta , suggesting leakage of the aneurysm ( fig .
she had a total white blood count of 20,600/mm , with a differential count of 94% neutrophils , 3% lymphocytes and 3% monocytes , and a c - reactive protein of 30.67 mg / dl .
the patient was prepared for surgery on an urgent basis and underwent endovascular surgery in a standard operating room equipped with a mobile c - arm and image intensifier . during the procedure ,
both groins were exposed , diagnostic angiography catheter was introduced via the left common femoral artery for intraoperative imaging .
the ruptured aorta was then covered with edurant aorto - uni - iliac graft ( medtronic inc .
, mineapolis , mn , usa ) via the right common femoral artery . a 10-mm amplatzer ( st .
paul , mn , usa ) vascular plug was placed in the left common iliac artery .
completion angiography showed a successful exclusion of the ruptured aortic segment without any signs of endoleak . and then fem - fem bypass was done with 8 mm polytetrafluoroethylene graft to restore blood flow to the left leg ( fig .
the patient had an uneventful postoperative recovery without complications , but constantly complained of back pain , which was revealed spondylitis at the level of aneurysm rupture area ( fig .
and then the blood culture had no growth and the c - reactive protein concentration returned to normal at discharge .
three - month follow - up ct showed near absorption of the periaortic hematoma ( fig .
the patient had been on oral antibiotics for one year and doing well at 2 years after discharge .
nearly 50% are seen at the infrarenal aorta , and 25% is involved the juxtarenal or pararenal aorta . despite advances in antibiotic treatment , pure medical management for mycotic aneurysms is often inadequate , and gold standard therapy remains surgical resection , debridement of the infected aorta and the surrounding tissue , and either an in situ interposition or extra - anatomical bypass .
however , these patients have high surgical risk and mortality , especially in non - salmonella infection and advanced age .
fillmore and valentine determined that sepsis is the leading cause of death for surgical infected aneurysm patients .
five independent variables are known to associate with operative mortality : extensive peri - aortic infection , female sex , staphylococcus aureus infection , aneurysm rupture and supra - renal location .
the negative effect of rupture on mortality is huge that the mortality rate of 13% with intact aneurysm increased to 33.3% in case of rupture .
however , placing an endovascular graft in an infected area remains counter - intuitive and against general surgical principles .
a literature review about endoluminal management of mycotic aneurysm revealed that evar seemed a possible alternative method for treating mycotic aneurysm and the only significant independent risk factors for persistent infection were rupture of aneurysm and fever .
evar has significant advantages over surgical resection as it avoids a large incision , full heparinization , extracorporeal circulation , aortic cross - clamping , interference with respiratory function , and the need for massive transfusion .
age is a predictor of poor outcome with major surgery , with one report finding that individuals of 80 years old compared with patients 65 to 69 had a twofold higher operative mortality .
the perioperative mortality in evar ranges from 1.9 to 5% ; however , perioperative mortality in open procedures ranges from 5 to 10% . for octogenarians ,
one of the most important factors in recovery is rapid return to baseline and prompt discharge from hospital .
thirty - six percentage of individuals who underwent open repair of abdominal aortic aneurysm did not return to be ambulatory ( preoperatively 100% ambulatory ) .
antibiotic treatment of infected aneurysms ranges anywhere from a few weeks to lifelong after surgery . and
the regimens should be based on cultures and sensitivities when a pathogen can be isolated .
if no pathogen is identified , the regimens should be broad , covering g(+ ) , g(- ) , anaerobic and fungus . | the generally accepted treatment for infected aortic aneurysms involves open surgical resection and debridement , with in situ or extra - anatomical bypass .
occasionally , endovascular management can be substituted for the standard operation dependent on the patient 's condition .
we report the case of an 81-year - old female with a ruptured infected aortic aneurysm and sepsis , successfully treated endovascularly .
she had been on oral antibiotics for one year and is doing well 2 years after discharge . |
vesicoureteral reflux ( vur ) in children has been referred as the prostate cancer of pediatric urology , as the 2 conditions share many features such as the high prevalence rates in their respective populations , the recent decline in utilization of diagnostic studies for these conditions , the historically high utilization of surgical intervention in the 2 conditions , as well as other features .
vur has a relatively high prevalence in children , where it occurs in approximately 1% of the general pediatric population , similar to the high prevalence of prostate cancer in men , one of the most common cancers in the world .
in addition , the screening or diagnostic tests for both diseases also appear to be in decline due to recently released guidelines .
the updated american academy of pediatrics ( aap ) guidelines on the diagnosis and management of urinary tract infections ( utis ) in febrile infants and young children has led to the delay of voiding cystourethrography ( vcug ) until the second uti , which has decreased the diagnostic incidence of vur .
similarly , in 2012 , the u.s . preventive services task force recommended against prostate - specific antigen ( psa ) screening for prostate cancer , as they concluded that the potential benefits of screening were not greater than the expected risks ; however , the debate on psa screening is still continuing .
furthermore , the historically high utilization of surgical intervention in the 2 conditions has led to the introduction of minimally invasive surgical options for both conditions .
robot - assisted laparoscopic radical prostatectomy has already become the new standard surgical treatment of prostate cancer for 90% of current prostate cancer surgical candidates as a result of its favorable and enduring oncologic outcomes comparable to open surgery and the relatively low complication rates for erectile dysfunction and postprostatectomy urinary incontinence . indications for surgical treatment of pediatric vur include breakthrough uti while on continuous antibiotics prophylaxis , renal scarring , and worsening or unresolving vur . determining which approach is most effective for treating a particular pediatric patient with vur involves weighing the pros and cons of various antireflux surgeries with respect to success rates , durability of results , avoidance of febrile utis , universal applicability , general availability , perioperative morbidity , wound cosmesis , and cost .
however , recently , minimally invasive techniques such as robot - assisted laparoscopic surgery has been increasingly utilized for the treatment of pediatric surgical conditions , as robotic surgical systems have provided surgeons with 3-dimensional vision , 10x magnification , and fully articulating endo - wrists suitable for dissection and suturing . as a result
, minimally invasive surgery options have become increasingly available for many complex reconstructive procedures in pediatric urology . since first introduced in 2004 , robot - assisted laparoscopic ureteral reimplantation ( ralur ) has been described as one of these new minimally invasive procedures that have been increasingly adopted and described in several clinical series . in this article
, we reviewed the literature over the past decade on pediatric ralur with a critical analysis of the perioperative outcomes with respect to reflux resolution and complications .
as a result of this review , based upon this analysis , we provide tips and tricks on how to facilitate the best possible outcomes with ralur to enable this minimally invasive surgical option to become a new standard treatment option for vur in children similar to the evolution of radical prostatectomy from an open procedure to become the new standard surgical treatment option for prostate cancer in adult men .
ralur has become more prevalent over the past decade but still remains relatively uncommon in many pediatric centers .
bowen et al . recently analyzed the use of pediatric open , laparoscopic and ralur in the united states from 2002 to 2012 .
the number of annual ureteral reimplantations decreased by 14.3% during this time period , but the minimally invasive ureteral reimplantations increased from 0.3% in 2000 to 6.3% in 2012 .
while robotic surgery has increased in volume over the past decade in the field of pediatric urology , ralur has lagged behind the increase of robot - assisted laparoscopic pyeloplasty ( ralp ) . in our personal surgical experience ( unpublished data ) ,
the number of ralur cases was actually the highest in case volume as of 2010 , while ralp was second highest as a result of the high prevalence of vur in the pediatric population .
however , the revised 2011 aap guideline for the workup of pediatric uti , where vcug was not recommended routinely after the first febrile uti led to decreases in the number of newly diagnosed vur patients and the subsequent number of ureteral reimplantation surgeries . of note , though , ralur still remains a relatively high volume procedure for a pediatric robotic surgery program ( fig .
another factor for why robotic ureteral reimplantation has not enjoyed as widespread adoption as quickly as robotic prostatectomy may be related to the necessary learning curve for the ralur procedure . while many centers have reported favorable outcomes and safety profiles for ralur ,
other centers have reported outcomes that did not reach the levels associated with traditional open reimplantation , indicating that the procedure 's complexity may have been underestimated .
a potential remedy for this is for surgeons early in their learning curve for robotic surgery to defer ralur procedures until a later point in their robotic experience .
the intravesical approach of ralur is composed of the following surgical steps : creation of pneumovesicum by the instillation of co2 gas into the bladder , intravesical ureteral mobilization , submucosal tunneling , and intravesical advancement of the mobilized ureters within the newly created submucosal tunnels .
woo first described this procedure using a cross - trigonal or cohen method , only 2 other series have reported their experiences with intravesical ralur ( table 1 ) .
peters and woo reported their clinical series of 6 children 5 to 15 years old without the need for open conversion .
five of 6 patients ( 83% ) had radiographic resolution of the vur , while 1 patient had a urine leak postoperatively secondary to inadequate bladder closure . in 2011 ,
. reported on their series of patients who underwent intravesical ralur ( n=19 ) with intravesical open ureteral reimplantation ( n=22 ) , as well as extravesical ralur ( n=20 ) with extravesical open ureteral reimplantation ( n=17 ) . in their series
, intravesical ralur was associated with shorter durations of urinary catheter drainage , fewer bladder spasms , and shorter hospital stays when compared to the open intravesical technique , but with a higher reported complication rate . in 2012 , chan et al .
reported their intravesical ralur experience in three children , all with high - grade bilateral vur .
all 3 patients ( 100% ) showed reflux resolution on postoperative vcug , and were free from uti during the follow - up period .
a notable point is that , despite the similar time of introduction of intravesical ralur in the literature with conventional laparoscopic intravesical ureteral reimplantation , no other reports on intravesical ralur have been published in the literature beyond the initial articles unlike the increasing number of articles of further procedural development with conventional laparoscopic intravesical ureteral reimplantation .
technical challenges with laparoscopic reimplantation and the expansion of the number of robotic systems worldwide may partially explain this evolution toward robotic reimplantation , as well as the multijointed angulation provided by the robotic instruments that facilitates the ureteral mobilization , submucosal tunneling , and suturing .
on the other hand , the larger size of the robotic camera ( 12 mm ) and instruments ( 8 or 5 mm ) have the potential for increased risk of port site bladder leakage , space limitation , and inferiority in cosmesis when compared to the conventional laparoscopic camera ( 5 mm ) and instruments ( 3 mm ) which are usually used during conventional laparoscopic reimplantation .
future development of smaller and less bulky robotic instruments may overcome these potential limitations and facilitate further adoption of the intravesical ralur procedure .
the extravesical approach of ralur is based on the open extravesical lich - gregoir procedure . since first described by peters in 2004 ,
the ralur experience appears to be following a similar historical experience to that of the open extravesical technique , where suboptimal results were noted in the early experience , with eventual widespread adoption worldwide . even though extravesical ralur has been utilized by many surgeons over a decade ,
the surgical outcomes of this procedure have been reported with variability among previously reported series ( table 2 ) . in 2008 , casale et al . reported their extravesical ralur experience of 41 children with vur .
all of the patients had bilateral reflux , and the reflux resolution rate was 97.6% .
no complications were reported except for one febrile uti episode which occurred in the patient with persistent vur postoperatively .
this group reported on their subsequent ralur case series with an increased number of 150 children with bilateral vur and at least 2 years of postoperative follow - up .
only one patient had persistent reflux after ralur which became downgraded from bilateral grade 5 vur to unilateral grade 2 vur .
no patients were reported to have de novo voiding dysfunction or urinary retention when measured by objective voiding parameters and validated questionnaire .
the authors concluded that bilateral nerve - sparing robotic - assisted extravesical reimplantation is associated with similar success rates as the traditional open approaches , with minimal morbidity and no voiding complications after surgery .
other subsequent series also reported similar favorable outcomes of ralur . in 2011 , smith et al .
compared surgical outcomes of 25 extravesical ralur patients with those of 25 open cross - trigonal ureteral reimplantation patients .
the overall success rate was 97% for ralur compared to 100% for open reimplantation , but with a reduced mean length of stay ( 33 hours vs. 53 hours ) and reduced pain medication usage in the ralur group . in 2015 ,
the complete resolution of vur was observed in 97.9% with only 2 patients ( 2.7% ) reporting development of temporary postoperative urinary retention that self - resolved within 2 weeks .
other series reported favorable reflux resolution rates with ralur but with uncommon but serious complications . in 2011 , marchini et al . reported 20 cases of extravesical ralur with a reflux resolution rate of 100% , but with 2 cases ( 10% ) of ureteral injury . in 2014 , akhavan et al . reported the outcomes of 78 extravesical ralur in 50 patients .
ten patients ( 20% ) had prior deflux injection , and 2 ( 4% ) had prior ureteroneocystostomy on the ipsilateral side .
the reflux resolution rate was 92.3% based on postoperative vcug , but the overall complication rate was 10% including 2 patients ( 4% ) with ureteral obstruction and 1 ( 2% ) with a ureteral injury , which necessitated ureteral stent placement in all 3 patients .
they found no association between persistent reflux and patient gender , age , presence of voiding dysfunction , history of prior reflux surgery , bilaterality , or stent placement during surgery .
two other series have reported suboptimal success rates with relatively high complication rates . in 2015 , grimsby et al . reported on the experience at 2 institutions with extravesical ralur .
of the 61 patients ( 93 ureters ) who underwent ralur , vur resolution was noted in only 44 of 61 patients ( 72% ) , while 6 major complications ( 10% ) were also noted including ureteral obstruction or ureteral leak .
no specific demographic or intraoperative factor was determined to be responsible for the lower success rate . in 2016 , herz et al .
reported the outcomes of extravesical ralur in 54 children with a total of 72 ureters .
overall surgical success was 85.2% of ureters and overall complication was 11% including ureteral obstruction ( 7.4% ) or ureteral injury ( 3.7% ) .
they concluded that bilateral ralur is associated with higher failure rates , higher complication rates , higher reoperation rates , and more postoperative utis and nonsurgical readmissions compared with unilateral ralur .
several possibilities may explain the variability in the reported surgical outcomes of extravesical ralur at different institutions .
as one potential explanation , similar to all new techniques , there is most likely variability in the patient populations and in surgeon experience , where the success rates after ralur may have been affected by case selection as well as by a surgeon 's learning curve . with the adoption of ralp ,
the operative time of ralp significantly decreased as surgeons progressed through their learning curve , and the reduction of operative time and the potential for major complications with ralur is expected as the learning curve is overcome .
similar to the open approach , essential surgical steps for ralur include the mobilization of the ureter proximally for approximately 4 to 5 cm to achieve a sufficient length for reimplantation which should include minimal usage of cautery and ureteral handling during distal ureteral dissection , with preservation of periureteral tissues and the delicate blood supply to the ureter .
manipulation of the ureter should also be minimized when the detrusor flaps are wrapped over the ureter during the reimplantation .
the most common complication is urinary retention , which can be considered a relatively mild complication since it usually does not require an extended hospital stay or additional surgical procedures .
replacement of the foley catheter for up to 2 weeks may be required , after which self - resolution of the retention usually occurs .
after foley catheter is removed , timed voiding and double voiding should be encouraged every 2 to 3 hours to promote bladder emptying .
while urinary retention is a known risk after bilateral open ureteral reimplantation as well with an incidence as high as 26% , the risk may be enhanced in ralur patients when voiding dysfunction or bladder bowel dysfunction is present
. however , the reported urinary retention rate after bilateral ralur overall appears to be lower than that for open extravesical ureteral reimplantation ( estimated at approximately 10% of bilateral cases ) .
casale et al . suggested that the enhanced robotic visualization allowed for detection and careful manipulation of the periureteral tissue at the bladder hiatus with identification of the pelvic plexus , and therefore a decrease in the incidence of postoperative urinary retention .
however , in another report , location of the pelvic plexus by recording muscle action potential of bladder smooth muscle after electric stimulation could not be identified during the assessment of bladder muscle response to electro - physiologic stimulation of the pelvic nerve plexus during ralur .
ureteral obstruction is commonly due to transient perioperative edema and often improves spontaneously over time .
placement of a ureteral stent can be considered when a reimplantation is performed in a solitary kidney .
if ureteral obstruction persists with signs of progressive obstruction or concomitant pyelonephritis , a ureteral stent should be placed .
ureteral injury can also be caused by aggressive dissection and devascularization of the ureter from excessive cauterization or an inadvertent injury to the ureter .
the lack of haptic feedback during robotic surgery may contribute to the incidence of this type of injury .
it may be noticed immediately during the robotic procedure , but also may have a delayed presentation with signs of abdominal distention and pain or ileus .
no consensus currently exists on which ralur surgical technique should be used to maximize surgical outcomes , as several techniques have been associated with outcomes similar to that of open reimplantation .
we previously described our modified top - down suturing technique of interrupted sutures without the need for ureteral elevation or stent placement that is associated with low complication rates and high success rates . unlike previously described
bottom to top techniques , where the detrusor muscle closure starts from the bottom to the top , our first detrusor closure stitch for the top - down approach is placed at the superior aspect in an interrupted fashion that creates the new muscle hiatus , and which also elevates the ureter without tension or manipulation to decrease instrument - related contact with the ureter for the subsequent sutures .
. also reported on a modified technique based on periodic review and critical analysis of video recordings of previous procedures , which increased the success rates for resolution of vur following their technique modifications .
some of their suggestions for improving surgical outcomes include adequate lengths ( 45 cm ) for the detrusor tunnel , and use of a u stitch .
some of the most important principles for operative success are based on principles commonly taught for all open reimplantation surgery such as a no - touch rule for handling the ureter , low cautery settings , and careful use of the robotic instruments that do not lead to inadvertent injury to the ureter and bladder .
concerns have been raised as to whether ralur is a cost - effective option when compared to open surgery due to its high capital costs and heterogeneous clinical outcomes , and several recent studies have attempted to compare the costs between ralur and open ureteral reimplantation . however , while the estimation of actual costs can be difficult , the overall conclusions from these studies noted that the direct cost including operating room charges may be higher for ralur , but the shorter hospital length of stay with lower hospitalization charges can result in equivalent total charges in comparison with open ureteral reimplantation .
this is especially applicable in the united states where daily charges for each hospital day are in the thousands of dollars .
compared the costs of 6 most frequently performed robotic surgeries with their similar nonrobotic procedures using patients identified in the pediatric health information system database .
the median hospitalization costs of ralur and open ureteral reimplantation were $ 13,096 ( range , $ 9,057$17,890 ) and $ 8,530 ( range , $ 6,901$11,148 ) for this group , respectively .
it should be noted that charges / costs can vary significantly between institutions and hence may limit the applicability of this comparison .
compared the cost and complications between 108 ralur and 1,494 open ureteral reimplantation cases from a nationwide sample .
the median hospital cost for ralur was $ 9,128 versus $ 7,273 for open ureteral reimplantation , however , there was a considerable variability from the differential experience among centers expressed as complications , and the higher cost of ralur was associated with a significantly higher rate of complications .
thus , we can expect that when the complication rate decreases with technical improvements in ralur , the costs will also reduce to the equivalent levels for open ureteral reimplantation . in our propensity - matched study
( publication pending ) comparing the hospital charges for robotic ( n=38 ) and open ( n=97 ) ureteral reimplantations , the higher operating room charges for the ralur were offset by the shorter hospital stays , lower hospitalization charges , and avoidance of major complications that led to equivalent overall hospital charges for the ralur and open cohorts ( $ 21,437 for ralur versus $ 21,461 for open reimplantation ) .
even though extravesical ralur has been utilized by many surgeons over a decade , the surgical outcomes of this procedure have been reported with variability among previously reported series ( table 2 ) . in 2008 , casale et al . reported their extravesical ralur experience of 41 children with vur .
all of the patients had bilateral reflux , and the reflux resolution rate was 97.6% .
no complications were reported except for one febrile uti episode which occurred in the patient with persistent vur postoperatively .
, this group reported on their subsequent ralur case series with an increased number of 150 children with bilateral vur and at least 2 years of postoperative follow - up .
only one patient had persistent reflux after ralur which became downgraded from bilateral grade 5 vur to unilateral grade 2 vur .
no patients were reported to have de novo voiding dysfunction or urinary retention when measured by objective voiding parameters and validated questionnaire .
the authors concluded that bilateral nerve - sparing robotic - assisted extravesical reimplantation is associated with similar success rates as the traditional open approaches , with minimal morbidity and no voiding complications after surgery .
other subsequent series also reported similar favorable outcomes of ralur . in 2011 , smith et al .
compared surgical outcomes of 25 extravesical ralur patients with those of 25 open cross - trigonal ureteral reimplantation patients .
the overall success rate was 97% for ralur compared to 100% for open reimplantation , but with a reduced mean length of stay ( 33 hours vs. 53 hours ) and reduced pain medication usage in the ralur group . in 2015 ,
the complete resolution of vur was observed in 97.9% with only 2 patients ( 2.7% ) reporting development of temporary postoperative urinary retention that self - resolved within 2 weeks .
other series reported favorable reflux resolution rates with ralur but with uncommon but serious complications . in 2011 , marchini et al . reported 20 cases of extravesical ralur with a reflux resolution rate of 100% , but with 2 cases ( 10% ) of ureteral injury . in 2014 , akhavan et al . reported the outcomes of 78 extravesical ralur in 50 patients
ten patients ( 20% ) had prior deflux injection , and 2 ( 4% ) had prior ureteroneocystostomy on the ipsilateral side .
the reflux resolution rate was 92.3% based on postoperative vcug , but the overall complication rate was 10% including 2 patients ( 4% ) with ureteral obstruction and 1 ( 2% ) with a ureteral injury , which necessitated ureteral stent placement in all 3 patients .
they found no association between persistent reflux and patient gender , age , presence of voiding dysfunction , history of prior reflux surgery , bilaterality , or stent placement during surgery .
two other series have reported suboptimal success rates with relatively high complication rates . in 2015 , grimsby et al . reported on the experience at 2 institutions with extravesical ralur .
of the 61 patients ( 93 ureters ) who underwent ralur , vur resolution was noted in only 44 of 61 patients ( 72% ) , while 6 major complications ( 10% ) were also noted including ureteral obstruction or ureteral leak .
no specific demographic or intraoperative factor was determined to be responsible for the lower success rate . in 2016 , herz et al .
reported the outcomes of extravesical ralur in 54 children with a total of 72 ureters .
overall surgical success was 85.2% of ureters and overall complication was 11% including ureteral obstruction ( 7.4% ) or ureteral injury ( 3.7% ) .
they concluded that bilateral ralur is associated with higher failure rates , higher complication rates , higher reoperation rates , and more postoperative utis and nonsurgical readmissions compared with unilateral ralur .
several possibilities may explain the variability in the reported surgical outcomes of extravesical ralur at different institutions .
as one potential explanation , similar to all new techniques , there is most likely variability in the patient populations and in surgeon experience , where the success rates after ralur may have been affected by case selection as well as by a surgeon 's learning curve . with the adoption of ralp ,
the operative time of ralp significantly decreased as surgeons progressed through their learning curve , and the reduction of operative time and the potential for major complications with ralur is expected as the learning curve is overcome .
similar to the open approach , essential surgical steps for ralur include the mobilization of the ureter proximally for approximately 4 to 5 cm to achieve a sufficient length for reimplantation which should include minimal usage of cautery and ureteral handling during distal ureteral dissection , with preservation of periureteral tissues and the delicate blood supply to the ureter .
manipulation of the ureter should also be minimized when the detrusor flaps are wrapped over the ureter during the reimplantation .
the most common complication is urinary retention , which can be considered a relatively mild complication since it usually does not require an extended hospital stay or additional surgical procedures .
replacement of the foley catheter for up to 2 weeks may be required , after which self - resolution of the retention usually occurs .
after foley catheter is removed , timed voiding and double voiding should be encouraged every 2 to 3 hours to promote bladder emptying .
while urinary retention is a known risk after bilateral open ureteral reimplantation as well with an incidence as high as 26% , the risk may be enhanced in ralur patients when voiding dysfunction or bladder bowel dysfunction is present .
however , the reported urinary retention rate after bilateral ralur overall appears to be lower than that for open extravesical ureteral reimplantation ( estimated at approximately 10% of bilateral cases ) .
casale et al . suggested that the enhanced robotic visualization allowed for detection and careful manipulation of the periureteral tissue at the bladder hiatus with identification of the pelvic plexus , and therefore a decrease in the incidence of postoperative urinary retention .
however , in another report , location of the pelvic plexus by recording muscle action potential of bladder smooth muscle after electric stimulation could not be identified during the assessment of bladder muscle response to electro - physiologic stimulation of the pelvic nerve plexus during ralur .
ureteral obstruction is commonly due to transient perioperative edema and often improves spontaneously over time .
placement of a ureteral stent can be considered when a reimplantation is performed in a solitary kidney .
if ureteral obstruction persists with signs of progressive obstruction or concomitant pyelonephritis , a ureteral stent should be placed .
ureteral injury can also be caused by aggressive dissection and devascularization of the ureter from excessive cauterization or an inadvertent injury to the ureter .
the lack of haptic feedback during robotic surgery may contribute to the incidence of this type of injury .
it may be noticed immediately during the robotic procedure , but also may have a delayed presentation with signs of abdominal distention and pain or ileus .
no consensus currently exists on which ralur surgical technique should be used to maximize surgical outcomes , as several techniques have been associated with outcomes similar to that of open reimplantation .
we previously described our modified top - down suturing technique of interrupted sutures without the need for ureteral elevation or stent placement that is associated with low complication rates and high success rates . unlike previously described
bottom to top techniques , where the detrusor muscle closure starts from the bottom to the top , our first detrusor closure stitch for the top - down approach is placed at the superior aspect in an interrupted fashion that creates the new muscle hiatus , and which also elevates the ureter without tension or manipulation to decrease instrument - related contact with the ureter for the subsequent sutures .
. also reported on a modified technique based on periodic review and critical analysis of video recordings of previous procedures , which increased the success rates for resolution of vur following their technique modifications .
some of their suggestions for improving surgical outcomes include adequate lengths ( 45 cm ) for the detrusor tunnel , and use of a u stitch .
some of the most important principles for operative success are based on principles commonly taught for all open reimplantation surgery such as a no - touch rule for handling the ureter , low cautery settings , and careful use of the robotic instruments that do not lead to inadvertent injury to the ureter and bladder .
concerns have been raised as to whether ralur is a cost - effective option when compared to open surgery due to its high capital costs and heterogeneous clinical outcomes , and several recent studies have attempted to compare the costs between ralur and open ureteral reimplantation . however , while the estimation of actual costs can be difficult , the overall conclusions from these studies noted that the direct cost including operating room charges may be higher for ralur , but the shorter hospital length of stay with lower hospitalization charges can result in equivalent total charges in comparison with open ureteral reimplantation .
this is especially applicable in the united states where daily charges for each hospital day are in the thousands of dollars .
compared the costs of 6 most frequently performed robotic surgeries with their similar nonrobotic procedures using patients identified in the pediatric health information system database .
the median hospitalization costs of ralur and open ureteral reimplantation were $ 13,096 ( range , $ 9,057$17,890 ) and $ 8,530 ( range , $ 6,901$11,148 ) for this group , respectively .
it should be noted that charges / costs can vary significantly between institutions and hence may limit the applicability of this comparison .
compared the cost and complications between 108 ralur and 1,494 open ureteral reimplantation cases from a nationwide sample .
the median hospital cost for ralur was $ 9,128 versus $ 7,273 for open ureteral reimplantation , however , there was a considerable variability from the differential experience among centers expressed as complications , and the higher cost of ralur was associated with a significantly higher rate of complications .
thus , we can expect that when the complication rate decreases with technical improvements in ralur , the costs will also reduce to the equivalent levels for open ureteral reimplantation . in our propensity - matched study
( publication pending ) comparing the hospital charges for robotic ( n=38 ) and open ( n=97 ) ureteral reimplantations , the higher operating room charges for the ralur were offset by the shorter hospital stays , lower hospitalization charges , and avoidance of major complications that led to equivalent overall hospital charges for the ralur and open cohorts ( $ 21,437 for ralur versus $ 21,461 for open reimplantation ) .
the trifecta of outcomes for the surgical treatment of prostate cancer are cancer control , urinary continence , and preservation of erectile function .
robot - assisted laparoscopic prostatectomy has become the standard surgical option for men with prostate cancer where over 90% of surgical patients are undergoing the robotic option , since the outcomes trifecta above for radical prostatectomy can be achieved at many centers .
similar to radical prostatectomy , a proposed trifecta for surgical treatment of vur can be equivalent success rates , equivalent hospital charges , and low complication rates similar to those of the gold standard , open ureteral reimplantation . during the past decade
, ralur has become an increasingly utilized option for the surgical management of vur , yet the reported outcomes and complication rates have been varied among institutions . as we enter into the second decade of ralur , it is well understood that the favorable outcomes and low complication rates for ralur need to be comparable to open ureteral reimplantation for all surgeons and centers , and we are well on the way to achieving these goals .
educational opportunities such as robotic hands - on course and expanded training during residency / fellowship training should help to standardize ralur techniques to those that are associated with comparable outcomes to open reimplantation . furthermore , multi - institutional collaborations are in progress that are identifying technical factors that hopefully will lead to widespread best practices for ralur .
in addition , we expect that the further evolution of robotic technology will continued to be applied to the pediatric field that will facilitate complex procedures such as ralur . however , the impetus to improve pediatric medical devices is difficult because of the smaller pediatric population and therefore smaller number of pediatric procedures when compared to the adult population .
an example is how the most recent da vinci robotic system ( xi ) has no 5-mm instruments , while they are available for the earlier models . as pediatric specialists , we need to continue to encourage as opposed to discourage the use of new technology in children , and especially for high volume procedures such as ralur , as only this will lead to the next big thing in pediatric surgical care .
from the lessons learned over the past decade , ralur for the treatment of pediatric vur should be reclassified as a complex reconstructive procedure in pediatric urology , as this procedure can be associated with significant complications and especially for surgeons early in their learning curve .
current efforts of identifying patient - related and intraoperative factors associated with optimal surgical outcomes provide an educational opportunity to improve the success rates associated with this procedure .
multiinstitutional collaborations are in the progress of identifying key surgical steps and technical factors with the goal of improving the surgical outcomes of ralur for all robotic pediatric urologic surgeons including surgeons early in their learning curve .
this should lead to similar outcomes and avoidance of complications for ralur that are already associated with open ureteral reimplantation , but with the advantages of a minimally invasive procedure . | the da vinci robotic system has improved surgeon dexterity , ergonomics , and visualization to allow for a minimally invasive option for complex reconstructive procedures in children . over the past decade , robot - assisted laparoscopic ureteral reimplantation ( ralur ) has become a viable minimally invasive surgical option for pediatric vesicoureteral reflux ( vur ) . however , higher - than - expected complication rates and suboptimal reflux resolution rates at some centers have also been reported .
the heterogeneity of surgical outcomes may arise from the inherent and underestimated complexity of the ralur procedure that may justify its reclassification as a complex reconstructive procedure and especially for robotic surgeons early in their learning curve .
currently , no consensus exists on the role of ralur for the surgical management of vur .
high success rates and low major complication rates are the expected norm for the current gold standard surgical option of open ureteral reimplantation .
similar to how robot - assisted laparoscopic surgery has gradually replaced open surgery as the most utilized option for prostatectomy in prostate cancer patients , ralur may become a higher utilized surgical option in children with vur if the adoption of standardized surgical techniques that have been associated with optimal outcomes can be adopted during the second decade of ralur . a future standard of ralur for children with vur
whose parents seek a minimally invasive surgical option can arise if widespread achievement of high success rates and low major complication rates can be obtained , similar to the replacement of open surgery with robot - assisted laparoscopic radical prostectomy as the new strandard for men with prostate cancer . |
clinical manifestations of toxicity are similar to those of cocaine and include sudden death due to cardiac arrhythmia , stroke , seizures , psychosis , and rhabdomyolysis .
neuropathologic alterations of amphetamine abuse include cerebral infarcts and hemorrhage , often caused by emboli associated with cardiac arrhythmias or myocardial infarction .
furthermore , vasospasm or vasculitis due to the pharmacologic action of amphetamines may be causative factors [ 1 , 2 ] .
we report a patient with an acute demyelination associated with amphetamine abuse which has not been previously described .
a tentative diagnosis of anorexia nervosa was rendered , as she reported weight loss of 60 kg within a few months .
the next day she developed cough , fever , and leukocytosis subsequently manifested dyspnea , cyanosis , and hypotension , and eventually required catecholamines . despite assisted breathing ,
the patient developed signs of brain edema ( anisocoria , dilated pupils ) and finally died due to multi - organ failure .
microscopic examination demonstrated moderate hypoxic neuronal damage , nerve cell loss , and moderate edematous changes .
in the white matter of the occipital lobe , two sharply demarcated areas of demyelination were evident ( figures 1(a ) and 1(b ) ) associated with reactive astrogliosis ( figure 1(c ) ) . the axons were well preserved on bodian silver impregnation ( figure 1(d ) ) .
there was a diffuse and perivascular infiltration of t - lymphocytes as seen on immunostaining with uchl ( cd45ro ) antibody ( figure 1(e ) ) as well as numerous cd68- and cr3/43-immunopositive macrophages / microglial cells ( figures 1(g ) and 1(h ) ) .
immunohistochemical investigations for the presence of infectious agents ( hiv-1 , vzv , cmv , and hsv-1 , toxoplasma gondii ) were negative .
toxicological analysis with gas chromatography ( gc ) and mass spectrometry ( ms ) of a white powder which was found in the bag of the patient on admission revealed an amphetamine content of 14.54% .
analysis of brain samples of the occipital region including the demyelinated area revealed an amphetamine content of 0.017 g / g determined by gc / ms ( figure 2 ) .
examined samples of brain tissue adjacent to the lesion of the same and of other brain slabs did not reveal any evidence for amphetamine .
although this is a very low level , which was determined after 6 weeks in the hospital , it indicates that a high blood level of amphetamine would have been possible before hospital admission .
however , on admission , the clinicians never had a suspicion of drug abuse , and therefore initial drug screening on blood or urine has not been performed .
clinical manifestations of toxicity are similar to those of cocaine and include sudden death due to cardiac arrhythmia , stroke , seizures , psychosis , and rhabdomyolysis .
amphetamines are anorectic drugs by virtue of their ability to cause the release and/or prevent the reuptake of noradrenaline and dopamine .
therefore , the rapid and extensive weight loss within a few months , as seen in our patient , might be due to a long - lasting amphetamine abuse .
neuropathologic alterations of amphetamine abuse include cerebral infarcts and hemorrhage , often caused by emboli associated with cardiac arrhythmias or myocardial infarction .
so far , to our knowledge , acute demyelination has not yet been described in cases of amphetamine abuse .
disorders affecting the white matter can be classified into inherited metabolic disorders , that is , the leukodystrophies ( including metachromatic leukodystrophy , krabbe disease , adrenoleukodystrophy , alexander disease , and pelizaeus - merzbacher disease ) and demyelinating disorders . based on their most probable cause
, the demyelinating disorders may be classified as immune - mediated ( multiple sclerosis ) , viral ( progressive multifocal leukoencephalopathy ) , toxic ( central pontine myelinolysis , marchiafava - bignami disease ) or chronic progressive subcortical demyelination ( binswanger disease ) and leuko - araiosis as well as ischemic . in general ,
demyelination may be induced by serum factors or degradative cell products secreted by activated macrophages , which , in turn , may be stimulated by products of activated lymphocytes [ 6 , 7 ] .
the process of demyelination is usually preceded by an increased permeability of the blood - brain barrier . in our case
, we could exclude most of the common viruses , toxic agents like alcohol , as well as ischemia and hypoxia .
thus , the two demyelinating foci found in the occipital lobe might have been incidental findings representing the early phase of ms which went undiagnosed . since
their size is quite small , they might not have been able to produce clinical features related to the specific brain region ( most probably visual dysfunctions ) . these functional disturbances could have been present but not noted as the patient was abusing drugs .
however , the presence of amphetamine within the demyelinating lesions makes it less likely that ms is the cause . as a reverse conclusion
, one would have to find also amphetamine traces in demyelinating lesions in cases clinically diagnosed as having ms in order to diagnose the present case as ms .
furthermore , amphetamine was only detected in the demyelinating foci , but not in adjacent or far distant brain regions .
based on the lack of other evidence , we conclude that the lesion is most probably due to the direct action of amphetamine .
data supporting this hypothesis are given by the observation that inhalation of preheated heroin causes a spongiform leukoencephalopathy with marked neurological deficits noted several weeks after exposure [ 8 , 9 ] .
ct and mri findings usually demonstrate myelin damage of the white matter of the cerebral hemispheres and cerebellum .
a lipophilic toxin - induced process was considered to be due to contaminants and induced or enhanced by cerebral hypoxia , but a definite toxin could not be identified [ 8 , 9 ] .
further support is given by the fact that the amphetamine derivatives 3,4-methylenedioxymethamphetamine ( mdma ) and 3,4-methylenedioxyamphetamine ( mda ) were demonstrated by immunohistochemistry in all cortical brain regions and the neurons of the basal ganglia , the hypothalamus , the hippocampus , and the cerebellar vermis of two amphetamine abuse fatalities .
one gene expression analysis of amphetamine administration in rats showed significantly altered gene expression for genes related to transcription factors , cellular stress / molecular chaperones , signalling pathways , synaptic function , protein synthesis / degradation , and others .
however , it was recently shown by microarray studies that the chronic abuse of cocaine in humans results in a significant decrease in the expression of myelin - related genes including myelin basic protein ( mbp ) , proteolipid protein ( plp ) , and myelin - associated oligodendrocyte basic protein ( mobp ) . in vivo administration of amphetamine resulted in a dose - related enhancement of neurological and histological signs of acute experimental allergic encephalomyelitis .
furthermore , widespread axonal damage with concomitant microglia activation has been shown in the brains of polydrug abusers .
it is noteworthy that , for the multitude of animal experiments done so far , damage to the white matter was never reported .
given the fact that the volume fraction of white matter in the mouse or rat brain is significantly lower than that in the human brain , it might be concluded that myelin is less vulnerable in these animals and , thus , no changes could be seen ; or possible white matter changes were never examined . in this context the use of amphetamine as a treatment for multiple sclerosis should be seen with caution .
this is the first report in which the demonstration of amphetamine in the affected brain region strongly suggests a direct link between amphetamine abuse and demyelination ; however , the pathogenetic mechanisms of the demyelination still remain unclear . | we report the case of a 31-year - old woman , admitted to the hospital for chest pain , dying a few days later from septic multiorgan failure , and showing at autopsy foci of acute demyelination in the occipital lobe .
gas chromatography / mass spectrometry analysis revealed the presence of amphetamine in the demyelinated area , which might be considered as the pathogenic agent , since other causes for demyelination could be excluded .
this case represents the first report showing a demyelinating process due to a street drug . |
biobanks collect and store human and non - human biological material for diagnostic and research purposes .
these biobanks keep sensitive data and biological samples also for long periods of time , and such data and samples can be used for research even many years after they have been deposited .
therefore , a well - designed and carefully applied informed consent is an essential document to help research purposes .
there has been a continuous growth in the activities of the biobanks yet a definitive informed consent model is still not available .
so , it is important i ) to make a systematic evaluation of the models of consent used , and this should also include a survey of public opinion in order to develop guidelines which can be shared in an international setting and ii ) to improve the quality of research projects .
biobanks collect and store human and non - human biological materials for diagnostic and research purposes .
some countries have identified their ideal operative context to be within institutes known as biological resource centers ( brc ) .
the creation of biobanks has required , among the various critical issues involved , the definition of adequate privacy protection systems to safeguard donors .
in fact , biobanks collect and hold sensitive data and biological samples also for long periods of time .
furthermore , since biobanks often operate through the internet , biological samples and their related data are often used for multicenter studies and can even be exchanged between brcs in different countries .
there is little homogeneity in the legislation regulating the exchange of biological materials and their documentation between countries .
for example , even within the european union there is no shared regulatory system concerning biobanking .
this means that the same kind of activity is allowed in some member states but not in others .
for this reason , some countries are starting to standardize their legislation and to share regulations concerning research and working methods . between 1999 and 2001
, the european commission set up the eurogenebank , a project which involved six countries and aimed to define the types of existing biobanks and the critical issues related to their regulation .
this has opened the debate on several issues , such as the methods used to collect biological samples , the objectives of biobanking , how the samples and related data should be managed and the access procedures . in 2009 ,
the german bmb - eu coop project analyzed the legal and ethical consequences of international biobanking , underlining how similar theoretical principles are applied in different ways in different countries .
one of the aspects to which legislators should pay attention is the issue of consent .
the american society of human genetics ( ashg ) has identified some essential items that must be covered by the donor s consent declaration for collecting samples for a specific research project : i ) the objective of the research , ii ) the limitations and possible consequences , iii ) risks and benefits , iv ) the type of information expected from the research , v ) how results will be made available , vi ) methods of maintaining confidentiality . according to the ashg , it is unacceptable to request informed consent for any possible research carried out using the samples at a later date . in cases of deceased donors , the indiscriminate use of samples and data
italian law concerning the donation of biological materials and treatment of personal data is presented in legislative decrees . the legislative decree n. 196 of 30 june 2003 ( code for the protection of personal data ) guarantees [ ] that the treatment of personal data is carried out with respect for the fundamental rights and liberties , and the dignity of the person concerned , with particular reference to confidentiality , personal identity and personal data protection rights . the legislative decree n. 191 of 6
november 2007 ( bringing into force of the 2004 ec directive 2004/23/ec on the definition of the regulations concerning donation quality and safety , the supply , control , processing , preservation , storing and distribution of human tissues and cells ) lists the essential information that must be given to donors , [ ] that is the objective and nature of the supply , its consequences and risks , analytical tests ( if carried out ) , registration and protection of donor data , medical confidentiality , therapeutic objective and potential benefits , together with information on guarantees that can be given to safeguard the donor . in order to validate the consent documentation
guidelines regarding this information have been published on the official journal n. 159 of 11 july 2011 .
recently , lombardy region has developed guidelines for the correct institution and regulation of biobanks , suggesting a possible organizational model and emphasizing the importance of the informed consent ; however , operative instructions are not provided .
the problems involved in informed consent were also examined at the poli - mi biobank of the fondazione ca granda ospedale maggiore policlinico of milan .
the poli - mi biobank offers a service of cryopreservation of biological material collected for research purposes by all the departments in which the fondazione is divided , the operative units ( ou ) . the center for transfusion medicine , cell therapy and cryobiology
is made up of 4 sectors : i ) the milano cord blood bank which performs characterization , storage and distribution of placental blood for hemopoietic transplantation ; the cell manipulation laboratory is responsible for the cryopreservation of hemopoietic stem cells from peripheral blood and bone marrow .
ii ) the franco calori cell factory , which is a certified laboratory compliant to the regulations and standards of good manufacturing practices ( gmp ) .
it is authorized to produce cell therapy products , and to carry out translational research on stem cells for transplantation and tissue repair .
iii ) the interdepartmental flow cytometry center carries out research and services for cell immunophenotyping and sorting ; the experimental hepatology laboratory studies cancer stem cells .
iv ) the poli - mi biobank preserves around 100,000 human biological samples ( serum , plasma , cells , dna , tissue ) collected for research purposes from 24 internal and external groups . in 2005 , the fondazione set up a workgroup with the aim of revising the methods of attaining informed consent relating to biological samples collected for research purposes on the basis of the italian legislation in force and in consideration of the international experiences reported so far .
the evaluation of the consent documentation used by the fondazione identified a variety of models . on the basis of the models in use , and taking into account the requirements reported in the literature , the workgroup drew up a single , standard model .
this model was proposed to all ou of the fondazione and was used experimentally from 2009 to 2011 .
since the immediate adoption of the new model by all ou in the fondazione who use the biobank services is unlikely , through 2011 it was decided to carry out a survey to evaluate its use by the various ous and at the same time to verify the completeness and the correct compilation of the old models that may still be in use .
all original consent documents collected at the fondazione are archived in the patient s case sheet .
the physician in charge provides the patient with information about the collection , preservation and use of human biological materials for research and obtains the patient s consent .
the physician presenting the consent form to the patient is responsible for its correct compilation in all its parts .
since the immediate adoption of the new model by all ou in the fondazione who use the biobank services is unlikely , through 2011 it was decided to carry out a survey to evaluate its use by the various ous and at the same time to verify the completeness and the correct compilation of the old models that may still be in use .
all original consent documents collected at the fondazione are archived in the patient s case sheet .
the physician in charge provides the patient with information about the collection , preservation and use of human biological materials for research and obtains the patient s consent .
the physician presenting the consent form to the patient is responsible for its correct compilation in all its parts .
the rexic project is a retrospective study that aims to verify the adoption of the single , standard consent model proposed for experimental use in 2009 , and to check the correct compilation and archiving of the informed consent documentation relating to biological samples deposited in the bank during 2011 .
the study protocol was drawn up together with the directors and staff of the center for transfusion medicine , cell therapy and cryobiology ( ctmc ) and the post - graduate school of hygiene and preventive medicine in milan .
for the study , we selected the ous or sectors which sent samples to the poli - mi biobank during 2011 .
all consent documentation was examined by those ous or sectors that collected at least 50 samples ; otherwise a 10% random sample of documents was examined . in order to collect data relating to the completeness and correct compilation of the consent documentation ,
an ad hoc evaluation form was provided based on the single , standard consent model proposed by the ctmc and established in november 2011 .
researchers confirmed the ou ( or sector ) on the form and a number code was used to identify the type of consent examined if this was not already identified by a quality control system code assigned by the fondazione .
the researcher had to verify whether each of these issues had been filled in and whether compilation was correct and/or complete .
in the first section we checked the presence of information relating to the ou where the samples were taken and its contact details ( telephone number and e - mail address ) . this information is useful for the accountability of donors . in the second section we verified the presence of 5 items : i ) the reasons
why and the methods by which the sample was taken ; ii ) information on the methods of preservation used for the biological samples ; iii ) information on the management of the documentation related to the samples deposited in the bank ( e.g. results of tests carried out , sensitive donor data , etc . ) ; iv ) information on the risks related to the collection and preservation of the biological material ; v ) information on the disposal of the stored material .
this kind of information is needed to justify the type of the collected sample and to explain the aim of the research .
we also examined whether the consent documentation included a note confirming that the donor may , at any time , withdraw consent to the use of his / her samples and data by writing to the director of the ou where the samples were taken , and if collected data were treated with the needed confidentiality and in respect of the donor s privacy .
lastly , the protocol set out the expression of consent for : i ) additional sample collection ; ii ) the preservation of samples ; iii ) the manipulation of biological material ; iv ) the management of sensitive data ; v ) the sharing of anonymous data ; vi ) the use of data for scientific publication ; vii ) the possible use of samples and data for further research ; viii ) the disposal of the samples once the research has been completed .
the collection of consent for future research is fundamental since the use of biological materials is not always immediate , and samples can be stored for an undefined time .
each consent document was checked to evaluate whether the following data had been provided : date , name , surname and signature of the person responsible for obtaining consent and of the donor . for those ous
which had still not adopted the single , standard model , evaluation of consent was made through a comprehensive checklist of all the above - listed items .
the project was presented by the directors of the involved ous during several meetings and in the presence of a specialist in public health and post - graduate public health students , who had been previously instructed on to how to fill in the evaluation form and who subsequently checked the consent documentation . the public health specialist and 4 students
were asked to verify whether the patients case sheets contained the consent documentation ; they also had to report the type of consent model used and to evaluate whether it had been filled in correctly and fully by those responsible for obtaining the patient consent .
a pilot evaluation was made on 10 patient case sheets at one of the ous , where the single , standard consent documentation adopted in november 2011 was already in use to test the evaluation method and to examine the level of agreement between the researchers .
the same 10 patient case sheets were evaluated separately by all the researchers and the results were then compared in order to establish if the level of agreement was acceptable . a level of agreement of over 95% was considered sufficient to then assign 2 researchers to each ou for subsequent evaluation of the patient case sheets : the 10 case sheets and consent forms obtained the same evaluation from all the researchers of the examination group .
the remaining 40 ( 23.3% ) patient case sheets , the absence of any consent documentation meant that no evaluation of content could be made .
the researchers identified that 48 consent declarations were obtained using the single , standard model adopted in november 2011 and other 84 consent documents were obtained using other models .
the consent declarations , obtained using the single , standard model were shown to be the most complete and were filled in better than the other models used .
in fact , the standard consent model always included information relating to the ou and the physician responsible for obtaining consent while the other items were filled in in 85 - 90% of cases .
the missing items were : use of the information for scientific publication and possible sharing of data acquired anonymously from other research centers .
furthermore , items relating to taking additional samples and their preservation were only included in less than 20% of the consent documents .
finally , 7 consent documents were not signed and/or the date was missing ( table 2 ) .
as far as the 84 consent documents obtained using other models are concerned , only items relating to the reason for the sample collection and the use of material for research were nearly always included and filled in fully and correctly ( 95% and 98% of cases , respectively ) .
compilation of items relating to consent for the sample to be taken was also satisfactory ( 99% ) , while consent to sample preservation and data availability was compiled in 64% of cases .
some types of consent models had sections that were preprinted and were , therefore , already filled in , such as information relating to the physician responsible for obtaining consent ( table 3 ) .
finally , it should be noted that examination of the patient case sheets and , therefore , evaluation of the consent were carried out directly at each individual ou .
this provided an opportunity to meet the staff involved and to raise their awareness as to how important it is to correctly and fully fill in and archive the documentation .
before discussing the results of our study , we feel appropriate to point out its main limitations , due to the relatively limited number of forms that could be examined . in this regard , it was disappointing to note that , independently from the type of informed consent form , in several instances the form was missing or partially filled .
however , although disappointing and negative in its nature , this finding is itself an interesting result of our investigation , which stresses the importance of regular staff education and careful auditing of informed consent practices related to the collection of biological materials for research biobanking . in spite of the above limitations ,
an analysis of the results shows that the adoption of the single , standard consent model improved the quality of consent collection .
the only information missing from this type of consent model was the expression of consent , while the items relating to taking and preserving additional samples was included in only 20% of cases . given the retrospective nature of the study , it was not possible to clarify whether in the majority of cases these items were missing because they were not relevant and this will be discussed with the ous involved .
however , critical issues still remain and it is hoped that these problems will gradually be resolved by more ous adopting and using this standard model , and by its subsequent revision and improvement .
the model currently being used was drawn up on the basis of the european masters in bioethics . over the last decades
, questions have been raised concerning which type of informed consent should be considered the gold standard from an ethical point of view , and this is of particular concern in fields such as biobanking .
some authors emphasize that consent to biobanking can never cover everything and can not , therefore , be informed .
this is because at the moment in which consent is obtained , it is probably not known for what type of future research the data will be used .
it is , therefore , often impossible to inform the donor as to the detailed objectives , methodology , risks and financial sources of subsequent research .
one example of a good organizational model is provided by the united kingdom biobank that has been set up in such a way as to offer an important resource capable of sustaining a wide range of research aimed at the prevention , diagnosis and treatment of diseases and promotion of health issues across all social groups .
the biobank does , however , encourage much greater participation on the part of donors in its operations , also through the creation of a specific interactive website . another question , which is often discussed in the scientific community , is the possibility of having the patient consent model renewed each time it is proposed that the preserved samples are used for a new research project .
there is a general agreement on the fact that blank consent is not acceptable , even though this is still used in some centers . in many cases , the option of asking consent for a wider use of material and data
this allows the researcher to carry out a wide range of activities but the consent is too vague to be considered informed .
various institutions are wondering how to draw up a consent model that respects those issues identified as essential .
it is interesting to mention the experience of the s. giovanni di dio center in brescia , northern italy . here
they have revised the main criteria related to the preservation and use of human - derived material from an ethical point of view ; sixteen critical areas were identified that must be respected by the consenting individual .
these regard the type of material taken , the location and duration of preservation , the reasons for the biobanking , the consequences on donor health , the voluntary nature of the donation , donor privacy , control of access to the deposited material and any possible transfer .
defining the requirements for giving consent to the collection , storage and use of biological samples and related information for research is still a subject of controversy at international level .
samples preserved in biobanks represent an important resource for future research , but their use can raise ethical questions regarding the consent obtained .
some groups are developing regulatory systems for the biobanks using a self - administered questionnaire to evaluate the attitudes and perceptions of potential donors of biological samples ; the majority of those interviewed have shown a positive attitude towards genetic research . in spite of
rapid expansion of the biobanks and their related activities , a definitive consent model is still not available .
this can probably be achieved by modifying and adapting the models in current use and by taking into consideration the perceptions and attitudes of donors . indeed ,
general population s confidence in research is essential in order to achieve important benefits for society at large .
it is to be hoped , therefore , that other experiences of this type are reported in the literature by those who are involved with ethical , legal and social issues related to research biobanking .
it can be expected that larger studies could provide useful information to improve the quality of current practices of research biobanking .
recently , critical aspects of the above issues have been thoroughly discussed in an editorial on hela and wi-38 cells recently published in nature , that invites for a rethinking of the ethical issues concerning the use of human tissue in research . | backgroundthe center for transfusion medicine , cell therapy and cryobiology , milan , northern italy , is the headquarter of the poli - mi biobank . it co - ordinates the biobank activities of the fondazione ca granda ospedale maggiore policlinico of milan .
such activities require specific safeguarding of donors rights and protection of sensitive and genetic data .
the fondazione ca granda ospedale maggiore policlinico has set up a project on informed consent with the aim of developing awareness and understanding of this issue .
within this project , it has been decided to evaluate how consent for biobanking material is expressed.design and methodsthe aim of the study was to evaluate the quality and completeness of consent to biobanking in the poli - mi biobank .
this was a retrospective study carried out in 2012 on samples of consent declarations collected by biobank units in 2011 .
some units used a single , standard consent model available from a previous poli - mi biobank workgroup .
other units used models which had been previouly formulated . evaluation was made using a form that indicated the essential elements of consent.resultsa total of 48 consent declarations were collected using the single , standard model and 84 were collected using other models .
the consent declarations that used the single , standard model were found to be the most complete and were filled in better than other models.conclusionsprogressive adoption of a simple , standard consent model is expected to improve the quality of consent acquisition .
regular audit of the compliance of consent practices with ethical and legal requirements is mandatory to improve the quality of research biobanking . |
all the authors provided substantial contributions to conception and design , acquisition of data , or analysis and interpretation of data ; they assisted in drafting the article or revising it critically for important intellectual content ; and they provided final approval of the version to be published .
extramural funding was provided by the us army medical research & materials command , cooperative agreement w81xwh-08 - 2 - 0056 .
all of the authors are employed by the uniformed services university of the health sciences or another entity of the us federal government .
there are no author financial affiliations associated with this manuscript other than those related to their government employment .
if the manuscript is accepted for publication , we are required by policy to include the following disclaimer : the views expressed in this manuscript are solely those of the authors and not necessarily reflect the opinion or position of the uniformed services university of the health sciences , the us army medical research & materials command , the national library of medicine , the us department of defense , or the us department of health and human services .
| background and objective the clinical note documents the clinician 's information collection , problem assessment , clinical management , and its used for administrative purposes .
electronic health records ( ehrs ) are being implemented in clinical practices throughout the usa yet it is not known whether they improve the quality of clinical notes . the goal in this study was to determine if ehrs improve the quality of outpatient clinical notes.materials and methods a five and a half year longitudinal retrospective multicenter quantitative study comparing the quality of handwritten and electronic outpatient clinical visit notes for 100 patients with type 2 diabetes at three time points : 6 months prior to the introduction of the ehr ( before - ehr ) , 6 months after the introduction of the ehr ( after - ehr ) , and 5 years after the introduction of the ehr ( 5-year - ehr ) .
qnote , a validated quantitative instrument , was used to assess the quality of outpatient clinical notes .
its scores can range from a low of 0 to a high of 100 .
sixteen primary care physicians with active practices used qnote to determine the quality of the 300 patient notes.results the before - ehr , after - ehr , and 5-year - ehr grand mean scores ( sd ) were 52.0 ( 18.4 ) , 61.2 ( 16.3 ) , and 80.4 ( 8.9 ) , respectively , and the change in scores for before - ehr to after - ehr and before - ehr to 5-year - ehr were 18% ( p<0.0001 ) and 55% ( p<0.0001 ) , respectively .
all the element and grand mean quality scores significantly improved over the 5-year time interval.conclusions the ehr significantly improved the overall quality of the outpatient clinical note and the quality of all its elements , including the core and non - core elements . to our knowledge , this is the first study to demonstrate that the ehr significantly improves the quality of clinical notes . |
the different forms of contamination come from different sources and are dealt with in different ways .
contamination can occur at the source , between source and storage points or in storage tanks .
defective joints , syphonage , rusted pipelines , and crossing over of sewage pipelines are other causes of contamination .
so , drinking water distribution systems may contain a diverse microbiological community that may include opportunistic pathogens and it is the most important single source of human exposure to gastroenteric diseases , mainly as a result of the ingestion of microbial contaminated water .
water contamination of respirators and incubators of newborns are the main sources of transfer [ 6 , 7 ] .
the cooling tower and the air condition system have also been reported to be sources of several outbreaks .
a wide variety of opportunistic pathogens such as pseudomonas can be incorporated into the piped potable water , colonizing the surfaces of pipelines with high numbers of biofilm - forming bacteria .
in addition , in bacteriological assessments , particularly for coliforms , the indicators of contamination by fecal matter must be routinely performed by many public health authorities to ascertain the quality of water to ensure prevention of further dissemination of pathogens through the agency of water .
addition of chlorine in different water treatment plants is a common practice , but it is not sufficient to ensure the safety of water .
there is adverse association between bacterial count of water samples and free residual chlorine but the failure of conventional treatment processes to eliminate critical waterborne pathogens in drinking water demands that improved and/or new disinfection technologies should be developed .
the aim of the present study was to determine the contamination rate of cold and warm water as well as water of containers of newborn incubators in hospitals of gilan province , iran , by pseudomonas aeruginosa , legionella pneumophila , and e. coli , using molecular approach .
during april june 2014 , one hundred and thirty - five water samples ( about 50 ml for each ) were aseptically collected in 60 ml plastic containers from 30 hospitals of the area ( 30 cold water samples , 32 warm water samples , and 73 incubator water samples ) .
cold water and warm water samples were taken after the water had run for 5 min .
potable water is supplied to the hospitals by the municipal system where it receives no additional treatment .
all water samples were concentrated 20-fold ( by dividing any sample into 5 falcon tubes , centrifugation with 12000 rpm for 30 min in 4c , discarding supernatant , dissolving precipitant in 0.5 ml remaining water , and collecting them in a tube ) . after discarding supernatant ,
sediment was resolved in 1 ml of the remaining solution and was divided into two different parts .
one part was treated in 50c for 30 min to reduce other contaminants for effective recovering of legionella . after heat treatment , each sample was inoculated in 5 ml liquid medium containing yeast extract 10 g / l , l - cysteine 0.04% , ferric
pyrophosphate 0.25 g / l , glycine 0.3% , and vancomycin 5 g / ml with final ph 6.9 and incubated in 35c for 3 days .
it should be noted that vancomycin and glycine are added to the medium after autoclave and when the temperature was around 40c .
the selectivity of the medium was subsequently improved by the incorporation of vancomycin and glycine .
the second part of each sample was inoculated in trypticase soy bean broth and incubated in 35c for 3 days for recovering p. aeruginosa and e. coli .
all cultured samples were divided over 1 ml in volume and centrifuged in a sigma model 3k30 centrifuge at 12.000 rpm in 4c for 20 min .
the supernatant was discarded , and the pellet was suspended with 1 ml of te buffer before dna extraction .
dna was extracted by using commercial procedure ( genomic dna purification kit , thermo scientific , lot : 00155557 , fermentas , lithuania ) .
the extracted dna was either used immediately for pcr or stored at 20c until analysis .
several rounds of pcr were performed to detect p. aeruginosa , integron 1 , metallo--lactamases gene , legionella pneumophila , mip gene , and e. coli as the following procedure : ( 1 ) conventional pcr by using specific primers for l. pneumophila and mip gene separately , ( 2 ) multiplex pcr by using specific primers for p. aeruginosa , integron 1 , and metallo--lactamases gene , ( 3 ) conventional pcr for detecting e. coli using specific primers , and ( 4 ) final round of pcr to investigate total presence of bacteria by using universal primers .
we used genomic dna of l. pneumophila type strain nctc 11192 as positive control for the first round of pcr .
a standard strain of p. aeruginosa ( atcc 27853 ) was used as positive control for the second round of pcr , and e. coli ( atcc 44338 ) was used as positive control for third and fourth rounds of pcr .
two microliters of the extracted template dna was used in a 20 l reaction mixture that included 10 l of pcr premix [ prime taq premix ( 2x ) , chorea lot number 201208 ] , 0.5 l of each primer , and 7 l of ddh2o .
cycling program for first round amplification of l. pneumophila ( species specific fragment ) was as follows : an initial denaturation at 94c for 5 min and then 35 cycles consisting of 94c for 60 s , 45c for 50 s , and 72c for 60 s were followed by a final extension at 72c for 10 min .
cycling program for second round of pcr was the same but the annealing temperature was 50c .
annealing temperature for third round of pcr was 58c and for 16s rrna amplification with the universal primers was 47c with the same cycling program .
electrophoresis of amplified product was performed on agarose gel ( 2% w / v ) by 125 volts for 45 minutes and analyzed by geldoctransluminator system ( vilberlourmat model ) .
one hundred and thirty - five water samples were aseptically collected from 30 hospitals of the area ( 30 cold water , 32 warm water , and 73 incubator water samples ) .
contamination rates of cold , warm , and incubator water samples with p. aeruginosa were 16.6% , 37.5% , and 6.8% consequently ( tables 24 and figure 1 ) .
degrees of contamination with l. pneumophila were 3.3% , 9.3% , and 10.9% ( tables 24 and figure 2 ) and with e. coli were zero , 6.2% , and zero ( tables 24 and figure 3 ) .
total bacterial contamination of cold , warm , and incubator water samples was 93.3% , 84.4% , and 89.0% consequently ( tables 24 and figure 4 ) .
metallo--lactamases gene was found in 20.0% of cold water samples , 15.6% of warm water samples , and 21.9% of incubator water samples ( tables 24 ) .
about 60.0% of p. aeruginosa in incubator samples had metallo--lactamases gene , 42.0% of warm water samples had p. aeruginosa with this gene , and 10.0% of p. aeruginosa in cold water samples had the same gene . about 8.3% of p. aeruginosa in warm water samples had integron 1 but we did not find p. aeruginosa with integron 1 in any sample of incubators and cold water .
we also did not find p. aeruginosa with both integron 1 and metallo--lactamases gene in incubator and cold water samples and just 1 of 12 p. aeruginosa of warm water sample had both integron 1 and metallo--lactamases gene .
about 25.0% of l. pneumophila in incubator samples had mip gene and 33.3% of warm water samples had l. pneumophila with mip gene .
we did not find l. pneumophila with mip gene in cold water samples . in total , 88.9% of all samples showed bacterial contamination as revealed by pcr with universal primers , 16.3% of all samples had p. aeruginosa , 8.9% had l. pneumophila , and 1.5% had e. coli .
the metallo--lactamases gene was found in 20.0% and integrin 1 was found in 3.0% of all samples .
the ability of microbes to survive in hospital water reservoir was described more than 30 years ago , and numerous studies have confirmed hospitals water as a source of nosocomial infection .
the bacterial contamination level related to the tap water was a point of dispersion of bacterial species , including potentially pathogenic organisms .
modes of transmission for waterborne infections include direct contact , ingestion of water , indirect contact , inhalation of aerosols dispersed from water sources , and aspiration of contaminated water .
several factors could be attributed to the contamination of surface water such as the age of the distribution system , the quality of the delivered water , defective chlorination , and improper maintenance .
contamination can occur at the source , between source and storage points or in storage tanks .
the bacterial contamination occurs because of regrowth of microorganisms in biofilms which are formed on interior surfaces of water pipes .
biological activity in biofilms is controlled by nutrient content of water , temperature , and residual chlorine .
microorganisms are widely distributed in nature and their abundance and diversity may be used as indicators for suitability of water .
certain types of microorganisms are characteristics of human fecal contamination . in the present study we used three bacteria ( e. coli , p. aeruginosa , and l. pneumophila ) to evaluate quality of studied water supplies because river and underground waters are main sources of tap water in the area and these bacteria are main human pathogens with water sources
actually in this study , e. coli was used as a representative of the enteric bacterial pathogens .
we also used l. pneumophila as ubiquitous waterborne microorganism and p. aeruginosa as ubiquitous waterborne opportunistic pathogen .
e. coli is a type of fecal coliform bacteria commonly found in the intestines of animals and humans and the presence of it in water is a strong indication of recent sewage or animal waste contamination .
we did not find e. coli in cold water and incubator water samples and just two warm water samples showed presence of e. coli . in some similar studies water distribution systems of examined hospitals were totally free of gram - negative bacteria belonging to family enterobacteriaceae .
although coliforms are known as the best index for monitoring water microbial quality , in some cases this index is not very efficient .
some believe that the excess growth of heterotrophic bacteria results in suppression of the coliforms . therefore , it is recommended to use other bacteria as an alternative index in water microbial quality control and pseudomonas can be one of these indicators .
p. aeruginosa is part of a large group of free - living bacteria that are ubiquitous in the environment .
aeruginosa is of major concern in hospitals , since it can cause severe secondary infections , such as pneumonia in burn victims or immunocompromised persons .
its occurrence in drinking water is probably related more to its ability to colonize biofilms in plumbing fixtures ( i.e. , faucets and showerheads ) than its presence in the distribution system or treated drinking water .
p. aeruginosa can survive in deionized or distilled water ; hence , it may be found in low nutrient or oligotrophic environments such as distilled water which is used for incubators of newborn ward . in the present study contamination degree of warm water with
p. aeruginosa was higher ( 37.5% ) than cold water ( 16.6% ) and incubator water ( 6.8% ) .
it also can be isolated even in the presence of 3.00 ppm residual free chlorine .
a number of laboratory - based studies have demonstrated the colonization of heterogeneous biofilms developed from tap water microorganisms by pathogens such as e. coli , l. pneumophila , and p. aeruginosa .
a number of studies showed that these bacteria are important opportunistic pathogens which can be involved in biofilm - associated contamination of domestic plumbing systems [ 3437 ] .
de abreu et al . presented that biofilm tap water was the major environmental source of pseudomonads in the healthcare facility . in the present study
it should be noted that metallo--lactamases producing strains have been increasingly reported worldwide and have been responsible for large outbreaks in several asian , european , and american hospitals [ 3842 ] .
genes encoding metallo--lactamases are generally located within class 1 integrons , together with those encoding aminoglycoside - modifying enzymes that confer multidrug resistance .
in addition , the integrons harboring metallo--lactamases determinants are frequently located on plasmids , certainly facilitating their intra- and interspecies spread . high prevalence of metallo--lactamases producing epidemic or endemic strains is also found in other mediterranean countries , such as italy and greece .
this bacterium can also survive at lower temperatures in drinking water environments [ 46 , 47 ] . l. pneumophila , particularly strains within serogroups 1 and 6 , is known as cause of both community acquired and hospital associated pneumonia [ 48 , 49 ] .
several reports have demonstrated that the major sources for legionnaires ' disease are the potable water systems of large buildings including hospitals , nursing home , and hotels . in a similar study ,
yaslianifard et al . found considerable contamination rate of l. pneumophila and p. aeruginosa in hospital water samples in tehran , iran .
shamabadi and ebrahimi found low rate contamination with gram - negative flora , moderate concentrations of legionella , and potentially pathogenic nonenterobacterial species . in the present study we found the degree of presence of l. pneumophila in cold water was lower than in warm water and incubator water .
it seems that high loads of p. aeruginosa can mask the presence of legionella , leading to an underestimation in some cases .
many authors agree that different microbe species as well as high microbial loads can play an important role in limiting or even inhibiting the growth of legionella [ 52 , 53 ] . in our study
actually overall pollution of hospital water systems with legionella found in the present work was smaller compared to earlier studies made in germany and italy [ 5457 ] . in a usa national surveillance study of 20 hospitals in 13 states , 14 hospitals were colonized with legionella in the water systems in which 43% of hospitals had environmental positive rates for legionella . in a study in spain on 12 hospitals ,
the present study showed that care should be taken concerning cleanliness and decontamination of water supplies for pathogenic organisms .
chlorine has been widely used as disinfectant due to its low cost and effectiveness in many countries but it is not sufficient to ensure the safety of water .
the maintenance of chlorine residue is needed at all points in distribution system supplied with chlorine as a disinfectant .
the failure of conventional treatment processes to eliminate critical waterborne pathogens in drinking water demands that improved and/or new disinfection technologies be developed .
recent research has disclosed that nanotechnology may offer solutions in this area , through the use of nanosorbents , nanocatalysts , bioactive nanoparticles , nanostructured catalytic membranes , and nanoparticle - enhanced filtration .
our study demonstrated that contamination degree of studied water samples with p. aeruginosa was considerable , with l. pneumophila was moderate , and with e. coli was very low .
we also found frequent presence of metallo--lactamases gene in the studied water samples indicating widespread multiple drug resistance bacteria . | this study is designed to determine the contamination degree of hospital water supplies with pseudomonas aeruginosa , legionella pneumophila , and e. coli in gilan , iran .
samples were collected directly into sterile containers and concentrated by centrifuge .
half part of any sample transferred to yeast extract broth and the second part transferred to trypticase soy broth and incubated for 3 days .
dna was extracted by using commercial kit .
four rounds of pcr were performed as follows : multiplex pcr for detecting pseudomonas aeruginosa , integron 1 , and metallo--lactamases gene ; pcr for detecting legionella pneumophila and mip gene separately ; pcr for detecting e. coli ; and another pcr for detecting whole bacterial presence .
contamination rates of cold , warm , and incubator water samples with p. aeruginosa , were 16.6% , 37.5% , and 6.8% consequently .
degrees of contamination with l. pneumophila were 3.3% , 9.3% , and 10.9% and with e. coli were zero , 6.2% , and zero .
total bacterial contamination of cold , warm , and incubator water samples was 93.3% , 84.4% , and 89.0% consequently .
metallo--lactamases gene was found in 20.0% of all samples .
contamination degree with p. aeruginosa was considerable and with l. pneumophila was moderate .
metallo--lactamases gene was found frequently indicating widespread multiple drug resistance bacteria .
we suggest using new decontamination method based on nanotechnology . |
the selective modulation of eukaryotic
gene expression by small
molecules may have important implications in the field of chemical
biology and human medicine .
pyrrole - imidazole polyamides are a class
of synthetic ligands that can be programmed to bind the minor groove
of specific dna sequences .
antiparallel ,
side - by - side n - methylpyrrole ( py ) and n - methylimidazole ( i m ) carboxamide ( im / py ) pairs distinguish gc
from cg base pairs , n - methyl-3-hydroxypyrrole
( hp)/py shows specificity for ta over at , whereas py / py
pairs are specific for both ta and at . by linking two strands of these
heterocyclic oligomers via a -amino butyric acid ( gaba ) turn
unit , hairpin py - im polyamides can be programmed to bind a large library
of dna sequences with affinities comparable to natural dna binding
proteins .
hairpin polyamides have been shown to localize to
the nuclei of living cells and regulate endogenous gene expression
by disrupting protein / dna interfaces .
cyclic polyamides containing a second gaba turn unit exhibit further
enhanced dna binding properties .
we have recently demonstrated their gene
regulatory effects on ar - activated gene expression in prostate cancer
models .
this discovery has opened
a new area of research toward transcriptional
regulation with small molecules , but the relative synthetic inaccessibility
of cyclic polyamides has remained a bottleneck for examining libraries
of structural variants that would modulate affinity , cell uptake ,
and biological activity .
initial solid - phase methods were low yielding
and required substantial premodifications of the pam resin . while the solution - phase synthesis of cyclic polyamides remains
useful in large - scale target - oriented synthesis , it has limited practicality
toward libraries for screening biological activities . the recent report by morinaga et al . offers a modular approach
to achieve cyclic polyamides by intramolecular coupling of a cysteine
and a chloroacetyl residue , but the modification of the optimal three - carbon
gaba turn into a sulfur - containing four - atom linker compromises its
dna binding affinity and
we report here a solid - phase polyamide
synthesis of a key hairpin
amino acid oligomer intermediate , which followed by intramolecular
cyclization affords cyclic polyamides 18 in good yields .
the modular approach led to rapid access
of a focused library of cyclic polyamides 17 ( scheme 1 ) with various core sequences
and turn unit modifications .
the utilization of fmoc chemistry allowed
for differentially protected turn units , which were modified selectively
to complement existing cellular imaging and cell uptake enhancement
technologies .
we examined the dna binding
properties and cytotoxicity profiles of compounds 111 and the cellular localization of cyclic polyamides 1214 by fluorescence microscopy .
all pybop - mediated
coupling conditions
were performed under microwave - assisted conditions ( see table 1 ) .
reagents and conditions : ( i ) 50% piperidine ,
dmf ; ( ii ) fmocpyoh , pybop , diea , dmf ; ( iii ) 50% piperidine , dmf ; ( iv )
fmocpyoh , pybop , diea , dmf ; ( v ) 50% piperidine , dmf ; ( vi ) fmocimoh ,
pybop , diea , dmf ; ( vii ) 50% piperidine , dmf ; ( viii ) z--dab(fmoc)-oh ( for 16 )
or boc--dab(fmoc)-oh ( for 7 ) , pybop , diea , dmf ;
( ix ) 50% piperidine , dmf ; ( x ) fmocpyoh , pybop , diea , dmf ; ( xi ) 50%
piperidine , dmf ; ( xii ) fmocpyoh , pybop , diea , dmf ; ( xiii ) 50% piperidine ,
dmf ; ( xiv ) fmocpyoh ( for 13 ) or
fmocimoh ( for 47 ) , pybop , diea ,
dmf ; ( xv ) 50% piperidine , dmf ; ( xvi ) fmocimoh , pybop , diea , dmf ; ( xvii )
50% piperidine , dmf ; ( xviii ) z--dab(fmoc)-oh
( for 1 and 4 and 7 ) or fmoc - gaba - oh
( for 2 and 5 ) or boc--dab(fmoc)-oh
( for 3 and 6 ) , pybop , diea , dmf ; ( xix ) 30%
hfip , dcm ; ( xx ) 20% piperidine , dmf ; ( xxi ) dppa , diea , dmf ; ( xxii )
10% tfmsa , tfa ( for 1 , 2 , 4 , and 5 ) or tfa ( for 3 , 6 ,
and 7 ) .
due to previously
observed decomposition of the conjugated c - terminal free carboxylic
acid in polyamide intermediates , 2-chlorotrityl chloride ( 2-cl - trt - cl )
resin was chosen for its mild synthesis and cleavage conditions .
polyamide
synthesis on this resin has been previously reported by aldrich - wright
and co - workers , but a resin - bound -alanine linker was used
in both instances , and a new loading procedure was therefore needed .
2-cl - trt - cl resin was first loaded with the fmoc - protected py monomer
in n , n - dimethylformamide ( dmf ) and
capped with methanol .
resin substitution levels were determined by
the fmoc test and confirmed by weighing the dry mass of the loaded
resin .
fmoc deprotection was achieved using a 50% piperidine in dmf
solution . in light of the recent improvements in both efficiency and
yield ,
the couplings were performed under microwave - assisted conditions
using the desired pybop - activated monomers .
initial syntheses performed at 60 c led to premature cleavage
of intermediates off the 2-cl - trt resin , and 50 c couplings
were therefore preferable .
the challenging i m to py coupling required
an fmocpyimoh dimer , which was obtained via an optimized procedure
by weltzer and wemmer .
this two - step deprotection coupling procedure was repeated
until the desired polyamide sequence was achieved . to build the small
library of polyamides in a modular fashion ,
the resin was split into
different batches at corresponding steps for further derivatization .
upon completion ,
the n - terminal fmoc - protected polyamide oligomer
was cleaved from the resin with 30% hexafluoroisopropanol ( hfip ) in
dichloromethane ( dcm ) , concentrated in vacuo , and the resulting residue
was subjected to a 20% piperidine solution to remove the fmoc group .
direct cleavage of the free amine polyamide oligomer was attempted ,
but found to be ineffective due to poor solubility of the zwitterion
intermediate in the cleavage solution .
after purification by high - performance
liquid chromatography ( hplc ) , the desired polyamide intermediates 1824 were obtained in 3140% yields
( table 2 ) .
all coupling reactions were conducted
under microwave - assisted conditions at 50 c with a 0.3 m solution
of the activated monomers ( 3 equiv of monomer acid , 3 equiv of pybop ,
8 equiv of diea , dmf ) .
fmocpyoh
coupling onto resin with
n - terminal i m was incomplete even at 60 c for up to 1 h. synthesis
of polyamide sequences that require this linkage should use the fmocpyimoh
dimer instead , demonstrated later in the synthesis of 8 .
reagents and conditions :
( i )
bzoh , pybop , diea , dmf ; ( ii ) 10% tfmsa , tfa .
the polyamide macrocyclization
step was achieved by a dppa - mediated ring - closing reaction between
the n - terminal amino group and the c - terminal carboxylic acid .
this
method was first employed by cho et al . in the synthesis of cyclic
polyamides . in order to obtain a general workup procedure applicable to polyamides
of various lipophilicities , diisopropylethylamine ( diea ) was used
as the base in place of sodium bicarbonate ( nahco3 ) in
the original conditions .
deprotection
of the turn units with trifluoromethanesulfonic acid ( tfmsa ) or trifluoroacetic
acid ( tfa ) , followed by hplc purification , afforded polyamides 17 in 3743% yields over two steps . by taking advantage of the fmoc protection scheme ,
this is further
highlighted in 6 and 7 , which share the
same asymmetric polyamide core targeted to the 5-wggwcw-3
sequence , and allowed for the selective conjugation of a benzoic acid
moiety on a single turn unit in polyamides 911 that has been recently developed to enhance the cellular
localization properties of hairpin polyamides ( scheme 2 ) .
monosubstituted benzyl carbamate
( cbz ) polyamides 3 , 6 , and 7 were chosen as targets based on unpublished results indicating that
cbz - functionalized polyamides are biologically active ( see supporting information ) .
cyclic polyamides 1214 with a fluorescein dye were synthesized
in a similar fashion and imaged in living cells via fluorescence microscopy
( scheme 3 ) .
furthermore , both the solubility
and the pharmacokinetic profiles of cyclic polyamides have been shown
to be highly dependent on subtle structural modifications , and this
method allows for the modular synthesis of these structural variants
in an efficient manner .
reagents and conditions :
( i )
fitc , diea , dmf ; ( ii ) 10% tfmsa , tfa .
previously established solid - phase polyamide synthesis
methods have been generally limited to sequences beginning with a
pyrrole monomer .
solid - phase synthesis of polyamides
starting with imidazole units on the commonly used kaiser oxime resin
have been low yielding , mainly attributed to the sensitivity of the
oxime
the addition of an aliphatic linker ( e.g. ,
boc--ala - pam resin ) circumvents this issue , but previous studies
on hairpin polyamides with c - terminal -alanine motifs have
shown reduced cellular uptake properties and thus diminished gene
regulatory effects of these products . using
the microwave - assisted conditions reported above , cyclic polyamide 8 targeted to the 5-wcgwgw-3 sequence found
in e - box binding sites has been synthesized in 13% yield overall .
initial attempts starting with fmocimoh - loaded resin resulted in
undesired cleavage during subsequent steps , and so syntheses began
with loading of the fmocpyimoh dimer onto 2-chlorotrityl resin .
after
deprotection coupling of the corresponding monomer units , followed
by resin cleavage and fmoc removal , polyamide intermediate 25 was isolated by hplc purification in 34% yield .
dppa - mediated cyclization
of 25 , followed by cbz deprotection afforded cyclic polyamide 8 in 39% yield over two steps ( scheme 4 ) .
hairpin polyamide 17 was synthesized in a similar
stepwise manner to afford the c - terminal acid intermediate , which
was then coupled to a 3,3-diamino - n - methyldipropylamine
linker , followed by isophthalic acid conjugation , boc deprotection ,
and isolated in 24% yield over 16 steps ( see scheme s1 ) .
this is a
step forward which allows for the synthesis of both cyclic and non--alanine - linked
hairpin polyamides with sequences beginning with an imidazole unit ,
further expanding the scope of targetable dna sequences and inhibition
of transcription - factor - mediated gene expression by py - im polyamides .
all pybop - mediated
coupling conditions
were performed under microwave - assisted conditions ( see table 1 ) .
reagents and conditions : ( i ) 50% piperidine ,
dmf ; ( ii ) fmocpyoh , pybop , diea , dmf ; ( iii ) 50% piperidine , dmf ; ( iv )
fmocpyoh , pybop , diea , dmf ; ( v ) 50% piperidine , dmf ; ( vi ) z--dab(fmoc)-oh , pybop , diea , dmf ; ( vii ) 50% piperidine ,
dmf ; ( viii ) fmocimoh , pybop , diea , dmf ; ( ix ) 50% piperidine , dmf ;
( x ) fmoc--ala - oh , pybop , diea , dmf ; ( xi ) 50% piperidine , dmf ;
( xii ) fmocpyimoh , pybop , diea , dmf ; ( xiii ) 50% piperidine , dmf ; ( xiv ) z--dab(fmoc)-oh , pybop , diea , dmf ; ( xv ) 30% hfip ,
dcm ; ( xvi ) 20% piperidine , dmf ; ( xvii ) dppa , diea , dmf ; ( xviii ) 10%
tfmsa , tfa .
py - im polyamide dna binding affinities and specificities have
historically been measured by quantitative dnase i footprinting assays . as previously reported , however , cyclic polyamides
have exceptionally high dna binding affinities that exceed the detection
limit of this experiment ( i.e. , ka
2 10 m ) . the dna binding affinities of cyclic polyamides 114 have been rank ordered by magnitude
of dna thermal stabilization ( tm ) and compared to the corresponding hairpin polyamides 1416 .
spectroscopic measurements were performed
on 12-mer dna duplexes with sequences 5-cgatgttcaagc-3 , 5-cgatggtcaagc-3 ,
and 5-cgatcgtgaagc-3 , each
containing a match binding site for the corresponding polyamides .
, the tm value for bisamino cyclic polyamide 1 ( tm = 26.1 c ) was
calculated to be significantly higher than that of hairpin polyamide 15 ( tm = 22.0 c ) .
so while 15 has an established
binding affinity to the match androgen response element ( are ) half - site
( 5-wgwwcw-3 ) in the subnanomolar range , cyclic polyamide 1 provides even greater stabilization to such dna duplexes .
mono - unsubstituted cyclic polyamide 2 provides less dna
stabilization compared to 1 ( tm = 20.4 c ) , which is likely due to the loss of
a positive charge and thus the loss of favorable electrostatic interactions
with the negatively charged dna backbone .
perhaps more surprising
is the high tm values retained
by monoprotected cyclic polyamides 3 ( tm = 27.3 c ) and 9 ( tm = 28.0 c ) , each containing a lone free
amino group and net + 1 charge . as shown in figure 1 , the benzoyl ( bz ) group in 9 projects straight
down the minor groove , avoiding unfavorable steric interactions with
the groove wall , and may offer insight into the high degree of dna
stabilization by 3 and 9 comparable to the
bisamino cycle 1 .
fluorescein - conjugated 12 ( tm = 21.8 c ) has a dna
binding affinity lower than 3 and 9 , perhaps
due to increased steric clashes from the larger substitution group
and unfavorable electrostatic interactions from the negatively charged
fluorescein group , but still binds dna at a similar level to 15 ( table 3 ) .
the magnitude of
stabilization provided by cyclic polyamides 47 , 1011 , and 1314 targeted to estrogen
response element ( ere ) half - sites ( 5-wggwcw-3 ) follows
a similar pattern to the aforementioned are targeting series .
mono - unsubstituted
cycle 5 ( tm = 18.6
c ) stabilizes the duplex at a comparable level to hairpin polyamide 16 ( tm = 16.7 c ) ,
whereas bisamino compound 4 ( tm = 23.2 c ) has a higher tm value . the mono - cbz cycles 6 ( tm = 23.6 c ) and 7 ( tm = 24.3 c ) , mono - bz - substituted 10 ( tm = 26.0 c )
and 11 ( tm = 25.1
c ) , and monofluorescein conjugates 13 ( tm = 22.2 c ) and 14 ( tm = 21.0 c ) each bind dna similar to 4 .
cyclic polyamide 8 ( tm = 13.4 c ) binds the match 5-wcgwgw-3
sequence at an elevated level compared to hairpin 17 ( tm = 6.6 c ) , which may prove important
toward targeting oncogenic transcription factors such as c - myc that
act through binding canonical e - box ( 5-cacgtg-3 ) sequences . molecular
model of benzoyl - substituted cyclic polyamide turn along
the dna minor groove , based on published crystal structure by chenoweth
et al .
the cytotoxicity of compounds 111 was assessed in a549 human lung carcinoma
and t47d human breast cancer cell lines ( tables 4 and 5 ) .
the cyclic polyamides targeted to
the 5-wgwwcw-3 sequence generally exhibit a higher
level of cytotoxicity than the 5-wggwcw-3 series ,
which is consistent with the trends observed in the dna thermal denaturation
analysis and confocal microscopy studies .
detailed inspection of the
ic50 values within the series of compounds and across the
cell lines , however , offers some unanticipated insights into the different
biological properties of these minor structural variants .
bisamino
cycle 1 , which has previously been shown to be biologically
active , did not display any significant level of cytotoxicity in either
cell line ( ic50 > 30 m ) .
mono - unsubstituted compound 2 ( ic50 = 4.9 m ) and mono - bz 9 ( ic50 = 1.0 m ) were comparably cytotoxic to hairpin
polyamide 15 ( ic50 = 3.1 m ) in a549
cells but an order of magnitude more cytotoxic ( ic50 =
74 and 79 nm , respectively ) than 15 ( ic50 =
710 nm ) in t47d cells .
the mono - cbz cycle 3 was consistently
the most cytotoxic compound in both a549 ( ic50 = 160 nm )
and t47d ( ic50 = 25 nm ) cell lines .
for the 5-wggwcw-3
targeting polyamides , the only
compound that exhibited an ic50 value lower than 30 m
in a549 cells was the mono - cbz compound 6 ( ic50 = 1.9 m ) . in t47d cells ,
consistent with the 5-wgwwcw-3
series , bisamino cycle 4 ( ic50 > 30 m )
was found to be not significantly cytotoxic and 6 ( ic50 = 460 nm ) was the most cytotoxic compound .
mono - unsubstituted
cycle 5 ( ic50 = 0.82 m ) again shares
a comparable level of cytotoxicity with the reference hairpin 16 ( ic50 = 1.1 m ) , whereas mono - bz compounds 10 ( ic50 = 13.3 m ) and 11 ( ic50 = 7.5 m ) are both an order of magnitude less cytotoxic .
perhaps most interestingly , mono - cbz compound 7 did
not exhibit observable levels of cytotoxicity ( ic50 >
30
m ) in either a549 or t47d cells . considering that 7 is a regioisomer of 6 , where the cbz group is simply
swapped onto the other turn , and that 7 only differs
from 3 by a single ch to n substitution ,
it is rather surprising that 7 is more than 15- to 65-fold
less cytotoxic than 6 and at least 180- to 1200-fold
less cytotoxic than 3 in the two examined cell lines .
given the comparable dna stabilization properties between 6 and 7 , and their common core sequence , we would not
have predicted this vast discrepancy in cytotoxicity .
this study
has demonstrated the large and somewhat unpredictable
effects in biological activity induced by small structural variations
of cyclic polyamides . on the basis of our preliminary work ,
the aggregation
and pharmacokinetic properties of polyamides also vary greatly depending
on structural modifications .
all this combines to highlight
the importance of a fast and reliable method to generate focused libraries
of cyclic polyamides for future research . to directly examine the
cellular localization of cyclic polyamides , fluorescein conjugates 1214
were synthesized and visualized
in living cells via confocal microscopy ( figures 2 and 3 ) .
the selective conjugation
of a single fluorescein molecule not only helped retain a free amino
group for solubility purposes but also allowed for the qualitative
comparison of the two fluorescein conjugates 13 and 14 that share the same asymmetric polyamide core . in
each of the cases examined ,
cyclic polyamides 1214 appear to permeate through the cellular membrane and localize
in the cell nucleus , which was confirmed by the colocalization with
hoechst 33258 dna stain .
for ease of qualitatively assessing compound
uptake , all a549 images were taken at a 660 fluorescence gain level ,
and all t47d images were taken at 600 fluorescence gain . comparing
figures 2 and 3 , the
fluorescence levels of compounds 1214 in t47d cells are all significantly higher than in a549 , which is
only further amplified by this difference in gain levels .
compound 12 matched to the 5-wgwwcw-3
sequence exhibits the highest level of nuclear localization in both
cell lines .
among the two 5-wggwcw-3 targeting cycles ,
polyamide 14 qualitatively appears to have a relatively
higher fluorescence signal in the cell nuclei in both a549 and t47d
cells .
this may help explain the cytotoxicity data reported above ,
where compounds 6 and 11 with cbz and bz
substitutions on the same side as the fluorescein in 14 consistently display larger biological effects than 7 and 10 that are more structurally similar to cyclic
polyamide 13 .
fluorescein
conjugates 12 ( top ) , 13 ( middle ) , and 14 ( bottom )
in a549 cells . in order to confirm nuclear localization , the fluorescence
panel ( left )
was compared with hoechst 33258 staining ( middle ) and
overlay ( right ) .
fluorescein
conjugates 12 ( top ) , 13 ( middle ) , and 14 ( bottom )
in t47d cells . in order to confirm nuclear localization , the fluorescence
panel ( left )
was compared with hoechst 33258 staining ( middle ) and
overlay ( right ) .
we have described a modular solid - phase
synthesis method , which ,
when combined with an established dppa - mediated macrocyclization step ,
afforded cyclic polyamides in a high - yielding and time - efficient manner .
using this method ,
we have overcome previous limitations and synthesized
both cyclic and hairpin polyamides that start with an imidazole unit .
the binding affinities of all synthesized cycles have been assessed
by dna thermal denaturation assays and compare favorably to hairpin
polyamides that bind their match dna sequences at subnanomolar concentrations .
furthermore , the protection strategy of our method allows for selective
modification of the gaba turn units , which we have used to rapidly
generate a focused library of compounds .
the cytotoxicity and uptake
analysis of the cyclic polyamides revealed unexpected properties that
further highlight the need for an efficient method to synthesize structural
variants of cyclic polyamides for future studies .
2-chlorotrityl chloride
( 2-cl - trt - cl ) resin was purchased from bachem .
microwave - assisted
coupling reactions were conducted on a biotage initiatior eight synthesizer .
polyamide concentrations were measured in 20% mecn in 0.1% ( v / v ) aqueous
tfa using an approximated extinction coefficient of 69 200
m cm at max near 310 nm , unless otherwise specified .
prior to manual
microwave - assisted synthesis , 2-cl - trt - cl resin ( 1.0 g , 1.59 mmol / g )
was first loaded by mixing with 576 mg ( 1.59 mmol , 1 equiv ) of fmocpyoh
monomer , followed by addition of 6 ml of dimethylformamide ( dmf ) and
1.38 ml of diisopropylethylamine ( diea ) ( 7.59 mmol , 5 equiv ) .
the
suspended mixture was stirred for 18 h , then capped by addition of
1 ml of methanol ( meoh ) and stirred for 1 h. the orange - colored , loaded
resin was then collected on a fritted peptide synthesis vessel , washed
with dmf ( 2 ) , meoh ( 2 ) , dmf ( 2 ) , meoh ( 2 ) ,
and diethylether ( et2o ) .
[ owing to the sensitivity of the
2-cl - trt resin toward hydrolysis / methanolysis , this final et2o wash was found to be essential and all loaded resin was sealed
and stored at 20 c . ]
the loading efficiency was quantitated
via the fmoc test and confirmed by measuring the mass of the dried
resin .
all solid - phase polyamide coupling reactions
were performed manually on a biotage initiator eight microwave synthesizer
on a 200500 mg scale of loaded resin .
prior to each monomer
coupling reaction , the n - terminal fmoc group was first removed in
a piperidine solution .
the fmoc deprotections were performed in a
fritted peptide synthesis vessel at room temperature , and the specific
conditions for each n - terminal monomer are as follows : ( a ) swell
resin in dcm ; ( b ) wash with dmf ; ( c ) add 50% piperidine in dmf ; ( d )
shake suspension for 10 min ; ( e ) wash with dmf ; ( f ) repeat steps a e
twice .
( a ) swell
resin in dcm ; ( b ) wash with dmf ; ( c ) add 50% piperidine in dmf ; ( d )
shake suspension for 5 min ; ( e ) wash with dmf ; ( f ) repeat steps a
following fmoc removal , the resin was deswelled in meoh ,
washed with et2o , dried in vacuo , and transferred to a
microwave synthesis vessel as a dry powder .
the corresponding monomer
acid ( 3 equiv ) was activated with pybop ( 3 equiv ) and diea ( 6 equiv )
in dmf ( 0.3 m concentration of monomer ) , and added to the resin .
the
coupling reactions were then setup in the microwave reactor at 50
c for the time durations described in table 1 .
after the listed microwave - assisted coupling times , the
reaction mixture was filtered into a peptide synthesis vessel , and
the collected resin was washed with dmf ( 3 ) , meoh ( 3 ) ,
et2o , and dried in vacuo . to ensure completion of each
deprotection and coupling step ,
analytical hplc spectra were taken
by cleaving a small resin sample in 30% hexafluoroisopropanol ( hfip )
in dcm .
the polyamide core was synthesized on 2-cl - trt resin
in an iterative
manner by repeating the deprotection
coupling procedures described
above using the corresponding monomeric units . upon completion of
the sequence , 100200 mg of the resin was suspended in 1 ml
of 30% hfip in dcm and stirred for 1 h to yield the crude n - terminal
fmoc - protected polyamide intermediate .
the reaction mixture was then
run through a cotton filter to remove the resin , and the filtrate
was concentrated in vacuo .
the residual oil was resuspended in 5 ml
of a 1:1 meoh / dcm mixture and reconcentrated in vacuo to give an off - white / beige
solid . to remove the n - terminal fmoc group , the solid
was redissolved
in 800 l of dmf , followed by addition of 200 l of piperidine ,
and the solution was stirred for 30 min . upon confirmation of complete
deprotection by analytical hplc
, the solution was added to 4 ml of
30% mecn in 0.1% aqueous tfa .
the precipitated 9-methylenefluorene
side product was then removed by centrifugation and washed twice with
2 ml of 30% mecn in 0.1% aqueous tfa .
the combined aqueous solution
was purified by reverse - phase hplc and lyophilized to dryness to yield
precyclic polyamide intermediates 1824 .
all dried samples of 1824 were
stored at 20 c prior to dppa - mediated macrocyclization .
synthetic yields and maldi - tof characterization data for 1824 are summarized below : ( 18 ) : 12.6 mol recovered ( 38.0 mol theoretical ,
33% yield ) .
maldi - tof [ m + h ] calcd for c70h77n22o15 = 1465.4 ,
observed = 1465.9 .
( 19 ) : 15.2 mol recovered
( 38.0 mol theoretical ,
40% yield ) .
maldi - tof [ m + h ] calcd for c62h70n21o13 = 1316.5 ,
observed = 1316.9 .
( 20 ) : 12.8 mol recovered
( 38 mol theoretical ,
34% yield ) .
maldi - tof [ m + na ] calcd for c67h79n22nao15 = 1453.6 ,
observed = 1453.9 .
( 21 ) : 15.1 mol recovered
( 49 mol theoretical ,
31% yield ) .
maldi - tof [ m + h ] calcd for c69h76n23o15 = 1466.5 ,
observed = 1466.9 .
( 22 ) : 17.2 mol recovered
( 51 mol theoretical ,
34% yield ) .
maldi - tof [ m + h ] calcd for c61h69n22o13 = 1317.5 ,
observed = 1317.2 .
( 23 ) : 16.2 mol recovered
( 51 mol theoretical ,
32% yield ) .
maldi - tof [ m + na ] calcd for c66h77n23nao15 = 1454.5 ,
observed = 1455.0 .
( 24 ) : 8.2 mol recovered
( 25 mol theoretical ,
33% yield ) .
maldi - tof [ m + na ] calcd for c66h78n23nao15 = 1454.5 ,
observed = 1454.9 .
intermediates 1824 were first dissolved in dmf ( 0.25 mm ) in a round - bottom flask equipped
with a magnetic stir bar , followed by addition of diea ( 200 equiv ) ,
and purged with argon for 15 min .
diphenylphosporyl azide ( dppa ) ( 50
equiv ) was then added to the reaction mixture in a dropwise manner ,
while rapidly stirring . upon full addition of the dppa
, the solution
was allowed to react and stirred at room temperature for 1620
h. after confirmation of reaction completion by analytical hplc , the
reaction mixture was concentrated in vacuo and the resulting oil residue
was dissolved in 3 ml of mecn and transferred to a 15 ml falcon tube .
the mecn was then removed with air flow , and 3 ml of 0.1% aqueous
tfa was added to the remaining oil layer to yield an off - white suspension ,
which was isolated via centrifugation and lyophilized to dryness . for reactions starting with 18 , 19 , 21 , and 22 , the lyophilized residue was submitted
to 1 ml of 10% trifluoromethanesulfonic acid ( tfmsa ) in tfa , stirred
for 5 min , frozen in ln2 , and thawed by layering 1 ml of dmf . for
reactions starting with 20 , 23 , and 24 ,
the lyophilized residue was submitted to 1 ml of neat
tfa , stirred for 15 min , frozen in ln2 , and thawed by layering 1 ml
of dmf .
all of the thawed solutions were then diluted with 6 ml of
0.1% aqueous tfa , purified by reverse - phase hplc , and lyophilized
to dryness to yield cyclic polyamides 17 .
synthetic yields and maldi - tof characterization data
for 17 are summarized below : ( 1 ) : 4.5 mol recovered ( 12.9 mol theoretical ,
35% yield ) .
maldi - tof [ m + h ] calcd for c54h63n22o10 = 1179.5 ,
observed = 1179.9 .
( 2 ) : 0.84 mol recovered
( 2.0 mol theoretical ,
42% yield ) .
maldi - tof [ m + h ] calcd for c54h62n21o10 = 1164.5 ,
observed = 1164.6 .
( 3 ) : 3.2 mol recovered
( 6.7 mol theoretical ,
47% yield ) .
[ m + h ] calcd for c62h69n22o12 = 1313.6 ,
observed = 1314.0 .
( 4 ) : 3.1 mol recovered
( 8.0 mol theoretical ,
39% yield ) .
maldi - tof [ m + h ] calcd for c53h62n23o10 = 1180.5 ,
observed = 1180.9 .
( 5 ) : 0.96 mol recovered
( 2.0 mol theoretical ,
48% yield ) .
maldi - tof [ m + h ] calcd for c53h61n22o10 = 1165.5 ,
observed = 1165.5 .
( 6 ) : 3.7 mol recovered
( 10.0 mol theoretical ,
37% yield ) .
[ m + h ] calcd for c61h68n23o12 = 1314.5 ,
observed = 1314.5 .
( 7 ) : 2.2 mol recovered
( 5.7 mol theoretical ,
38% yield ) .
maldi - tof [ m + h ] calcd for c61h68n23o12 = 1314.5 ,
observed = 1314.8 .
a solution of benzoic acid ( 3.0
mg , 0.025 mmol , 25 equiv ) and pybop ( 13 mg , 0.025 mmol , 25 equiv )
in dmf ( 0.5 ml ) and diea ( 44 l , 0.25 mmol , 250 equiv ) was stirred
at room temperature for 10 min .
the activated solution was then added
to 3 ( 1.0 mol ) and stirred for 3 h. after confirmation
of complete reaction by analytical hplc , 12 ml of cold et2o was added to the reaction mixture and cooled at 20 c
for 16 h. the precipitate was then isolated by centrifugation and
allowed to air - dry .
the resulting residue was submitted to 1 ml of
10% trifluoromethanesulfonic acid ( tfmsa ) in tfa , stirred for 5 min ,
frozen in ln2 , and thawed by layering 1 ml of dmf .
the thawed solution
was then diluted with 6 ml of 0.1% aqueous tfa , purified by reverse - phase
hplc , and lyophilized to dryness to yield cyclic polyamide 9 ( 684 nmol , 68% yield ) . using the same procedure described above ,
starting with 6 ( 1.60 mol ) and 7 ( 750
nmol ) , yielded monobenzoyl - substituted cyclic polyamides 10 ( 1.05 mol , 67% yield ) and 11 ( 367 nmol , 49%
yield ) , respectively .
( 9 ) : maldi - tof [ m + h ] calcd for c61h67n22o11 = 1283.5 , observed = 1284.1 .
( 10 ) : maldi - tof [ m + h ] calcd for c60h66n23o11 =
1284.5 , observed = 1284.5 . ( 11 ) : maldi - tof [ m +
h ] calcd for c60h66n23o11 =
1284.5 , observed = 1284.9 .
a solution of fluorescein isothiocyanate
( fitc ) ( 2.7 mg , 7.0 mol , 25 equiv ) in dmf ( 0.2 ml ) and diea
( 12 l , 0.07 mmol , 250 equiv ) was added to 3 ( 0.28
mol ) and stirred for 2 h. after confirmation of complete reaction
by analytical hplc , 12 ml of cold et2o was added to the
reaction mixture and cooled at 20 c for 16 h. the precipitate
was then isolated by centrifugation and allowed to air - dry .
the resulting
residue was submitted to 1 ml of 10% trifluoromethanesulfonic acid
( tfmsa ) in tfa , stirred for 5 min , frozen in ln2 , and thawed by layering
1 ml of dmf .
the thawed solution was then diluted with 6 ml of 0.1%
aqueous tfa , purified by reverse - phase hplc , and lyophilized to dryness
to yield cyclic polyamide 12 ( 65 nmol , 23% yield ) . using
the same procedure described above , starting with 6
( 0.40
mol ) and 7 ( 0.40 mol ) , yielded cyclic polyamide fluorescein - conjugate 13 ( 345 nmol , 86% yield ) and 14 ( 118 nmol , 29%
yield ) , respectively . ( 12 ) : esi - ms [ m + h ] calcd for c75h74n23o15s = 1568.6 , observed = 1568.3 .
( 13 ) : esi - ms [ m + h ] calcd for c74h73n24o15s = 1569.5 ,
observed = 1569.2 .
( 14 ) : esi - ms [ m + h ] calcd for c74h73n24o15s = 1569.5 ,
observed = 1569.3 .
2-cl - trt - cl
resin ( 200 mg , 1.59 mmol / g ) was first loaded with fmocpyimoh dimer
( 96 mg , 0.20 mmol ) , which was obtained from published procedures .
the obtained fmoc - py - im-(2-cl - trt )
resin ( 265 mg , 0.59 mmol / g ) was subjected to the previously described
microwave - assisted solid - phase synthesis conditions to build the corresponding
polyamide sequence .
a quarter of the resin ( 0.15
mol theoretical ) was then cleaved and purified by reverse - phase
hplc to yield precyclic polyamide intermediate 25 as
an off - white powder ( 13.3 mol , 34% yield ) .
the isolated 25 ( 2.0 mol ) was subjected to dppa - mediated macrocyclization
conditions analogous to that for compounds 17 , with the cbz groups removed with 10% tfmsa in tfa , and
purified by reverse - phase hplc to afford cyclic polyamide 8 ( 773 nmol , 39% yield ) .
( 25 ) : maldi - tof [ m
co2 + h ] calcd for c65h675n22o13 = 1371.6 , observed = 1371.7 .
( 8) : maldi - tof [ m + h ] calcd for c61h67n22o11 =
1129.5 , observed = 1130.0 .
detailed experimental procedures
and characterization data for 17 are provided in the supporting information .
melting temperature analysis
was performed on a varian cary 100 spectrophotometer equipped with
a thermocontrolled cell holder possessing a cell path length of 1
cm .
a degassed aqueous solution of 10 mm sodium cacodylate , 10 mm
kcl , 10 mm mgcl2 , and 5 mm cacl2 at ph 7.0 was
used as analysis buffer .
dna duplexes and polyamides were mixed in
1:1 stoichiometry to a final concentration of 2 m for each
experiment .
prior to analysis , samples were heated to 95 c and
cooled to a starting temperature of 25 c with a heating rate
of 5 c / min for each ramp .
denaturation profiles were recorded
at = 260 nm from 25 to 95 c with a heating rate of 0.5
c / min .
the reported melting temperatures were defined as the
maximum of the first derivative of the denaturation profile .
cell lines were cultured at 37 c
under 5% co2 using standard cell culture and sterile techniques .
f-12k ( kaighn s ) medium was used for a549 cells , and rpmi 1640
was used for t47d cells .
for each experiment , cells were
plated in 200 l of the proper medium onto glass - bottom cell
culture plates at a density of 1 10 ( a549 ) or 1.5
10 cells / ml ( t47d ) .
cells were grown for 24 h ,
and media were replaced with fresh media containing polyamide to give
a final dmso concentration of 0.1% .
next , cells were incubated for
16 h , followed by removal of media , washing , and addition of fresh
media .
imaging was performed
at the caltech beckman imaging center using a zeiss lsm 510 meta nlo
two - photon inverted laser scanning microscope equipped with a 40
oil - immersion objective lens .
fluorescein conjugates 1214 were imaged in multi - track mode
using 488 nm laser excitation at 15% output with a pinhole of 375
m and a standard fluorescein filter set .
hoechst was imaged
using 800 nm two - photon excitation with an hft kp680 dichroic and
a 390 to 465 nm band - pass filter with a fully open pinhole .
for cytotoxicity assays ,
cell lines were plated in 96-well cell culture plates in 100 l
of media at a density of 1 10 ( a549 ) or 5
10 cells / ml ( t47d ) .
ic50 values were determined
using the sulforhodamine b ( srb ) colorimetric assay as previously
described .
cells were grown for 24 h ,
before polyamides in 100 l of media were added in serial dilution ,
in quadruplicate for each concentration .
after incubation for 72 h ,
cell medium was replaced with 100 l of fresh media , and cells
were allowed to recover for an additional 24 h. cells were then fixed
by adding 100 l of 10% trichloroacetic acid directly to each
well and stored at 4 c for 1 h , before being washed , dried ,
stained with 100 l of 0.057% srb solution per well for 30 min ,
and washed and dried again as described .
after solubilizing the bound
dye with 200 l of 10 mm tris ( ph 10.5 ) per well , absorbance
at 490 nm was measured on a perkinelmer victor microplate reader .
the data are charted as a percentage of untreated controls , corrected
for background absorbance .
these values were determined
by nonlinear least - squares regression fit to y = a + ( b
+ 10((log ec50 x ) h ) ) , where a = max , b = min , and h = hill slope . | cyclic py - im polyamides containing two gaba turn units
exhibit
enhanced dna binding affinity , but extensive studies of their biological
properties have been hindered due to synthetic inaccessibility .
a
facile modular approach toward cyclic polyamides has been developed
via microwave - assisted solid - phase synthesis of hairpin amino acid
oligomer intermediates followed by macrocyclization .
a focused library
of cyclic polyamides 17 targeted
to the androgen response element ( are ) and the estrogen response element
( ere ) were synthesized in 1217% overall yield .
the fmoc protection
strategy also allows for selective modifications on the gaba turn
units that have been shown to improve cellular uptake properties .
the dna binding affinities of a library of cyclic polyamides were
measured by dna thermal denaturation assays and compared to the corresponding
hairpin polyamides .
fluorescein - labeled cyclic polyamides have been
synthesized and imaged via confocal microscopy in a549 and t47d cell
lines .
the ic50 values of compounds 17 and 911 were determined ,
revealing remarkably varying levels of cytotoxicity . |
an osteochondroma or exostosis is a benign bone tumour consisting of a bony outgrowth covered by a cartilage cap that occurs commonly in the metaphysis of long bones ( distal femur , proximal tibia , proximal humerus ) and pelvis .
osteochondromas are rarely localized in the foot and ankle , except in cases of multiple hereditary exostoses .
we describe an unusual case of a distal tibia osteochondroma affecting the lateral malleolus of a young girl .
a thirteen - year - old female came to us with a palpable lump in her right ankle . on physical examination , there was mild restriction of motion of the ankle and a regular swelling over the anterolateral aspect of the ankle , hard in consistency , painless and without neurovascular impairment .
an x - ray was performed showing a well - defined exostosis arising from the distal aspect of the tibia , causing pressure erosion and impending fracture of the distal fibula .
the ct scan clearly depicted the lesion and the erosion of the fibula ( figs . 1 and 2 ) .
we performed a thorough physical examination of the patient 's limbs in order to rule out another exostoses . due to the risk of having a fibula fracture we decide to perform a surgical excision of the lesion .
the patient underwent simple removal through an anterior approach ; it was not necessary to perform a fibular osteotomy .
intraoperative , we found a sessile exostosis with a broad base resembling a cauliflower , eroding the fibula , which was quite thin ( fig .
the histopathology exam showed an osteochondroma with a 1.2 cm thick cap of benign hyaline cartilage .
after the operation the patient was put in a below knee non weight bearing plaster cast for 6 weeks with a gradual transition to partial full weight bearing cam - walker . at six months after the operation ,
osteochondroma or osteocartilagenous exostosis is a benign surface lesion of bone consisting of a bony outgrowth covered by a cartilage cap .
it is considered the most common benign bone tumour , although the true incidence is unknown because most of the lesions are asymptomatic and never identified . incidentally discovered osteochondromas in asymptomatic patients are managed with observation .
the patient should be informed of the rare possibility of malignant change ( < 1% ) and should return for evaluation if the lesion becomes larger or painful .
the main symptoms are related to its size and location : irritation of nearby structures , bursitis due to chronic friction or stalk fracture secondary to traumatisms .
resection is indicated for patients with a symptomatic lesion secondary to irritation of the surrounding soft tissue , for a lesion in a location that is subjected to minor trauma , for a lesion causing a cosmetic deformity or potential damage to surrounding joints or neurovascular structures , and for a lesion that has characteristics of malignant transformation . if possible
, resection of an osteochondroma in a child should be postponed until skeletal maturity because the cartilage cap will become smaller and will be farther from the growth plate 6 .
if they affect the ankle , they are mainly found arising from the interosseous border , deforming distal tibia and fibula and occurring prior to physeal fusion , as have been reported by wani et al . , .
plastic deformation of tibia and fibula , mechanical blocking of joint motion , syndesmotic problems ( synostosis or diastasis ) , varus or valgus deformities of the ankle and subsequent degenerative changes in the ankle joint are some of the documented complications in the neglected cases , so most of the authors prefer a surgical resection for osteochondromas in this location .
surgical treatment of osteochondromas consists of simple removal ; mirra reiterated the importance of complete resection of the cartilaginous cap to prevent recurrence .
the anterior approach used in this case is associated with the least amount of postoperative morbidity , as have been used by wani et al .
there is still little information of the natural evolution after treatment of osteochondromas arising from the distal aspect of either the tibia or the fibula .
although most of the osteochondromas in children should be treated conservatively until skeletal maturity , those affecting the distal tibia or fibula should be treated with surgical excision in order to prevent ankle deformity , syndesmotic lesions or even fracture due to the expanding nature of this benign tumour .
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 .
| introductionan osteochondroma or exostosis is a benign bone tumour consisting of a bony outgrowth covered by a cartilage cap that occurs commonly in the metaphysis of long bones , mainly the distal femur , proximal tibia and proximal humerus.presentation of casewe describe an unusual case of a distal tibia osteochondroma affecting the lateral malleolus of a young girl.discussionmost osteochondromas are asymptomatic and seen incidentally during radiographic examination .
osteochondromas are rarely localized in the foot and ankle.conclusionalthough most of the osteochondromas in children should be treated conservatively until skeletal maturity , those affecting the distal tibia or fibula should be treated with surgical excision in order to prevent ankle deformity , syndesmotic lesions or even fracture due to the expanding nature of this benign tumour . |
intraductal papillomas are relatively common benign breast lesions , and represent about 5% of proliferative benign breast lesions
1 . they may clinically present either as mammographically detected or palpable breast masses , with or without nipple discharge .
giant intraductal breast papillomas are uncommon and to our knowledge , only seven cases have been reported in the literature 27 . of these cases ,
six of the cases were reported by institutions outside the united states 26 and only one case occurred in the united states 7 .
herein , we report a unique case of giant intraductal papillomas of the breast in a woman aged 80 years highlighting that the differential diagnosis of a large breast mass includes both benign and malignant conditions .
we also emphasize that , although benign , papillomas presenting as a large breast mass affecting the skin requires extensive breast surgical treatment .
an 80-year - old woman presented to the breast clinic with a left breast mass .
the patient reported previous benign excisional biopsy of a breast lump around age 50 with no breast - related concerns since that time .
about two years ago , she noticed a lump in the left breast that appeared to increase in size over time . around the same time , she had a single episode of spontaneous bloody discharge from the left nipple .
due to various personal reasons , she delayed her own care and had not sought breast evaluations until the size of the mass had reached a point that prompted her to have it assessed .
she had not experienced any pain , discharge , or skin breakdown in her breast , other than some discomfort related to the fullness .
she had no family history of breast or ovarian cancer and no risk factors for breast cancer other than her age .
clinical examination revealed a large left breast mass measuring 20 cm involving the inferior and medial left breast .
the overlying skin was bluish - purple in color , with a lateral deviation and distortion of the nipple ( fig.1 ) .
upon palpation , the mass measured 20 15 cm with thickening of the nipple .
the patient also had a small mass on the left upper anterior chest that had remained unchanged for several years .
breast imaging work up included mammography , ultrasound , and magnetic resonance imaging ( mri ) .
diagnostic mammography revealed a large mass in the left breast and a few scattered coarse calcifications ( fig.2a ) .
ultrasound examination demonstrated a complex vascular mass with a large homogenous fluid component occupying the majority of the left breast with features suspicious of malignancy ( fig.2b ) .
bilateral mri was performed to further characterize the mass , revealing a 13 11 13 cm mass with both cystic and enhancing solid components abutting the chest wall and the lateral aspect of the mass demonstrating skin involvement ( fig.2c ) . additionally , the small palpable mass on the superior left chest wall was consistent with a lipoma .
( a ) mammogram ( medial - lateral oblique view ) of the left breast demonstrating a large central breast mass . ( b ) ultrasound with color doppler of the solid , vascular portion of the left breast mass .
( c ) mr image of the left breast mass demonstrates the large cystic portion and one of the enhancing solid portions of the complex mass .
imaging features were suggestive of malignancy and the differential diagnosis included locally advanced breast cancer , papillary cancer , angiosarcoma , intracystic papilloma or hematoma secondary to a lesion . in order to facilitate breast biopsy ,
the radiologist performed an aspiration of the left breast mass removing 600 cc of brown - gray fluid ; cytology negative for malignancy .
an ultrasound - guided core - needle breast biopsy was performed with biopsies taken from the mass at the subareolar location and at 10 o'clock position , posterior depth against the chest wall .
both revealed sclerosing papilloma with focal usual ductal hyperplasia and minimal focal cytologic apocrine atypia ( fig.3 ) .
a prominent left axillary lymph node was subject to fine needle aspiration biopsy and was negative for malignancy .
40 5 high magnification view of core biopsy sample demonstrates apocrine atypia .
the mastectomy specimen was finely sliced at 45 mm intervals throughout and the firm areas were submitted for microscopic examination ( fig.4 ) .
pathology revealed a giant papilloma in the subareolar left breast with multiple dark red mural papillary nodules lining the cyst wall .
the postoperative phase was uncomplicated and the patient was advised routine follow - up care .
an 80-year - old woman presented to the breast clinic with a left breast mass .
the patient reported previous benign excisional biopsy of a breast lump around age 50 with no breast - related concerns since that time .
about two years ago , she noticed a lump in the left breast that appeared to increase in size over time . around the same time , she had a single episode of spontaneous bloody discharge from the left nipple .
due to various personal reasons , she delayed her own care and had not sought breast evaluations until the size of the mass had reached a point that prompted her to have it assessed .
she had not experienced any pain , discharge , or skin breakdown in her breast , other than some discomfort related to the fullness .
she had no family history of breast or ovarian cancer and no risk factors for breast cancer other than her age .
clinical examination revealed a large left breast mass measuring 20 cm involving the inferior and medial left breast .
the overlying skin was bluish - purple in color , with a lateral deviation and distortion of the nipple ( fig.1 ) .
upon palpation , the mass measured 20 15 cm with thickening of the nipple .
the patient also had a small mass on the left upper anterior chest that had remained unchanged for several years .
breast imaging work up included mammography , ultrasound , and magnetic resonance imaging ( mri ) .
diagnostic mammography revealed a large mass in the left breast and a few scattered coarse calcifications ( fig.2a ) .
ultrasound examination demonstrated a complex vascular mass with a large homogenous fluid component occupying the majority of the left breast with features suspicious of malignancy ( fig.2b ) .
bilateral mri was performed to further characterize the mass , revealing a 13 11 13 cm mass with both cystic and enhancing solid components abutting the chest wall and the lateral aspect of the mass demonstrating skin involvement ( fig.2c ) . additionally , the small palpable mass on the superior left chest wall was consistent with a lipoma .
( a ) mammogram ( medial - lateral oblique view ) of the left breast demonstrating a large central breast mass . ( b ) ultrasound with color doppler of the solid , vascular portion of the left breast mass .
( c ) mr image of the left breast mass demonstrates the large cystic portion and one of the enhancing solid portions of the complex mass .
imaging features were suggestive of malignancy and the differential diagnosis included locally advanced breast cancer , papillary cancer , angiosarcoma , intracystic papilloma or hematoma secondary to a lesion . in order to facilitate breast biopsy
, the radiologist performed an aspiration of the left breast mass removing 600 cc of brown - gray fluid ; cytology negative for malignancy .
an ultrasound - guided core - needle breast biopsy was performed with biopsies taken from the mass at the subareolar location and at 10 o'clock position , posterior depth against the chest wall .
both revealed sclerosing papilloma with focal usual ductal hyperplasia and minimal focal cytologic apocrine atypia ( fig.3 ) .
a prominent left axillary lymph node was subject to fine needle aspiration biopsy and was negative for malignancy .
40 5 high magnification view of core biopsy sample demonstrates apocrine atypia .
the mastectomy specimen was finely sliced at 45 mm intervals throughout and the firm areas were submitted for microscopic examination ( fig.4 ) .
pathology revealed a giant papilloma in the subareolar left breast with multiple dark red mural papillary nodules lining the cyst wall .
the postoperative phase was uncomplicated and the patient was advised routine follow - up care .
intraductal papillomas are benign breast lesions that are usually asymptomatic but may be diagnosed as mammographically detected abnormalities or clinically present as breast lumps , or nipple discharge .
single intraductal papillomas are most commonly centrally located in the breast in a subareolar location and present with nipple discharge varying in color from milky to green to brown 8 .
multiple intraductal papillomas are generally peripherally located , presenting incidentally as mammographically detected masses or as palpable breast masses 9 .
we present an uncommon clinical presentation of a papilloma with an unusually large size at presentation ( 20 cm 15 cm ) and bluish - purple discoloration of the overlying skin .
, three imaging modalities were used to image the breast mass : mammography , ultrasound , and mri . in general , papillomas do not have a classic appearance on mammography , rather they present as mammographic densities 10 . on ultrasound ,
they are often seen as an intraductal mass with or without ductal dilatation , intracystic mass , or a solid pattern with the intraductal mass completely filling the duct 11 .
there is limited data describing mri features of papillomas that may include enhancing nodules with or without intraductal components 10 of note , as papillary breast lesions enhance on mri , this test can not differentiate between benign and malignant papillary lesions making tissue diagnosis necessary . in this patient ,
ultrasound showed a large complex partially cystic mass in the left breast , raising the suspicion for malignancy .
mri revealed a large mass with both solid and cystic components that was highly suggestive of malignancy .
due to the concern for malignancy and the uncommon presentation of this mass , ultrasound - guided core - needle biopsy was necessary for diagnostic evaluation .
the biopsy finding of sclerosing papilloma with cytologic atypia was discordant with the imaging findings that suggested malignancy .
therefore , the final diagnosis was made after surgical excision of the mass , demonstrating the difficulties in diagnosis with an atypical presentation of a benign condition such as giant intraductal papilloma .
benign papillomas often show apocrine metaplasia , some of which are cytologically atypical with enlarged nuclei and prominent nucleoli .
focal minimal cytologic atypia , as in this case , usually has no prognostic significance .
however , severe and extensive apocrine atypia in a core biopsy of a papilloma may indicate the existence of possible carcinoma in a large papillary tumor . although the standard of care for large papillomas of the breast is excision 3 , the size of the mass and severe stretching of the skin limited the opportunities for excision with optimal closure in this patient .
hence , the patient underwent mastectomy of the left breast . due to the benign findings
based on her excellent overall good health and good 10-year life expectancy , she was advised screening mammogram of the contralateral breast in one year . in our patient ,
the giant nature of the mass provided unique complications in diagnosis , imaging , and the necessity to pursue mastectomy rather than surgical excision .
when approaching the differential diagnosis for a large breast mass , this case highlights the importance of considering the benign entity of giant papillomas of the breast .
| key clinical messagethe differential diagnosis of a large breast mass in a post - menopausal woman can include both benign and malignant etiologies . although rare , diagnosis of giant intraductal papilloma must be considered in the differential .
furthermore , although benign , papillomas presenting as a large breast mass affecting the skin require extensive breast surgical treatment . |
immortalized human nscs using a retroviral vector carrying v - myc and vascular endothelial growth factor ( hb1.f3.vegf ) were maintained at a density of 1 10 cells/100-mm dish ( sarstedt , sparks , nv ) in dulbecco 's modified eagle 's medium with high glucose ( dmem , hyclone , logan , ut ) containing 10% fetal bovine serum ( fbs , hyclone ) and 1% penicillin - streptomycin ( hyclone ) .
cells were grown to 80% confluence in a 37 humidified incubator with 5% co2 and 95% air environment , and were then harvested in trypsin containing edta ( hyclone ) .
the cells were cultured in 100-mm dishes ( sarstedt ) coated with poly - l - lysine ( sigma , st louis , mo ) .
ten spraque - dawley rats ( 250 - 300 g ; samtako , osan , korea ) were used in this study .
after an intraperitoneal injection of 1% ketamine ( 30 mg / kg ) and xylazine hydrochloride ( 4 mg / kg ) , the rats were placed in a stereotaxic frame ( lab standard , stoelting , wood dale , il ) . a burr hole was made , and a 26-gauge needle was inserted through the burr hole into the striatum ( ap : + 1.0 mm , ml : -2.6 mm , dv : -5.0 mm ) .
ich was induced by the administration of collagenase type iv ( 1 l saline containing 0.23 u , sigma ) over a period of five minutes .
the needle was then gently removed , the burr hole was sealed with bone wax , and the wound was sutured .
animals were maintained in separate cages at room temperature ( 25 ) , with free access to food and water under a 12-hour light - dark cycle .
super - paramagnetic iron oxide particles ( spio , feridex , berlex imaging , wayne , in ) were used to label the nscs ( hb1.f3.vegf ) .
protamine sulfate ( ps , sigma ) was used as a transfection agent ( ta ) .
five g/l spio and 0.25 g/l ps were added to the culture medium , which was incubated at room temperature for 10 minutes with occasional stirring .
the growth medium for hb1.f3.vegf cells was replaced with this solution and was incubated for 48 h. labeled cells were harvested for transplantation by gentle trypsinization . to remove excess iron oxide particles ,
trypsinized cells were washed in phosphate buffered saline and the cells were concentrated by centrifugation , resuspended in phosphate buffered saline , and kept on ice . for transplantation of labeled nscs ,
rats were stereotaxically fixed under anesthesia with intraperitoneal injection of 1% ketamine ( 30 mg / kg ) and xylazine hydrochloride ( 4 mg / kg ) a week after ich induction .
two microliters of 1 10 labeled nscs were delivered to the transplantation site ( left striatum ) through a hamilton syringe using a microinjector ( microinjector model 310 plus , kd scientific , holliston , ma ) at the speed of 0.5 l / min .
needles were left in the transplantation site for several minutes in order to minimize cell damage caused by negative pressure . to determine the sensitivity of 3 t mri for the detection of labeled nscs , duplicate aliquots of decreasing numbers of spio - labeled nscs ( 2 10
, 5 10 , 5 10 , 250 ) were injected into a 2% gelatin tube using a microinjector with a hamilton syringe .
each of the gelatin tubes were then placed in a custom - made holder for the experiments performed using a 3 t mr scanner ( magnetom tim trio , siemens medical solutions , erlangen , germany ) and clinically available wrist coil .
the imaging parameters were as follows : tr / te = 231/10 msec , flip angle = 25 , field of view ( fov ) = 7.0 cm , matrix number = 256 192 , slice thickness = 2 mm and number of excitation = 2.0 .
rats with grafted spio - labeled nscs were examined for period of 1 - 6 weeks ( 1 , 2 , 3 , 4 , and 6 weeks ) post - transplantation using a 3 t mr scanner and clinically available wrist coil .
rats were anesthetized for imaging using general inhalation anesthesia ( 1.5% isoflurane in a 1:2 mixture of o2/n2 ) during the mri procedures . to reduce artifacts during respiration ,
t2-weighted images ( tr / te = 3,000/100 msec , flip angle = 150 , acquisition time = 2 ' 42 " ) and t2-weighted images ( tr / te = 231/10 msec , flip angle = 25 , acquisition time = 2 ' 56 " ) were obtained .
other imaging parameters were as follows : fov = 7.0 cm , matrix number = 256 192 , slice thickness = 2 mm , slice gap = 0.1 mm and number of excitation = 2.0 .
mr images were reviewed on a picture archiving and communication systems ( pacs ; marotech , seoul , korea ) workstation .
t2-weighted images were used for detection of ich and t2-weighted images were used to track the labeled nscs .
the labeled nscs were supposed to present as hypointense foci in brain parenchyma because of a magnetic susceptibility artifact .
the change of signal intensity ( si ) ratio on a t2-weighted image was measured in defined regions of interest in both periventricular regions .
after in vivo mri , two rats were sacrificed by intravenous injection of pentobarbitone sodium each week .
a coronal section was made through the needle entry site and was then fixed in paraffin .
hematoxylin and eosin ( h & e ) stain , prussian blue stain , and an immunohistochemical stain using an anti - nestin human specific monoclonal antibody ( chemicon , temecula , ca ) were used for a histological examination by one pathologist .
immortalized human nscs using a retroviral vector carrying v - myc and vascular endothelial growth factor ( hb1.f3.vegf ) were maintained at a density of 1 10 cells/100-mm dish ( sarstedt , sparks , nv ) in dulbecco 's modified eagle 's medium with high glucose ( dmem , hyclone , logan , ut ) containing 10% fetal bovine serum ( fbs , hyclone ) and 1% penicillin - streptomycin ( hyclone ) .
cells were grown to 80% confluence in a 37 humidified incubator with 5% co2 and 95% air environment , and were then harvested in trypsin containing edta ( hyclone ) .
the cells were cultured in 100-mm dishes ( sarstedt ) coated with poly - l - lysine ( sigma , st louis , mo ) .
ten spraque - dawley rats ( 250 - 300 g ; samtako , osan , korea ) were used in this study .
after an intraperitoneal injection of 1% ketamine ( 30 mg / kg ) and xylazine hydrochloride ( 4 mg / kg ) , the rats were placed in a stereotaxic frame ( lab standard , stoelting , wood dale , il ) . a burr hole was made , and a 26-gauge needle was inserted through the burr hole into the striatum ( ap : + 1.0 mm , ml : -2.6 mm , dv : -5.0 mm ) .
ich was induced by the administration of collagenase type iv ( 1 l saline containing 0.23 u , sigma ) over a period of five minutes .
the needle was then gently removed , the burr hole was sealed with bone wax , and the wound was sutured .
animals were maintained in separate cages at room temperature ( 25 ) , with free access to food and water under a 12-hour light - dark cycle .
super - paramagnetic iron oxide particles ( spio , feridex , berlex imaging , wayne , in ) were used to label the nscs ( hb1.f3.vegf ) .
protamine sulfate ( ps , sigma ) was used as a transfection agent ( ta ) .
five g/l spio and 0.25 g/l ps were added to the culture medium , which was incubated at room temperature for 10 minutes with occasional stirring .
the growth medium for hb1.f3.vegf cells was replaced with this solution and was incubated for 48 h. labeled cells were harvested for transplantation by gentle trypsinization . to remove excess iron oxide particles ,
trypsinized cells were washed in phosphate buffered saline and the cells were concentrated by centrifugation , resuspended in phosphate buffered saline , and kept on ice .
for transplantation of labeled nscs , rats were stereotaxically fixed under anesthesia with intraperitoneal injection of 1% ketamine ( 30 mg / kg ) and xylazine hydrochloride ( 4 mg / kg ) a week after ich induction .
two microliters of 1 10 labeled nscs were delivered to the transplantation site ( left striatum ) through a hamilton syringe using a microinjector ( microinjector model 310 plus , kd scientific , holliston , ma ) at the speed of 0.5 l / min .
needles were left in the transplantation site for several minutes in order to minimize cell damage caused by negative pressure .
to determine the sensitivity of 3 t mri for the detection of labeled nscs , duplicate aliquots of decreasing numbers of spio - labeled nscs ( 2 10 , 5 10 , 5 10 , 250 ) were injected into a 2% gelatin tube using a microinjector with a hamilton syringe .
each of the gelatin tubes were then placed in a custom - made holder for the experiments performed using a 3 t mr scanner ( magnetom tim trio , siemens medical solutions , erlangen , germany ) and clinically available wrist coil .
the imaging parameters were as follows : tr / te = 231/10 msec , flip angle = 25 , field of view ( fov ) = 7.0 cm , matrix number = 256 192 , slice thickness = 2 mm and number of excitation = 2.0 .
rats with grafted spio - labeled nscs were examined for period of 1 - 6 weeks ( 1 , 2 , 3 , 4 , and 6 weeks ) post - transplantation using a 3 t mr scanner and clinically available wrist coil .
rats were anesthetized for imaging using general inhalation anesthesia ( 1.5% isoflurane in a 1:2 mixture of o2/n2 ) during the mri procedures . to reduce artifacts during respiration , each rat head
t2-weighted images ( tr / te = 3,000/100 msec , flip angle = 150 , acquisition time = 2 ' 42 " ) and t2-weighted images ( tr / te = 231/10 msec , flip angle = 25 , acquisition time = 2 ' 56 " ) were obtained .
other imaging parameters were as follows : fov = 7.0 cm , matrix number = 256 192 , slice thickness = 2 mm , slice gap = 0.1 mm and number of excitation = 2.0 .
mr images were reviewed on a picture archiving and communication systems ( pacs ; marotech , seoul , korea ) workstation .
t2-weighted images were used for detection of ich and t2-weighted images were used to track the labeled nscs .
the labeled nscs were supposed to present as hypointense foci in brain parenchyma because of a magnetic susceptibility artifact .
the change of signal intensity ( si ) ratio on a t2-weighted image was measured in defined regions of interest in both periventricular regions .
after in vivo mri , two rats were sacrificed by intravenous injection of pentobarbitone sodium each week .
a coronal section was made through the needle entry site and was then fixed in paraffin .
hematoxylin and eosin ( h & e ) stain , prussian blue stain , and an immunohistochemical stain using an anti - nestin human specific monoclonal antibody ( chemicon , temecula , ca ) were used for a histological examination by one pathologist .
nscs were efficiently labeled and spio located inside cells was visualized by prussian blue staining .
the iron oxide nanoparticles in colonies of nscs in culture were observed as blue spots on microscopy ( fig .
1 ) . to check the sensitivity of the mri technique and to mimic the signal behavior in brain tissue , we performed in vitro mri of labeled cells .
nscs were labeled with spio as described above , and a cell suspension ( at concentration of 2 10 , 5 10 , 5 10 , 250 cells/l ) was suspended in 2% gelatin .
mr images showed a clear hypointense signal at all concentrations greater than 5 10 cells ( fig .
one week after implantation of nscs , t2-weighted imaging showed ich in the right striatum .
hypointensity on mri was found only at the injection site of labeled nscs and migration of labeled nscs was not detected .
migration of nscs into the corpus callosum or the right periventricular region was not seen on the histopathological findings ( fig .
3 ) . on mri two weeks after implantation , the ich finding in the right striatum had not changed .
migration of nscs did not occur except a hypointense spot at the injection site of the nscs , decreased slightly in size as seen on t2-weighted imaging .
prussian blue staining showed fewer blue spots along the corpus callosum , and immunohistochemical staining confirmed migrating nscs ( fig .
4 ) . on mri three weeks after implantation , t2-weighted imaging showed a further decrease in the size of ich in the right striatum .
t2-weighted imaging showed a decreased size of the hypointense spot in the left striatum and linear hypointensity along the corpus callosum , which is considered to represent labeled nscs .
prussian blue staining and immunohistochemical staining showed a large number of labeled nscs along the corpus callosum and right periventricular region ( fig .
mri after four and six weeks showed similar findings of decreased size of ich in the right striatum on t2-weighted imaging and an increased hypointense spot in the right periventricular region on t2-weighted imaging . on t2-weighted imaging ,
prussian blue staining and immunohistochemical staining showed labeled nscs in the right periventricular region ( fig .
changes of the si ratio in the right periventricular region showed a rapid drop between two and three weeks , which was well correlated with the histological findings of large - scale migration of labeled nscs . on the contrary , the si ratio at the implantation site of the left striatum gradually increased ( fig .
nscs were efficiently labeled and spio located inside cells was visualized by prussian blue staining .
the iron oxide nanoparticles in colonies of nscs in culture were observed as blue spots on microscopy ( fig .
to check the sensitivity of the mri technique and to mimic the signal behavior in brain tissue , we performed in vitro mri of labeled cells .
nscs were labeled with spio as described above , and a cell suspension ( at concentration of 2 10 , 5 10 , 5 10 , 250 cells/l ) was suspended in 2% gelatin .
mr images showed a clear hypointense signal at all concentrations greater than 5 10 cells ( fig .
one week after implantation of nscs , t2-weighted imaging showed ich in the right striatum .
hypointensity on mri was found only at the injection site of labeled nscs and migration of labeled nscs was not detected .
migration of nscs into the corpus callosum or the right periventricular region was not seen on the histopathological findings ( fig .
3 ) . on mri two weeks after implantation , the ich finding in the right striatum had not changed .
migration of nscs did not occur except a hypointense spot at the injection site of the nscs , decreased slightly in size as seen on t2-weighted imaging .
prussian blue staining showed fewer blue spots along the corpus callosum , and immunohistochemical staining confirmed migrating nscs ( fig .
three weeks after implantation , t2-weighted imaging showed a further decrease in the size of ich in the right striatum .
t2-weighted imaging showed a decreased size of the hypointense spot in the left striatum and linear hypointensity along the corpus callosum , which is considered to represent labeled nscs .
prussian blue staining and immunohistochemical staining showed a large number of labeled nscs along the corpus callosum and right periventricular region ( fig .
mri after four and six weeks showed similar findings of decreased size of ich in the right striatum on t2-weighted imaging and an increased hypointense spot in the right periventricular region on t2-weighted imaging . on t2-weighted imaging ,
prussian blue staining and immunohistochemical staining showed labeled nscs in the right periventricular region ( fig .
changes of the si ratio in the right periventricular region showed a rapid drop between two and three weeks , which was well correlated with the histological findings of large - scale migration of labeled nscs . on the contrary , the si ratio at the implantation site of the left striatum gradually increased ( fig .
in our study , clinically available 3 t mri was acceptable to use for the detection of implanted nscs crossing the corpus callosum to the periventricular region , and this finding correlated with the histopathologic findings .
migration through the corpus callosum to the lesion site in a stroke model has been reported in several studies ( 11 , 14 - 15 ) .
almost all of the reported studies used experimental mri ( > 4.7 t ) that enabled in vivo imaging with near microscopic resolution ( 9 - 11 , 16 , 17 ) .
detection of nscs using experimental mri has also been achieved using extremely long measurement times of several hours to achieve the necessary signal - to - noise ratio and contrast ( 9 , 16 ) .
one type is an agent containing gadolinium - diethylenetriamine penta - acetic acid ( gd - dtpa ) , and the other type is an agent containing spio . as stem cells labeled with gd - dtpa can be monitored for up to seven days after injection , it is not appropriate for long - term monitoring . stem cells labeled with spio are appropriate for long - term , in vivo monitoring because of the stability and high contrast of spio ( 14 , 18 ) .
we used spio ( feridex ) in our study as it is a contrast agent based on dextran - coated spio nanoparticles and the fda clinically approves it as a blood pool agent .
particles of spio can be as small as 7 - 10 nm and are located in unmagnetized cell cytosol .
spio can be detected with high magnetic mri t2- as well as on t2-weighted imaging because of its susceptibility artifact ( 14 ) .
spio nanoparticles have been used for labeling cells for in vivo mr cell tracking ( 15 ) .
frank et al . ( 14 ) showed that magnetic labeling of mammalian cells using ferumoxides and tas is possible and may enable cellular mri and tracking in both experimental and clinical settings .
labeling a cell with a contrast agent based on spio nanoparticles before transplantation has been widely investigated for use in human medicine ( 19 - 20 ) . widely used tas are poly - l - lysine ( pll ) and protamine sulfate .
pll is not approved by the fda and is cytotoxic at a concentration of 10 m ( 21 - 22 ) .
the fda has approved the use of protamine sulfate as an antidote of heparin , and it is safe and effective as a ta ( 22 ) . in our study , we could label nscs effectively with spio and protamine sulfate .
spio is detectable with at least 500 cells in a plane by the use of 4.7 t mri and 100 cells in a plane by the use of 9.4 t mri ( 10 ) .
high magnetic field mri ( > 4.7 t ) has a higher sensitivity than 3 t mri for the detection of spio due to the high susceptibility effect of high magnetic field mri . in our study , 5 10 cells with a 2-mm slice thickness could be detected on 3 t mri .
it is difficult to compare the results of various reports because the sensitivity of labeled stem cells varies according to the magnetic strength , surface coil , and various parameters .
various routes have been tested for the administration of nscs ( 8 - 10 , 12 , 23 , 24 )
. a greater number of nscs were recruited around the lesion site when ipsilateral intraparenchymal or intraventricular injection was used rather than contralateral intraparenchymal injection ( 2 ) .
there was no difference in the recruited cell numbers between ipsilateral intraparenchymal injection and intraventricular injection .
this finding was thought to be due to the greater probability of survival of the normal parenchyma than of the injured parenchyma .
however , intravenous injection required the use of more stem cells and increased the risk of occlusive venous disease caused by thrombus formation and an obstructive lesion or mass formation in the spleen , kidney , or lung ( 25 , 26 ) .
we used contralateral intraparenchymal injection of nscs to increase survival of stem cells and to monitor the intraparenchymal route of the implanted nscs ( 2 ) .
the optimal method of implantation should be further investigated in order to be able to deliver stem cells safely and effectively in the future . in our study , mr findings three weeks after implantation showed hypointensity along the corpus callosum , and a drop in the si in the periventricular region , both of which suggest that migration of nscs is prominent from two weeks to three weeks after implantation .
a histological examination showed inhomogenous distribution of the nscs at the lesion site , especially in the periventricular region ; this correlated well with the mr findings .
( 9 ) , who reported that spio - labeled embryonic stem cells injected intracerebrally could migrate into an ischemic lesion induced by occlusion of the middle cerebral artery .
however , in our study , 3 t mri could not detect migration along the corpus callosum at two weeks after implantation despite the presence of a small number of nscs in the corpus callosum , as detected on the histological findings .
this difference can be explained by the lower spatial resolution of 3 t mri than experimental high magnetic field mri .
on 3 t mri , acceptable images could be obtained with a 2-mm slice thickness as the signal - to - noise ratio is decreased with a slice thickness less than 2-mm .
therefore , small numbers of nscs may not be detectable on 3 t mri . in conclusion
, we have demonstrated that clinically available 3 t mri can detect the migration of nscs along the corpus callosum in rats with ich .
nscs labeled with iron oxide nanoparticles , remained viable and were able to migrate into an ich site .
therefore , in the future , 3 t mri may be useful to monitor the migration of nscs in the clinical setting of stem cell therapy . | objectiveto access the feasibility of clinically available 3 t mri to detect the migration of labeled neural stem cells ( nscs ) in intracerebral hemorrhage ( ich ) in a rat model.materials and methodsthe ethics committee of our institution approved this study .
ich was induced by the injection of collagenase type iv into the right striatum of ten sprague - dawley rats .
human nscs conjugated with feridex ( super - paramagnetic iron oxide : spio ) were transplanted into the left striatum one week after ich induction .
mri was performed on a 3 t scanner during the first , second , third , fourth , and sixth weeks post - transplantation .
mri was obtained using coronal t2- and t2 * -weighted sequences .
two rats were sacrificed every week after in vivo mri in order to analyze the histological findings.resultsich in the right striatum was detected by mri one and two weeks after transplantation without migration of the nscs .
there was no migration of the nscs as seen on the histological findings one week after transplantation .
the histological findings two weeks after transplantation showed a small number of nscs along the corpus callosum . on mri
three weeks after transplantation , there was a hypointense line along the corpus callosum and decreased signal intensity in the right periventricular region .
histological findings three weeks after transplantation confirmed the presence of the hypointense line representing spio - labeled nscs .
mri four and six weeks after transplantation showed a hypointense spot in the right periventricular region . the histological findings
four and six weeks after transplantation showed the presence of prominent nscs in the right periventricular region.conclusion3t mri can detect the migration of nscs in rats with ich along the corpus callosum .
therefore , 3 t mri could be feasible for detecting the migration of nscs in the clinical setting of stem cell therapy . |
alpha spectrometry is the most widely used method for the determination of plutonium isotopes in environmental samples .
however , this method does not permit the determination of beta - emitting pu , a radionuclide abundant in the environment .
the best way to determine pu in an environmental sample is by liquid scintillation ( ls ) after the isotope has been removed from the sample by a radiochemical procedure .
the following procedure can be used for the simultaneous determination of alpha and beta plutonium activity [ 24 ] .
an important consideration is choosing an extracting agent that is both efficient and minimizes quenching effects during ls measurements .
the aim of this study is to illustrate a rapid method for the determination of pu in soil samples using a simple solvent extraction method followed by ls measurements .
the procedure described here is applied after the determination of other pu isotopes in the sample by a technique of alpha spectrometry .
there are many commonly known extracting agents for the extraction of heavy metal traces , such as : trioctylphosphine oxide , topo [ 57 ] , bis-(2-ethylhexyl ) phosphoric acid , hdehp [ 8 , 9 ] , methyltrioctylammonium chloride ( aliquat 336 ) [ 10 , 11 ] , n , n - diethyldodecanamide , deda or thenoyltrifluoracetone , htta .
these compounds have been tested to find those of high plutonium recovery from the environmental samples .
the ultra low - level spectrometer quantulus 1220 ( wallac , perkin - elmer ) was used for ls measurements .
permablend iii ( packard ) , consisted of 91 % ppo and 9 % bis - msb dissolved in toluene was applied as a scintillation cocktail .
samples were measured for 5 h , after 24 h stabilization in darkness in the apparatus to avoid chemiluminescence .
standard solutions of pu ( ~0.02 bq ) in 3 m nitric acid were used as tracers .
alpha spectrometric measurements were performed using the alpha spectrometer ( model 7401 canberra ) with pips detector of 17 kev fwhm resolution , the 1520 mixer - router , s-100 multichannel analyzer , and genie 2000 software for quantitative analysis ( canberra ) . before the ls measurements
, the experiments were performed to choose a proper extractant which is characterized by the best efficiency and low quenching effects .
the following extractant solutions were tested : 0.2 m hdehp dissolved in toluene , 0.2 m topo in cyclohexane , 0.05 m aliquat 336 in xylene , 0.3 m deda in toluene and 0.01 m htta in toluene .
the extraction procedure was described previously [ 14 , 15 ] . for the verification of the procedure
the samples were collected in 1991 near bragin village from two successive layers of soil ( 05 and 510 cm ) .
the samples were ashed , co - precipitated , separated by ion exchange and then electrodeposited on small steel plates , as described recently .
after the alpha - spectrometry measurements , the electro - deposited plutonium was washed off of the steel plates with nitric acid and extracted into the organic phase of 0.2 m hdehp solution in toluene .
an aliquot of the organic phase was mixed with the scintillation cocktail and its activity was measured with a quantulus spectrometer .
the characteristics of a useful extractant include the following : acceptable counting efficiency , resistance to decomposition by nitric acid , miscibility with the scintillation cocktail , low background counts , and low quenching characteristics .
the activities of ten soil samples are shown in figs . 1 , 2 .
figure 1 illustrates the activities of five random soil samples taken from a soil depth 05 cm deep .
figure 2 illustrates another set of five samples taken from 5 to 10 cm soil depth.fig .
1activity concentration of plutonium ( bq / kg ) in soil layer 05 cm ( five sample replicates)fig .
2activity concentration of plutonium ( bq / kg ) in soil layer 510 cm ( five sample replicates ) activity concentration of plutonium ( bq / kg ) in soil layer 05 cm ( five sample replicates ) activity concentration of plutonium ( bq / kg ) in soil layer 510 cm ( five sample replicates ) figure 1 illustrates that the surface layer of soil ( 05 cm deep ) displayed the following activities : pu of 2.88 bq / kg , 6.98 bq / kg of pu and 60.8 bq / kg of pu .
figure 2 illustrates that the deeper layer of soil ( 510 cm deep ) displayed the following activities : 0.066 bq / kg of pu , 0.50 bq / kg of pu and 0.53 bq / kg of pu .
the estimated uncertainties of these results are about 10 % for both soil depths .
calculation of the isotopic ratios : pu / pu and pu / pu allows the estimation of the plutonium origin . calculated isotope ratios
they can be interpreted in a historical context : what is the origin of the plutonium in the soil samples ?
global fallout plutonium reveals a very small share of pu ( about 4 % ) . on the contrary ,
the chernobyl fallout includes almost 50 % of pu [ 17 , 18].table 1isotopic ratios of plutonium in analyzed chernobyl contaminated samples ( the last column ratio has been recalculated back on the date of the chernobyl accident)layer
pu / pu
pu / pu
pu / pu in 198605 cm0.429.532510 cm0.131.13.6 isotopic ratios of plutonium in analyzed chernobyl contaminated samples ( the last column ratio has been recalculated back on the date of the chernobyl accident ) table 1 shows the calculated isotopic ratios of the two soil layers .
according to the literature data , the typical global fallout ratio pu / pu is ranged 0.030.05 , and for chernobyl fallout is 0.30.65 .
the observed ratio indicates that the upper soil layer is contaminated mainly with chernobyl fallout and the lower rather with a global one .
the data from the literature is as follows , the ratio pu / pu for global fallout is about 4.2 ( based on the year 1986 ) , and for the chernobyl fallout it is about 94.8 and varies widely . as seen in table 1 , the calculated average ratio pu / pu in the surface layer is 9.5 and after calculation on the date of the chernobyl disaster it will be equal to 32 . in the deeper layer the pu / pu ratio was 1.1 .
, one can conclude that in the deeper soil layer plutonium from global fallout predominates and in the surface layer the contribution of plutonium from chernobyl fallout is undoubtedly the largest .
the presented procedure which combines the alpha spectrometry and the ls measurements after solvent extraction may be useful for the rapid determination of all plutonium isotopes present in the environment ( pu , pu and pu ) . it was found that among five various extractants , the hdehp reveals the best properties to be applied as an extracting agent during the sample preparation to ls measurement .
the elaborated method used for the determination of plutonium in the samples contaminated with chernobyl fallout shows its usefulness .
calculated isotopic ratios confirmed a large share of this source of contamination in the analyzed samples , especially in those taken from the upper layer of soil . | a simple and rapid procedure for the determination of plutonium isotopes in the environment is presented .
the procedure combines alpha spectrometry , solvent extraction and liquid scintillation measurements to ensure that both alpha- and beta - emitting isotopes are determined . of five tested extractants , bis-(2-ethylhexyl )
phosphoric acid was found to be the best choice .
the procedure was applied to soil samples contaminated with chernobyl fallout . |
the groups of suprahyoid muscles include four muscles : the digastric , the geniohyoid , stylohyoid and mylohyoid .
the bellies of the digastric are joined together by an intermediate tendon that passes through a fibrous sling that is attached to the body of the hyoid bone .
these muscle groups anchor the hyoid bone against the traction of the infrahyoid muscles . the anterior belly of the digastric muscle inserts to the digastric fossa , in the lower interior of the mandible .
the posterior belly originates in the mastoid notch , on the medial side of the mastoid process of the temporal bone .
two bellies of the digastric muscle are inserted in the body of the hyoid bone by means of a tendon .
it is important to mention that when the mandible is fixed , the digastric muscle raises the hyoid bone , while when the hyoid is fixed , the digastric muscle opens the mouth by lowering the mandible .
the bellies of the digastric muscle are the borders of the submandibular , submental and carotid triangles .
the submandibular ( digastric ) triangle is limited by the anterior and posterior bellies of the digastric muscle and by the inferior margin of the mandible .
they are the submandibular gland , submandibular lymph nodes , hypoglossal nerve , facial vein , nerves and vessels for the mylohyoid muscle and the carotid sheath , which contains the external and internal carotid arteries , internal jugular vein and vagus nerve .
the submental triangle is limited by the anterior belly of the digastric muscle , the hyoid bone and the anterior median line .
the submental lymph nodes and the anterior jugular vein are located in the submental triangle .
the carotid triangle is limited by and the superior belly of the omohyoid muscle , the posterior belly of the digastric muscle , and by the anterior margin of the sternocleidomastoid muscle .
this triangle contains the carotid sheath , with the common carotid artery , branches of the external carotid artery , the internal jugular vein and its tributaries , the deep cervical lymph nodes and the vagus , accessory and hypoglossal nerves [ 3 , 10 , 11 ] .
the embryonal development of the anterior belly of the digastric muscle begins in the fourth week of pregnancy , other hand originates from the second pharyngeal arch .
the anterior belly of the digastric muscle is innervated by the trigeminal nerve ( fifth cranial pair ) , while the posterior belly is innervated by the facial nerve ( seventhcranial pair ) . in the literature
there have been data of anatomical variations in the digastric muscle ever since 1847 , as pointed out by bergman et al . in 1905
testut pointed some variations in the anterior belly , with the presence of a supernumerary fascicle inserted in the raphe of the mylohyoid muscle , or in the hyoid bone or the digastric fossa of the opposite side .
other variations were described in accordance with the classifications of zlabek , which took into account their phylogenetic and ontogenetic development , and the classifications of yamada , which enumerated six different types of variations in the anterior belly : atavistic type , origin type , insertion type , mixed type , complex type and defect type .
proposed that the classification of anatomical variations in the anterior belly of the digastric muscle should be based on the position of the attachments of the bellies of the muscle .
this work is a collaboration between the department of anatomy and histology , the department of endocrine surgery , the department of forensic medicine and deontology from sofia s medical university , and the department of general surgery , state hospital , biala slatina city , bulgaria .
digastric muscles of human material ( of 80 cadavers and patients aged 21 to 82 years , 40 males and 40 females ) were used for this morphological study .
experiments were conducted in strict compliance with the ethical principles for handling human and cadaveric material applied at the medical university of sofia and the legislative requirements for the protection of human rights .
varieties in sublingual muscles were not found , except in one cadaver , in which we came across the described variety .
fixation of cadaveric materials in the dissecting room used for training for students and graduates of each medical university in the european union is very specific and is regulated by the state law .
all legislative requirements to protect human rights have been observed as well . during a routine dissection in february 2013 in the section hall of the department of anatomy and histology ,
after extraction of the skin from the neck , digastrics muscles were carefully analyzed from the some variation .
the digastric muscles that presented anatomical variations were photographed using a sony cyber - shot dsc - t1 camera with a carl zeiss vario - tessar lens .
sofia , establish a very rare variation . as seen on the figures 1 , 2 , 3 , and 4 , the anterior belly of the digastric muscle originates from the original mesoderm of the first pharyngeal arch .
the deficiency in the differentiation of this layer on one side may be responsible for unilateral variations , whereas deficiencies on both sides may be responsible for bilateral variations . in our case
thus we came to the conclusion that the deficiency in the differentiation of the first pharyngeal arch was bilateral .
this does not correspond to the literature data according to which the unilateral variations are more frequent . in some cases unilateral anatomical variations
may be responsible for the asymmetry in the anterior region of the neck or even in the movement of the floor of the mouth or the temporomandibular joint .
thus they may present greater clinical importance because this may lead to an imbalance in the movement of the larynx .
however , more recentl investigations showed a balance between unilateral and bilateral variations . according to this
it is important to note that the unilateral type of asymmetry may be able to be considered in clinical examinations and in imaging examinations like ultrasound , tomography and magnetic resonance , with lymph nodes , benign cervical masses like thyroglossal cysts , or neoplasia . in conclusion , it is important that our case refers only to the variation of the anterior belly
. other studies in literature also refer mainly to the anterior belly of the digastric . during some surgical procedures in the anterior region of the neck especially in the submandibular and submental triangles
for example when performing submandibulectomy the digastric muscle and its tendon serve for anatomical orientation . | the digastric muscle is composed by two muscle bellies : an anterior and a posterior , joined by an intermediate tendon . this muscle is situated in the anterior region of the neck .
the region between the hyoid bone and the mandible is divided by an anterior belly into two triangles : the submandibular situated laterally and the submental triangle which is located medially .
we found that the anatomical variations described in the literature relate mainly to the anterior belly and consist of differences in shape and attachment of the muscle . during routine dissection in february 2013 in the section hall of the department of anatomy and histology in medical university
sofia we came across a very interesting variation of the digastric muscle .
the digastric muscles that presented anatomical variations were photographed using a sony cyber - shot dsc - t1 camera , with a carl zeiss vario - tessar lens .
we found out bilateral variation of the digastric muscle in one cadaver .
the anterior bellies were very thin and insert to the hyoid bone .
two anterior bellies connect each other and thus they formed a loop .
the anatomical variations observed of our study related only to the anterior belly , as previously described by other authors .
it is very important to consider the occurrence of the above mentioned variations in the digastric muscle when surgical procedures are performed on the anterior region of the neck . |
staphylococcus aureus is a main source of hospital - acquired infections causing pneumonia , endocarditis , osteomyelitis , soft - tissue , and skin infections .
s. aureus also causes serious nail infections ( paronychia ) and is a common cause of food poisoning due to the production of enterotoxin .
a main problem concerning s. aureus infections is its ability to become resistant to multiple antibiotics including -lactams ( mrsa ) and glycopeptides and also to more recent molecules such as linezolid and daptomycin [ 1 , 3 , 4 ] . in the mid-1990s ,
the emergence of community - acquired antibiotic - resistant staphylococcal infections in individuals with no identified risk factors raised new concerns .
the underlying factors of s. aureus pathogenicity relate to the coordinated expression of numerous virulence factors .
the combined risks of disease and diminishing efficacy of antibiotic treatments have incited the scientific community to investigate staphylococcal transcriptional and posttranscriptional regulation in detail .
in contrast to eukaryotes , bacterial mrnas are usually short - lived with a half - life ranging from a few seconds to over one hour .
rna processing is a cleavage leading to functional transcripts , while rna degradation results in rnas transformed into oligonucleotides and nucleotides .
transcriptional and post - transcriptional regulatory pathways control protein production and contribute to homeostasis and adaptation to environmental stress . in bacteria ,
the first step of rna decay is generally thought to involve removal of the rna 5-end pyrophosphate .
exonuclease activity is solely 3 to 5 while in bacillus subtilis , 5 to 3 exonuclease activity was uncovered .
extensive studies on rna processing and degradation reveal that while several rnases are present in other species of their respective taxons ( e.g. , rnase iii , pnpase , rnase r , rnase p , and rnase z ) ( figure 1 and table 1 ) , rnase e , while essential in e. coli , is absent in b. subtilis . instead , rnase j1 , rnase j2 , and rnase y are present in b. subtilis and for some aspects are functional homologs of rnase e. since rnases were primarily studied in the proteobacteria e. coli and the firmicutes b. subtilis , results obtained for these species will be presented to discuss the recent knowledge on rna decay in s. aureus .
rnase iii is a double - strand ( ds ) specific rnase discovered in e. coli extracts more than forty years ago .
its activity is divalent cation - dependent and is inhibited in vitro by metal chelators .
rnase iii - family enzymes show a large diversity in terms of primary protein structure , ranging from the b. subtilis mini - iii rnase ( 143 amino acids ; uniprot o31418 ) to the large homo sapiens dicer1 protein ( 1,922 amino acids ; uniprot q9upy3 ) .
however , all family members possess a common riiid - like domain that includes a nine - residue signature motif . through its ds - rna specificity
these roles include the maturation of ribosomal rnas ( rrna ) by cleaving stem - loops inside the primary rrnas [ 810 ] and mrna processing including its own mrna by cleaving a stem - loop involved in a feedback autoregulation .
one of the first discovered roles of rnase iii was its implication in the lifestyle of temperate bacteriophage . rnase iii cleaves a stem - loop in the 5utr region of the n gene transcript , thus releasing the shine dalgarno ( sd ) sequence and permitting recruitment of ribosomes .
due to its ds - specific rnase activity , rnase iii is also involved in cleaving small regulatory rna ( srna)/mrna duplexes [ 13 , 14 ] .
recent studies in streptococcus pyogenes show that rnase iii acts in concert with the crispr csn1 protein to mature crispr rnas ( crrna ) , resulting in prophage silencing . in b. subtilis , amounts of 470 transcripts , representing 11% of total transcripts ,
were shown to be altered by rnase iii depletion . however , rnase iii essentiality was due neither to its global role on bulk rna level nor to rrna maturation , but to its role in the elimination of toxins encoded by type i toxin / antitoxin ( ta ) systems .
the deletion of txpa / rata and yont / as - yont tas was sufficient to suppress the rnase iii essentiality .
rnase iii is the most studied s. aureus rnase ; its role was mainly determined through the characterization of virulence genes regulated by the agr system [ 1720 ] .
rnaiii , a 514 nucleotide regulatory rna which base - pairs with numerous targets , is the agr system effector ( figure 2(a ) ) [ 20 , 21 ] . the staphylococcal protein a , encoded by the spa gene , inhibits phagocytic engulfment ;
the regulation of spa involves the formation of an rnaiii - spa mrna duplex that is then degraded by rnase iii .
duplex formation is sufficient to prevent translation of spa mrna ; spa mrna degradation by rnase iii contributes to the irreversibility of the process .
other examples where mrna - rnaiii duplex formation leads to a translational arrest and consequent mrna degradation include ( i ) rot mrna ( encoding a regulator of toxins ) through imperfect base pairings involving two loop - loop interactions and of ( ii ) coa mrna ( encoding the staphylococcal coagulase ) via the binding of two distant regions of coa mrna ( figure 2(b ) ) [ 17 , 22 ] . toeprinting and rnase cleavage assays demonstrated that rnase iii cleaves at the bottom of a stem loop and also inside loop - loop interactions ( figure 2(b ) ) .
two recent studies gave novel insights at a genome scale on the function of the staphylococcal rnase iii [ 23 , 24 ] .
a first approach was based on sequencing of cdna libraries obtained by coimmunoprecipitation assays with either wild - type rnase iii or catalytically inactive but binding - efficient rnase iii [ 24 , 25 ] .
these experiments elucidated the roles of rnase iii in different cellular processes including ( i ) rrna and trna processing , ( ii ) rnase iii autoregulation by self - cleavage , and ( iii ) processing / cleavage of mrnas and mrna - srna duplexes .
the first step is a cleavage within a long hairpin in the cspa mrna 5utr ( figure 2(c ) ) . as a consequence
, the mrna 5utr is shortened giving rise to a more stable transcript and rendering the sd sequence accessible for a higher rate of translation .
this case exemplified the role of rnase iii in stimulating translation efficiency as was demonstrated for the n gene in phage [ 7 , 12 ] .
in addition to mrna targets , 58 noncoding rnas ( ncrnas ) were coimmunoprecipitated with rnase iii .
the use of a catalytically inactive rnase iii allows capturing of ds - rnas , including srnas base - paired to mrnas , so that srna targets can be identified at a genome scale .
a second study focusing on the role of rnase iii at a genome scale was performed using a comparative transcriptomic analysis of wild - type and rnase iii deficient ( rnc ) strains .
the authors sequenced cdna of both long and short ( < 50 nt ) transcripts .
a collection of short transcripts covering more than 75% of all mrnas throughout the s. aureus genome was identified . in the absence of rnase iii , an accumulation of antisense transcripts and a decrease of short transcripts
were observed , suggesting that rnase iii likely eliminates a basal level of pervasive transcription . to assess whether this pervasive transcription is common to different bacteria ,
sequencing of short rnas was performed for b. subtilis , enterococcus faecalis , listeria monocytogenes , and salmonella enterica . a correlation between the absence of rnase iii and an increase of short transcripts
was observed in all the tested gram - positive bacteria , but not in the sole gram - negative species tested ( s. enterica ) .
it will be interesting to test whether pervasive transcription is mainly associated with gram - positive bacteria .
first , interactions between antisense and sense transcripts could be fine - tuned via rnase iii , which consequently could control cellular protein levels .
an rnase iii paralog , named mini - iii due to its small size ( 143 amino acids in b. subtilis ) , has been described in low gc content gram - positive bacteria .
mini - iii plays a role in the maturation of 23s rrna in b. subtilis [ 2628 ] .
however , it can be replaced by the combined activity of rnase j1 , rnase ph , and yham .
a mini - iii ortholog is present in s. aureus but to date has not been characterized ( table 1 ) .
the endonuclease rnase y of b. subtilis ( encoded by rny , formerly ymda ) was identified as an rnase that cleaves single - stranded a- or au - rich sequences .
it cleaves sam - dependent riboswitches , including the yitj riboswitch , but only in the presence of sam , which contributes to forming a terminator structure .
the initial rate of 5 monophosphorylated rna degradation is faster than for 5 triphosphorylated rnas .
however , after prolonged in vitro incubation , the same amount of yitj cleaved product was observed even for 5 end triphosphorylation .
these results indicate that rnase y shows a preference for 5 monophosphorylated substrates , as observed for rnase e. 5-dependent and 5-independent endonuclease activities were observed for rnase y .
moreover , as rnase y can bind rna 5 ends , it may compete with rnase j for the same substrate ( see the following ) .
rnase y is involved in the decay of polycistronic infc - rpmi - rplt mrnas encoding the elongation factor if3 and ribosomal proteins l35 and l20 .
when rnase y is absent , at low l20 concentrations , a longer transcript is stabilized .
when processed by rnase y , the transcript is subsequently degraded by rnase j thanks to an entry site for its 53 exonucleolytic activity . the presence / absence of rnase y thus influences the level of translation of if3 , l35 , and l20 .
rnase y via its processing activity is also involved in regulation of the gapa operon and bsrg / sr4 type i toxin / antitoxin system [ 33 , 34 ] .
rnase y depletion increases the half - life of bulk rna levels in b. subtilis . according to two studies , mrna abundance is ,
respectively , increased and decreased for 795 and 309 mrnas or 550 and 350 mrnas .
the proportion of rnase y targets in the different studies is similar ; however , only 263 candidates were common to both studies maybe due to the use of different depletion mutants .
rnase y depletion has diverse effects , including decreased biofilm formation ( due to the stabilization of sinr mrna resulting in the sinr repressor accumulation ) , modifications in folate and amino acid biosynthesis , extracellular polysaccharide synthesis , and an increase in penicillin - binding protein 2a mrna stability .
overall , these studies revealed the important role of rnase y in b. subtilis physiology and metabolism . in s. aureus , the rny ortholog ( aka cvfa )
was discovered as a regulator of virulence genes using silkworm and mouse infection models [ 36 , 37 ] .
rnase y has a transmembrane domain , an rna binding domain ( kh domain ) , and a metal - dependent phosphohydrolase domain ( hd domain ) .
the integrity of the hd domain is required for the rny - dependent phenotypes . as is the case for b. subtilis , rny is not essential in s. aureus , as tested in the nctc8325 and newman strains [ 36 , 39 ] .
a recent microarray study in the newman strain revealed differential expression of about 570 genes between a rny mutant and its corresponding isogenic wild - type strain . in a similar experiment ,
functional classification of the affected genes indicates that the downregulated genes are mostly involved in pathogenicity or proteolysis whereas the upregulated genes are mainly involved in transport and metabolism .
the downregulation of virulence gene expression is linked to processing of the saepqrs operon and expression of the two - component system saes / saer .
rnase y is the key player for the endonucleolytic cleavage of t1 leading to a more stable t2 transcript and resulting in enhanced saers translation .
the threonyl - trna synthetase leader region from b. subtilis expressed in e. coli is processed by rnase e , suggesting that an rnase e functional equivalent exists in b. subtilis . however , no rnase e homolog is present in the b. subtilis genome .
therefore , enzymes having rnase e - like activities ( i.e. , a role in the maturation of 16s/23s rrnas and cleavage of the t - box of threonyl - synthetase ) were searched in b. subtilis leading to the discovery of rnase j1 and rnase j2 ( formerly ykqc and ymfa ) encoded by rnja and rnjb , respectively [ 44 , 45 ] .
as demonstrated by copurification and bacterial double - hybrid techniques , rnase j1 and j2 exhibited strong interactions forming heterodimers and heterotetramers .
these enzymes are bifunctional with endonuclease and 5 to 3 exonuclease activities , this latter property being until recently considered to be restricted to eukaryotes . however , rnase j2 has poor 5 to 3 exonuclease activity compared to the rnase j1 or rnases j1/j2 complex .
the exonuclease activity is 5 monophosphate - end - dependent and single - strand - specific ; it is completely inhibited by triphosphorylated ends [ 47 , 48 ] .
the absence of rnase j2 had no effect on bulk rna level ; however , rnase j1 depletion in the absence of rnase j2 resulted in an increase in total mrna half - life from 2.6 min to 3.6 min .
this increase is smaller in comparison to what was observed for rnase e depletion in e. coli but comparable to what was observed for pnpase deletion in b. subtilis ( see the following ) .
thirty percent of total transcripts are targeted by rnase j1 revealing a wide action of this rnase . in b. subtilis ,
the paradigm for rna decay is an endonucleolytic cleavage by rnase y , giving access to rnases j for 5 to 3 exonucleolytic activity , whereas pnpase performs a 3 to 5 exonucleolytic activity .
as studies were performed on depleted strains but not on a null - mutant , the global role of rnase js could be underestimated as a residual rnase j activity may still be present .
a recent study showed that it is possible to inactivate both rnja and rnjb genes in b. subtilis .
the rnja mutant is viable with a long doubling time ( 76 min instead of 26 min ) with defects in sporulation , competence , and cell morphology , while the rnjb mutant has a growth rate similar to that of the wild - type strain .
results from a saturated transposon mutagenesis suggested that rnja and rnjb genes were essential in s. aureus .
however , linder and colleagues succeeded in deleting both rnja and rnjb . each mutant exhibits poor growth at 42c .
the fact that transposon mutagenesis is carried out at 42c explains the discrepancy between the two studies .
inactivation of the rnase j2 active site by site - directed mutagenesis did not affect cell growth rate .
this finding may indicate that rnase j2 is needed for rnase j1 efficiency but have a minor role in rna processing .
overexpression of rnase j1 can partially compensate the lack of rnase j2 , suggesting that a homodimer rnase j1/j1 could be used in the absence of rnase j2 .
more experiments are needed to explain this compensation . a methodology for sequencing 5 rna ends
specific mrnas are enriched in rnase j mutants , and clear mapping of the 5 mrna ends has led to the identification of rnase j roles in rna processing .
it processes 16s rrna after endonucleolytic cleavage by rnase iii as observed in sinorhizobium meliloti and b. subtilis [ 44 , 53 ] . in s. aureus , the maturation of acpp ( acyl - carrier protein ) mrna exemplifies the role of rnase j1 and j2 . in the absence of rnase j1
, this essential mrna is cleaved by rnase j2 , thus demonstrating the activity of this enzyme in s. aureus .
rnase j is a complex enzyme made of two partners ( j1 and j2 ) forming heteromers ( either dimers or possibly tetramers ) in vivo .
rnase j2 has a narrower role than rnase j1 ; some functions of rnase j2 were observed in the rnase j1 mutant ( e.g. , acp mrna processing ) .
the polynucleotide phosphorylase ( pnpase ) is a 3 to 5 exonuclease with a peculiar mechanism for rna decay ; whereas classical rnases cleave rna molecules by hydrolysis , the pnpase cleaves rnas by phosphorolysis involving an inorganic phosphate and releasing a nucleotide diphosphate .
this contributes to the dual role of pnpase which also acts as a polymerase when the concentration of inorganic phosphate is lower than those of nucleotide diphosphate .
s. aureus encodes three 3 to 5 exonuclease orthologs , pnpase , rnase r , and yham .
the global role of pnpase in rna decay was determined at a genome scale , comparing wild - type and pnpa isogenic strains . while in the wild - type strain about 51% of total transcripts were degraded after five minutes ,
pnpase may be important for the degradation of the large amount of csp ( cold shock protein ) transcripts after a cold shock induction as demonstrated in e. coli .
a recent study showed that s. aureus pnpase also interacts with rnase y to degrade transcripts .
as an example , agr expression is decreased by the absence of rnase y and this effect is suppressed in a pnp mutant .
this phenotype is reversed when pnpase is expressed from an ectopic gene suggesting a direct role of pnpase in this process .
the rna degradosome was discovered during purification of rnase e from e. coli by two independent teams [ 58 , 59 ] .
copurification of rnase e with pnpase provided a clue for the existence of a multiprotein complex involved in mrna degradation . in e. coli ,
the major components of this complex are ( i ) rnase e , ( ii ) pnpase , ( iii ) rna helicase b ( rhlb ) , and ( iv ) the glycolytic enzyme enolase .
thus , rnase e is an endonuclease sensing 5 monophosphate ends and pnpase is a 3 to 5 exonuclease ( see the above ) .
interestingly , rhlb is a dead box helicase belonging to a ubiquitous protein family often possessing rna - dependent atpase activity .
rhlb unwinds rna structures that can block pnpase action , as exemplified by the effect of the repetitive intergenic region ( rep ) on pnpase .
this role has been further supported by the demonstration that the rna degradosome requires atp hydrolysis to degrade rna containing rep .
as the enolase belongs to glycolytic metabolism , it might sense ( i ) the energetic state of the cell or ( ii ) phosphosugar stress .
the existence of an rna degradosome in b. subtilis was proposed with rnase y as the central partner instead of rnase e. protein - protein interactions demonstrated by a bacterial two - hybrid system led to the identification of rnases j1/j2 , pnpase , a rna helicase csha , enolase , and phosphofructokinase , another glycolytic enzyme , as rnase y partners .
phosphofructokinase interacts with major partners of the degradosome ( pnpase , rnase y , and the rnases j1 , j2 ) but also with rnase iii which has not been identified as part of the degradosome .
a similar approach was used to identify the s. aureus degradosome and led to the identification of the same partners , that is , enolase ( sar0832 ) , phosphofructokinase ( sar1777 ) , a dead box rna helicase ( sar2168 ) , pnpase ( sar1250 ) , rnase j1 ( sar1063 ) , rnase j2 ( sar1251 ) , and rnase y ( sar1262 ) , with an additional partner , the rnase rnpa ( see the following ) .
rnpa interacts only with csha interacting itself with enolase , phosphofructokinase and rnase y. s. aureus and b. subtilis degradosome components are similar ; however , the interactions between the different partners seem to be simpler in s. aureus . in b. subtilis
, each partner interacts with at least three other partners whereas , in s. aureus , each partner seems to interact with two only other partners .
until now , few rnases have been studied directly in s. aureus , and putative roles for the others are predicted based on assignments from other organisms . among them , rnase p , a nucleoprotein complex shared by all kingdoms of life , removes 5 extra - nucleotides from trna precursors . where known in bacteria ,
it is composed of a ribozyme ( m1 rna alias rnpb ) , rna possessing catalytic activity , and a protein ( protein c5 alias rnpa ) expressed from the rnpb and rnpa genes , respectively . besides its impact on the maturation of trna 5 ends , rnase p is involved in the maturation of 4.5s rna precursor , polycistronic mrna of histidine operon , tmrna and some rna phages .
a paralog of the rnpa moiety of rnasep was identified in s. aureus sharing only 24% amino acid identity with e. coli rnpa ; all conserved amino acids proved to be essential ( table 1 ) .
interestingly , a recent study searching for new antimicrobial compounds led to a compound interacting with rnpa , suggesting that essential rnases might be effective drug targets .
other rnases such as the 3 to 5 exonuclease rnase r that processes 3 trna ends and the endonuclease rnase z that removes the 3 trna termini are conserved in s. aureus .
potential non - trna targets of rnase z have been searched in e. coli by microarrays ; the amount of more than 150 mrnas had been increased in the rnz mutant as compared to the wild - type isogenic strain , possibly indicating a role of rnase z in processing of a more wide range of rnas than just trnas or indirect effect .
however , so far , nothing is known concerning the role of rnase z in s. aureus . the 5s rrna precursor in bacteria with low gc content is matured by the specific rnase m5 [ 73 , 74 ] .
the ribosomal protein l18 is proposed to alter precursor conformation , stimulating 5s rrna processing , whereas the ribosomal protein l5 inhibits cleavage .
however as for rnase z and r , it has not been studied in s. aureus and its impact remains to be established .
these enzymes perform diverse fundamental cellular processes , including dna recombination , replication and repair , and rna interference .
the family is divided in three subclasses , hi to hiii , which are expressed in b. subtilis from paralog genes rnha , rnhb , and rnhc , respectively . in b. subtilis , only rnases hii and hiii possess rnase h activity and are essential .
even if crystallographic structure of rnase hiii was obtained by diffraction , the rnha , rnhb , and rnhc , genes are also present in s. aureus and await characterization .
a 3 to 5 exonuclease degrading single strand rnas , encoded by the yham gene , was purified from a b. subtilis strain lacking pnpase and rnase r .
the yham deletion alone did not affect growth in the tested conditions nor the bulk mrna half - life ; however , strains lacking yham and either rnase r or pnpase were unable to grow at low temperature .
the s. aureus yham ortholog gene expresses cbf1 which was initially shown to be a dna - binding protein involved in plasmid replication .
purified cbf1 has rnase activity , but to date , its role at a genomic level was not well understood . in e. coli , degradation of short oligonucleotides
b. subtilis lacks an orn ortholog , but the corresponding activity is performed by two paralogs named nanornase a and nanornase b ( encoded by nrna and nrnb , resp . ) , which act together to scavenge and recycle nucleotides for new rna transcripts .
genome sequence analyses indicate the existence of an nrna orthologous gene in s. aureus , while to date , no study concerning this rnase family has been performed .
for instance , rnase iii , rnase j1 , rnase j2 , and rnase y are found in all isolates of the s. aureus species . however , several acquired rnases have been described .
these enzymes are , so far , part of toxin / antitoxin ( ta ) systems .
the two main ta systems are type i ta , in which the antitoxin is a small antisense rna that base - pairs with toxin mrna , and type ii ta , in which the antitoxin is a protein acting on a posttranslational step . several toxins or antitoxins exhibit rnase properties as is the case for the well characterized ta system maze / mazf .
the mazf ribonuclease recognizes a specific sequence that may vary between species . in e. coli
, mazf recognizes the 5 end of aca and cleaves just before the cytosine ( aca with represents the cleavage site ) , whereas in s. aureus , samazf cleaves inside a five - base sequence uacau [ 86 , 87 ] . up to now , three ta systems exhibiting rnase activity were described in s. aureus , samaze / f , sapemi / k , and yefm - yoeb [ 8891 ] . in e. coli ,
expression of mazf causes global mrna degradation leading to reprogramming and growth arrest ; cell death is rescued by maze . however , maze can not rescue cells in the presence of a quorum - sensing - induced pentapeptide that competes with maze and thus cell death is induced .
the quorum - sensing allows communication between bacteria and this pentapeptide acts as a death inductor . in e. coli ,
mazf is involved in the cleavage of ( i ) mrnas at aca sequences in the vicinity of the aug start codon and ( ii ) 16s rrna within the 30s subunit .
modified ribosomes are required for translation initiation of these leaderless mrnas , which are likely involved in stress adaptation . in s. aureus ,
mazf cleaves at uacau which is a relatively abundant sequence , for instance , inside the srap gene , coding for a protein involved in the cell adhesion and thus virulence .
recently , another role of ribonuclease - encoding ta system has been described for sapemi / k .
this plasmid - encoded ta system , in addition to its role in plasmid maintenance , seems to play a global regulatory role in virulence by altering the translation of a large pool of genes .
the last system , yefm - yoeb , has a ribosome - dependent rnase activity .
the toxin binds the a site of the 50s ribosomal subunit and then cleaves the mrna three base pairs after the start codon .
in addition , sayoeb exhibits a ribosome - independent rnase activity in vitro by cleaving free mrna consistent with that previously observed in e. coli .
enzymes without rnase activities , such as rpph and hfq , can be involved in the decay of bulk rna .
rpph , for rna pyrophosphate hydrolase , triggers rna degradation by removing the 5 pyrophosphate of mrna .
the remaining 5 monophosphate rnas are then more efficiently targeted by rnase e. rpph belongs to the nudix ( nucleoside diphosphate linked to x ) protein family , which exhibits phosphohydrolase activity . in e. coli , rpph is responsible for the acceleration of the decay of hundreds of transcripts , demonstrating its importance in rna stability .
the purified e. coli rpph protein did not present any substrate specificity , at least in terms of the 5-end nucleotide .
however , unexpected substrate specificity was recently reported for rpphbs , the b. subtilis rpph ortholog [ 96 , 97 ] .
rpphbs drives pyrophosphate hydrolysis of a synthetic rna when ( i ) at least two and preferably three or more nucleotides are unpaired at the 5 end and ( ii ) if the second nucleotide is a guanosine and the third nucleotide is preferentially a purine .
this observation has been further explained by a rpphbs crystallographic study revealing a binding pocket that fits a guanosine in the second position of substrates . yet
the crystal structure of bdellovibrio bacteriovorus rpph leads to a different interpretation , rpph recognizes the first nucleotide of the sequence .
these results prompted us to inactivate and identify possible substrate specificity in s. aureus ( bonnin and bouloc , unpublished data ) .
based on protein sequence identity ( 38% amino acid identity with rpphbs ) and conserved synteny , we identified saouhsc_01913 as the gene expressing the s aureus rpph ortholog ( rpphsa ) .
total rnas of s. aureus hg003 and hg003 rpph in exponential phase were extracted , sequenced by rna - seq and transcriptomes of these strains were compared using deseq tools .
unexpectedly , very few differences were observed between the two transcriptomes with only four transcripts stabilized in the rpph mutant ( figure 3 ) .
none of them had a guanosine in the second position and they did not share any apparent common features .
these results indicate a minor role of rpphsa in the tested condition possibly due to the presence of a second rpph - like enzyme that could compensate for the absence of rpphsa ( saouhsc_01913 ) .
a key non - rnase player in rna processing and decay is the rna - binding protein hfq
. discovered more than forty years ago in e. coli , hfq was identified as an essential host factor for bacteriophage q . in many bacteria
, hfq promotes activity of regulatory rnas by protecting them against degradation and stimulating pairing with their targets .
consequently , srna - regulated genes can be both posttranscriptionally up- or downregulated and the absence of hfq can generate numerous phenotypes ; for an in - depth review , see . while the first solved hfq crystal structure was that of s. aureus ( hfqsa ) , its function in s. aureus remains unknown .
in contrast to the multiple hfq phenotypes reported for enteric bacteria , the absence of hfq in s. aureus as well as in b. subtilis has no impact on srna - mediated regulation , reviewed in . it is conceivable that hfqsa is poorly or not expressed in the studied strains . in one report , hfqsa deletion in strains where hfq was detected resulted in decreased toxicity and virulence , and over a hundred genes showed differential expression in an hfq - mutant compared to a wild - type strain using microarrays .
the observed discrepancy between the different studies may lie in the fact that the strains used for these experiments are different in some aspects even if they belong to the same lineage , that is , s. aureus nctc8325 .
surprisingly , the hfqsa gene fails to substitute for the hfq of salmonella in srna - mediated regulation .
altogether , these results suggest that hfqsa does not play a central role in posttranscriptional regulation
. further investigation will be necessary to understand the exact role of hfq in s. aureus .
recent studies on the rnase functions in s. aureus indicate that the scheme for rna decay is similar to that in the low g+c content firmicutes model b. subtilis .
for instance , the absence of rnase e , replaced by rnase js and y , is also observed in s. aureus .
rnases are key players of posttranscriptional regulation and therefore are involved in virulence factor regulation . as an example , rnase iii controls the expression of factors involved in cell adhesions or factor involved in immunity escape via the degradation of srna / mrna duplexes . up to now ,
the impacts of only three rnases , that is , rnase iii , y , and js , have been studied on the genome scale .
further studies will be needed to elucidate the precise roles of the other rnases present in s. aureus and their potential effects on virulence gene regulation . | the regulation of rna decay is now widely recognized as having a central role in bacterial adaption to environmental stress . here
we present an overview on the diversity of ribonucleases ( rnases ) and their impact at the posttranscriptional level in the human pathogen staphylococcus aureus .
rnases in prokaryotes have been mainly studied in the two model organisms escherichia coli and bacillus subtilis . based on identified rnases in these two models ,
putative orthologs have been identified in s. aureus .
the main staphylococcal rnases involved in the processing and degradation of the bulk rna are ( i ) endonucleases rnase iii and rnase y and ( ii ) exonucleases rnase j1/j2 and pnpase , having 5 to 3 and 3 to 5 activities , respectively . the diversity and potential roles of each rnase and of hfq and rpph are discussed in the context of recent studies , some of which are based on next - generation sequencing technology . |
partial gonadal dysgenesis ( pgd ) , one of the 46,xy disorders of sex development ( dsd ) , is a rare disorder characterized by sex ambiguity due to variable degrees of testicular dysgenesis in individuals without a syndromic picture who have a normal male karyotype .
the histology of dysgenetic testes may vary from gonads with a few tubular structures and predominance of fibrous tissue to those with mild abnormalities , such as reduction of mean tubular diameter and mean number of germ cells and sertoli cells per tubular profile .
dysgenetic testes may be found bilaterally or may be associated with streak gonads , and the degree of embryonic sertoli and leydig cell dysfunction determines the degree of virilization of the internal and external genitalia .
thus , the genital phenotype may range predominantly from male to female , including cases of marked sex ambiguity [ 35 ] .
pgd was initially considered by many authors as a variant of 46,xy complete gonadal dysgenesis ( cgd ) , which is characterized by bilateral streak gonads and female internal and external genitalia .
however , mutations in sry ( sex determining region y ) gene , which have been described in many cases of xy cgd [ 6 , 7 ] , are rarely seen in pgd [ 810 ] . in recent years , both heterozygous and homozygous mutations in nr5a1 ( nuclear receptor subfamily 5 , group a , member 1 ) gene , which codifies the sf1 ( steroidogenic factor 1 ) protein , have been found in about 15% of patients with pgd [ 1113 ] . there is a high risk of germ cell neoplasia in the streak gonads of these individuals , which may reach 35% ; as a consequence ,
tumors may also arise in the dysgenetic testes , particularly those with marked dysgenesis , which are not located in the scrotum [ 1 , 15 ] .
thus , when the patients are raised as males , preservation of testes must be carefully evaluated .
the main differential diagnosis of pgd is mixed gonadal dysgenesis ( mgd ) , one of the dsd associated with sex chromosome abnormalities .
pgd and mgd share similar gonadal and genital features ; however , in mgd there is mosaicism with a 45,x cell line and one or more lineages with a normal or structurally abnormal y chromosome . as a consequence
, patients with mgd may show clinical features of turner syndrome , including short stature , dysmorphisms , and cardiovascular and renal malformations .
distinguishing pgd from mgd depends on the karyotype , which must include the analysis of a sufficient number of cells to rule out mosaicism with high degree of confidence [ 17 , 18 ] .
most studies on pgd focused on its gonadal and genital and sex hormone features and also on the search for mutations in genes involved in testis differentiation ; however , little is known about other aspects of its clinical picture , including growth , puberty , and possible associated clinical conditions . as a consequence , when diagnosis is made , it is difficult to provide complete information to the parents on prognosis . between 1996 and 1998
we had the opportunity to evaluate 13 patients with pgd , all reared as males , using the same clinical and histopathological criteria .
these patients had also been subject to the same cytogenetic and molecular evaluations [ 2 , 10 ] , and many were followed in our university hospital since then .
the aim of this study was to analyze long - term follow - up of these patients , in order to better establish the prognosis of this condition .
ten of 13 patients previously reported by our group ( scolfaro et al . ) were followed up in the university hospital and were included in this study .
seven of them were regularly followed up in the pediatric endocrinology service and the other three were seen recently by us .
the patients were first seen by us in the 1990s with ages ranging from 14 days to four years ; nine were referred in the first year of life . in scolfaro
's study , the diagnosis of pgd was supported by the findings of ambiguous genitalia , a g - banded 46,xy karyotype with analysis of 1632 cells , negative response of testosterone to hcg test without increase in precursors of testosterone synthesis ( progesterone , dehydroepiandrosterone , and androstenedione ) , low amh levels , presence of a streak gonad in two patients , and , in all cases , at least one gonad with histopathological features compatible with testicular dysgenesis ( abnormal mean tubular diameter , severe tubular hypoplasia , low tubular fertility index , severe germinal hypoplasia , and/or hyperplasia of sertoli cells ) . at the time of the first visit to our service , assessment of luteinizing hormone ( lh ) ,
follicle - stimulating hormone ( fsh ) , progesterone , androstenedione , and dehydroepiandrosterone had been performed by radioimmunoassay in our service and anti - mllerian hormone ( amh ) by an enzyme - linked immunosorbent assay , using antibodies against human recombinant amh in the laboratory of the unit de recherches sur l'endocrinologie du developpement ( inserm ) , montrouge , france .
the human chorionic gonadotropin ( hcg ) test had been performed with measurement of total testosterone levels before and 24 hours after the last of a series of 3 daily intramuscular injections of 2000 iu of hcg ( profasi , serono ) and was considered normal when the patient presented an increase in testosterone level of more than 4.9 nmol / l ( 1.4 ng / ml ) .
maternal age at birth ranged from 19 to 46 years ( mean 28 years ) and paternal age from 16 to 52 years ( mean 31.1 years ) .
within nine full - term gestations , birth weight ranged from 2470 to 3750 g ( mean 3097 g ) and length from 46 to 51.5 cm , with an average of 48.4 cm .
no patients had consanguineous parents and only two had a family history of genital ambiguity : case 2 ( a first cousin once removed ) and case 5 ( maternal aunt and great - aunt ) .
bilateral dysgenetic testes were found in 6/10 cases and dysgenetic testis with contralateral streak was found in two . in the remaining cases
there was unilateral dysgenetic testis ; in one of the cases the contralateral gonad was absent and in the other it had not been biopsied .
all patients were raised as boys and had at least one testis in the scrotum . all had absence of mutations in sry or wt1 ( wilms tumor 1 ) genes .
two of them ( cases 2 and 3 ) had heterozygous nr5a1 mutations , a p.lys38 and a p.ser32asn , respectively . at the time of the last clinical evaluation in our service , patients ' ages ranged from 15.5 to 19.8 years ( mean 18 years ) ; the mean time between first and last clinical evaluation in our service was 17.3 years ( range 13.519.7 years ) .
follow - up data were obtained from the medical files and included neuromotor development , learning disabilities , congenital malformations , acquired diseases , concentrations of follicle - stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , and total testosterone ( determined by electrochemiluminescence ) , semen analysis , and occurrence of testicular neoplasia . the normal pubertal male range for fsh , lh , and testosterone was , respectively , 1.512.4
iu / l , 1.78.6 iu / l , and 2.868.10 ng / ml . semen analysis was done according to the latest world health organization 's guidelines .
growth data were also collected from medical records , as well as data on pubertal development , which was evaluated considering the age of onset of the first signs of puberty and its progression .
patients ' heights were expressed as standard deviation score ( sds ) using reference data from cdc ( nchs / cdc 2000 ) .
final height was considered when growth rate was 1.0 cm / year in patients who had completed pubertal development ( at least tanner stage 4 ) . whenever measurement of biological parents was available
, the target height was calculated as ( maternal height + paternal height + 12.5 cm)/2 6,5 cm .
all patients had normal motor development but three presented learning disabilities of unknown etiology ( which were mild in two cases and moderate in one ) .
five had various associated conditions , including vesicoureteral reflux in two cases and facial dysmorphisms in one patient .
primary hypothyroidism with negative antithyroid antibodies was diagnosed in one of the patients with a nr5a1 mutation when he was 12 years old ( case 2 ) ; hearing loss due to middle ear infections , psychiatric problems , and obesity were also observed ( one case each ) ( table 2 ) .
results of measurements of gonadotropins ( fsh and lh ) and testosterone during follow - up were also analyzed .
data on the measurement of gonadotropins at the end of the prepubertal period , between ten and 12 years , were available for six patients ( cases 2 , 4 , 6 , 7 , 8 , and 10 ) .
fsh levels were in the upper limit of the normal male pubertal range or higher in 3/6 patients ( cases 2 , 6 , and 7 ) , while lh levels were normal in all cases ( figures 1 and 2 ) . at the last hormone evaluation ( mean 16.5 years ; range 12.620.5 years ) fsh concentrations were high in 4/10 patients ( cases 2 , 3 , 7 , and 9 ) , in the upper limit in four ( 4 , 5 , 6 , and 10 ) , and normal in two ( 1 and 8) ( figure 1 ) . in turn ,
lh concentrations were high in 4/10 patients ( cases 3 , 6 , 7 , and 9 ) , in the upper limit in one ( case 1 ) , and normal in five ( 2 , 4 , 5 , 8 , and 10 ) ( figure 2 ) .
the levels remained normal in seven cases , remained at the low limit of normality in one , and decreased in one patient ( case 10 ) , who received androgen replacement therapy ( figure 3 ) .
nine patients had reached final height , which ranged from 1.57 to 0.80 sds , and in two of the eight cases for whom information on parental height was available it was lower than their genetically predicted range ( target height 6.5 cm ) .
one patient ( case 4 ) was still growing , with normal velocity and within the parental target range ( table 3 ) .
eight patients had complete and spontaneous pubertal development ( tanner stage 4 ) , one was at stage g4p4 at 15.5 years ( case 4 ) , and another ( case 10 ) received androgen therapy at 17.6 years , when pubertal development was g3p3 ; a few months later he reached stage g4p4 .
the seven patients who were regularly followed up in our pediatric endocrinology service had normal progression of puberty ; most had low testicular volume .
three patients had a sperm count ; all had severe oligozoospermia and low motility , and two had also abnormal semen viscosity .
in dsd , sex assignment should be based on a precise diagnosis of the condition 's underlying etiology . together with genital appearance and surgical options , this will allow the establishment of a prognosis on the need for lifelong replacement therapy , potential fertility , and malignancy risk and also possible associated conditions . in the case of pgd , however , prognosis is not yet clearly established .
our results showed that all patients had normal neuromotor development , that most had normal growth , and that there was no consistent pattern of associated conditions .
however , though learning disabilities are usually not a feature of dsd , in this sample it was observed in a significant proportion of cases ( almost one - third ) , including a patient with moderate difficulties .
although this association may be casual , one may also consider the possibility that both conditions , testicular dysgenesis and cognitive impairment , have the same origin .
interesting findings regarding our two patients bearing a nr5a1 mutation are hypothyroidism in one of them and schizophrenia in the other . acquired primary
hypothyroidism has not been described as a feature of patients with pgd , with or without nr5a1 mutations .
the low prevalence of these conditions in young people aged 1118 years ( 0.113% ) and the fact that it is even rarer in males , with a 1 : 2.8 male to female ratio , make this finding noteworthy , even though expression of nr5a1 mrna in thyroid gland is very low . on the other hand
, there is a recent publication of two women with mutations in this gene who had psychiatric symptoms .
the absence of testicular tumors in this sample demonstrates that maintenance of testes in the labioscrotal folds of patients reared as males is a relatively safe procedure , at least until the end of puberty .
though positive results from hcg stimulation tests in infancy or childhood were obtained in 4/9 patients , spontaneous pubertal development occurred in all cases .
pubertal delay was not observed , and in 9/10 cases there was normal progression of puberty , which strongly indicates that there is a good prognosis regarding spontaneous puberty in pgd patients reared as males when at least one testis may be kept in the scrotum . in fact , in 7/9 cases testosterone levels were in the normal range during adolescence , though a progressive rise of lh in half of the cases raises the possibility that leydig cell dysfunction may become evident in adulthood . on the other hand , high levels of fsh in most patients ,
sometimes observed early in adolescence , indicated that reproductive function was impaired , which could be shown in those patients whose sperm count was obtained .
some patients with nr5a1 mutations and 46,xy pgd have been shown to have normal testosterone production in adolescence inducing spontaneous virilization [ 2426 ] , though follow - up indicated a progressive gonadal failure with elevated fsh in such cases .
a similar picture was observed in our two patients with nr5a1 mutations ( cases 2 and 3 ) and also in the other eight cases without mutations in this gene .
however , to the best of our knowledge , no other studies on long - term follow - up of patients with pgd reared as males are available to allow comparison with our sample .
patients with pgd raised as males who have at least one testis in the labioscrotal region have a good prognosis for growth and spontaneous pubertal development but not for spontaneous fertility .
though additional studies are still required , our results also indicated that management of individuals with this condition should include screening for learning disabilities . | background / aims .
studies on 46,xy partial gonadal dysgenesis ( pgd ) have focused on molecular , gonadal , genital , and hormone features ; little is known about follow - up .
our aim was to analyze long - term outcomes of pgd .
methods .
retrospective longitudinal study conducted at a reference service in brazil .
ten patients were first evaluated in the 1990s and followed up until the 2010s ; follow - up ranged from 13.5 to 19.7 years .
all were reared as males and had at least one scrotal testis ; two bore nr5a1 mutations .
main outcomes were : associated conditions , pubertal development , and growth
. results .
all patients had normal motor development but three presented cognitive impairment ; five had various associated conditions . at the end of the prepubertal period ,
fsh was high or high - normal in 3/6 patients ; lh was normal in all . at the last evaluation ,
fsh was high or high - normal in 8/10 ; lh was high or high - normal in 5/10 ; testosterone was decreased in one .
final height in nine cases ranged from 1.57 to 0.80 sds .
all had spontaneous puberty ; only one needed androgen therapy .
conclusions .
there is good prognosis for growth and spontaneous pubertal development but not for fertility . though additional studies are required , screening for learning disabilities
is advisable . |
the ingestion of instruments or materials used in various dental procedures may occur accidentally in dentistry .
foreign bodies vary in size and shape and range from burs , posts , root pieces , teeth , orthodontic brackets , endodontic instruments ( files , broaches ) , impression materials , implant components , and restorations [ 1 , 2 ] . some objects are made of materials that lack radiopacity , which makes them impossible to identify and locate ; diagnostic bronchoscopy / endoscopy or computed tomography for localization is then required [ 3 , 4 ] .
foreign body ingestion is a commonly seen accident in emergencies , usually in children ( 80% ) , elderly , mentally impaired , or alcoholic individuals ; whereas it may occur intentionally in prisoners or psychiatric patients [ 58 ] .
rarely foreign body ingestion results in serious complications , such as intestinal perforation , bleeding , obstructions or impactions .
the patient position in the dental chair as well as the patient s medical history is important in preventing serious complications .
usually instruments entered the gi tract pass asymptomatically and atraumatically within 2 days to 4 weeks .
however there are many potential sites for impactions , among them the ileocecal valve is the most common site .
approximately seventy five percent of perforations occur at or near this site or at the rectosigmoid junction [ 6 , 9 ] . only 1 percent of ingested foreign bodies cause an intestinal obstruction requiring surgery [ 10 , 11].the aim of this article is to document implant screwdriver ingestion along with its consequences and to offer guidance for prevention and management .
a 69 year - old male patient presented with a chief complaint of missing upper left second molar and mandibular left first molar teeth ; for which implant placement was planned .
implant placement was done successfully and patient was recalled after 4 months for second stage surgery . during second stage surgery while placing a gingival former on the implant in the region of upper left second molar , the screw driver accidentally slipped from the operator s hand .
the operator made an unsuccessful attempt to retrieve the instrument by making the patient spit .
the case was attended by a gastroenterologist and immediately pa chest and abdominal radiograph were taken confirming the screwdriver to be in stomach ( fig 1 ) .
an endoscopy was planned , which was performed under local anesthesia but the instrument could not be retrieved .
patient was kept under observation and advised to consume fiber - rich food to enhance intestinal motility and to regularly examine his stools .
on the second day again radiographs were repeated and this time screwdriver was located in the intestine ( fig 2 ) ; the very same day the patient passed the ingested screwdriver in his stool ( fig 3 ) .
it must be emphasized that preventing complications of foreign body ingestion and aspiration is of great importance .
this applies to the identification of at risk patients by means of comprehensive clinical examination and thorough patient history taking . in our case
the patient was senile and apprehensive ; there was difficulty in screwing the gingival former in the maxillary posterior region of upper left second molar , from where the instrument slipped . as the patient was in the supine position direct access to oropharynx was eminent
although rare , ingestion / inhalation of a foreign body may lead to serious complications ; therefore , immediate radiographic evaluation is a must including pa / lateral chest , lateral neck and abdominal radiographs .
usually , most ingested foreign bodies are expelled in stools without causing any complications in several days to several weeks ; for which conservative management of serial radiographs and fiber rich diet is an initial protocol [ 14 , 15 ] .
if serial radiographs depict the same location of foreign body or there is a sign of abdominal tenderness or hemorrhage then most likely there is retention , obstruction or intestinal perforation for which an invasive procedure is indicated and has to be done as early as possible through gastroscopy for its retrieval .
the mallampati score is assessed by asking the patient ( in a sitting posture ) to open his / her mouth and protrude the tongue as much as possible .the anatomy of the oral cavity is visualized ; specifically , whether the base of the uvula , faucial pillar and soft palate are visible .
depending on whether the tongue is maximally protruded and/or the patient asked to phonate , the scoring may vary .
class iv : only hard palate visible it is emphasized that all practitioners should take preventive steps during treatment like placement of gauze screen across the oropharynx , tying ligature ( dental floss ) to instruments , adjusting chair position ( sitting position prevents aspiration / ingestion and supine position increases the risk of swallowing ) and should be able to manage the patients in emergency situations . sending a patient home in the belief that a foreign body that slipped into the oropharynx has been swallowed and
should a foreign body be ingested / aspirated , the patient must be examined clinically and radiographically ; diagnosis must be performed immediately by a specialist and patient and his family members must be informed .
class iv : only hard palate visible it is emphasized that all practitioners should take preventive steps during treatment like placement of gauze screen across the oropharynx , tying ligature ( dental floss ) to instruments , adjusting chair position ( sitting position prevents aspiration / ingestion and supine position increases the risk of swallowing ) and should be able to manage the patients in emergency situations . sending a patient home in the belief that a foreign body that slipped into the oropharynx has been swallowed and will pass through the gut may be associated with complications and lead to litigation .
should a foreign body be ingested / aspirated , the patient must be examined clinically and radiographically ; diagnosis must be performed immediately by a specialist and patient and his family members must be informed .
this case report illustrates a case of foreign body ingestion in an anxious senile patient having mallampati class 1 , during implant restoration in the posterior maxilla ; proper preventive measures may prevent such complications . | one of the complications during a routine dental implant placement is accidental ingestion of the implant instruments , which can happen when proper precautions are not taken .
appropriate radiographs should be taken to locate the correct position of foreign body ; usually the foreign body passes asymptomatically from gastrointestinal tract but sometimes it may lead to intestinal obstruction , perforations and impactions .
the aim of this article is to report accidental ingestion of 19 mm long screw driver by a senile patient . |
clinical studies of the efficacy of fingolimod ( fty ) investigated fty as first line medication in relapsing - remitted multiple sclerosis ( rrms ) in comparison to placebo or once weekly intramuscular interferon--1a showing its superiority regarding relapse rate , progression of disability and end points of mri .
while the united states food and drug administration ( fda ) and other countries registered fty as first - line therapy in rrms , fty was registered in europe by the european medicines agency ( ema ) only as a second line therapy in rrms .
so far the only registered second line medication in rrms in europe had been natalizumab ( n ) .
clinical trials with n showed its superiority in comparison with placebo and with ongoing interferon therapy alone .
this is the first study comparing clinical efficacy of fty as a second line drug in rrms with n in a real - life cohort .
this is an observational cohort - study using health data routinely collected in outpatient neurology practices throughout germany who are members of the neurotransconcept network .
sex , age , relapses , edss and medication are documented digitally in - time during clinical visits at least once within 3-month periods in all patients with ms in the participating practices .
all neurologists are trained to document these data in a standardized way in the digital data source and are certified edss - raters .
this data acquisition protocol is approved by the ethical committee of the bavarian medical board ( bayerische landesrztekammer , 14.06.2012 ) .
the data of the participating neurology practices are pooled anonymously to form the database of the study .
this cohort analysis includes all rrms patients starting on either fty or n in the two years between 1 february 2009 and 31 january 2011 and who completed at least 12 months of treatment by 31 january 2012 . the decision to change from first - line to second line therapy and the choice of treatment were at the discretion of the treating neurologist and the patient in accordance with the label of the two second - line therapy drugs in germany .
the primary outcome parameters were progression , as measured by worsening in the edss and relapse rate during the first 12 months of treatment with either n or fty . differences between treatment groups for demographic parameters , edss , relapse rate were tested with a t test . kaplan
meyer survival curves were calculated analyzing the proportion of patients without progression of edss , new relapses as well as for the composite parameter freedom of clinical disease activity , combining lack of relapse and progression .
progression of edss was defined as an increase of the edss score by one point , if baseline edss was smaller than 5.5 , or 0.5 points if baseline edss was equal or higher than 5.5 .
two hundred and thirty - seven patients starting on n and 190 patients on fty were identified and included .
their demographic characteristics are shown in table 1.table 1demographic data of patient groupsparameternatalizumabfingolimod
p valuenumber of patients fully documented237190mean age ( years)37.39 9.640.47 8.710.0007sex female / male percentage69.62/30.3867.89/32.110.7028mean duration of ms ( years)9.15 6.99.89 6.90.3577mean edss at baseline3.3 1.82.3 1.6<0.0001mean annualized relapse rate0.42 0.840.34 0.690.3083mean relapses 3 months prior to treatment0.56 0.80.25 0.6<0.0001 demographic data of patient groups patients with fty had a significantly lower edss at baseline and less relapses during the three months prior to treatment than n , while the annualized relapse rate did not differ significantly between groups .
mean edss scores in both treatment groups showed a slight tendency to improve , without statistically significant change over time within groups or difference of change between groups ( fig .
1mean edss score and relapse rate in three months intervals of therapy mean edss score and relapse rate in three months intervals of therapy mean relapse rate decreased drastically in both treatment groups within three months of therapy in a similar degree and remained on a low level over the documented observation time ( fig . 1 ) .
both treatment groups saw a similar proportion of patients with unchanged and improved edss ( 80.53 % in fty , 79.32 % in n ) and deteriorated edss .
the higher proportion of patients free of clinical disease activity in fty was not statistically significant compared to n ( fig .
2number and percentages of patients in edss strata and free of clinical disease activity number and percentages of patients in edss strata and free of clinical disease activity both treatments achieved a similar proportion of patients being relapse free ( p value 0.35 ) , progression free ( chi square p value 0.18 ) or relapse and progression free ( p value 0.05 ) at 12 months ( fig .
3kaplan meyer survival curves of patients free of relapse , free of progression , with freedom of clinical disease activity over one year of therapy with n or fty kaplan meyer survival curves of patients free of relapse , free of progression , with freedom of clinical disease activity over one year of therapy with n or fty to address potential effects of different baseline demographics of both cohorts , adjusted linear regression analyses have been performed .
all clinical and demographic baseline parameters revealed low and non - significant correlation coefficients for the outcome parameter edss progression ( proportion of patients without progression ) . regarding the outcome parameter relapse activity between months 9 and 12 , age , edss at baseline and number of relapses at baseline revealed weak but significant correlations ( see table 2 ) .
these weak correlations account for 3.2 % of the variance at most.table 2spearman correlation coefficientsparameteragesexedss baselinerelapse rate baselinetherapy fty vs. n12 months
r value
r value
r value
r value
r valueedss progression0.0380.0460.0170.0170.065relapse probability0.149*0.0100.136*0.179 * 0.046 * p < 0.05 spearman correlation coefficients to balance observed covariates between subjects from this observational study we employed the propensity score method in addition . in brief ,
propensity score stratification was performed according to the method published by rosenbaum and rubin .
the same baseline characteristics ( age , sex , treatment group , relapses at baseline , edss score at baseline ) , as considered in the adjusted regression model , were also considered in the propensity score stratification method . for
the results of the propensity score regression analysis ( see table 3 ) are fully in line with the adjusted linear regression model already presented in the paper . no significant effect of baseline differences on clinical parameters , like relapses and progression ,
this is not surprising as this fact has already been stated by senn et al .
.table 3propensity score method to analyze influence of baseline characteristicsodds ratio95 % ci
p valuesummary of estimated odds ratios for relapses ps regression ( continuous)1.39(0.84 , 2.29)0.20summary of estimated odds ratios for edss progression ps regression ( continuous)0.74(0.41 , 1.34)0.32summary of estimate odds ratios for relapses or edss progression ps regression ( continuous)0.94(0.60 , 1.47)0.78 propensity score method to analyze influence of baseline characteristics
both primary outcome parameters edss and quarterly relapse rate stabilized and improved , respectively , within three months after initiating treatment with fty or n to a similar extent .
the differences in baseline clinical and demographic parameters between treatment groups in this real - life dataset reflect the change in attitude of neurologists and ms patients to optimize therapy at an earlier stage with a lower disability and only single relapses , if a safe and efficient therapeutic option is available .
anyhow , these differences did not influence comparability of the two strata as shown by regression analysis and propensity scores .
these data show that fty and n positively influence the course of rrms regarding degree of stabilization of edss and reduction of relapses at a similar rate .
both drugs achieve freedom of clinical disease activity in about two - thirds of patients whose disease activity had not been sufficiently controlled under first - line medication .
longer observation periods with higher patient numbers will show whether this trend toward improvement of edss over 12 months in both treatment groups is real and which patient strata will benefit most .
these data underline the importance of an early change in therapy of rrms if medication with interferons or glatirameracetate does not achieve sufficient control of disease activity . | although fingolimod is registered as a second - line drug in relapsing - remittend multiple sclerosis ( rrms ) in europe there are no clinical studies available comparing fingolimod ( fty ) and natalizumab ( n ) .
this observational cohort - study used health data routinely collected in outpatient neurology practices throughout germany completing a treatment period of 12 months included 237 patients starting on n and 190 patients on fty because of failure of the first - line treatment .
mean relapse rate drastically decreased in both treatment groups within three months of therapy in a similar degree and remained on a low level .
both treatment groups saw a similar proportion of patients with unchanged and improved edss ( 80.53 % in fty , 79.32 % in n ) .
there was no statistically significant difference between the proportion of patients being relapse free ( 75.79 % in fty , 71.73 % in n ) , progression free ( 87.39 % in fty , 82.70 % in n ) or relapse and progression free ( 71.05 % in fty , 62.03 % in n ) at 12 months in both strata .
clinical efficacy of fty and n in rrms second - line - therapy was similar during the first 12 months of treatment . |
spinal epidural angiolipomas are uncommon , benign tumors accounting for 0.041.2% of spinal axis tumors and 23% of extradural spinal tumors .
all angiolipomas show iso- or hyperintensity on t1-weighted images and hyperintensity on t2-weighted images of magnetic resonance imaging ( mri ) and most lesions enhance with gadolinium administration .
a 56-year - old male presented with history of numbness below the chest of 3 months duration and heaviness in bilateral lower limbs of 2 months duration and difficulty in walking for 2 months . on clinical examination , multiple subcutaneous lipomas were present .
there was decreased sensation below d5 dermatome and spasticity in both lower limbs with hyperreflexia .
mri of dorsal spine revealed a sausage - shaped homogenous lesion at d4 - 5 level .
it was showing hypointensity on t1-weighted images and hyperintensity on t2-weighted images of mri [ figure 1a c ] .
( a ) t1-weighted image of mri dorsal spine showing hypointense epidural lesion at d4 - 5 level , compressing the spinal cord ; ( b ) t2-weighted images of mri dorsal spine showing hyperintense epidural lesion ; ( c ) t2-weighted mri axial images demonstrating severe cord compression d4 - 6 laminectomy was done and a reddish vascular epidural lesion of size 2 1 cms was seen compressing the cord [ figure 2 ] .
intraoperative photograph showing epidural angiolipoma pathological examination of the resected lesion showed mature adipose tissue and abundant vascular channels , the caliber of which was variable , ranging from nearly capillary size to cavernous , but mostly several times larger than the fat cells [ figure 3 ]
histopathology of angiolipoma showing admixture of mature adipose tissue and vascular elements ( h and e , 200 ) postoperative course was uneventful .
paraparesis gradually improved . neurological examination at 60 months follow - up revealed full power in the legs with normal tone and no hyperreflexia .
a 56-year - old male presented with history of numbness below the chest of 3 months duration and heaviness in bilateral lower limbs of 2 months duration and difficulty in walking for 2 months . on clinical examination , multiple subcutaneous lipomas were present .
there was decreased sensation below d5 dermatome and spasticity in both lower limbs with hyperreflexia .
mri of dorsal spine revealed a sausage - shaped homogenous lesion at d4 - 5 level .
it was showing hypointensity on t1-weighted images and hyperintensity on t2-weighted images of mri [ figure 1a c ] .
( a ) t1-weighted image of mri dorsal spine showing hypointense epidural lesion at d4 - 5 level , compressing the spinal cord ; ( b ) t2-weighted images of mri dorsal spine showing hyperintense epidural lesion ; ( c ) t2-weighted mri axial images demonstrating severe cord compression
d4 - 6 laminectomy was done and a reddish vascular epidural lesion of size 2 1 cms was seen compressing the cord [ figure 2 ] .
pathological examination of the resected lesion showed mature adipose tissue and abundant vascular channels , the caliber of which was variable , ranging from nearly capillary size to cavernous , but mostly several times larger than the fat cells [ figure 3 ] .
histopathology of angiolipoma showing admixture of mature adipose tissue and vascular elements ( h and e , 200 )
paraparesis gradually improved . neurological examination at 60 months follow - up revealed full power in the legs with normal tone and no hyperreflexia .
spinal epidural angiolipomas are rare , benign tumors , with only 128 cases reported in the literature till now.[12612 ] the most common location of angiolipomas is in the soft tissues of the extremities , trunk , or neck .
howard and helwig established angiolipoma as a clinicopathological entity containing vascular and mature adipose elements .
location in spinal canal is rare and the commonest location in spinal canal is in dorsal epidural region .
spinal epidural angiolipomas commonly occur in adults in the fourth and fifth decades and have female preponderance .
angiolipomas are iso- or hyperintense on t1-weighted images and variable in t2-weighted images of mri , but usually hyperintense .
the degree of central hypointensity on t1-weighted images is predictive of the degree of vascularity . in the current case ,
the non - infiltrating type of spinal angiolipoma is more common and is usually well encapsulated and generally arises from the posterior epidural space .
spinal infiltrating angiolipomas are generally located in the anterior epidural space and can invade the vertebral body .
the common clinical presentations of spinal epidural angiolipomas are progressive paraparesis , back pain without radiculopathy , lower extremity sensory changes , and hyperreflexia .
pregnancy can aggravate the symptoms probably because of impaired spinal venous drainage or hormonal changes .
non - infiltrating angiolipomas are well defined and separate from the surrounding tissue and can usually be easily removed by laminectomy . in case of infiltrating spinal angiolipomas , wider
most of the patients have good postoperative outcome after surgical excision of spinal angiolipomas in spite of severe preoperative neurological deterioration .
adjuvant radiation has not been recommended to patients with this benign pathological entity , since the prognosis is very good even in the infiltrating variety . | spinal epidural angiolipomas are rare , benign tumors composed of mature lipocytes admixed with abnormal blood vessels
. only 128 cases of spinal epidural angiolipomas have been reported in literature till now .
spinal angiolipomas are predominantly located in the mid - thoracic region .
we report a case of dorsal epidural angiolipoma in a 56-year - old male who presented with paraparesis and was diagnosed to have d4 - 5 epidural angiolipoma .
total surgical excision of the epidural angiolipoma was done and his paraparesis gradually improved . |
advances in three - dimensional imaging resulted in a better understanding of the craniofacial structures .
one of the modern techniques , cone beam computed tomography ( cbct ) , which was invented for angiography in 1982 , was then used for maxillofacial imaging .
in contrast to the common cephalometric radiographies , cbct produced images with the actual anatomic size .
the reconstructed three - dimensional images showed the cranium in all angles and views and provided helpful information on the teeth and other structures for diagnostic and descriptive purposes .
cbct has many advantages over conventional ct , including a lower radiation dose , better image quality ( less than a millimeter resolution ) , lower price , less physical space , and a better capability of restoration . regarding the bone structures , the effect on the type of the bone
is recognized on its tolerance to the imposed forces , and it is stated that the low quality of the bone is related to higher complication after dental treatments .
fanuscu and chang state that the bone mass , structure , and characteristics are effective factors in bone quality .
therefore , it is necessary for the clinician to have appropriate knowledge of the bone quality , prior to any treatment .
ct is one of the medical imaging methods that is useful for acquiring information from the structure , as also the density of the tissue .
accordingly , based on the hounsfield unit , the density of the bone is in direct relationship with the radiation attenuation coefficient of the tissue . due to its higher radiation dose , ct is not applicable for evaluating the density of the alveolar bone ; for instance , in orthodontic treatments , the patient may need several ct images in several months .
application of cbct in dentistry is rapidly growing and it seems that evaluating the quality and quantity of the bones by using the information acquired from cbct will be practical . although studies have been done on the accuracy of cbct in comparison with intraoral radiography , panoramic tomography , and conventional ct , yet few studies have dealt with the precision of computation of bone density and the effect of the position of the object , that is , the position of the patient in different cbct units , on the acquired density . in 2011 , nackaert , et al . evaluated the variety of densities in cbct images in comparison to multislice ct , based on the hounsfield number .
the results indicated that evaluation of the density in multislice ct images showed a stable hounsfield number , but the quantity of density was variable in the cbct images . in 2011 , chang , et al . evaluated the density changes on all sides of the tooth during orthodontia , by use of cbct
the findings indicated that cbct is a suitable method of evaluating the density changes of the bone in the teeth under orthodontic treatment . in 2009 , nomura ,
et al . investigated the validity of cbct - voxel values in evaluating the bone mineral density ( bmd ) .
the findings of that study indicated that there was a strong relationship between the cbct - voxel values and the hounsfield number in the multislice ct . in 2008 , lagravere , et al .
investigated the effect of the object position on density , in cbct ( new tom 3 g ) , based on the hounsfield number .
the results demonstrated that there was no statistically meaningful relationship between the object position in cbct and the effect on the measured density .
therefore , considering the limitations of the present study and availability of different types of cbct units , we decided to investigate the precision of computation and the effect of object location on the density value in three systems of cbct ( proma 3d , galileos 3d , and new tom 3 g ) in comparison to the multislice ct , as the gold standard .
in an experimental study done in the torabinejad dental research center and department of oral and maxillofacial radiology , school of dentistry , esfahan university of medical science , esfahan , iran , three cylindrical samples with different densities , including polyethylene ( pe ) ( 914/0 = g cm ) , polyamide ( pa ) ( 115/1 = g cm ) , and polyvinyl chloride ( pvc ) ( 433/1 = g cm ) were made with 20 mm height and 7.47 diameter .
in addition , three bone pieces harvested from a cadaver , including the anterior mandibular bone from the central teeth position , posterior mandibular bone from the premolar teeth position , and posterior mandibular bone from the molar teeth position all with 10 mm width , were examined . for each sample , a thin plastic rectangular - shaped cylinder ,
10 cm in length , 8 cm in width , and 4.5 cm in depth , filled with water was prepared and located in another thin plastic rectangular - shaped cylinder , 12 cm in length , 9.5 cm in width , and 7 cm in depth , filled with oil , to simulate the x radiation absorption of the soft tissues . in order to determine the position of the samples , two perpendicular lines were drawn on the floor of the smaller cylinder in such a way that their conjunction was on the center of the cylinder .
position 1 : on the transversal line , 2 cm anterior to the center of the interior box .
position 2 : on the right side of the box , 1 cm posterior to the center of the longitudinal line on the right side .
position 3 : on the left side , 1 cm anterior to the center of the longitudinal line on the left side of the box [ figure 1 ] .
a view of the prepared box and positions of material on its floor each of the sample objects was located in all positions in each of the units and they were exposed to radiation : galileos 3d ( germany , kvp = 85 , mas = 28 ) .
newtom 3 g ( italy , kvp = 110 , mas = 1.25 ) , proma 3d ( finland , kvp = 84 , mas = 16 ) , and multislice ct ( germany , 16 slice , kvp = 85 , mas = 100 ) .
subsequently , three cross - sectional slices were prepared of each sample ( with 1 mm width and 1 mm distance ) .
five millimeters of the top and 5 mm of the bottom of each sample were ignored in computations and only 10 mm of the central part of each sample was considered for computations of density .
two lines were drawn on the image acquired from each cross - sectional slice , to divide it into four equal parts .
the density of each of the four parts and the central point was computed . for each slice
five values and for each sample 15 values were computed [ figure 2 ] . for bone samples ,
five cross - sectional slices were prepared ( with 1 mm width and 1 mm distance ) .
the central point was determined on the buccal cortex and 2 mm above it , as the points of computing the density , in such a way that for each slice two values and for each sample 10 values were computed .
the computed method for evaluation of density in each cross - sectional slice this process was performed for each sample in all three cbct units , multislice ct unit , and all the positions .
( a ) cbct images of cylindrical samples and bone pieces in galileos 3d ( b ) newtom 3 g ( c ) promax 3d then the density values acquired from all samples were converted into hounsfield numbers by use of the regression equation of each unit , for statistical purposes , and then a single variable t analysis was performed in the spss ( 9.01 version ) .
the regression equation of each unit was : galileos 3dy ( hounsfield unit ) = 2152.9 + 1.694 ( density ) proma 3dy ( hounsfield unit ) = 358.99 + 1.236 ( density ) newtom 3gy ( hounsfield unit ) = 146.97 + 1.316 ( density ) to compute the regression equation related to each unit , the average of the density values of the polyviny l chloride ( pvc ) in position 1 of the central slice was compared with the hounsfield number computed in the same position and the same slice in the ct unit .
the regression equation was computed for converting the density values in each unit to a hounsfield number .
in our study , the densities obtained for each of the materials and bone pieces in each position and for each unit were entered onto the check list , to be compared with the multislice ct .
then the obtained results were compared with the gold standard through a single variable t test . in table 1 , the average density of each sample , with its position changes in the galileos 3d unit is presented . the average density of each sample with its position in galileos 3d in all cases , a p value of < 0.05 was considered significant .
therefore , in all cases , the average density showed a statistically meaningful difference in the reference value , and a change in position affected the density value .
the results of the effect of the change in position on the density in proma 3d are demonstrated in table 2 . the average density of each sample with its position in promax 3d the results indicate statistically meaningful differences of the test in all cases in a way that the resulting average density changes are evident with the changes in position .
the results obtained from newtom 3 g are presented in table 3 . in all cases
( except for pe in position 1 and the molar sample in position 1 ) the average density has a statistically meaningful difference in the reference value and the p value < 0.05 , and the change in position affects the quantity of density .
the average density of each sample with its position in newtom 3 g the findings of the surveys revealed that in galileos 3d and proma 3d units , the density had a statistically significant difference from the reference value , therefore , these two units were not reliable for determining the density . on the other hand , in newtom 3 g ,
the p value is higher than 0.05 , and therefore , it is not significant . consequently , the average density in this unit does not have a statistically meaningful difference in the reference value and this unit is reliable for determining the density [ table 4 ] . the average density in every type of cone beam computed tomography
the clinical success of dental implants is affected by the quality and quantity of the bone .
a true understanding of the quality of the bone is very helpful before implantation . it can help the clinician to predict the result of the treatments and provide the patient with very good treatment
lekholm and zarb classified the bone density in terms of radiography into four types , based on the density of the cortical bone in contrast to the trabecular bone .
mish considers that the density of the bone is related to clinical stiffness of the bone , which is visible in drilling the bone before implantation . in a new method , johansson and strid
have introduced cutting resistance before implantation as a method of evaluating the bone . on the other hand , in evaluating the density of the bone ,
although these techniques demonstrate the reliable quality of the bone , they are not practical methods for surgeons and clinicians , in terms of clinical considerations .
there is the possibility of evaluating the density of the bone in ct and this quantity is shown with the hu number , which is reliable and can be used as the gold standard for determining density , but regarding the high radiation dose in ct , finding an appropriate substitute for it can be highly helpful .
regarding the increasing application of cbct in dentistry , evaluating the quality of the bone may be practically possible .
in each cbct unit , computing density is possible , but the reliability of the obtained values and the effect of the change in the position of the patient , or in fact , the position of the area to be observed in the unit , on the quantity of density have been always questioned . for instance , zou h considers that cbct is a helpful tool for evaluating density , but hua y maintains that cbct is not reliable for this purpose . in the present study
the effect of the position of the object on the density value was investigated in cbct in comparison to multislice ct .
the findings indicated that in contrast to the values obtained from multislice ct , only the values obtained from the newtom 3 g unit did not have statistically meaningful differences , and therefore , it was reliable
. on the other hand changing position affected the density value in all three units in comparison to the multislice ct , which was in accordance with the findings of nackaert , et al . , who investigated the effect of changing position on the density value in five cbct units .
this demonstrated the stability of the hounsfield number in the multislice ct and the change of density value due to the change of position in all cbcts .
in addition , the findings of the present study were in contrast to that of the study of lagravere mo , in 2008 , on the newtom 3 g unit .
in that study , no finding demonstrated a meaningful relationship between the position of the object in cbct and its density value . in that study two
software systems were used in order to determine the linear relationship between the density and the hounsfield number , but in the present study , the regression equation acquired from the previous studies and the present study was applied for converting the density values obtained from each unit , because it was not possible to access such software systems , to download them from the internet . in justifying the effect of the object position on density , we can refer on the one hand to the cone beam effect of the radiation in cbct , which resulted in more noise , artifact , and distortion of the environment , and on the other hand to a high quantity of scatter rays and difference in homogeneity of the x - ray ( quantum mottle ) , which resulted in lack of uniformity in the radiated ray as well as the absorbed ray . in the study conducted by chang , et al .
, the density change on all sides of the teeth , during orthodontia , was evaluated using cbct and the findings confirmed that cbct was reliable and useful for evaluating such changes . in this study , although cbct was able to show the changes of density in a given position , the reliability of the acquired values in comparison to the gold standard values was not studied and only the density values in a given position were evaluated during the time . in the present study , the change in position had a significant effect on the density value and it seemed that in order to evaluate the density of a given area it was necessary to provide a repeatable position to eliminate the effect of the position on the density value .
for all three types of cbct units , the findings indicated that regardless of the change in position in evaluating the density values , only the newtom 3 g type of cbct units were reliable .
however , even in this type of unit , the change in position affected the density values .
the results of the present study demonstrated that regardless of the method of study and data collection , a correct relationship between the values obtained from the cbct units and the hounsfield numbers obtained from the multislice ct was of great importance .
naitoh , in his study , also mentioned this fact and stated that the cbct values were not absolute , and using the regression line was useful in estimating the mineral density of the bone .
in addition , in nomura y 's study , in 2009 , the relationship between the voxel values of cbct and ct numbers was nonlinear and the suggestions of the researcher for more future studies to determine the appropriate relationship , confirmed this fact .
evaluating the quality and quantity of bone has been done visually so far , through clinical diagnoses and radiographic images , in order to evaluate the bone trabecula and the space of the marrow .
however , the present study demonstrates that although the change in position affects the density value , in the case of providing a repeatable position , it is possible to evaluate the quantity and quality of the bone in comparison to other areas of the mandible in the same patient and to determine the point with a higher density , to place the implant . in other words , in addition to radiographic diagnosis and evaluation , besides its high ability in linear evaluations , cbct can show the clinician the points with higher density and the values that are exclusive to each unit , and are comparable to multislice ct , in cases like the newtom 3 g type of cbct . | background : bone density measurement in a radiographic view is a valuable method for evaluating the density of bone quality before performing some dental procedures such as , dental implant placements . it seems that cone - beam computed tomography ( cbct ) can be used as a diagnostic tool for evaluating the density of the bone , prior to any treatment , as the reported radiation dose in this method is minimal .
the aim of this study is to investigate the effect of object location on the density measurement in cbct versus multislice computed tomography ( ct).materials and methods : in an experimental study , three samples with similar dimensions , but different compositions , different densities ( polyethylene , polyamide , polyvinyl chloride ) , and three bone pieces of different parts of the mandibular bone were imaged in three different positions by cbct and multislice ct sets .
the average density value was computed for each sample in each position .
then the data obtained from each cbct was converted to a hounsfield unit and evaluated using a single variable t analysis .
a p value < 0.05 was considered to be significant.results:the density in a multislice ct is stable in the form of a hounsfield number , but this density is variable in the images acquired through cbct , and the change in the position results in significant changes in the density . in this study , a statistically significant difference ( p value = 0.000 ) has been observed for the position of the sample and its density in cbct in comparison to multislice ct.conclusions:density values in cbct are not real because they are affected by the position of the object in the machine . |
neuroendocrine tumors ( nets ) of the pancreas are rare , comprising 12% of all pancreatic malignancies .
the vast majority of these are primary pancreatic malignancies , as metastases to the pancreas are rare , and metastases of neuroendocrine tumors from other primary sources to the pancreas are even rarer .
pulmonary neuroendocrine tumors comprise 2030% of all nets and 1114% of all lung cancers and are classified into four subtypes , typical and atypical carcinoid , small cell lung carcinoma and large cell neuroendocrine carcinoma . depending on their degree of differentiation , these tumors have been well - documented to metastasize to intrathoracic lymph nodes , liver , central nervous system ( cns ) , skeletal bones , adrenals , and mammary glands . there are reports of pulmonary nets rarely metastasizing to other rare locations .
for example , birker et al . published a case report of a metastasis to the testis from a primary net of the lung .
choi et al . showed in a case report that atypical lung nets in an asymptomatic patient can metastasize to the breast .
we have only found one other case of metastatic net to the pancreas arising from the lung in the literature .
similarly , in studies of carcinomas metastatic to the pancreas , pulmonary origin is rare . in an autopsy study of 1740 japanese patients with 690 malignant tumors of non - primary pancreatic origin , nakamura et al
. found only 103 secondary pancreatic tumors , 17% of which originated from the lung , and none of which were nets .
kim et al . performed a review of 371 patients with nets from various sites , in which none of the pancreatic nets had their origin in the lung .
a 58-year - old female presented to our clinic in june 2013 with an incidentally diagnosed pancreatic neuroendocrine tumor .
she was previously enrolled in a clinical trial in 2011 for low dose computed tomography ( ct ) surveillance protocol for lung cancer . at that time , she was found to have a 1.4 cm carcinoid tumor of the right lung .
she denied having carcinoid symptoms at the time , with the exception of occasional diarrhea . reportedly , her urine 5-hydroxyindoleacetic acid ( 5-hiaa ) was within normal limits .
she underwent a thoracotomy with lobar resection of the carcinoid tumor , with post - operative follow - up ct scans at 6-month intervals . on the
last scheduled surveillance in june 2013 , an incidental 1 cm pancreatic tail mass was discovered .
endoscopic ultrasound ( eus ) with biopsy of the tumor was performed with findings consistent with a pancreatic neuroendocrine tumor .
a gallium scan was negative , though a positron emission tomography ( pet ) scan demonstrated increased uptake in the tumor area .
a ct scan in our institution revealed a 3.4 cm 2.1 cm area of hypoattenuation in the pancreatic tail , best visualized on the arterial phase images , corresponding to the patient 's recent eus and biopsy proven neuroendocrine tumor in the pancreatic tail ( fig .
interestingly , the mass itself demonstrated no appreciable internal hypervascularity on the arterial phase , and moreover , it was relatively poorly defined .
in addition , there was no evidence of distant metastatic disease or any suspicious lymphadenopathy .
the patient 's medical history was otherwise only notable for breast cancer in 2001 and hypertriglyceridemia .
the decision was made with the patient 's involvement , to have this mass surgically resected , and a distal pancreatectomy and splenectomy was performed .
the surgical pathology results demonstrated a neuroendocrine carcinoma ( who grade 3 ) , 1.9 cm in size , involving the pancreas and the peripancreatic tissue ( fig .
background pancreas showed pancreatic intraepithelial neoplasia , and twenty - three lymph nodes that were taken were negative for tumor .
this neuroendocrine tumor in the pancreas was morphologically similar to the patient 's previous lung tumor .
the neoplastic cells in the pancreas immunolabeled with antibodies to synaptophysin and chromogranin and were negative for ttf1 and pax8 , with a high ki67 index of nearly 25% .
the pathology from the patient 's right lower lobe lung mass was reviewed , and it demonstrated atypical carcinoid ( grade 2 well - differentiated neuroendocrine neoplasm ) measuring 1.4 cm with lymphovascular invasion .
focal necrosis was observed and up to five mitoses per 10 high power fields were identified .
submitted immunostains showed the neoplastic cells to be focally positive for ttf-1 and negative for cdx-2 and pax-8 , and a ki-67 proliferation index of 8% . to ensure that the lung was the primary source for the pancreatic net
, we had to rule out a metastasis from the patient 's previous breast cancer .
a panel of breast cancer immunomarkers applied to the tumor in the pancreas demonstrated positive immunostaining for ck7 , while stains for er , pr , ck20 , gata-3 , gcdfp and mammoglobin were all negative .
given the similar morphology and otherwise identical immunolabeling profiles , we would strongly favor that the lesion in the pancreas is a metastasis from the patient 's previous lung atypical carcinoid tumor .
the second case is a 59-year - old female of ashkenazi descent , who was diagnosed with small cell lung cancer in 2003 . of note ,
she was treated with a cisplatin - based chemotherapy regimen , lung irradiation , and partial prophylactic radiation to the brain at another hospital . in 2006 ,
she was also diagnosed with stage iiic serous adenocarcinoma of the ovary , which was surgically managed and followed with 6 cycles of taxol and carboplatin chemotherapy . in subsequent follow - up , a rise in her ca-125
was noted , however , imaging studies have failed to demonstrate a definitive ovarian cancer recurrence .
imaging revealed an abnormality in the tail of the pancreas , and a positron emission tomography ( pet ) scan demonstrated uptake in this area .
an endoscopic ultrasound and biopsy of this pancreatic tail lesion demonstrated neoplastic cells consistent with either a new primary or possibly metastatic disease from the lung . a distal pancreatectomy , splenectomy , and paraortic lymphadenectomy were performed .
surgical pathology showed atypical neuroendocrine neoplasm , most consistent with a metastatic atypical carcinoid tumor , 2.5 cm in size , involving the parenchyma of the pancreas .
the lesion was composed of spindle shaped neuroendocrine cells with an increased mitotic rate , which focally reached the threshold for atypical carcinoid ( 520 mitoses per 20 hpf ) .
a panel of immunostains was performed , and the neoplasm in the pancreas was strongly positive for cytokeratin ae1/ae3 , synaptophysin and chromogranin . while it was also positive labeling for somatostatin , this stain is known to have a very high background and it is challenging to identify how much of this labeling is specific .
the neoplasm also weakly immunolabelled for ttf-1 and demonstrated a sustentacular pattern of labeling for s-100 protein .
this pattern of labeling is most certainly consistent with an atypical carcinoid tumor of the lung metastatic to the pancreas .
a 58-year - old female presented to our clinic in june 2013 with an incidentally diagnosed pancreatic neuroendocrine tumor .
she was previously enrolled in a clinical trial in 2011 for low dose computed tomography ( ct ) surveillance protocol for lung cancer . at that time , she was found to have a 1.4 cm carcinoid tumor of the right lung .
she denied having carcinoid symptoms at the time , with the exception of occasional diarrhea . reportedly , her urine 5-hydroxyindoleacetic acid ( 5-hiaa ) was within normal limits .
she underwent a thoracotomy with lobar resection of the carcinoid tumor , with post - operative follow - up ct scans at 6-month intervals . on the
last scheduled surveillance in june 2013 , an incidental 1 cm pancreatic tail mass was discovered .
endoscopic ultrasound ( eus ) with biopsy of the tumor was performed with findings consistent with a pancreatic neuroendocrine tumor .
a gallium scan was negative , though a positron emission tomography ( pet ) scan demonstrated increased uptake in the tumor area .
a ct scan in our institution revealed a 3.4 cm 2.1 cm area of hypoattenuation in the pancreatic tail , best visualized on the arterial phase images , corresponding to the patient 's recent eus and biopsy proven neuroendocrine tumor in the pancreatic tail ( fig .
interestingly , the mass itself demonstrated no appreciable internal hypervascularity on the arterial phase , and moreover , it was relatively poorly defined .
in addition , there was no evidence of distant metastatic disease or any suspicious lymphadenopathy .
the patient 's medical history was otherwise only notable for breast cancer in 2001 and hypertriglyceridemia .
the decision was made with the patient 's involvement , to have this mass surgically resected , and a distal pancreatectomy and splenectomy was performed .
the surgical pathology results demonstrated a neuroendocrine carcinoma ( who grade 3 ) , 1.9 cm in size , involving the pancreas and the peripancreatic tissue ( fig .
background pancreas showed pancreatic intraepithelial neoplasia , and twenty - three lymph nodes that were taken were negative for tumor .
this neuroendocrine tumor in the pancreas was morphologically similar to the patient 's previous lung tumor .
the neoplastic cells in the pancreas immunolabeled with antibodies to synaptophysin and chromogranin and were negative for ttf1 and pax8 , with a high ki67 index of nearly 25% .
the pathology from the patient 's right lower lobe lung mass was reviewed , and it demonstrated atypical carcinoid ( grade 2 well - differentiated neuroendocrine neoplasm ) measuring 1.4 cm with lymphovascular invasion .
focal necrosis was observed and up to five mitoses per 10 high power fields were identified .
submitted immunostains showed the neoplastic cells to be focally positive for ttf-1 and negative for cdx-2 and pax-8 , and a ki-67 proliferation index of 8% . to ensure that the lung was the primary source for the pancreatic net
, we had to rule out a metastasis from the patient 's previous breast cancer .
a panel of breast cancer immunomarkers applied to the tumor in the pancreas demonstrated positive immunostaining for ck7 , while stains for er , pr , ck20 , gata-3 , gcdfp and mammoglobin were all negative .
given the similar morphology and otherwise identical immunolabeling profiles , we would strongly favor that the lesion in the pancreas is a metastasis from the patient 's previous lung atypical carcinoid tumor .
the second case is a 59-year - old female of ashkenazi descent , who was diagnosed with small cell lung cancer in 2003 . of note ,
she was treated with a cisplatin - based chemotherapy regimen , lung irradiation , and partial prophylactic radiation to the brain at another hospital . in 2006 ,
she was also diagnosed with stage iiic serous adenocarcinoma of the ovary , which was surgically managed and followed with 6 cycles of taxol and carboplatin chemotherapy . in subsequent follow - up , a rise in her ca-125
was noted , however , imaging studies have failed to demonstrate a definitive ovarian cancer recurrence .
imaging revealed an abnormality in the tail of the pancreas , and a positron emission tomography ( pet ) scan demonstrated uptake in this area .
an endoscopic ultrasound and biopsy of this pancreatic tail lesion demonstrated neoplastic cells consistent with either a new primary or possibly metastatic disease from the lung . a distal pancreatectomy , splenectomy , and paraortic lymphadenectomy were performed .
surgical pathology showed atypical neuroendocrine neoplasm , most consistent with a metastatic atypical carcinoid tumor , 2.5 cm in size , involving the parenchyma of the pancreas .
the lesion was composed of spindle shaped neuroendocrine cells with an increased mitotic rate , which focally reached the threshold for atypical carcinoid ( 520 mitoses per 20 hpf ) .
a panel of immunostains was performed , and the neoplasm in the pancreas was strongly positive for cytokeratin ae1/ae3 , synaptophysin and chromogranin . while it was also positive labeling for somatostatin , this stain is known to have a very high background and it is challenging to identify how much of this labeling is specific .
the neoplasm also weakly immunolabelled for ttf-1 and demonstrated a sustentacular pattern of labeling for s-100 protein .
this pattern of labeling is most certainly consistent with an atypical carcinoid tumor of the lung metastatic to the pancreas .
our two cases establish the possibility of lung neuroendocrine tumors metastasizing to the pancreas , even years after the diagnosis of the primary lung tumor .
clinicians should consider this possibility when evaluating newly discovered pancreatic masses in patients with a history of lung neuroendocrine neoplasms . in our experience ,
endoscopic ultrasound with fine needle aspiration should be instrumental in the diagnostic evaluation of these patients , when technically feasible . obtaining tissue diagnosis
enables the comparison of the pancreatic mass 's morphologic characteristics and immunohistochemical profile to those of the previous lung net .
in particular , immunohistochemistry plays a vital role in differentiating primary pancreatic net versus neuroendocrine metastasis and expert opinion on choosing and interpreting immunohistochemical markers should be sought .
while the management of primary pancreatic neuroendocrine tumors is well outlined , there is scarcity of data on long - term outcomes of pancreatic neuroendocrine metastases from the lung .
therefore , establishing this diagnosis pre - operatively could be instrumental in the treatment decision - making .
although surgical resection of the metastases was feasible in both of our cases and is likely to be technically feasible in the majority of similar cases in the absence of widespread disease , there are no published data describing long - term outcomes following resection of pulmonary neuroendocrine metastases to the pancreas . by definition , patients with lung net metastases to the pancreas have systemic disease with likely micro - metastases present , but not yet clinically evident , in other organs .
therefore , the benefit of metastasectomy via pancreatectomy in this setting remains to be defined .
in conclusion , these two cases highlight that metastases to the pancreas from lung net are possible , although they remain very rare . clinicians should be aware of this possibility during the follow - up of patients with a history of lung net .
furthermore , our case reports identifies the subtleties of diagnosis and management in those patients and highlights that expert opinion should be sou
written informed consent was obtained from the patients 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 .
matthew weiss : study design , data analysis , writing.key learning pointsneuroendocrine tumors of the lung can metastasize to the pancreas.tissue biopsy by eus and immunohistochemical staining is paramount in differentiating primary neuroendocrine tumors of the pancreas from neuroendocrine metastasis.expert opinion should be sought if neuroendocrine metastasis to the pancreas is suspected.the value of pancreatectomy for neuroendocrine metastasis to the pancreas remains unclear .
neuroendocrine tumors of the lung can metastasize to the pancreas.tissue biopsy by eus and immunohistochemical staining is paramount in differentiating primary neuroendocrine tumors of the pancreas from neuroendocrine metastasis.expert opinion should be sought if neuroendocrine metastasis to the pancreas is suspected.the value of pancreatectomy for neuroendocrine metastasis to the pancreas remains unclear .
tissue biopsy by eus and immunohistochemical staining is paramount in differentiating primary neuroendocrine tumors of the pancreas from neuroendocrine metastasis . | introductionprimary pancreatic neuroendocrine tumors are a well - established disease entity , however , neuroendocrine metastases to the pancreas from other sites have been scarcely documented .
specifically , pancreatic metastases from a pulmonary carcinoid tumor have only previously been described in a single case report.presentation of casewe sought to outline our institutional experience of two patients with pulmonary neuroendocrine tumors that developed metastases to the pancreas , confirmed by gross pathology and immunohistochemistry . in both cases ,
the pancreatic metastases were surgically resected and their pulmonary origin were discovered post-operatively.discussionour findings should raise awareness to the possibility of metastatic disease when evaluating a pancreatic mass in a patient with a clinical history of pulmonary carcinoid tumor .
expert opinion on immunohistochemically differentiating a primary pancreatic neuroendocrine malignancy from a metastasis should be employed in these cases.conclusionestablishing this diagnosis pre - operatively could affect the decision to proceed with surgical resection , given the morbidity of pancreatectomy and the unknown long - term clinical outcome of patients with pulmonary carcinoid tumors metastatic to the pancreas . |
obesity , an excessive accumulation of fat in an individual , is an important public health issue and contributes to serious health problems and extensive economic costs worldwide .
a common measure of adult obesity is body mass index , defined as a person 's weight ( in kilograms ) divided by the square of his or her height ( in meters ) , above 30 .
although weight loss is associated with clear health benefits , the prevention of weight regain has remained a challenge . even with the help of professionals and extended behavioral treatments ,
weight regain typically occurs when professional contact ends . by 35 years posttreatment , about 85% of the patients would have regained weight or even exceeded their pretreatment weight .
recommendations for obese individuals include losing and maintaining a loss of 510% of body weight in order to reduce the risk of developing chronic medical conditions .
although a 10% weight loss may not return an obese to a nonobese state , the positive health benefits of a 10% weight loss are well documented .
some studies show correlations between long - term weight loss and continued consumption of low - fat diets , increased physical activity , self - monitoring weight , and maintaining a consistent eating pattern across weekdays and weekends [ 79 ] .
it is unclear though how some formerly obese individuals are able to persevere with healthy behaviors when the majority find it difficult to do so . a randomized controlled trial explicitly designed to minimize posttreatment weight regain investigated the results of cognitive - behavioral therapy ( cbt ) on one hundred and fifty female obese participants but found little evidence to support a cognitive - behavioral approach to the treatment of obesity .
the literature therefore has been sparse on the psychological predictors of obesity maintenance and few baseline characteristics have been consistently predictive of subsequent outcomes .
a frequent criticism of obesity research is also the lack of a sound theoretical background , and researchers have stated the need for theory - based formulations in obesity research [ 1416 ] . in the absence of robust empirical evidence ,
current interventions for obesity have largely relied on anecdotal clinical evidence [ 17 , 18 ] .
hence an informed understanding of how to best treat this group of patients remains elusive .
this paper reviews and discusses extant literature on the psychological and neuropsychological aspects of obesity - management and aims to elucidate important influences on obesity maintenance .
in particular , past research has overlooked the role of executive function ( higher - level cognitive processes ) interacting with obesity maintaining mechanisms .
current studies have indicated a negative association between obesity and cognition , especially in the area of executive function , irrespective of the presence of binge eating .
a novel theoretical framework by way of a clinical obesity maintenance model ( comm ) is proposed . in the final sections of the paper ,
a review of recent findings in the field of behavioral and cognitive psychology is synthesized in this section to provide the rationale for the proposed clinical obesity maintenance model ( comm ) .
it is proposed that a network of interrelated psychological mechanisms accounts for the maintenance of obesity with or without an accompanying a binge eating disorder .
executive function has been described as encompassing a range of cognitive processes facilitating initiation , planning , regulation , inhibition , sequencing , and attainment of complex goal - oriented behavior and thought , all of which may impact on eating behavior .
recent studies have indicated a negative association between obesity and cognition , especially in the area of executive function , irrespective of the presence of binge eating . a systematic review of 34 studies by smith et al
. found a consistent association between obesity and low scores on executive function in children , adolescents , and adults , even after controlling for socioeconomic status or medical comorbidities .
for example , studies have shown that obese participants show a pronounced impairment in decision making in the iowa gambling task ( igt ) , a neuropsychological task of executive function designed to simulate real - life decision making and learning in terms of reward and punishment [ 22 , 23 ] .
obese individuals perform similarly on this task compared to those with anorexia nervosa and to those with orbitofrontal dysfunction and worse than those who are dependent on substance use .
in addition , similar to individuals with anorexia nervosa , obese individuals have impaired central coherence ( a detail - focused processing style , defined by not being able to see the big picture ) , but in contrast to those with anorexia nervosa they are very impulsive .
finally , obesity has been shown to be associated with poorer performance on tests of global cognitive function and memory ( e.g. , ) .
the mechanism(s ) by which obesity results in cognitive impairment however are uncertain . postulated mechanisms include the effects of hyperglycemia , hyperinsulinemia , and vascular damage to the central nervous system .
two recent studies have shown that low cognitive performance predicts increased levels of adiposity [ 31 , 32 ] .
, no trials have yet investigated the effects of remediation of cognitive deficits on weight loss and/or weight loss maintenance .
empirical evidence indicates that when behaviors are underpinned by habits , they are autonomically enacted by the presence of cues or internal drive states [ 33 , 34 ] .
when aiming at changing these behaviors , the intentions need to be powerful enough to override the existing habits .
habitual behaviors are maintained because of ( a ) mental efficiency , that is , speed and ease with which past patterns of behavior can be initiated and executed , ( b ) automaticity , that is , unawareness of performing the behavior ,
( c ) susceptibility to cues by prevailing environmental events , and ( d ) these patterns are consistent with short - term goals [ 33 , 35 ] . the ongoing persistence required to shift well - established behavior patterns is exemplified in the field for weight control . a systematic review and meta - analysis by gardner et al
eight of nine studies in this meta - analysis provided evidence that habits moderated the intention - behavior relationship such that the impact of intention on behavior diminished as habit strength increased .
few studies of behavior change in the field of obesity have attempted to formally incorporate the role of habit . in a field study of healthy eating , on a sample of 320 university students , verplanken and faes confirmed the deleterious effects of counterintentional habits ( e.g. , eating fatty foods ) .
recent studies have begun to employ a validated measure of habit in both dietary behaviors and physical activity .
results have indicated that habit strength considerably adds to the extent of variance in behavior across various age groups ( e.g. , fruit consumption , saturated fat consumption , adolescent screen - viewing behavior associated with consumption of sugar - sweetened beverages , and physical activity ) .
one reason why long - term behavior change may be difficult for the obese population is that the behaviors that individuals want to change are relatively habitual , such as poor dietary habits , sedentary behaviors , and environmental behaviors .
clustering refers to the cooccurrence of life - style - related risk factors that can be expected based on the prevalence of the separate behaviors . among adults , smoking and alcohol consumption
have been shown to correlate with consumption of fewer fruits and vegetables and more fat [ 44 , 45 ] .
smoking , excessive alcohol use , an unhealthy diet , and physical inactivity are the
empirical evidence indicates that these unhealthy behavior clusters tend to occur in combination within individuals [ 4749 ] and among those who are obese .
sedentary activity , in particular tv viewing , has been associated with unhealthy eating practices and may in part explain the relationship between sedentary behavior and obesity [ 51 , 52 ] .
kushner and choi investigated the prevalence of unhealthy eating , exercise , and coping pattern traits among a large sample comprising of 1865-years - old , 446,608 healthy - weight , overweight , and obese adults .
this study found that the prevalence of these patterns rose with increasing bmi and that unhealthy lifestyle patterns in diet , exercise , and coping were highly prevalent among the overweight and obese population .
the authors concluded that pattern recognition represents a new method to analyze the cluster of behaviors , attitudes , and traits seen among this population .
a better understanding about the prevalence , co - occurrence , and correlates of behaviors of obese individuals is needed to inform the design of interventions for weight management .
presently , there is no empirical evidence regarding the maladaptive behavioral clusters in the obese that are extended with habit strength .
emotional regulation requires a person to be aware of internal experiences ( neutral , negative , and positive internal emotional states ) , identify the emotion , and effectively cope with or tolerate the emotion [ 55 , 56 ] .
in contrast , obese individuals demonstrate a dysregulated physiological response to intense emotion by tending to increase their food intake during periods of emotional arousal and/or stress , a response known as emotional eating [ 5760 ] .
negative mood states are triggers for overeating in obese / overweight samples and overeating / binge eating may function as strategies to escape , avoid , or minimize negative affect [ 6164 ] . for example , a study by jansen et al .
, on overweight / obese and normal weight participants , clustered into high- and low - negative - affect subtypes , found that negative mood induction and food exposure elicited overeating in the overweight / obese high negative affect subtype when compared to overweight / obese low negative affect subtype and normal - weight controls .
thus , while negative affect alone does not contribute to overeating and obesity , there is certainly a subsample of obese individuals who overeat when they are feeling low .
although there is substantial empirical support for a direct link between emotional dysregulation and binge eating disorder ( see ) , at present , there is little understanding of the maladaptive ways in which obese individuals cope with deficits in emotion regulation . a major depressive episode is characterized by periods of 2 weeks or more of depressed mood and/or anhedonia , in conjunction with other symptoms , including significant disturbance in appetite , sleep , psychomotor retardation or agitation , loss of energy , concentration problems , feelings of worthlessness or guilt , and/or suicidal ideation or intent .
an association between depression and obesity has been observed in both epidemiological and clinical studies [ 68 , 69 ] with recent meta - analyses on both cross - sectional and prospective studies giving evidence for such an association [ 70 , 71 ] . a study by petry et al
differences were apparent for past - year depression as well with prevalence rates of 11.4% for class 1/ii obesity and 16.2% for class iii obesity . among weight - loss - seeking obese individuals ,
prevalence of depression is even higher , with 19% to 50% reporting a lifetime history of depression [ 74 , 75 ] . a meta - analysis by luppino et al
obesity was found to increase the risk of developing depression and depression was found to be predictive of obesity .
a study by renn et al . , also found bidirectional associations between depression and obesity : obese individuals had a 55% increased risk of developing depression over time , whereas depressed persons had a 58% increased risk of becoming obese .
this study found a dose - response gradient in that the association between depression and obesity was stronger than the association between depression and overweight .
although the causes for the obesity - depression link are uncertain , several possible theories have been discussed including the following : ( a ) obesity exacerbates inflammatory markers and these markers in turn have a role in the development and maintenance of depression ; ( b ) obesity contributes to body dissatisfaction and low self - esteem , putting the overweight individual at risk for depression , especially in treatment - seeking and morbidly obese individuals [ 70 , 75 ] ; ( c ) depression may increase weight by through central neuroendocrine effects either primary or secondary to antidepressant use [ 70 , 76 , 77 ] .
the exact nature of the relationship between depression and obesity maintenance remains unclear , perhaps because clinical depression is a common exclusion criterion in weight - loss intervention trials [ 78 , 79 ] .
it is important to further elucidate how these two major health problems interact , so that prevention and treatment strategies are improved .
one other construct that has been included in the proposed comm is that of health literacy . in order to understand and respond to the difficulties presented by a chronic condition , individuals construct their own common - sense model of their condition comprising of their belief sets and attitudes [ 80 , 81 ] . in the area of mental health , researchers [ 82 , 83 ] have argued that poor mental health literacy , that is , poor awareness and understanding of the nature and treatment of health problems , is a major factor in the individual , social , and economic burden of mental health problems .
poor health literacy also includes attitudes and beliefs likely to be conducive to stigmatization of and discrimination against a condition or illness [ 84 , 85 ] . in the field of obesity
, it has been postulated that interventions that include health literacy will produce greater effects than standalone behavioral approaches ( e.g. , ) .
the health literacy construct in the proposed model will add to the multidimensional nature of the obesity experience by including ( a ) risk awareness / consequences of binge eating / overeating , ( b ) perceived personal control over condition / treatment , ( c ) perception of barriers to recovery and maintenance of successful weight management , and ( d ) other obesity - related beliefs and attitudes .
it is not however understood how much this changes attitudes and beliefs of people with weight disorder and the efficacy of more specific health literacy interventions in obesity is unknown .
in line with the above discussion , the proposed clinical obesity maintenance model ( comm : figure 1 ) draws upon previous research and includes the following components from the cognitive and behavioral principles that are hypothesized to regulate weight management behaviors : ( a ) executive function , ( b ) habitual cluster behaviors , ( c ) emotion dysregulation , ( d ) depression , and ( e ) health literacy .
it is reasoned that by delineating the role of emotional processes , the strength of habitual cluster behaviors , and executive functioning , the multidimensional nature of obesity maintaining behaviors is elucidated more clearly to inform scientific literature and clinical practice .
the suggested constructs may not necessarily operate simultaneously , nor may they be active in every case .
their partial independence may in part account for the heterogeneity and complexity that is found in the maintenance of obesity .
( see 1a in figure 1 ) , there is an ample evidence that depressive disorders are associated with deficits in attentional functions , executive control , lowered cognitive flexibility , verbal learning , memory , and interpretation biases [ 8992 ] . due to the impaired cognitive processes often associated with depression
, obese individuals with depression may be less able to adhere to the recommended dietary and physical activities [ 69 , 93 ]
. a converse relationship may also apply ( see 3d , 3e in figure 1 ) .
habit strength may mediate the relationship between depression and obesity maintaining behaviors through cognitive means .
for example , studies have shown that obese individuals who regain weight show a perceived lack of personal control over their weight management and a dichotomous thinking style , that is , an all or nothing thinking style about their eating behaviors [ 95 , 96 ] .
hence when they are unable to resist strong habits , they may tend towards depressogenic thought patterns .
in addition , the comm proposes that poor health literacy ( see 3c in figure 1 ) is a logical prerequisite for the development and maintenance of depressogenic beliefs and attitudes about the obesity condition .
studies that have examined the impact of health literacy on the obese have generally relied on the quality of life as an outcome measure ( e.g. , [ 97 , 98 ] ) .
little research has been done on the link between poor health literacy and depression with regard to obesity maintaining behaviors .
the comm aims to bridge this gap by proposing a cognitive - behavioral pathway ( see 3c in figure 1 ) in which poor health literacy contributes to the development of information - processing biases that increase the risk of depression in obese individuals .
research has shown that when cognitive resources become limited ( i.e. , deficits in executive function ) , individuals are inclined to make heuristic - based choices .
this is reflected in obesity maintaining dietary habits ( see 1b in figure 1 ) . given the habitual nature of eating and the rapidity with which people make eating decisions , they are likely to be the consequence of automatic responses to contextual food cues , many of which lead to increased caloric consumption and poor dietary choices [ 99 , 101 ] . in addition , emotion regulation has been described as a continuous , dynamic system responsive to all emotional experience , consisting of both autonomic and controlled processes [ 102 , 103 ] , perhaps mediated by executive function ( see 1c in figure 1 ) . as it relates to obesity , problems in attentional and inhibitory control
although deficits in attentional control in obesity may contribute to behavioral impulsivity related to eating , emotion regulation as a self - regulatory process may control the affective processes that relate to eating ( see 3f in figure 1 ) .
thus when obese and depressed individuals have a poor repertoire of emotion regulation strategies ( e.g. , deficits in inhibitory control ) , they may be unable to withhold inappropriate prepotent responses that have accumulated habit strength ( see 3f in figure 1 ) .
furthermore , there may be a bidirectional association between depression and emotional dysregulation ( see 3 g in figure 1 ) .
evidence indicates that depression is characterized not only by poor mood levels ( i.e. , low positive affect and high negative affect [ 105 , 106 ] , but also by emotion dysregulation ( for a meta - analysis , see [ 107 , 108 ] ) .
psychologically oriented research in the context of obesity maintenance is noteworthy for its tendency to focus on a limited range of psychological constructs , its limited conceptualization of psychological difficulties , and for its inconsistent findings .
researchers ( e.g. , [ 95 , 110 , 111 ] ) have reiterated the need to examine the behavioral and psychological mechanisms that underpin longer - term weight management . as cooper et al
. stated , the cognitive - behavioral model of obesity as a standalone model does not fully explain nor address the mechanisms that contribute to weight regain . the clinical obesity maintenance model ( comm )
contrasts with earlier models focused on the cognitive and behavioral pathways maintaining obesity , by taking into account additional salient constructs such as habitual cluster behaviors , emotion dysregulation , health literacy , and executive deficiencies .
overlooking important cognitive and psychological barriers in the weight loss maintenance phase may perpetuate a sense of failure in the obese people and further undermine the low sense of self - esteem and self - efficacy of these individuals .
the comm is presented as a psychological model and does not aim to include all the medical and sociocultural features associated with obesity .
this model contains psychological features on the basis of extant empirical literature implicating a strong association between the proposed constructs .
it is suggested that an integrative , multiprong approach may prove remedial in the assessment and treatment of obesity maintenance .
for example , obesity - specific health literacy may improve the attitudes and beliefs regarding their weight management .
teaching emotion regulation strategies [ 55 , 56 ] that affect weight management behaviors and employing cognitive - behavioral therapy for depression may enable better coping skills with negative emotion and improve mood levels . as proposed by smith et al .
the comm is planned to be further evaluated in empirical studies which include structural equation modeling and randomized controlled trial testing on interventions based on aspects of the model . | psychological distress and deficits in executive functioning are likely to be important barriers to effective weight loss maintenance .
the purpose of this paper is twofold .
first , in the light of recent evidence in the fields of neuropsychology and obesity , particularly on the deficits in the executive function in overweight and obese individuals , a conceptual and theoretical framework of obesity maintenance is introduced by way of a clinical obesity maintenance model ( comm ) .
it is argued that psychological variables , that of habitual cluster behaviors , emotional dysregulation , mood , and health literacy , interact with executive functioning and impact on the overeating / binge eating behaviors of obese individuals .
second , cognizant of this model , it is argued that the focus of obesity management should be extended to include a broader range of maintaining mechanisms , including but not limited to cognitive deficits .
finally , a discussion on potential future directions in research and practice using the comm is provided . |
the term comorbidity was introduced in 1970 by feinstein , a doctor of internal medicine and epidemiologist who studied cooccurrence of diseases among persons with diabetes .
any distinct clinical entity that has existed or that may occur during the course of a patient who has the index disease under study , in which
later , it has been recommended to reserve the term comorbidity to conditions that are not known as a direct consequence of the index disease , while use of the term complication should imply a strong evidence of causality .
today , comorbidity is often used to describe the cooccurrence of separate distinct diseases at one specific point of time , regardless of the direction between them , and when the association observed is not expected to be explained by causality .
the international diabetes federation estimates the global prevalence of diabetes in 2013 to approximate 8% , while the world mental health survey undertaken from 2001 to 2007 found lifetime and 12-month prevalence of major depressive episode to reach 15% and 6% , respectively .
major depression and diabetes are ranked the 4th and 9th most important causes of disability adjusted life years ( dalys ) in the developed world , reflecting the high prevalence and , for particularly depression , the early onset of disease .
further , the effect of comorbid diabetes and depression on decrements in health has been shown to be interactive , suggesting a negative effect on health beyond that expected by adding the effect of the two disorders . perhaps due to this large impact on public health , numerous studies have investigated various aspects of the comorbidity between depression and diabetes , in particular type 2 diabetes .
for instance , previous work has found evidence of a bidirectional association between type 2 diabetes and depression [ 811 ] . as outlined in figure 1
, three possible directions of the association between type 2 diabetes and depression can underlie the observation of comorbidity between the disorders .
first , common etiology can increase a person 's risk of both diseases ; second , persons with type 2 diabetes have increased prevalence or risk of future development of depression ; or third , persons with depression have increased prevalence or risk of development of type 2 diabetes .
pathophysiology is defined as the study of mechanisms of disease , that is , how changes in normal physiology can cause diseases .
some literatures have in general terms sought to describe the pathophysiology underlying the comorbidity between type 2 diabetes and depression [ 1316 ] ; however , to our best knowledge , no previous review has addressed possible direction specific pathophysiological mechanisms for why we observe comorbidity between type 2 diabetes and depression .
this review therefore aims to give an overview over possible pathophysiological mechanisms for each of the directions of the association between type 2 diabetes and depression , with special emphasis on stress - theory . in this review , the term depression not necessarily equals the diagnostic criteria for various affective disorders according to dsm - v or icd-10 but should rather be understood broadly as a heterogeneous group of disorders with core symptoms of impaired mood and lack of interest and energy .
cooccurrence of type 2 diabetes and depression more often than expected by chance can be a result of common etiology , overall considered to be either shared genetic or environmental factors .
theoretically , comorbidity between type 2 diabetes and depression could be explained by common genetic factors ; that is , genes associated with one of the disorders could be associated with or inherited together with genes associated with the other disorder .
most likely due to large heterogeneity of the disorders , no previous genome wide association studies ( gwas ) have explored the comorbidity between type 2 diabetes and depression , while only one twin study has investigated the comorbidity between depression and diabetes , types 1 and 2 combined . in this latter study including about 1200 middle - aged male twins , about 50 percent homo- and heterozygote , respectively , the estimated genetic correlation between depression and diabetes was r = 0.19 ( 95% ci : 0.000.46 ) .
the authors concluded that they found overall little evidence of common genetic factors accounting for the comorbidity between diabetes and depression among middle - aged men .
a range of environmental factors is associated with both type 2 diabetes and depression , and it is likely that these factors can explain at least part of the association between the diseases .
examples of such factors include , but are not limited to , unfavorable lifestyle such as inactivity , poor sleep , and diet [ 1820 ] , psychological and social factors including early life stress [ 21 , 22 ] and demanding socioeconomic position [ 23 , 24 ] , and more strict medical
factors such as other conditions ( e.g. , obesity ) [ 25 , 26 ] and medical treatment ( e.g. , corticosteroids ) . as an example ,
early life stress in terms of evacuation abroad to temporary foster care during world war 2 has later in life been associated with increased risk of type 2 diabetes ( odds ratio 1.4 ( 95% ci : 1.1 , 1.9 ) ) , as well as increased risk of depressive symptoms ( beck depression inventory score 10 ) ( odds ratio 1.7 ( 95% ci : 1.1 , 2.6 ) ) .
further , a meta - analysis from 2011 showed that low socioeconomic position measured in terms of education , income , and occupational status was associated with increased risk of type 2 diabetes relative to high status ( rr education 1.41 ( 95% ci : 1.28 , 1.51 ) , rr occupation 1.31 ( 95% ci : 1.09 , 1.57 ) , and rr income 1.40 ( 95% ci : 1.05 , 1.88 ) ) , while cohort studies have found up to a doubled risk of depression among children of parents with low socioeconomic status compared with children of parents with high status .
as both type 2 diabetes and depression are highly prevalent diseases in the developed world , the absolute effect of this relative increased risk at population level is particularly high .
the common denominator for these environmental factors is that they represent a threat to the homeostasis of the stress systems of the body . to a large extent ,
the body is able to regulate the effect of acute stress , mainly via the hormone system , the autonomous system , and the inflammatory system [ 28 , 29 ] .
however , if these systems are highly activated over time , the stress response may become unintentional and eventually harmful . in the following section and further illustrated in figure 2 , we outline possible pathophysiological mechanisms illustrating how chronic stress responses can increase the risk of development of both type 2 diabetes and depression .
stress activates the hypothalamus pituitary adrenal axis , often abbreviated hpa - axis , increasing the production of cortisol from the adrenal cortex [ 28 , 30 ] . additionally , stress increases stimulation of the sympathetic part of the autonomous nervous system ( sns ) , further increasing the production of adrenalin and noradrenalin from the adrenal medulla [ 28 , 30 ] .
it is well established that these stress hormones exert an overall catabolic effect on the body by mobilizing glucose as well as fat and proteins to the blood , counteracting the effect of insulin which mainly acts as an anabolic hormone facilitating uptake and storage of glucose .
this represents a mechanism by which chronic stress activation can lead to insulin resistance and eventually type 2 diabetes .
either directly , or via the hpa - axis and the autonomous nervous system , the inflammation system is additionally activated by stress , evident by increased production of proinflammatory cytokines from immune cells and tissues , such as il-6 , tnf- , and interferon- [ 32 , 33 ] .
the cytokines can directly stimulate receptors in the hypothalamus and the pituitary , thereby facilitating more production of cortisol .
it has further been suggested that proinflammatory cytokines can contribute to development of insulin resistance directly by modulation of the insulin receptor in periphery tissues .
for example , grimble proposes in a review in 2002 over inflammatory status and insulin resistance that tnf- , via reduced phosphorylation of a subunit of the insulin receptor , contribute to development of insulin resistance .
population based studies have further underlined the importance of inflammatory factors in the development of type 2 diabetes . in the british whitehall ii study ,
the association between low socioeconomic status and risk of incident type 2 diabetes was attenuated by up to one - third after adjustment for inflammatory factors measured during follow - up .
in addition to the contribution of cortisol and cytokines on development of insulin resistance , they are proposed to also have a negative impact on areas or systems in the brain associated with alterations in depression , such as the monoamine system and hippocampus [ 37 , 38 ] .
first , hippocampus exhibits glucocorticoid receptors , and it has been demonstrated that stimulation over time with high levels of cortisol can result in reduced neurogenesis and synaptic plasticity as well as apoptosis in distinct areas of hippocampus .
second , an effect of cytokines on hippocampus mediated by increased levels of oxidative stress resulting in lower levels of bdnf ( brain derived neurotropic factor ) has been proposed .
bdnf is a neurotropic peptide essential for survival and proliferation of neurons in the brain , and low levels have been found among persons with depression , while use of antidepressant agents has been associated with upregulation of bdnf production [ 37 , 38 ] .
finally , it has been proposed that certain proinflammatory cytokines can increase degradation of tryptophan , an essential amino acid .
this degradation takes place in the kynurenine pathway mainly in liver , kidneys , and immune cells .
it has been shown that cytokines induce one rate limiting enzyme in this pathway , the indoleamine dioxygenase ( ido ) , while cortisol can upregulate another rate limiting enzyme , tryptophan 2,3-dioxygenase ( tdo ) , resulting in lower concentrations of available tryptophan and higher concentrations of other metabolites in the kynurenine pathway .
serotonin , maybe the most important monoamine implicated in the pathophysiology of depression [ 37 , 38 ] , can solely be produced by tryptophan , and theories exist that low concentration of tryptophan consequently will result in less available serotonin in the brain .
moreover , some of the additional metabolites further down the kynurenine pathway , such as quinolinic acid , are suggested to have an additional negative impact on hippocampus in terms of reduced neurogenesis and induction of apoptosis .
an overview over theories on possible pathophysiological mechanisms linking cytokines with depression can be found in a review by myint and kim from 2014 . in summary , this hypothesis on chronic and unintentional stress response is a suggestion on how common etiology in terms of environmental factors via distinct pathophysiological mechanisms possibly can contribute to the development of both type 2 diabetes and depression .
several population based studies have established that persons with type 2 diabetes have both increased prevalence and incidence of depression relative to persons without diabetes [ 43 , 44 ] . while ali et al . found an odds ratio of 1.6 ( 95% ci
: 1.2 , 2.0 ) for comorbid depression among persons with type 2 diabetes in a meta - analysis of cross - sectional studies , nouwen et al
. showed a relative risk of 1.24 ( 95% ci : 1.09 , 1.40 ) for incident depression among persons with type 2 diabetes in a meta - analysis of prospective studies .
first , it is likely that several unspecific factors associated with chronic diseases can increase the risk of depression .
it is easy to imagine it being demanding to maintain stable blood glucose over time , to monitor glucose levels and calculate dosage of medication .
further , type 2 diabetes is often associated with an unfavorable lifestyle in terms of inactivity , overweight , and obesity . in a large population based , cross - sectional study from norway ,
persons with both types 1 and 2 diabetes reported lower levels of activity and had higher bmi than persons without diabetes .
these psychological and lifestyle factors can again be viewed as a type of unspecific stress threatening the homeostasis of the body and therefore possibly increase the risk of depression via the pathophysiological mechanisms outlined earlier .
second , the majority of persons with type 2 diabetes need oral antidiabetic medication to maintain stable blood sugar within reference range . hypothetically , increased prevalence and risk of depression could be caused by antidiabetic treatment . according to the literature
, there is little evidence that depression is a plausible side effect of insulin , biguanides , and/or sulfonylurea .
however , use of insulin and sulfonylureas is associated with weight gain , and if one views weight gain and possibly overweight and obesity as a type of unspecific stress , the pathophysiological mechanisms addressing how stress can cause depression might also be relevant in this context .
a systematic review over brain imaging studies on persons with diabetes from 2006 concluded that diabetes was associated with cerebral atrophy and lacunar infarcts , as well as regional alterations in cerebral blood flow . a more recent study on middle - aged persons with type 2 diabetes of less than 10-year duration showed reductions in brain volume restricted to the hippocampus relative to controls with similar age , sex , and level of education .
in multivariate regression analysis , an inverse association between hba1c and hippocampal volume was found , particularly interesting since the patients had fairly well controlled diabetes with a mean hba1c of 6.9% .
the finding of an inverse association between hba1c and structural changes in hippocampus has also been confirmed in studies of patients with type 1 diabetes , supporting a theory of a possible effect of glucose levels on the brain . when compared to a nondiabetic control group , persons with well controlled type 1 diabetes aged 2540 years without complications showed lower density of gray matter in several brain regions , including hippocampus
. further , an inverse association with lower density and higher hba1c and number of severe hypoglycemic events were found , leading the authors to conclude that persistent hyperglycemia and severe hypoglycemia likely have impact on brain structure .
further , a research group from us has conducted several studies comparing changes in brain structure , function , and biochemistry among persons with type 2 diabetes and the combination of type 2 diabetes and depression with healthy controls [ 5153 ] . for several outcomes , such as cortical gray matter thickness and level of the excitatory neurotransmitter glutamate
, the most pronounced effect was found among persons with both type 2 diabetes and depression , and the authors consequently suggest that there is a strong subcortical neurobiological component to depression in patients with type 2 diabetes .
finally , we might observe comorbidity between type 2 diabetes and depression because persons with depression have increased prevalence and incidence of type 2 diabetes .
interestingly , the association has in several studies tended to be stronger when considering depression as the exposure and diabetes as the outcome than the other way around [ 56 , 57 ] .
for example , a meta - analysis exploring this bidirectional association found a relative risk of 1.60 ( 95% ci : 1.37 , 1.88 ) for type 2 diabetes among persons with depression , while persons with type 2 diabetes had a modest increased risk of depression with an relative risk of 1.15 ( 95% ci : 1.02 , 1.30 ) .
obviously , psychological and lifestyle factors associated with depression can again be viewed as a type of stress and increase the risk of type 2 diabetes via the pathophysiological mechanisms outlined earlier . illustrating the negative impact of depression on insulin resistance even in persons free of diabetes , asghar et al .
have shown that the impaired insulin sensitivity among women with major depression was improved after successful treatment with antidepressant agents .
potential risk of diabetes after use of antidepressant agents is a hot topic in the literature on comorbidity between diabetes and depression .
a systematic review on antidepressant medication as a risk factor for type 2 diabetes and impaired glucose regulation was published in 2013 in diabetes care .
the authors sought to conduct a meta - analysis , but this was not feasible due to large heterogeneity and discrepancies in quality between the single studies .
the findings were mixed ; use of some antidepressants , particularly those with strong noradrenergic properties , was associated with worsening of glucose control , while use of other agents in fact was associated with improved glycemic control , such as selective serotonin reuptake inhibitors .
additionally , a meta - analysis on antidepressant use and risk of diabetes was published in 2013 in korean journal of family medicine , including a majority of the studies included in the review in diabetes care .
stratified analyses on type of antidepressant agents showed increased risk of type 2 diabetes after use of both selective serotonin reuptake inhibitors ( ssri ) and tricyclic antidepressant agents ( tcas ) ( relative risk ssri : 1.35 ( 95% ci : 1.15 , 1.58 ) , relative risk tca 1.57 ( 95% ci : 1.26 , 1.96 ) ) .
further analyses adjusted for bmi , severity of depression , and physical activity showed a significantly increased risk of type 2 diabetes of 14% ( rr : 1.14 ( 95% ci : 1.01 , 1.28 ) ) .
animal studies have shown that , for example , selective serotonin reuptake inhibitors directly can inhibit both the production and the effect of insulin [ 61 , 62 ] .
cultivating hepatic rat cells incubated with paroxetine and sertraline , israeli researchers found weaker response of insulin on the insulin receptor , an effect mediated via reduced phosphorylation of some of the subunits within the receptor complex .
a few years later , the same group demonstrated that incubating beta - cells from rat pancreas with paroxetine , sertraline , and fluoxetine attenuated the production of insulin and in some of the samples induced apoptosis of the beta - cells .
if use of antidepressant agents independently of weight gain and severity of depression increases the risk of type 2 diabetes , this direct effect on the insulin receptor could be one of the mechanisms possibly explaining it .
epigenetics is the study of factors influencing the long term expression of the genes , that is , how one set of genes can be transcribed differently depending on environmental factors .
these changes in transcription are stable over time and can be inherited . in brief , the change is facilitated via methylation of the dna or via modification of the histones , around which the dna is packed .
it has been suggested that epigenetic changes , at least in theory , can contribute to the occurrence of comorbidity between diseases .
epigenetics could therefore represent a new element added to each of the three presented theories for why we observe comorbidity between type 2 diabetes and depression and should consequently be viewed as a direction independent approach . to our best knowledge , no study has addressed if or how epigenetic changes might explain the established comorbidity between type 2 diabetes and depression ; however , studies have documented epigenetic changes after stress and the changes in epigenetics can be of importance in the pathogenesis of both type 2 diabetes and depression .
alterations in gene expression were found in postmortem specimens from persons with type 2 diabetes compared to controls without diabetes matched on age , in addition to corresponding changes in expression of synaptic proteins .
the authors suggested that such studies might elucidate possible epigenetic mechanisms explaining the increased risk of dementia among persons with diabetes ; however , one might argue that it could shed light on possible epigenetic mechanisms explaining the increased prevalence and incidence of depression among persons with diabetes as well .
due to the diversity of the reviewed studies , the term depression not necessarily equals the diagnostic criteria for various affective disorders according to dsm - v or icd-10 in this review but should rather be understood broadly as a heterogeneous group of disorders with core symptoms of impaired mood and lack of interest and energy .
this understanding of depression represents a limitation , mainly because we can not exclude that symptoms interpreted as depression in this review rather are symptoms of another psychiatric disorder , for example , anxiety disorders .
additionally , it limits us from exploring the association between type 2 diabetes and the severity of depression and consequently further limits a discussion on how this could be related to the various pathophysiological mechanisms .
several different pathophysiological mechanisms can underlie the comorbidity between type 2 diabetes and depression , most likely interacting with each other , thereby possibly reinforcing a negative effect on the development of the diseases .
however , the unspecific mechanisms involved in the stress response might , at least to some extent , explain each of the directions of the association between type 2 diabetes and depression . in order to determine the impact of the stress response due to common etiology at population level , a prospective study on the association between type 2 diabetes and depression adjusting for information on various forms of stressors such as lifestyle , psychological , and social factors early in life could be conducted . to determine the contribution of the different pathophysiological mechanisms at person level , the best approach is probably to first determine the temporal sequence between the disorders . | type 2 diabetes and depression are regarded as comorbid conditions , and three possible directions of the association between the diseases can underlie this observation of comorbidity .
first , common etiology can increase a person 's risk of both diseases ; second , persons with type 2 diabetes have increased prevalence or risk of future development of depression ; or third , persons with depression have increased prevalence or risk of development of type 2 diabetes .
this review gives an overview over possible pathophysiological mechanisms for each of the directions of the association between type 2 diabetes and depression and further discusses epigenetics as an additional , direction independent approach .
we argue that unspecific pathophysiological mechanisms involved in the stress response might , at least to some extent , explain each of the directions of the association between type 2 diabetes and depression , while changes in brain structure and function among persons with diabetes and possible increased risk of development of type 2 diabetes after use of antidepressant agents could represent more disease specific mechanisms underlying the comorbidity . |
nearly all types of malignant cells and many types of diseased tissue cells demonstrate alterations in their glycosylation patterns when compared to their normal counterparts .
alterations in glycosyltransferases expression , sugar nucleotide donors , and disruption of the golgi contribute to the development of diseases , such as hereditary disorders , immune deficiencies , cardiovascular disease , and cancer [ 2 , 3 ] . in the tumor environment ,
underexpression , truncation or altered branching patterns of certain glycans , and/or changes in glycosylation allow neoplasm cells to usurp many of the events that occur in development such as receptor activation , cell adhesion , and cell motility , which permits tumor cells to invade and spread throughout the organism .
the investigation of lectin - carbohydrates interactions is necessary for the understanding of both the glycophenotype and the behavior of the tumor during its differentiation and confirms the hypothesis that biochemical changes in the cell are events that may signal the cell differentiation .
lectin histochemistry based on colorimetric detection has been used as a supporting tool for differential diagnosis for skin lesions , but this technique only permits qualitative or semiquantitative analyses of carbohydrates expression .
thus , quantitative analyses require a sensitive detection that permits an objective and quantitative evaluation of the bound lectin .
chemiluminescence ( cl ) analyses represent a powerful tool in life sciences , offering high detectability and specificity [ 10 , 11 ] and allowing low limits of detection ( attomole - zeptomole ) and versatility .
it is a promising and effective technique for quantitative analysis of proteins , nucleic acids , and carbohydrates [ 1315 ] .
therefore , this approach combined with lectin histochemistry could be valuable to reduce the subjective evaluation .
acridinium ester ( ae ) compounds quickly outperformed luminol and isoluminol in chemiluminescence for their low detection limits ( attomolar range ) and specificity .
exposure of ae label to an alkaline hydrogen peroxide solution triggers a flash of light .
ae forms an unstable dioxetane yielding n - methylacridone and produces light at a wavelength of 470 nm .
the skin tumors comprise mainly basal cell carcinoma ( bcc ) , squamous cell carcinoma ( scc ) , keratoacanthoma ( ka ) , and actinic keratosis ( ak ) .
nonmelanoma skin cancers are the most common form of cancer and their incidence has been increasing considerably .
these tumors are rarely fatal but are considered to be fast growing and if neglected may be locally and functionally destructive .
the diagnosis of skin neoplasias becomes inaccurate , in some cases , due to a variety of factors that affect the test accuracy , such as a huge spectrum of tumors and their variants and the lower differentiation . here
lectins conjugated to ae were used to evaluate the glycophenotype in tissues from cutaneous tumors based on the chemiluminescence approach .
the biological relevant carbohydrate residues -d - glucose / mannose , gal-(1 - 3)-galnac , -l - fucose , and neu5ac-(2,3 ) were evaluated , respectively , by using the lectins concanavalin a ( con a ) , peanut agglutinin ( pna ) , ulex europaeus agglutinin - i ( uea - i ) , and maackia amurensis agglutinin ( maa ) .
n - hydroxysuccinimide - activated dimethyl acridinium ester ( ( dmae - nhs)/1966 - 1 - 53 - 2/organic lab ) was kindly supplied by dr .
con a , pna , uea - i , maa , n , n - dimethylformamide , trypsin from porcine pancreas , methyl--d - mannoside , d - galactose , -l - fucose , n - acetylneuraminic acid , and sephadex g-25 were purchased from sigma - aldrich ( st .
chemiluminescent detection was performed using siemens reagent tsh 500 t ( siemens medical solutions diagnostics , malvern , pa , usa ) composed of 0.5% h2o2 in 0.1 n hno3 and 0.25 m naoh .
skin fragments were obtained through paraffin - embedded biopsies from the tissue bank of the clinic hospital at the federal university of the state of pernambuco ( ufpe ) , northeastern region of brazil , and diagnosed as basal cell carcinoma ( bcc = 17 ) , squamous cell carcinoma ( scc = 13 ) , keratoacanthoma ( ka = 13 ) , and actinic keratosis ( ak = 9 ) .
the normal human epidermis fragments ( ne = 14 ) were obtained from individuals submitted to plastic surgery and supplied from hospital santa clara , recife , pe , brazil .
this study was approved by the health science centre bioethical board from the federal university of pernambuco , brazil ( cep / ccs / ufpe no .
195/09 ) . lectins ( 1 ml containing 2 mg of protein ) were incubated with 10 l of acridinium ester solution ( 0.2 mg diluted in 400 l of n , n - dimethylformamide ) for 1 h at 25c under rotary stirring .
the conjugate ( lectin - ae ) was applied to a column of sephadex g-25 ( 10 1 cm ) , previously equilibrated with 10 mm phosphate buffer , containing 0.15 m nacl ( pbs ) , ph 7.2 , and eluted with this buffer .
aliquots ( 1 ml ) were collected and their protein content was spectrophotometrically determined at 280 nm .
after conjugation , con a - ae , pna - ae , and maa - ae were evaluated regarding the maintenance of their carbohydrate recognition property ( hemagglutinating activity ) using glutaraldehyde treated rabbit erythrocytes and human erythrocytes for uea - ae according to beltro et al . .
paraffin sections ( 8 m ) of samples were cut , transferred to glass slides , deparaffinized in xylene , and rehydrated in graded alcohols ( 3 100% and 1 70%10 dips each ) .
slices were treated with 0.1% ( w / v ) trypsin solution at 37c for 2 min and washed ( twice , 5 minutes each time ) with 10 mm phosphate buffer saline containing 0.15 m nacl ( pbs ) .
afterwards , tissue slices were incubated with lectins - ae ( 100 l100 g ml ) for 2 h at 4c followed by washings ( 3 5 min ) with 100 ml of pbs .
the area corresponding to the tissue section ( square - shaped ) was defined as 0.25 cm . then , the tissues were cut and transferred to polypropylene test tubes containing 50 l of pbs .
finally , solutions of 0.5% h2o2 in 0.1 n hno3 ( 50 l ) and 0.25 m naoh ( 50 l ) were added for chemiluminescent measurement using a luminometer modulus single tube 9200 - 001 ( turner biosystems , usa ) .
the emission intensity was determined as relative light units ( rlu ) with a counting time of 5 seconds per sample .
lectin binding inhibition assays were accomplished by incubating each lectin solution with 300 mm methyl--d - mannoside ( con a ) , d - galactose ( pna ) , -l - fucose ( uea - i ) , and n - acetylneuraminic acid ( maa ) for 45 min at 25c prior to their incubation with tissues .
the software originpro 8 ( originlab corporation , one roundhouse plaza , northampton , ma , usa ) was used for the statistical analysis , and data were expressed as mean standard deviation ( s.d . ) .
obtained data were compared using anova and parametric statistical test of tukey ( p < 0.05 ) through sigmaplot ( ca , usa ) .
lectins conjugated to acridinium ester ( con a - ae , pna - ae , uea - ae , and maa - ae ) were collected by sephadex g-25 chromatography and their profiles were identical to those previously reported .
the superposition of the protein and chemiluminescence peaks indicated that all studied lectins were labeled with the ae .
the lectin derivatives were also capable of recognizing their specific carbohydrates according to the hemagglutinating activity .
these lectin derivatives were used to investigate the glycophenotype of cutaneous tumor tissues and the results are displayed in figure 1 .
lower expression of -d - glucose / mannose was presented for all tumors compared with the normal tissue .
ak , ka , scc , and bcc showed values of rlu equal to 143,694 28,626 rlu ; 140,704 29,009 rlu ; 148,238 38,333 rlu , and 134,007 30,537 rlu , respectively , whereas the normal tissue was 271,435 64,329 rlu .
the -l - fucose expression pattern was also lower for cutaneous tumors ( rlu values of 11,831 1,935 rlu ; 28,773 6,468 rlu ; 14,586 3,464 rlu , and 13,880 2,926 rlu for ak , ka , scc , and bcc , resp . ) compared with the normal epidermis ( 33,743 5,182 rlu ) .
on the other hand , they presented higher expression of gal-(1 - 3)-galnac compared with the normal skin tissues .
ak , ka , scc , and bcc presented rlu values of 23,766 2,525 rlu ; 32,567 6,127 rlu ; 31,172 5,134 rlu ; and 18,007 4,618 rlu , respectively , whereas a value of 7,567 1,799 rlu was found for the normal tissue .
actinic keratosis and scc exhibited higher expression of neu5ac-(2,3)gal than normal epidermis ( 35,687 8,226 rlu , 65,370 16,811 rlu , and 44,864 11,644 rlu , resp . ) .
however , ka ( 26,927 6,942 rlu ) and bcc ( 29,836 6,179 rlu ) showed equal rlu values compared with the normal tissue .
the standard deviation of all measurements varied from 10.6% to 25.9% of the mean value .
for all lectins , the mean among rlu values of cutaneous tumors and normal epidermis was significantly different , as observed using parametric statistical test of tukey .
however , statistically significant variations were not observed among rlu values of cutaneous tumors labeled with cona - ae , ak and ka labeled with pna - ae , ak and scc labeled with uea - ae , and bcc and ka labeled with maa - ae .
lectins - ae inhibition binding assay using specifics carbohydrates ( 300 mm ) resulted in a decrease in rlu values ( from 65% to 93% ) indicating that unspecific binding between the conjugates and cell surface molecules did not occur .
in our laboratory , the acridinium ester was used as a marker to trypanosoma cruzi and schistosoma mansoni antigens in chemiluminescent immunoassay . by this method ,
small amounts ( nanogram scale ) of circulating antibodies were detected in the patients ' serum .
the glycocode of normal , fibroadenoma , and invasive duct carcinoma tissues was investigated using lectins labeled with ae [ 15 , 22 ] .
anti - her-2-ae allowed a reduction in the subjectivity in prognostic and predictive information intrinsic to her-2 status evaluation in breast tumors .
cell surface glycans are highly related to cell - cell communication , host - pathogen interaction , and cell matrix interactions .
tumor invasion is dependent on a loss of intercellular adhesion and transmigration of cells through the basement membrane ( bm ) as well as the surrounding extracellular matrix ( ecm ) .
metastasis is a multistep phenomenon , which involves the loosening of the tumor cells from the primary tumor , the degradation of the extracellular matrix , and the invasion of blood vessels at the site of the primary tumor .
these steps require adhesive interactions , which are mediated by cell surface glycans and their interactions with endogenous carbohydrate binding proteins ( lectins ) .
aberrant glycosylation is a key feature of malignant transformation and the glycans involved influence the adhesive interactions of cancer cells often providing favorable conditions for tumor dissemination .
the molecular organization and the stability of adherent junctions formation are influenced by the presence of altered glycans on the cell surface , phenotypic change commonly observed in malignancy .
alterations in the expression of glycophenotype between benign and malignant skin lesions have been previously reported through lectin histochemistry and computer image analysis .
it is worthwhile to register that they did not compare with normal epidermis and included trichoepithelioma and seborrheic keratosis that were not studied in this work .
these authors reported higher expression of -d - glucose / mannose ( except in scc ) and d - galactose residues in cutaneous tumors .
the chemiluminescent -d - glucose / mannose detection ( con a - ae ) corroborated these findings but they were statistically lower than those found for the normal epidermis .
they also found high d - galactose residues ( pna ) as well as our results except that lower rlu was detected compared to the normal epidermis .
another contradiction was found as far as fucose is concerned . here fucose was chemiluminescently detected in the tumors that were statistically lower than that estimated for normal epidermis .
therefore , these lectins can be used as markers for distinction among skin lesions ; however , the chemiluminescent approach may be more sensitive than that based on computer image analysis of lectin histochemistry .
reported that laminins , a principal component of bm , exhibit unique saccharidic epitopes ( mannose - rich oligosaccharides ) .
therefore , aberrant -d - glucose / mannose expression in this protein may modify the adhesive interactions between transformed cells , contributing for the detachment of cancer cells from the primary tumor mass and the acquisition of a more motile and invasive phenotype .
several common structural changes occur in tumor glycans and can affect interactions between tumor cell - surface glycans and endogenous lectins , which may determine the metastatic potential of the tumor cell , including increases in the level of truncation and branching of structures as well as an increased expression of unusual terminal sequences .
these changes often result in an increased exposure of terminal galactose residues such as those found in the cancer - associated t antigen ( gal 1 - 3 galnac ) and the lewis x trisaccharide ( gal1 4(fuc1 - 3)glcnac )
. the higher expression of galactose residues observed by high rlu could be related to the potential role of galactose ligands in tumor cell metastasis by the correlation between increased expression of galectin , a family of galactose binding proteins , and the ability of many tumor cell types to metastasize .
in this study , fucosylation pattern changes were observed by the decrease of chemiluminescence signal using uea - i conjugated to ae for the labeling of cutaneous tumors .
ak showed fucosylation pattern similar to malignant lesions and different when compared to benign lesion ( ka ) .
this suggests a biochemical similarity with malignant neoplasms and supports the studies claiming that ak and cec are the same lesion in different degrees of development .
it is known that both integrins and e - cadherin are associated with cancer cells characteristics through regulation of the cell - extracellular matrix interaction and homotypic cell - cell adhesion , respectively .
. showed that core fucosylation is essential for the function of 31 integrin , a protein that connects many biological functions such as development , control of cell proliferation , and protection against apoptosis and malignant transformation .
therefore , based on the information above , it can be concluded that fucose would be involved in the biological behavior of cancer cells through functions regulation of many membrane associated proteins .
sialic acids ( sias ) are typically the outermost monosaccharide units on the glycan chains of glycolipids and glycoproteins and usually occur as a terminal component at the nonreducing end of their carbohydrate chains . due to its terminal localization on glycans and the variety of linkages to the underlying sugar chain , sialic acid has been utilized by a wide variety of sia - binding proteins , mainly viral and bacterial pathogens proteins , selectins , and siglecs . the increase in the expression of neu5ac-(2,3)gal between benign and malignant lesions of skin , ak , and scc , respectively , was observed by the increase of rlu values .
the differences in the sialylation degree of glycoconjugates on a tumor cell surface of scc may play an important role in the process of cell malignization and metastasis .
vural et al . reported the increase of total sialic acid ( tsa ) levels between patients with ak and controls , probably related to increased turnover of cells .
observed that differentiated squamous cell carcinoma expresses 2,3-linked - neuac residues ( recognized by lectin maa ) and the poorly differentiated squamous cell carcinoma 2,6-linked - neuac ( recognized by sambucus nigra agglutinin ) .
sialyl - glycoconjugates may regulate the interaction of cancer cells with other cells and with the cell matrix .
these molecules may be responsible for adhesion as well as antiadhesion and for extending the survival time of cancer cells in the blood stream .
moreover , sialic acid may also be involved in growth regulation . from the results presented in this work
one can assume that the chemiluminescent lectin - histochemistry approach allows the glycophenotype direct quantitative evaluation of skin tumors combining the specificity lectin - carbohydrates interaction and sensitivity of chemiluminescent assay . despite
that the number of analyzed samples ( n values ) does not permit a definitive assumption as far as diagnosis is concerned the statistical analyses showed glycophenotype alterations according to the cutaneous tumors . furthermore , although paraffin - embedded skin fragments were obtained from different individuals and sections were cut from them , acceptable mean dispersion values ( standard variation range of 10.6%25.9% ) were observed .
higher expression of -gal ( 1 - 3)-galnac and neu5ac-(2,3)gal as well as lower expression of -d - glucose / mannose and -l - fucose residues was evidenced by differences in rlu values .
these results confirm the importance of lectins as potential biomarkers for detection of changes in glycophenotype of skin neoplasms .
furthermore , this procedure is an effective tool to eliminate subjective analyses of microscopic preparations . | background .
tumor cells show alterations in their glycosylation patterns when compared to normal cells .
lectins can be used to evaluate these glycocode changes .
chemiluminescence assay is an effective technique for quantitative analysis of proteins , nucleic acids , and carbohydrates due to its high sensitivity , specificity , and rapid testing . objective . to use histochemiluminescence based on lectin
conjugated to acridinium ester ( ae ) for the investigation of glycophenotype changes in cutaneous tumors .
methods .
concanavalin a ( con a ) , peanut agglutinin ( pna ) , ulex europaeus agglutinin - i ( uea - i ) , and maackia amurensis agglutinin ( maa ) were conjugated to acridinium ester .
biopsies of cutaneous tumors and normal skin were incubated with the lectins - ae , and chemiluminescence was quantified and expressed as relative light units ( rlu ) .
results .
actinic keratosis ( ak ) , keratoacanthoma ( ka ) , squamous cell carcinoma ( scc ) , and basal cell carcinoma ( bcc ) showed lower expression of -d - glucose / mannose and -l - fucose residues compared to normal tissue .
cutaneous tumors displayed higher expression of gal-(1 - 3)-galnac residues than normal tissue .
ak and scc exhibited higher expression of neu5ac-(2,3)gal residues than normal epidermis .
ka and bcc showed equivalent rlu values compared to normal tissue . conclusions .
lectin histochemiluminescence allowed quantitative assessment of the carbohydrate expression in cutaneous tissues , contributing to eliminate the subjectivity of conventional techniques used in the histopathological diagnosis . |
access to public ltss programs varies across states and across disability and age groups , resulting in inequities based on where one lives , whether one s disability is lifelong or acquired , and which functions it affects .
a complex , fragmented system is at the root of the problem , along with the institutional bias in medicaid that treats hcbs as an optional service while institutional ltss are mandatory .
most hcbs programs are offered at the discretion of the states , with eligibility criteria and the menu of services often varying by type of disability and age group .
the 288 hcbs waiver programs operating under section 1915(c ) of the social security act generally target a specific disability or age group ; a cross - disability hcbs alternative is offered in 32 states that have chosen to create a personal care services program ( ng et al .
some require a household income less than the supplemental security income ( ssi ) level , which is about three quarters of the federal poverty level , whereas many hcbs waiver programs allow incomes up to three times the ssi level ( ng et al .
some states expand eligibility by subtracting health care expenditures from the household s income . in 38 states ,
working people who exceed the usual income limits are allowed to buy in to medicaid with a subsidized premium ( kehn , 2013 ) . aside from income , limits on financial assets are as low as $ 2,000 for many people qualifying for medicaid on the basis of disability .
advocacy groups have encouraged policy makers to expand ltss eligibility to a uniform 300% of ssi and to greatly increase the asset limit ( howes , 2010 ) .
single point of entry , options counseling . to reduce confusion over available ltss programs , aging and disability resource centers serve as single points of entry in many states , offering options counseling to consumers and professionals seeking assistance on their behalf .
other states have innovative counseling programs run through state agencies ( summer & howard , 2011 ) .
the balancing incentive program in the aca requires states to adopt a single - point - of - entry ( or no wrong door ) approach .
for example , vermont s choices for care , a global waiver program under section 1115 of the social security act , consolidates all hcbs and institutional services under a single umbrella .
the program is unique in offering the two types of services on a completely equal footing , depending only on level of need ( kaiser commission , 2006 ) .
the shift of ltss to a managed care system also offers the potential to consolidate programs within the health plan s purview .
separate budgetary allocations for institutional and hcbs programs can work as a disincentive to rebalancing the ltss system .
a practice used in several states to foster rebalancing is global budgeting for all ltss programs , or , more broadly , flexible accounting so that savings in institutional expenditures can be seamlessly reallocated to hcbs programs ( hendrickson & mildred , 2012 ) .
assessment of need . uniform assessment of level of need across programs and disability categories helps promote greater equity in service provision ( aarp , 2013 ) .
development of a standardized ltss assessment is a requirement of the balancing incentive program in the aca , and promising assessment tools are in use in washington and wisconsin ( nsclc , 2012a ) , among other states .
cost - containment measures .
most state hcbs programs fail to fully meet the assessed needs of people who require a lot of help . in all but eight states , hcbs programs place limits either on the number of hours of help a person can receive or on the total amount that can be spent on the person s services .
another cause of unmet need is a waiting period between application and receipt of services .
more than 400,000 people are on waiting lists for hcbs waiver programs nationally , with some states having very long lists ( e.g. , 125,000 in texas ) , whereas others have no such lists ( ng et al . , in press ) .
many hcbs programs incorporate some level of consumer direction , such as consumer choice in the allocation of service budgets or in the hiring and firing of service providers . by 2010 , 44 states allowed consumer direction within some or all of their medicaid hcbs programs ( ng et al .
programs in 36 states allow consumer direction of services received from an independent ( nonagency ) provider ( ng et al .
, in press ) . in some state systems , most notably california , independent providers are the norm , while an agency model predominates elsewhere .
matching service registries .
first created in california in the 1990s , publicly funded registries exist in 19 states to help consumers find workers meeting their requirements .
consumers can either perform their own database searches or call the operating agency and request matches ( seavey & marquand , 2011a ) .
fiscal intermediary or financial management services to shield self - directing consumers from the paperwork and other burdens of being an employer .
these services can be provided by a quasi - governmental agency , a private payroll or similar business , or an independent living center , home health agency , or other provider ( scherzer , wong , & newcomer , 2007 ) . the ability to employ family members , who are often the consumer s preferred source of consumer - directed ltss ,
is allowed by pcs programs in 20 states , but only 4 states allow a spouse or legal guardian to serve as a paid helper ( ng et al .
california s in - home supportive services program is often seen as the model for such a system . the availability of backup help in case the scheduled worker does not arrive is a particular problem for consumers using independent providers , who may have nowhere to turn if the worker does not show up .
a few small - scale programs serve as models of this type of service ( see , e.g. , access living , 2008 ) .
consumer training in hiring , supervising , and firing workers , and , if necessary , keeping records , making payments , and so on .
such training might be provided by independent living centers , fiscal intermediary agencies , or the hcbs program .
flexible budgets with which to pay for services , along with support in using them .
the cash and counseling demonstrations pioneered this approach , with good results in terms of greater satisfaction and reduced unmet need ( carlson , foster , dale , & brown , 2007 ) .
more recently , the department of veterans affairs ( va ) has begun offering veteran - directed hcbs , which closely follows the cash and counseling model and offers support in budget planning and management ( department of veterans affairs , 2012a ) .
another important but often neglected aspect of state ltss systems is workforce development to promote a stable and sizeable workforce of appropriately skilled workers .
such activities could include the following :
worker training or certification requirements . in the absence of federal standards for training or certification ,
half of the states impose a training requirement specifying a set number of hours ( with a median of 40hr minimum training ) , and one quarter of states had either a state - sponsored curriculum or a requirement for certification ( marquand , 2013 ) .
although three fifths of consumers wanted their workers to be better trained ( consumer voice , 2012 ) , training requirements remain controversial , with some consumers preferring train their own workers rather than following instructions given by professionals .
furthermore , some advocates fear that required training would limit the pool of potential workers , especially by discouraging family members from seeking payment .
training could also be offered on a voluntary basis or at the discretion of the consumer .
personal assistance workers generally earn low wages that have not kept pace with inflations ( phi , 2012b ) , leading to high turnover , an inexperienced workforce , and , in some places , worker shortages .
wage increases in some programs have reduced turnover and markedly increased the availability of workers ( howes , 2005 ; seavey & marquand , 2011b ) .
working conditions could also be improved through offering health coverage as an employment benefit , which many workers do not have .
new federal regulations will soon require employers to pay overtime to many workers for the first time , which will likely improve job conditions , but perhaps at the expense of cutbacks in the amount of help received by consumers .
other efforts to improve recruitment and retention of personal assistance workers include developing career ladders , so that entry level workers could see a path to skill building and advancement to a more secure , more respected , and better - paid job ( phi , 2012a ) .
however , professionalization , like credentialing , runs the risk of reducing the level of consumer direction and control and thus is not necessarily favored by disability advocates .
with caregiver stress identified as an important predictor of nursing home placement ( spillman & long , 2009 ) , support for family caregivers is crucial to the ability of many ltss recipients to remain in the community over the long term .
caregiver support can take many forms : the department of veterans affairs ( 2012b ) offers perhaps the most comprehensive package of supports , available to the primary family caregiver of qualifying post-9/11 veterans .
the package includes a monthly stipend in lieu of payment , reimbursement of care - related travel expenses , health coverage , mental health services , training , and respite care .
helping family caregivers prepare for , give input into , and handle transitions out of hospitals and institutions and back into the community is the focus of another innovative practice ( levine , halper , rutberg , & gould , 2013 ) .
the ability of paid ltss workers to perform certain health care - related tasks is advocated as an important support for family caregivers , who would otherwise have to perform those tasks themselves .
sixteen states allow tasks such as administering oral medications and injections , ventilator care , and tube feeding to be performed by attendants or aides rather than nurses ( reinhard , kassner , houser , & mollica , 2011 ) . increased access to assistive technology and home modifications through public programs
is proposed as a strategy to reduce physical strain and overall burden among family caregivers , as well to help consumers maintain community living ( the lewin group , 2012 ) .
remote monitoring , often proposed to ease caregiver stress , is being piloted and evaluated by senior care organizations in several states ( magan , 2011 ) .
efforts to transition institutional residents back to the community have gained prominence with the money follows the person ( mfp ) demonstration , which was extended until 2016 in the aca .
mfp focuses on long - stay residents who are medicaid beneficiaries , offering services such as care coordination , personal assistance , and technology for up to 1 year following return to the community .
although some state mfp programs have transitioned large numbers of residents , the overall transition rate is very low ( irvin et al . , 2012 ) , reflecting the difficulty in returning to the community people who may have given up their housing and support systems , and even lost independent living skills .
innovative approaches to transition include the following :
early intervention strategies offer transitional supports to nursing home residents early in their stay . the minnesota return to community program targets residents staying 6090 days and provides care coordination following discharge , resulting in substantially increased transition rates ( arling , dennis , kane , woodhouse , & abrahamson , 2012 ) .
some states offer transitional assistance services or community transition services through medicaid hcbs programs , potentially making them available to a broader population and over a longer period than mfp .
several states , including texas , massachusetts , and new york , require health plans to offer transitional services as part of a managed ltss system .
peer mentoring and peer support programs have been successful in facilitating transition of long - stay institutional residents .
mentors can serve as role models for consumers dubious of their ability to thrive in the community ( san francisco ihss public authority , 2013 ) , and peer support programs are being implemented to promote community transitions of people with mental health disabilities ( sudders , 2013 ) .
diversion from institutional placement for people at high risk is the focus of innovative practices in several states , where rapid eligibility decisions hasten access to hcbs and avoid unnecessary institutionalization .
several states use posthospital assessment procedures that can be conducted within a few days , and washington has a provisional eligibility system that authorizes hcbs waiver services while the application is being completed ( summer & howard , 2011 ) .
assessments of state ltss systems often focus on expenditures on institutional services versus hcbs , numbers of participants in different programs , amounts of services received , and policies that have been put into place . although these characterizations are important , they are no substitute for measuring quality and outcomes , such as adequacy and appropriateness of care and the consumers level of integration , control , participation , and general quality of life .
several national , state , and university efforts are being undertaken to develop measures covering one or more of those areas ( for a summary , see dredf & nsclc , 2013 ) : the measure applications partnership ( 2012 ) , convened by the national quality forum , proposes ltss and health care quality measures including consumer choice , life satisfaction , and community participation .
the agency for healthcare research and quality ( 2010 ) identified measures of medicaid hcbs quality , including consumer choice , control , and satisfaction .
the centers for medicare and medicaid services ( 2012 ) funded the development of several personal experience surveys for use by hcbs programs .
the latest , developed by truven and air and released in 2012 , contains measures of met and unmet need for ltss , choice of and satisfaction with services , and community participation .
wisconsin s personal experience outcomes integrated interview and evaluation system ( peonies ) identifies domains of a conceptual framework for hcbs outcomes , rather than actual measures ( dredf & nsclc , 2013 ) .
three domains relate to choice and making one s own decisions , three to social and community participation , three to appropriate treatment by paid workers and others , and the remaining three to health , safety , and stability .
a decade ago , only a few states operated programs offering ltss under a managed care model . now there are 43 managed ltss systems or demonstration projects active , or proposed and awaiting approval , in 30 states ( nasuad , 2013 ) . in these
, ltss is offered as a component of an integrated , managed system that also includes acute health care services .
one goal is to provide better - coordinated and more effective services than does the traditional , highly fragmented , fee - for - service system .
eighteen of the 30 states have or plan to have so - called duals demonstration projects involving managed ltss .
these projects , for beneficiaries of both medicare and medicaid , are aimed at integrating the two programs into a single , nonduplicative package .
most states will use a financing model in which the mco is given a capitated payment , or a set amount for each member to provide all needed health care and ltss . in three of the programs , however , an alternative model known as managed fee - for - service is being used ( medicare - medicaid coordination office , 2013 ) . in this model ,
the state coordinates medicare and medicaid benefits to provide seamless integration and access to all necessary services and gets to share in any cost savings resulting from the integration ( mann & bella , 2011 ) .
perhaps the most important difference between the two models is that , in a capitated model , the mco assumes financial risk for all expenditures , providing a strong motivation to reduce costs . in the managed fee - for - service model , providers are not at risk , but the state has a financial incentive to reduce costs . whether these approaches result in better care coordination or
cutting corners in service provision , or both , is a key question for evaluation .
an appropriately structured capitated model , including ltss across all settings , could provide an incentive for mcos to favor hcbs over more costly institutional services , thus hastening rebalancing of the ltss system , and to work to transition nursing home residents back into the community ( ncd , 2013 ) .
in contrast , programs that carve out institutional services might offer the plans an incentive to unnecessarily send people into nursing homes ( dembner , 2012 ) . apart from the duals demonstrations , 21 states operate managed ltss programs for medicaid - only participants ( nasuad , 2013 ) . among both the duals demos and the other medicaid managed ltss projects
a few programs have statewide scope and serve people of all ages with all types of disabilities , but most are more limited : operating only in selected regions of the state , or offered only to specific age groups , or targeted to or excluding people with a particular type of disability .
the diversity of programs makes for an interesting test bed of practices that might prove effective in both service provision and cost containment .
one key distinction among the programs is whether enrollment is mandatory or voluntary , and , if the latter , whether consumers must opt in or are passively enrolled with the opportunity to opt out .
advocates prefer an opt - in approach ( nsclc , 2012b ) , which has been successfully used by large programs in wisconsin and minnesota ( dembner , 2012 ) . for their part , states probably prefer transitioning everyone to avoid having to maintain two parallel systems .
different enrollment processes also affect the ability of researchers to compare managed care models with fee - for - service or to make comparisons across types of models , due to possible self - selection bias .
assessment of level of need is of particular concern in a managed ltss framework . fearing that health plans would underassess people s needs , advocates have promoted third - party assessment that is free of conflicts of interest ( aarp , 2013 ; dembner , 2012 ) .
third - party assessments , performed by government agencies or community - based organizations , are part of the duals demonstration projects in several states .
quality and outcome measurement is even more important in managed ltss than in fee - for - service systems . in a managed care environment
, there is a risk that mcos will become a black box , providing services but not reporting even basic measures of expenditures and populations by setting .
closely related is the issue of who is keeping track of and reacting to these indicators .
when states transfer service provision to private entities , a state agency must be empowered and staffed to monitor and enforce contractual agreements and ensure quality and adequacy of service provision .
advocates encourage states to have an oversight and monitoring plan to track and resolve problems with mco performance ( nsclc & dredf , 2012 ) .
furthermore , ombudsman programs , whether run by a state agency or an independent , quasi - official body , can protect the interests of consumers in dealing with mcos .
advocacy organizations serving in such roles could prove a promising strategy for protecting consumer rights .
disability rights wisconsin operates the ombuds program for younger adults getting ltss in that state ( dembner , 2012 ) .
several issues in managed ltss must be assessed at the mco level , rather than programwide .
some of these are similar to those discussed with reference to the entire state ltss system , such as support for consumer - directed services , workforce development , support for family caregivers , and programs for transition out of institutions and diversion from institutional placement .
coverage issues , such as consumers ability to obtain durable medical equipment and other assistive technology , are also key .
care coordination is another important issue because the ability to coordinate services is one of the purported benefits of the managed , integrated care model .
advocates promote multidisciplinary care coordination including people knowledgeable about available community supports ( aarp , 2013 ) and with expertise in ltss rather than solely clinical matters ( nsclc & dredf , 2012 ) . as with assessment , coordination of services
might be optimally provided by a third party , free of conflicts of interest that could provide a financial incentive to cut services ( ncd , 2013 ) .
a related issue is the extent to which the mcos , health care organizations used to treating illness , gain a better understanding of the importance of ltss in fostering independent living and community participation . aside from meeting basic needs
, ltss enables many consumers to leave their homes , work , and engage in community activities .
states could require plans to incorporate employment and other community participation supports into their benefits ( bcil , ncil , community catalyst , dredf , & tri - county independent living center , 2012 ) .
training may also be needed in cultural competency to handle the influx of new members with disabilities and their accommodation needs .
in furthering its goal of strengthening the u.s . ltss system , hhs should encourage and support states in broadening hcbs offerings to better meet consumers needs , consolidating fragmented programs and standardizing eligibility to increase equity across populations , and assisting consumers in enrolling in appropriate ltss programs and in giving them greater choice and control over their services , including the ability to return to the community from an institutional setting if they want to .
federal and state policies should promote a stable and appropriately skilled ltss workforce by improving job quality and should find ways to support family caregivers in continuing to provide the help that consumers need .
hhs should require , and states should welcome , expanded efforts to measure ltss quality and outcomes , relying not only on administrative data but also on direct feedback from consumers . although numerous existing programs and demonstration projects suggest potential strategies to achieve these purposes , further research is needed to identify and confirm promising practices in many of these policy domains .
several policy issues , such as quality and outcome measurement , are of particular concern as states continue the rapid process of transitioning ltss consumers into integrated , managed care programs .
state agencies be empowered to monitor quality and enforce requirements for high - quality services .
the needs of consumers must be protected by their getting assessed for services fairly by entities without a conflict of interest , getting support in resolving problems encountered in dealing with the mcos , and being given the option of remaining in or returning to a fee - for - service system if needed .
hhs and the states need to be especially vigilant in ensuring that mcos retain and enhance the ability for consumers to direct their own services and continue to receive services that are not strictly health care related but are more generally aimed at supporting people in participating fully in their communities .
this work was supported by the national institute on disability and rehabilitation research ( grant number h133b130034 ) and the administration for community living . | in response to a new federal initiative to improve the u.s . long - term services and supports ( ltss )
system , this commentary discusses an array of policies and practices that could potentially improve ltss provision by shifting from institutional to community - based services , increasing equity across populations , offering consumers more choice and control , improving conditions for workers and caregivers , and promoting improved consumer - level outcomes .
policy areas include access to publicly funded ltss , support for consumer direction , workforce development , caregiver support , transition from institutions to the community , diversion from institutional placement , and quality and outcome measurement .
policy considerations apply both to programs and to the managed care organizations that are increasingly responsible for ltss provision .
additional policy areas related to managed ltss include financial risk and capitation rates , enrollment strategies , assessment , outcomes monitoring , care coordination , and support for independent living goals . |
to evaluate the role of high resolution ultrasonography ( us ) and endoscopic ultrasonography ( eus)-elastography in predicting malignant lymphadenopathy .
this prospective study included 88 patients who underwent eus or us examination of different groups of lymph nodes ( lns ) .
the classification as benign or malignant based on the real - time elastography pattern and the b - mode us / eus images was compared to the final diagnosis obtained by eus or us guided fine - needle aspiration cytology ( fnac ) , tru - cut biopsy or excisional biopsy and follow - up in benign lesions not indicated for biopsy for at least 12 months .
regarding the echogenicity , 98.3% of the benign lns were hyperechoic , 1.7% were hypoechoic , while 89.7% of the malignant lns were hypoechoic , 3.4% were heterogenous and 6.9% were hyperechoic . with cut - off value of 1.93 , the sensitivity of longitudinal to transverse ratio was 73% and the specificity was 100% .
score 1 elastography had specificity of 100% in diagnosis of benign lns , sensitivity was 76.3% , positive predictive value ( ppv ) was 100% , negative predictive value ( npv ) was 84.7% while score 2 had a sensitivity of 60% , specificity of 31.5% , ppv of 15.3% , npv of 79.3% .
score 3 had a sensitivity of 70.2% , specificity of 100% , ppv of 13.8% , npv of 100% in detecting malignancy while score 4 had a sensitivity of 85.5% , specificity of 100% , ppv of 100% , npv of 65.5% .
elastography is a promising diagnostic modality that may complement standard ultrasound and eus and help guide fnac during staging of lns . | objective : to evaluate the role of high resolution ultrasonography ( us ) and endoscopic ultrasonography ( eus)-elastography in predicting malignant lymphadenopathy.methods:this prospective study included 88 patients who underwent eus or us examination of different groups of lymph nodes ( lns ) .
the classification as benign or malignant based on the real - time elastography pattern and the b - mode us / eus images was compared to the final diagnosis obtained by eus or us guided fine - needle aspiration cytology ( fnac ) , tru - cut biopsy or excisional biopsy and follow - up in benign lesions not indicated for biopsy for at least 12 months.results : regarding the echogenicity , 98.3% of the benign lns were hyperechoic , 1.7% were hypoechoic , while 89.7% of the malignant lns were hypoechoic , 3.4% were heterogenous and 6.9% were hyperechoic .
with cut - off value of 1.93 , the sensitivity of longitudinal to transverse ratio was 73% and the specificity was 100% .
score 1 elastography had specificity of 100% in diagnosis of benign lns , sensitivity was 76.3% , positive predictive value ( ppv ) was 100% , negative predictive value ( npv ) was 84.7% while score 2 had a sensitivity of 60% , specificity of 31.5% , ppv of 15.3% , npv of 79.3% .
score 3 had a sensitivity of 70.2% , specificity of 100% , ppv of 13.8% , npv of 100% in detecting malignancy while score 4 had a sensitivity of 85.5% , specificity of 100% , ppv of 100% , npv of 65.5%.conclusion : elastography is a promising diagnostic modality that may complement standard ultrasound and eus and help guide fnac during staging of lns . |
it classically occurs in the second decade of life , has its onset during puberty , and affects both sex and all ethnicities .
the estimated prevalence in the general population is 54 per 100,000 . using a combination of four indices , ie ,
central k ( descriptive of central steepening ) , the i - s value ( inferior - superior dioptic asymmetry ) , sim k , and the srax index ( skewed radial axes ) , rabinowitz was able to distinguish 98% of keratoconus videokeratographs from a group of normal controls,1 and in marked keratoconus , simple observation of the presence of keratometry over 47.20 d can make the diagnosis.2 sensory strabismus occurs due to temporary or permanent loss of vision in one or both eyes .
we present the case of a 17-year - old boy with bilateral keratoconus , exotropia , and a nodular lesion of the bulbar conjunctiva in the left eye . a prism cover test revealed a 45 prism d exodeviation ( figure 1 ) .
the aligned eye was the right eye and the keratoconus in this eye was of late onset .
vision in the left eye was poor and the keratoconus was advanced in this eye .
the patient has a corrected visual acuity of 6/9 in his better and aligned eye and an acuity of 1/60 in the affected eye .
keratometric measures were 42.75 d at an axis of 28 and 49.0 d at an axis of 118 in the right eye , and 52.0 d at an axis of 154 and 58.75 d at an axis of 64 in the left eye .
simk1 was 45.24 d at an axis of 113 and sim k2 was 42.75 d at an axis of 25 in the right eye .
sim k of the left eye could not be obtained because of a high steep keratometry value .
refraction in the right eye was + 1.5 , 7.5 25 and refraction in the left eye could not be obtained ( figure 2 ) .
a search of pubmed yielded only two studies reporting development of sensory strabismus after keratoconus . in these reports , binocular function in patients with longstanding asymmetric keratoconus was investigated by the authors .
the first of these reports was by sherafat et al , who investigated a database of approximately 350 patients with keratoconus.4 only 20 of these patients were identified as having abnormal binocular function.4 the abnormalities described in their study were central suppression of the deviating eye , reduction or loss of stereopsis with a microtropia when wearing a scleral lens , and a manifest exotropia with suppression when the poorer eye was uncorrected .
sherafat et al accepted the age of diagnosis as being the age of onset of keratoconus , and suggested that the adult visual system in these cases had been affected by prolonged visual deprivation .
however , it has generally been assumed that the sensitive period ends at about 7 years of age and that the adult human visual system is not affected by visual deprivation.59 it is possible that the age of onset in the patients reported on by sherafat et al may have been earlier than puberty and therefore their visual deprivation may have settled .
the second report was by khan and al - shamsi , who investigated a database of 103 patients with keratoconus and identified abnormal binocular function due to longstanding keratoconus in only seven patients.10 in their series , six patients had persistent exotropia .
after intervention to improve visual acuity , six patients complained of constant binocular diplopia that resolved after successful surgical alignment .
the authors explained that resolution of diplopia by realignment of the eyes enabled suppression of scotoma and freedom from diplopia .
the major limitation of their study was the lack of information provided on age at onset of keratoconus and a lack of explanation regarding the etiology of suppression of keratoconus . in our study
, neither the patient nor his parents knew when the keratoconus had started , but did give two clues , ie , a history of childhood - onset strabismus and vernal conjunctivitis with eye rubbing .
this lesion had invaded the cornea , was surrounded by injected blood vessels , and looked like pseudoepitheliomatous hyperplasia or a sequel of keratoconjunctivitis in childhood ( figure 3 ) .
pseudoepitheliomatous hyperplasia is an inflammatory lesion that usually occurs on the eyelid , but rarely in the bulbar conjunctiva .
however , there was no active inflammation of the conjunctiva in our patient , so this lesion may have been a sequel of keratoconjunctivitis in childhood .
the causes of poor eyesight are varied , with chorioretinal atrophy , congenital cataract , optic atrophy , retinal disease , complicated cataract , leucoma , coloboma , high myopia , congenital glaucoma , penetrating trauma , contusional eye trauma , and traumatic cataract being the more common causes.11 sensory strabismus also appears to have various causes , but is not a consequence of keratoconus,1,12 which typically commences at puberty and commonly affects both eyes .
however , when one eye is more severely affected than the other , visual loss can be prevented by glasses or contact lenses .
the visual loss is gradual and occurs primarily as a result of irregular astigmatism and myopia.13 the sensitive period for development of suppression ends when the keratoconus has settled .
the presence of a nodular lesion of the bulbar conjunctiva and a history of vernal conjunctivitis and eye rubbing in this patient confirms vernal keratoconjunctivitis in childhood .
rabinowitz1 and sugar12 consider that atopic conjunctivitis and eye rubbing are a cause of keratoconus .
the probable scenario in this patient is that vernal keratoconjunctivitis caused the childhood keratoconus , which became advanced in the left eye , and the prolonged visual loss was not corrected before the age of 7 years .
we believe that the abnormal binocular function due to keratoconus in our patient and in the cases reported by other authors occurred before the age of 7 years .
review of the relevant literature suggests that keratoconus seldom begins in the sensitive period during which abnormal visual experience affects visual development and may induce sensory exotropia . in our patient , sensory exotropia had developed because of prolonged visual loss based on the keratoconus in his poorer eye .
if keratoconus occurs before the age of 7 years and prolonged visual loss is not corrected , sensory strabismus can develop , as in our patient . | this case report describes a 17-year - old boy with sensory strabismus due to keratoconus and an ipsilateral nodular lesion of the bulbar conjunctiva . the aligned eye was the right eye and keratoconus in this eye was of late onset .
vision in the left eye was poor and keratoconus was advanced in this eye . due to the longstanding nature of the keratoconus and its occurrence in a developmentally sensitive period ,
sensory exotropia had developed in the left eye .
there was a nodular lesion of the bulbar conjunctiva in the ipsilateral eye .
if keratoconus occurs before the age of 7 years and the prolonged visual loss is not corrected , sensory strabismus can develop , as in this patient . |
in the current issue of critical care , venkatesh and colleagues present a novel study on adiponectin in critical illness .
this article , along with the concept of adipose tissue and adipocytokines being involved in the mechanisms of critical illness , requires some commentary .
historically , adipose tissue was thought to be an inert tissue that stores energy and protects the body from temperature and injury .
in contrast to this concept , it is now evident that adipose tissue is an active endocrine organ secreting many kinds of adipocytokines such as adiponectin , leptin , il-6 , and tnf that may affect metabolism , body weight regulation , and glucose and lipid homeostasis .
produced exclusively in the adipose tissue , adiponectin is the most abundant adipocytokine and circulates in the blood at high concentrations , accounting for approximately 0.01% of the total plasma protein [ 3 - 5 ] .
studies in rodents demonstrate a role for adiponectin in obesity and insulin resistance . in humans , reduced serum levels of adiponectin in obese subjects compared with nonobese subjects and negative correlations between adiponectin and body mass index ( bmi )
furthermore , serum concentrations of adiponectin have been found to be inversely correlated with insulin resistance in both nonobese and obese subjects and in patients with type 2 diabetes mellitus [ 11 - 13 ] . in spite of numerous clinical studies on adiponectin ,
however , its exact role in metabolism is unclear and remains to be elucidated , along with the as yet unsolved adiponectin paradox : a decreased concentration of adiponectin in obesity seems paradoxical , since fat tissue is the only known source of this protein . in viuew of that
, the paper by venkatesh and colleagues broadens knowledge of the subject and links the function of adipose tissue to the general defense against critical illness .
the authors ' suggestion that adiponectin may play a part in the inflammatory response in patients with critical illness is interesting , but it should be considered bearing in mind some limitations of the study . perhaps the major limitation is the lack of data on patients ' bmi .
associations between low and high bmi and mortality are well known , as are the correlations between bmi and adiponectin .
different bmi values between groups could consequently have influenced the adiponectin results . under these circumstances ,
furthermore , the associations between serum cortisol and adiponectin in critical care patients demonstrated in this study also require comment .
the high standard deviation for cortisol might have been caused by adrenal insufficiency in several patients with very low cortisol levels . in this setting , the group is not homogeneous with respect to adrenal response to stress , and consequently the overall statistical result may be confusing .
still , the authors have raised an interesting problem regarding the link between adrenal function and adipose tissue metabolism in critical illness .
an increased secretion of glucocorticoids by the adrenal gland is a well - known defense mechanism against hypotension and stress , whereas a decreased cortisol concentration is associated with a worse prognosis .
on the basis of this article , therefore , one can propose the hypothesis that a decreased adrenal response presumably inhibits protective adiponectin secretion , thus deteriorating the overall clinical condition .
obviously , in face of the above - mentioned study limitations , this tempting hypothesis requires further research .
although based on a preliminary study only , the idea shown by venkatesh and colleagues generates new hypotheses meaning we should have a rethink of the role of fat tissue in the critical state in humans . obviously the authors did not investigate the mechanism of decreased adiponectin in critical illness , as it was not the aim of their study .
however , the following fact is now evident : being a complicated organ and being spread throughout the body hormone - producing and cytokine - producing machinery , adipose tissue can not stand passively by in the face of severe threat to life , and is involved in numerous inflammatory responses including production of adipocytokines , tnfs , and metabolites of fat catabolism .
the nature of these responses remains to be elucidated , however , and noteworthy clinical studies should prompt further efforts in basic research to explain the mechanisms beyond clinical observations .
bmi : body mass index ; il : interleukin ; tnf : tumor necrosis factor .
| historically , adipose tissue was thought to be a passive tissue that stores energy and protects the body from temperature and injury .
in contrast to this concept , it is now evident that adipose tissue is an active endocrine organ secreting many kinds of adipocytokines , including adiponectin . presumably , adipose tissue and its products may have some impact on numerous pathways of response to trauma , sepsis and stress .
the discussion on a plausible role of adiponectin in critical illness has been raised by the fact of finding hypoadiponectinemia in critically ill patients .
the nature of this phenomenon , however , remains to be elucidated , and noteworthy clinical studies should prompt further efforts in basic research to explain the mechanisms beyond the clinical observation of low adiponectin levels in humans with severe illness . |
it has now been 20 years since the first reports appeared demonstrating that interleukin-2 is required to prevent the development of systemic autoimmune disease .
subsequent studies by sakaguchi and colleagues identified cd25 , the interleukin-2 receptor chain ( il2r ) , as one of the first useful markers for the identification of regulatory t cells ( tregs ) .
this led to the initial hypothesis that il2 is required for the development or function of tregs and more recently the implementation in the clinic of agonist il2:anti - il2 complexes for the treatment of autoimmune and inflammatory conditions . supporting the initial hypothesis that il2 is involved in treg development , work by malek and colleagues demonstrated that the autoimmune disease that developed in il2rb mice could be prevented by the transfer of cd25 tregs from wt mice into il2rb host mice .
additional work by this same group demonstrated that expression of an il2rb transgene that was expressed solely in the thymus was sufficient to rescue the defect in treg development suggesting that the defect in il2rb mice is due to a failure of treg development in the absence of il2 .
in contrast , lafaille and colleagues found that transfer of cd4 t cells from il2 mice into a mouse model of experimental autoimmune encephalomyelitis prevented disease , while cd4 t cells from il2ra mice did not .
these results suggested that cd4 t cells in il2 mice are capable of developing into and functioning as tregs . supporting this observation , two other groups used either foxp3-gfp reporter mice , or the ability to stain for intracellular foxp3 , to demonstrate that young il2 mice have foxp3 tregs and that the defect in these mice had to do with reduced function or fitness of these cells .
finally , work from our group and steve ziegler s was able to reconcile these findings by demonstrating that while young il2 mice do not lack foxp3 tregs , comparable il2rb mice have a substantial defect in treg development .
this latter result reflects redundancy between il2 and il15 as il2 x il15 mice mimic the defect in treg development observed in il2rb mice .
it is important to point out that under physiological circumstances il15 does not play a role in treg development or function as il2 signaling in tregs leads to downregulation of the il15r chain , thereby rendering these cells much less responsive to il15 .
thus , subsequent studies have demonstrated that the original experiments by malek and lafaille and colleagues were both correct as il2 plays an important role in both treg development and function .
cd4cd25foxp3 tregs that develop in the thymus ( also known as natural tregs ) constitute 24% of cd4 single positive ( cd4sp ) thymocytes , yet this relatively small population plays a critical role in maintaining peripheral tolerance and preventing autoimmunity .
the t cell receptor ( tcr ) repertoire of these natural tregs overlaps with that of non - regulatory t cell populations but is skewed to favor tcrs that interact with higher affinity to self - antigens in the thymus .
the molecular mechanisms that drive treg development have been tied to three primary signaling modules .
first , tcr signaling plays a key role as tcrs with higher affinity for self - antigen are preferentially selected into the treg lineage .
second , the costimulatory receptor cd28 also plays an important role as cd28 and b7 - 1/b7 - 2 mice both show clear defects in treg development .
third , signals emanating from the interleukin-2 receptor are also required for treg differentiation in the thymus .
these observations culminated in the development of a two - step model of thymic treg development , in which a tcr- and cd28-dependent , but cytokine - independent first step generates an il2-responsive intermediate treg progenitor that lacks foxp3 expression .
subsequently , a tcr - independent , il2/stat5-dependent second step results in the rapid conversion of treg progenitors into mature foxp3 tregs ( fig . 1 ) .
( a ) cd4sp thymocytes perceiving high affinity / avidity signals emanating from tcr / cd28 are first programmed via the nfb pathway to express il2r and il2r , rendering them highly responsive to il2
. a second step , which is tcr - independent , but cytokine - dependent , is completed when treg progenitors receive il2 signals transmitted via stat5 to subsequently drive expression of foxp3 .
( b ) cd4sp thymocytes plotted on the basis of cd25 and foxp3 expression can be categorized into ( 1 ) conventional or non - treg cells which are cd4cd25foxp3 ( gated in red ) , ( 2 ) cd4cd25foxp3 treg progenitors , which are also cd122 and gitr ( gated in green ) and ( 3 ) cd4cd25foxp3 mature tregs ( gated in blue )
. the representative tcr signal strength of each of these populations , reported via nur77-gfp expression , is shown in the histogram on the right . upon interacting with medullary antigen presenting cells ( apc ) presenting self - peptide : mhc ii complexes , strong tcr signals in a fraction of cd4sp thymocytes cause them to differentiate into treg progenitors , marked by elevated expression of the high - affinity il2r chain ( cd25 ) , the il2r chain ( cd122 ) , and the costimulatory tnf receptor superfamily member , glucocorticoid - induced tnf - related protein ( gitr ) .
the emergence of this cd4cd25cd122gitrfoxp3 treg progenitor population requires canonical activation of the nfb pathway downstream of tcr and cd28 ligation .
paired activation of lck from these receptors signals through the canonical nfb pathway to ultimately promote nuclear translocation of c - rel and rel - a .
the requirement for nfb activation in treg differentiation is demonstrated by the absence of thymic tregs and importantly , treg progenitors in animals deficient in cd28 , prkcq , carma1 , bcl10 and rel .
further studies revealed that c - rel binds the conserved non - coding sequence 3 ( cns3 ) located in the foxp3 gene to promote epigenetic modification of foxp3 rendering it permissive for subsequent transcription initiation .
the conversion of foxp3 treg progenitors into mature foxp3 tregs in the thymus occurs via a tcr - independent but il2/stat5-dependent process .
ligand binding by the high affinity il2r complex leads to phosphorylation of three key tyrosine residues located in the cytoplasmic domain of il2r by the kinases jak1 and jak3 .
phosphorylation of tyr-338 recruits the sh2-containing adaptor molecule , shc , facilitating activation of the ras / mapk / erk and pi3k / akt pathways via grb2 and gab2 , respectively .
phosphorylation of il2r at tyr-510 ( and to a lesser degree tyr-392 ) is critical for recruiting and activating stat5 .
the importance of il2r signaling in thymic treg differentiation is clearly demonstrated by the fact that the lethal autoimmunity in mice lacking il2rb is due to a failure to generate thymic tregs , and this phenotype is completely restored by adoptive transfer of small numbers of wild type tregs . moreover , retroviral transduction of il2rb bone marrow with wild type il2rb , or a mutant construct capable of activating only stat5 via tyr-510 , restored thymic treg generation in bone marrow chimeric mice .
in contrast , restoration of treg development did not occur when mutant constructs capable of activating ras / pi3k , but not stat5 , were transduced into il2rb bone marrow cells and engrafted into recipient mice .
likewise , crossing il2rb mice to transgenic mice expressing a constitutively active form of stat5b ( stat5b - ca mice ) restored treg development in the thymus .
additional support for the role of stat5 in treg development came from two studies that demonstrated that conditional deletion of stat5 in dp thymocytes ( i.e. , cd4-cre stat5a / b mice ) had minimal effects on cd4sp thymocytes with the exception of cd4foxp3 thymic tregs .
together , these findings indicate that stat5 activation downstream of il2r is required for thymic treg development .
two groups have demonstrated that cd4cd25foxp3 thymocytes are direct precursors of foxp3 tregs , which require only an additional il2r / stat5-dependent signal to express foxp3 ( fig . 1 ) .
first , hsieh and colleagues showed that adoptive transfer of cd4cd25foxp3 thymocytes , but not cd4cd25foxp3 thymocytes , into the thymii of wild - type hosts resulted in the development of cd4foxp3 tregs .
similar results were observed upon adoptive transfer into mhcii - deficient mice demonstrating that the conversion process did not require additional signals via the tcr .
in addition , stimulation of sorted treg progenitors with il2 in vitro led to induction of foxp3 mrna within a few hours followed by the development of cd4foxp3 tregs 24 h later .
several studies indicate that the treg tcr repertoire is biased toward self - reactivity , although there is some overlap with the conventional cd4foxp3 tcr repertoire .
initial studies by burchill et al . found that augmented stat5 signaling clearly altered the treg tcr repertoire .
specifically , this study evaluated the effect of forced stat5 activation on the treg tcr repertoire by tracking the frequency of cd4foxp3 cells specific for a peptide called 2w1s bound to i - a mhc class ii molecules using peptide : mhcii tetramers ( in this study the 2w1s : i - a tetramer ) in littermate control and stat5b - ca mice .
a simple comparison of the ratio of cd4foxp3 to cd4foxp3 cells among total and 2w1s - specific t cells revealed that this tcr is dramatically underrepresented in the treg pool of wild type mice .
the frequency of cd4foxp3 t cells specific for 2w1s : i - a in wt mice ( ~36% ) was much lower than that observed for the average of all other treg tcr specificities ( ~15% ) ; in contrast , in stat5b - ca mice the frequency of 2w1s : i - a specific tregs was identical to the average of all other treg tcr specificities .
this finding demonstrated that for at least one tcr specificity regulation of stat5 signaling had a profound effect on its distribution within the treg repertoire .
thus , ectopic stat5 activation removed the tcr selection bias that typically results in underrepresentation of 2w1s : i - a specific t cells in the treg tcr repertoire . to extend the observations on the role of stat5 and the treg tcr repertoire beyond a single tcr ,
a fixed tcr transgenic system was employed to partially restrict the repertoire , and sequencing was performed on over 1,000 productive v2 rearrangements from thymocytes isolated from stat5b - ca mice or their wt littermate controls .
tonic stat5 signaling in stat5b - ca mice led to a dramatic expansion in the diversity of productive rearrangements among cd4foxp3 tregs including substantial numbers of tcrs that were typically not found in the treg tcr repertoire .
these initial studies have been confirmed more recently by moran and hogquist using an independent approach .
this latter study used bac transgenic mice in which a gfp reporter had been knocked into the nur77 gene locus .
importantly , this study confirmed that signal strength for tcrs expressed by tregs is higher than that for conventional cd4 t cells ( fig . 1 ) .
when the nur77-gfp mice were crossed to stat5b - ca mice , it was observed that the treg tcr repertoire was much broader and included substantial numbers of t cells with tcrs that signaled with significantly lower signal strength as assessed using the nur77-gfp reporter .
/ stat5 signaling helps to focus the thymic treg tcr repertoire on tcrs with higher intrinsic signal strength .
this suggests that il2r / stat5 signaling plays an important role in ensuring the preferential development of tregs with higher reactivity to self - antigens , which may be important in preventing autoimmunity .
several questions remain unanswered pertaining to the role of il2 and stat5 in promoting thymic treg differentiation .
first , what cell subsets in the thymus actually synthesize the il2 required for completing the second step of treg development ?
il2 was originally described as a cytokine made by activated t cells to drive proliferation and survival of t cells , thus amplifying effector responses . in this regard
, a reasonable assumption might be that developing thymocytes produce the il2 needed for thymic treg development .
however , thymocytes make exceedingly low levels of il2 ( if any ) relative to splenocytes upon stimulation ( s.a.m . and m.a.f .
, unpublished observation ) . the observation that dendritic cells and b cells can also make il2 suggest that these cell subsets might play an important role in producing the il2 needed for treg development in the thymus
. further work in which il2 can be conditionally deleted in t cells , dendritic cells and b cells will be needed to definitively address this question .
a second question has to do with what signals actually induce or regulate il2 production in the thymus ; such signals remain undefined .
stat5 binding sites have been found in the foxp3 promoter region as well as within the cns2 region of intron 1 in the foxp3 gene and several studies have shown stat5 binding to those sites .
deletion of the entire cns2 region including the stat5 binding sites did not prevent treg development although it did have an effect on stability of foxp3 expression .
however , the cns2 region is highly methylated in non - tregs and typically completely demethylated in natural foxp3 tregs .
if methylation of this region normally represses foxp3 transcription , then deletion of the entire region would remove the need for any factors that typically reverse this methylation state .
thus , whether stat5 binding to cns2 plays a role in treg development is difficult to determine based on studies deleting the entire cns2 region . the critical co - factors that interact with stat5 to promote treg development are also poorly characterized .
stat5 is known to interact with a variety of both co - activators , such as cbp and p300 , and co - repressors such as ncor2 .
intriguingly , treatment of treg progenitors with two distinct histone deactylase ( hdac ) inhibitors prevented the il2/stat5-dependent conversion of treg progenitors into tregs .
although this result at first appears counterintuitive , it is consistent with several reports demonstrating that stat - dependent gene transcription frequently requires hdac activity .
whether ncor2 and associated hdacs are recruited to the foxp3 locus by stat5 during thymic treg differentiation , and if so , how this complex regulates foxp3 transcription , remains to be elucidated .
thus , the molecular mechanisms by which stat5 alters transcription of genes involved in treg differentiation remains to be established .
an important feature of peripheral tolerance is the conversion of nave cd4 t cells into induced regulatory t cells ( itregs ) in peripheral lymphoid organs .
itregs have important roles in protecting against chronic inflammatory conditions , and likely play a key role in regulating immune responses to commensal microorganisms .
the differentiation of itregs , like ntregs in the thymus , requires both tcr- and il2-dependent signals .
however , unlike ntregs , itregs require transforming growth factor- ( tgf ) for their differentiation . moreover , while the carma1/nfb pathway is required for the development of ntregs it actually antagonizes itreg differentiation . finally , the stability of itregs is lower than that of ntregs , a feature which correlates with the greater degree of dna methylation of the cns2 region of the foxp3 gene in itregs vs. ntregs .
a role for il2 in itreg development was established many years ago in studies documenting the role of both tgf and il2 in itreg differentiation .
more recent studies have demonstrated that il2 and stat5 also play critical roles in maintaining stability of the itreg lineage .
specifically , these studies demonstrated that transfer of itregs into congenic hosts resulted in loss of foxp3 expression in the transferred itregs .
this result could be blocked by co - administration of agonist il2 : anti - il2 complexes indicating that il2 was required to maintain foxp3 expression in itregs in vivo .
these studies further documented that the loss of foxp3 expression correlated with re - methylation of the cns2 region ( also referred to as regulatory t cell specific demethylated region or tsdr ) of the foxp3 gene , and that il2 stimulation prevented this re - methylation process . the mechanism by which this occurs remains unclear
however , stat5 binding sites are found in the cns2 region , which may be important for maintaining foxp3 expression .
it is also possible that stat5 directly initiates demethylation of this region in nave cd4foxp3 t cells as they are being converted into cd4foxp3 itregs . arguing against this possibility is evidence that stat5 binds poorly to its cognate dna binding site when it is methylated .
however , only one of the three potential stat5 binding sites found in the cns2 region contains a cpg motif that could be methylated .
for example , blazar and colleagues demonstrated that tregs expressing a constitutively active form of stat5b ( stat5b - ca mice ) are superior to wt tregs in protecting mice from graft - versus - host disease .
this was due to a number of factors including ( 1 ) improved homeostasis of transferred tregs , ( 2 ) augmented treg suppressor function and ( 3 ) reduced ability of stat5b - ca effector t cells to differentiate into th1 and th17 cells . supporting these observations , malek and
colleagues demonstrated that il2 and stat5 signaling are required for development of a population of klrg1 treg cells that appear to express elevated levels of many factors required for treg function , such as il10 .
interestingly , this report documented that quite modest levels of il2/stat5 signals are required for thymic treg development and peripheral survival .
in contrast , the development of klrg1 tregs , which represent a terminally differentiated form of treg with augmented suppressor function , require much stronger il2/stat5 dependent signaling . in many ways
this latter population resembles klrg1 effector cd8 t cells suggesting that these two populations may develop via similar mechanisms .
as mentioned above , stat5 has been shown to inhibit the production of il17 both in vivo and in vitro .
the mechanism underlying this has been attributed to the ability of stat5 to directly compete with il6-dependent stat3 binding to enhancers within the il17 gene locus .
stat5 binding within the il17 gene locus correlated with recruitment of the co - repressor ncor2 , which is known to interact with stat5 . furthermore ,
il2 downregulates il6 receptor expression on itregs which further acts to prevent itregs from differentiating into th17 cells .
thus the balance between stat5 and stat3 signaling plays an important role in directing itreg development .
these cytokines activate stat6 and stat4 respectively , which are required for inhibiting treg differentiation .
while the mechanism by which stat6 and stat4 inhibit treg development remains to be precisely defined , both of these factors have been shown to reduce stat5 binding to the promoter and/or cns2 region of the foxp3 gene .
thus , cytokine crosstalk plays an important role in directing the differentiation of itregs . as mentioned above with regard to thymic treg development , stat5 interacts with a number of potential binding partners .
interestingly , recent work from the oshea lab has shown that micro rnas activated by tgf and retinoic acid receptor ( rar ) suppress the expression of one of these binding partners , the co - repressor ncor2 .
specifically , this study demonstrated that conversion of itregs into t follicular helper cells ( tfh ) is limited when mir10a is expressed at high levels , such as is the case following tgf and rar signaling in the periphery .
it appears that mir10a may have a role in fixing the itreg cell lineage by suppressing conversion of itregs into either th17 or tfh cells .
additional studies are needed to more precisely define the role of ncor2 and other stat5 interacting partners on the development and maintenance of regulatory t cells .
work over the past 20 years has clearly documented an important role for il2 and stat5 in shaping the development of both natural and induced tregs .
more recent studies point to a critical role for il2/stat5 signals in modifying the functional activity of regulatory t cells
. however , the molecular mechanisms by which stat5 and its many potential binding partners influence treg biology are just beginning to be explored . a better understanding of how the il2/stat5 pathway governs treg biology may allow for more targeted approaches for augmenting or inhibiting treg function .
such information could be particularly useful in developing strategies to inhibit treg responses to tumors or to augment treg responses in autoimmunity . | interleukin-2 and its downstream target stat5 have effects on many aspects of immune function .
this has been perhaps best documented in regulatory t cells . in this review
we summarize the initial findings supporting a role for il2 and stat5 in regulatory t cell development and outline more recent studies describing how this critical signaling pathway entrains regulatory t cell differentiation and affects regulatory t cell function . |
the light microscopic study was performed on 40 human horizontal rectus muscles obtained from 3 normal subjects and 34 patients with oculomotor disorders .
six control horizontal rectus muscles were obtained from the right globe of 3 multiorgan donors ( 2 muscles from each ) a few hours after postmortem in conformity with legal requirements .
patients with oculomotor disorders were composed of 20 patients with acquired strabismus , 11 with infantile strabismus , and 3 with congenital nystagmus .
one horizontal rectus muscle from each patient with oculomotor disorder was obtained during resection of a rectus muscle .
the electron microscopic examination was carried out on 6 horizontal rectus muscles from the left globe of 2 multiorgan donors ( 8 month infant and 61-year - old female ) and 4 patients , including 10-year - old subject with intermittent exotropia , 3-year - old with infantile exotropia , 49-year - old with acquired paralytic strabismus and 4-year - old with congenital nystagmus .
methods for securing human tissues were humane , included proper consent and approval , and complied with the tenets of the declaration of helsinki and austrian federal transplantation law .
the 20 patients with acquired strabismus consisted of 6 with paralytic strabismus , 5 with constant exotropia , 5 with intermittent exotropia , 3 with sensory strabismus , and 1 with secondary esotropia after retinal reattachment .
the 11 patients with infantile strabismus consisted of 5 with infantile exotropia , 5 with infantile esotropia , and 1 with congenital third cranial nerve paralysis .
the mean age in patients was 29.4 ( range , 2 - 64 ) years in patients with acquired strabismus , 3.45 ( range , 1 - 10 ) years in patients with infantile strabismus , and 4.3 ( range , 3 - 6 ) years in patients with congenital nystagmus ( table 1 ) . before surgery
, all patients affected by strabismus demonstrated large - angle deviations ( 50 prism diopters ) , and all patients with congenital nystagmus showed large - angle face turn ( 30 degrees ) .
all samples from the eoms were obtained by excising a full width strip of rectus muscle during strabismus surgery .
eoms were placed on slight stretch and care was taken to include the distal myotendinous junction in the 8 - 12 mm long tissue strip .
all specimens were gently stretched with 2 forceps and fixed with pins to the bar during the fixation process to prevent folding and curling .
the specimens were fixed for at least 24 hours in 10% buffered formalin and then embedded in paraffin for routine histopathological processing .
complete series of paraffin cross sections ( 10 m ) were cut on a microtome and mounted on glass slides .
paraffin cross sections were stained with hematoxylin and eosin at approximately 200 m intervals , and we examined samples progressing from muscle to tendon to select the most distal section just before reaching the first evidence of tendon . from the distal part from this selected section , we made 5 serial sections at intervals of 50 m , which totaled to 200 m .
series of these 5 sections in the myotendinous junction were stained with hematoxylin and eosin .
total imc counts in each eom were evaluated based on an examination of these 5 serial sections with 50 m intervals in the distal myotendinous junction ( 200 m ) . when the imc was found , 2 nearest sections ( 10 m intervals ) were impregnated with silver for light microscopy . for conventional transmission electron microscopy
, tissues were immersed in a fixative solution consisting of 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) and allowed to remain in this solution for more than 12 hours . after rinsing in the same buffer ,
samples were postfixed in 1% osmium tetroxide in phosphate buffer 0.1 m ( ph 7.4 ) , dehydrated in ethanol at increasing concentrations , and embedded in epon .
when the imc was found , approximately 10 ultra - thin sections alternating with semi - thin ones were cut at appropriate intervals ( 50 - 60 nm ) , mounted on copper grids , stained with uranyl acetate and lead citrate , and examined under a hitachi h-7100 transmission electron microscope ( hitachi , tokyo , japan ) .
all specimens were gently stretched with 2 forceps and fixed with pins to the bar during the fixation process to prevent folding and curling .
the specimens were fixed for at least 24 hours in 10% buffered formalin and then embedded in paraffin for routine histopathological processing .
complete series of paraffin cross sections ( 10 m ) were cut on a microtome and mounted on glass slides .
paraffin cross sections were stained with hematoxylin and eosin at approximately 200 m intervals , and we examined samples progressing from muscle to tendon to select the most distal section just before reaching the first evidence of tendon . from the distal part from this selected section , we made 5 serial sections at intervals of 50 m , which totaled to 200 m .
series of these 5 sections in the myotendinous junction were stained with hematoxylin and eosin .
total imc counts in each eom were evaluated based on an examination of these 5 serial sections with 50 m intervals in the distal myotendinous junction ( 200 m ) . when the imc was found , 2 nearest sections ( 10 m intervals ) were impregnated with silver for light microscopy .
for conventional transmission electron microscopy , tissues were immersed in a fixative solution consisting of 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) and allowed to remain in this solution for more than 12 hours . after rinsing in the same buffer ,
samples were postfixed in 1% osmium tetroxide in phosphate buffer 0.1 m ( ph 7.4 ) , dehydrated in ethanol at increasing concentrations , and embedded in epon .
when the imc was found , approximately 10 ultra - thin sections alternating with semi - thin ones were cut at appropriate intervals ( 50 - 60 nm ) , mounted on copper grids , stained with uranyl acetate and lead citrate , and examined under a hitachi h-7100 transmission electron microscope ( hitachi , tokyo , japan ) .
small nerves enclosed by a loose capsule of connective tissue cells terminated in the myotendinous region .
the imcs were identified with a light microscope by two investigators ( sep , hss ) .
golgi tendon organs or muscle spindles were not observed in any stained eom region . in the control eoms ,
nerve ending and muscle fibers were sheeted by thin capsules , which constituted the imcs ( fig . 1a ) .
the presence of nerve fibers within the imcs was revealed by silver staining ( fig .
examinations of the distal myotendinous junctions of eoms of patients with acquired strabismus showed normal features . in the congenital strabismus eoms ,
the distance of neighboring nerve fibers increased gradually from distal to proximal muscle fibers ( fig .
a single myelinated nerve fiber ran without branching , and the axon was completely sheathed by a schwann cell .
most imcs possessed normal features , however some schwann cell degenerations were observed in the eoms of patient with intermittent exotropia ( fig .
examination also found atrophic muscle fiber and flattened schwann cell process in some of the imcs of patient affected by paralytic strabismus ( fig .
most nerve fibers at the distal myotendinous junction in patient with infantile strabismus were observed to be altered .
these modifications consisted of schwann cell degeneration and axon alteration with maintenance of a practically normal general architecture ( fig .
. examinations of the distal myotendinous junction of patient with congenital nystagmus revealed small , anomalous nerve terminal endings . some schwann cells and axons lacked neurotubules and neurofilaments , and golgi apparatuses and mitochondria were also absent .
axonal myelin was anomalous , discontinuous , and unevenly distributed , and unmyelinated nerve fibers demonstrated fragmented neurilemma , swelling , and degeneration .
small nerves enclosed by a loose capsule of connective tissue cells terminated in the myotendinous region .
the imcs were identified with a light microscope by two investigators ( sep , hss ) .
golgi tendon organs or muscle spindles were not observed in any stained eom region . in the control eoms ,
nerve ending and muscle fibers were sheeted by thin capsules , which constituted the imcs ( fig . 1a ) .
the presence of nerve fibers within the imcs was revealed by silver staining ( fig .
examinations of the distal myotendinous junctions of eoms of patients with acquired strabismus showed normal features . in the congenital strabismus eoms ,
the distance of neighboring nerve fibers increased gradually from distal to proximal muscle fibers ( fig .
a single myelinated nerve fiber ran without branching , and the axon was completely sheathed by a schwann cell .
most imcs possessed normal features , however some schwann cell degenerations were observed in the eoms of patient with intermittent exotropia ( fig .
examination also found atrophic muscle fiber and flattened schwann cell process in some of the imcs of patient affected by paralytic strabismus ( fig .
most nerve fibers at the distal myotendinous junction in patient with infantile strabismus were observed to be altered .
these modifications consisted of schwann cell degeneration and axon alteration with maintenance of a practically normal general architecture ( fig .
. examinations of the distal myotendinous junction of patient with congenital nystagmus revealed small , anomalous nerve terminal endings . some schwann cells and axons lacked neurotubules and neurofilaments , and golgi apparatuses and mitochondria were also absent .
axonal myelin was anomalous , discontinuous , and unevenly distributed , and unmyelinated nerve fibers demonstrated fragmented neurilemma , swelling , and degeneration .
small nerves enclosed by a loose capsule of connective tissue cells terminated in the myotendinous region .
the imcs were identified with a light microscope by two investigators ( sep , hss ) .
golgi tendon organs or muscle spindles were not observed in any stained eom region . in the control eoms ,
nerve ending and muscle fibers were sheeted by thin capsules , which constituted the imcs ( fig . 1a ) .
the presence of nerve fibers within the imcs was revealed by silver staining ( fig .
examinations of the distal myotendinous junctions of eoms of patients with acquired strabismus showed normal features . in the congenital strabismus eoms ,
the distance of neighboring nerve fibers increased gradually from distal to proximal muscle fibers ( fig .
at the distal myotendinous junction of control eoms , several imcs were observed near the muscle fibers . within the imc capsule , a single myelinated nerve fiber ran without branching , and the axon was completely sheathed by a schwann cell .
most imcs possessed normal features , however some schwann cell degenerations were observed in the eoms of patient with intermittent exotropia ( fig .
4 ) . examination also found atrophic muscle fiber and flattened schwann cell process in some of the imcs of patient affected by paralytic strabismus ( fig .
most nerve fibers at the distal myotendinous junction in patient with infantile strabismus were observed to be altered .
these modifications consisted of schwann cell degeneration and axon alteration with maintenance of a practically normal general architecture ( fig .
. examinations of the distal myotendinous junction of patient with congenital nystagmus revealed small , anomalous nerve terminal endings . some schwann cells and axons lacked neurotubules and neurofilaments , and golgi apparatuses and mitochondria were also absent .
axonal myelin was anomalous , discontinuous , and unevenly distributed , and unmyelinated nerve fibers demonstrated fragmented neurilemma , swelling , and degeneration .
although still under debate , proprioceptive input from the eoms probably plays a role in the control of ocular alignment .
steinbach and smith5 have given supports for a proprioceptive contribution to spatial localization from the study of strabismus patients .
they have demonstrated patients undergoing strabismus surgery for a second time showed more errors in perceiving shifts of eye position when compared with those undergoing their first surgery , and suggested that some nervous organs may be important for eye position proprioception . palisading nerve endings in imcs
ruskell denied not only the existence of imcs at birth in human,21 but also their role in proprioception in his review article.22 however , lukas et al.23 have given important arguments against ruskell 's study , and suggested that imcs may serve as proprioceptors as well as effectors . despite many studies on the subject
, uncertainty persists of the exact roles of imcs in visual spatial perception and ocular motor behavior .
several groups have suggested that imcs are sensory,16,21,22,24 - 29 but others have considered them as motor30 - 32 structures , or both.23 billig et al.26 identified sensory nerve endings in cat extraocular muscles by injecting neuronal tracers into the trigeminal ganglion , which contains the sensory cells that innervate the eye muscles . after the application ,
three types of nerve endings were labeled within eoms , one type confirmed palisading endings of imcs at the myotendinous junction of each eom , whilst the other types were sensory terminals within the muscle belly .
they reported imcs establish contacts with tendon fibrils and attach muscle fibers , and reveal sensorylike nerve terminals .
however , they also presented the morphological features of motor terminals , which were confirmed by -bungarotoxin staining .
based on these features , they proposed that imcs might function as " propriocept - effectors " , by combining sensory and motor qualities .
another view have highlighted imcs as motor structures and suggested that visual spatial perception might be served by efference copy.14,30 - 32 sas and schb30 observed degenerated palisade endings in eoms after removing small stereotactic lesions in oculomotor nuclei .
blumer et al.31 studied imcs in rabbit eoms , and based on fine structure and -bungarotoxin binding suggested that myoneural contacts in imcs are exclusively motor . a recent study on cat eoms also provided evidence that palisade endings are exclusively motor .
specifically , it was found that palisade endings arise from nerve fibers that establish motor contacts on muscle fibers , and also that neuromuscular contacts in palisade endings exhibit -bungarotoxin staining.32 specific research attention has also been focused on the possible correlation between oculomotor disorders and structural modifications of proprioceptors at the myotendinous junction .
however , data on the possible morphological alterations of imcs in the various forms of strabismus is still lacking .
corsi et al.7 observed alterations in proprioceptors located at the scleral myotendinous junction of the eoms of patients suffering from congenital strabismus .
another study20 on congenital esotropia also reported the presence of altered sensory nerve endings at the myotendinous junction , and of normal motor nerve endings in the muscle body .
this damage to nerve endings consisted of alterations to both contractile structures and mitochondria , and resulted in severer lesions at the myotendinous junction than in the muscle body.20 in patients with congenital nystagmus , the myotendinous and tendinoscleral area of eoms showed not only anomalous imcs but also anomalous vascular endothelial cells.33 all of these alterations support the hypothesis that the most important eom functional alteration in congenital oculomotor disorders concerns the distal myotendinous junction , and that imcs , as proprioceptors , play a prominent role in disease pathogenesis . in this study
, we observed that almost normal features in the nerve endings at the myotendinous junction are demonstrated in acquired strabismus , including constant exotropia , intermittent exotropia , paralytic strabismus , sensory strabismus , and secondary esotropia after retinal reattachment .
however it is important to note that some imcs in noninfantile strabismus exhibited the morphologic alterations , which should be interpreted as rather acquired abnormalities from strabismus than the causes of oculomotor disorders .
, it appears that congenital and early - onset oculomotor disorder may be related to a degeneration and/or dysgenesis of imcs .
furthermore , when considering the normal features of most imcs in acquired strabismus our study supports the hypothesis that some disturbance of imcs may play an important role in the pathogenesis of oculomotor disorder , and that this is not the consequence of strabismus due to other causes . we speculate that imcs are supposed to provide proprioceptive information , and a proprioceptive feedback system should be stimulated and calibrated early in life for the development of binocular vision .
our study provides evidence that some disturbances in the proprioceptive feedback network , such as degeneration and/or dysgenesis of imcs , could result in oculomotor disorders .
this concept is supported by the findings of previous studies,34,35 in which neonatal de - efferentation in the rat resulted in a reduced size of proprioceptors , whereas de - afferentation in young rats caused the formation of receptors that maintained their general architecture , but completely lacked the nerve component . in conclusion ,
imcs are supposed to function as proprioceptors , although they may have also effector properties .
it should be emphasized in strabismus surgery that imcs , as ocular proprioceptors , are located in the myotendinous region . | purposeto analyze innervated myotendinous cylinders ( imcs ) in the extraocular muscles ( eoms ) of normal subjects and strabismic patients.methodsthe rectus muscles of 37 subjects were analyzed .
distal myotendinous specimens were obtained from 3 normal subjects , 20 patients with acquired strabismus , 11 with infantile strabismus , and from 3 with congenital nystagmus , and were studied by using light microscopy . some specimens ( 6 rectus muscles )
were also examined by transmission electron microscopy.resultsimcs were found in the distal myotendinous regions of eoms .
the imcs of patients with acquired strabismus showed no significant morphological alterations .
however , significant imcs alterations were observed at the distal myotendinous junction of patients with congenital strabismus and congenital nystagmus.conclusionsthis study supports the notion that imcs in human eoms function mainly as proprioceptors , along with effector properties , and a disturbance of ocular proprioceptors plays an important role in the pathogenesis of oculomotor disorder .
we suggest that a proprioceptive feedback system should be stimulated and calibrated early in life for the development of binocular vision . |
incomplete spinal cord injury ( sci ) is characterized by partial functional loss , with
varying degrees of voluntary motor and sensory functions remaining in the three segments
below the injury at a neurological level1 , 2 .
the majority of patients with incomplete
sci showed impaired upper extremity function due to damage in motor and sensory , decreasing
the ability to perform independent daily activities and , concomitantly , quality of life3 .
constraint - induced movement therapy
( cimt)4 is a rigorous training method ,
designed to improve function of the upper extremities by exploiting neural plasticity5 .
cimt is the preferred treatment for stroke
patients , in addition to those with diverse diseases , including cerebral palsy , hip joint
fracture , and incomplete sci .
nevertheless , original cimt protocols are problematic to implement , due to physical and
psychological pressures resulting from a 6-hour usage restriction on unaffected upper
extremities . as a result ,
the modified cimt ( m - cimt ) protocol was developed6 , 7 . to
preserve and enhance effects of m - cimt on the function of the upper extremities , subjects
are emphasized the necessity of using both hands and encouraged to do so in their daily
activities8 .
this study applied m - cimt , developed to treat patients with neurological repercussions of
stroke , to a subject with incomplete sci as a method of promoting mobility of an upper
extremity exhibiting impaired function .
moreover , this study aimed to enhance performance of
daily activities and overall quality of life for the subject , through functional bimanual
training .
the subject was a 35-year - old patient with an incomplete central sci ( tetraplegia , c6/c6(m )
c4/c4(s ) asia - d ) , hospitalized in a rehabilitation facility .
the incomplete central cord
syndrome was diagnosed by a specialist and exhibited mobility differences between functions
of the left and right upper extremities . before participation , the procedures , risks , and
potential benefits
the
participant s rights were protected according to the guidelines established by the
university of hanseo . the subject was capable of holding and putting down objects using the
hand with weaker function ( left ) ( i.e. , capable of at least 20 rotation in wrist adduction
and flexion condition and at least 10 rotation of the metacarpophalangeal and carpal bone
joints ) .
the subject had no cognitive damage , as evidenced by a korean mini - mental state
examination ( mmse - k ) score of 29 , and was capable of independent walking .
the intervention
was implemented for 4 hours a day , comprising m - cimt for 3 hours combined with functional
bimanual task training for 1 hour , five times a week , for 3 weeks . for m - cimt ,
the subject
wore a mitten on the unaffected ( right ) side to restrict movement and performed daily
activity tasks through exclusive use of the affected ( left ) side .
functional bimanual task
training was performed in a treatment room for 1 hour following m - cimt , five times a week .
during this process
, daily activities were performed with an emphasis on using both affected
and unaffected upper extremities simultaneously .
the bimanual task training consisted of
pouring water in a glass , fastening a button , putting on or removing a shirt , folding
towels , and wiping windows . to assess function of the upper extremities ,
the performance of
daily activities , and quality of life before and after training , the manual function test
( mft ) , box & block test ( bbt ) , spinal cord independence measure version iii ( scim - iii ) ,
and short form 36 health survey ( sf-36 ) were used .
the mft score of the affected upper extremity increased from 28 before the intervention to
30 following the intervention . the bbt score increased from an average of 47.7 before the
intervention to 54 after the intervention .
the scim - iii score increased from 95 to 97 after
the intervention , with an increase of one score in each of bathing ( upper body ) and outdoor
movement ( over 100 m ) .
the sf-36 score increased by 3 points from 74 to a total of 77
following the intervention .
through exclusive use of the subject s affected side , in combination with bimanual task
training , this study aimed to observe an improvement in the functional ability of the
affected upper extremity in a patient with an incomplete sci .
results suggest enhanced upper
extremity mobility following intervention , with a positive outcome for the patient s quality
of life and performance of daily activities .
these results are consistent with observations
of improved upper extremity function following application of m - cimt to patients with
neurological insufficiencies as a result of stroke9 . studies of cimt have previously focused on patients with stroke or
children with cerebral palsy , with limited research on patients with spinal cord damage . in
general ,
active use of the affected extremities in daily life is necessary to generalize the
effects of cimt , and cimt reduces the gap between the existing performance and functional
levels of patients .
notwithstanding , the cimt protocol presents problems in that it limits
posture balance and daily activities for some time with restriction on use of the unaffected
side . to alleviate this problem , the subject in this study was required to perform
functional bimanual task training for 1 hour after applying the m - cimt protocol for 3 hours ,
to complement functional aspects of the unaffected side .
most studies have reported a
greater increase in favorable outcomes when applying bimanual training in the rehabilitation
process of the upper extremities . in part
, this facilitates recovery of the ability to carry
out daily activities correctly , since most independent daily activities require bilateral
coordination10 , 11 .
general m - cimt protocol involves 2-hour daily intensive
training on the affected side , with a 6-hour restriction on the use of the unaffected
side12 .
this study limited the use of
the subject s unaffected side to only 3 hours , accommodating the hospitalized patient s
characteristics .
moreover , the use of only a single subject means the results can not be
generalized .
future research is required to ascertain whether observed changes in the
function of upper extremities and quality of life hold consistent with larger test groups
and more rigorous training protocols . | [ purpose ] in this study , we examined effects of modified constraint - induced movement
therapy ( m - cimt ) and functional bimanual training , when applied to a patient with
incomplete spinal cord injury , on upper extremity function and daily activities .
[ subject
and methods ] one patient , diagnosed with c4 incomplete spinal cord injury , underwent
physical therapy with constraint - induced movement therapy for 3 hours and task - oriented
bimanual training for 1 hour , per day .
this combined 4-hour session was performed five
times a week , for 3 weeks , totaling 15 sessions .
upper extremity function was measured
using the manual function test ( mft ) and box & block test ( bbt ) .
additionally , spinal
cord independence measure version iii ( scim - iii ) and short form 36 health survey ( sf-36 )
were used to assess functional outcomes .
[ results ] mobility of the hand and overall
function of upper extremities were enhanced following intervention .
moreover , the
subject s quality of life and ability to carry out daily activities also improved .
[ conclusion ] modified constraint - induced movement therapy and bimanual training was
effective in enhancing upper extremity function and performance of daily routines in a
patient with incomplete spinal cord injury .
further studies , recruiting multiple subjects ,
should focus on m - cimt using diverse methods , performed during the course of daily
activities . |
the classic scenario for crc progression is the lymphatic , hematogenous ( to the liver , the lungs , etc ) and peritoneal metastases .
there have been standard treatment strategies for the first two forms of metastases , but a unified treatment guideline is yet to be formulated for the third form of metastasis , which is typically referred to as peritoneal carcinomatosis ( pc ) . characterized by the implantation of tumor nodules throughout the peritoneal cavity and production of refractory ascites , pc is found in about 815% crc patients at first treatment 1 . at present , the conventional therapeutic approach including systemic chemotherapy , with or without palliative surgery , provides limited clinical benefit , with median overall survival ( os ) no more than 6 months 24 .
knowledge on pc mechanisms and coping strategies has evolved considerably over the past three decades , and pc is no longer universally considered as terminal cancer metastasis , but regional tumor progression , and proactive therapeutic strategies with cytoreductive surgery ( crs ) and hyperthermic intraperitoneal chemotheropy ( hipec ) are hopeful to bring significant survival benefit in selected patients .
major technical advantages of this treatment approach are to maximally reduce the visible tumor burden by crs , and to eradicate residual tumor nodules , micrometastases and free tumor cells by hipec 5 .
superiority of this strategy has been demonstrated by a high - level clinical study 6 .
however , there has been no data from well designed studies from china . to address this clinical problem ,
we have conducted a series of preclinical and clinical studies on the feasibility , efficacy , and safety of this multidisciplinary treatment approach in animal models 7 and in clinical setting 8,9 , and established a designated crs + hipec program at our institution .
this case - control study was to compare the efficacy and safety of crs + hipec versus crs alone for the treatment of pc from crc , so as to provide rationale for more evidence - based clinical studies in chinese patients .
this study included 62 consecutive patients of crc pc treated from january 2004 to december 2013 at the department of oncology , zhongnan hospital of wuhan university .
the inclusion criteria were : ( 1 ) age 2075 years old ; ( 2 ) karnofsky performance status ( kps ) score > 50 ; ( 3 ) life expectancy > 8 weeks ; ( 4 ) peripheral white blood cells count 3,500/mm and platelet count 80,000/mm ; ( 5 ) acceptable liver function , with bilirubin , aspartic aminotransferase , and alanine aminotransferase levels < 2 upper limit of normal ( uln ) ; ( 6 ) acceptable renal function , with serum creatinine level < 1.2 uln ; ( 7 ) cardiovascular pulmonary and other major organ functions could stand major operation ; and ( 8) with definite histological diagnosis .
the exclusion criteria were : ( 1 ) age < 20 or > 75 years ; ( 2 ) any lung , liver , or prominent retroperitoneal lymph node metastases during preoperative assessment ; ( 3 ) serum bilirubin or liver enzymes 2 uln ; ( 4 ) serum creatinine level 1.2 uln ; and ( 5 ) prominent mesentery contracture as revealed by medical imaging studies .
although these patients were treated in the same period at our center , they were not strictly randomized , this study was therefore defined as a case - control study , which included 29 patients receiving crs alone ( control group ) and 33 patients receiving crs + hipec treatment ( study group ) .
crs + hipec were performed by a designated team focusing on pc treatment .
after general anesthesia , a midline xiphoid - pubic incision was made , and the pci was evaluated according to sugarbaker principles 10 .
subsequently , maximal crs was performed to remove the primary tumor with acceptable margins , any involved adjacent tissue and organs , regional lymph nodes , and peritonectomy 10 .
unresectable tumors were cauterized with ball - tipped electrosurgical device at the maximal electric power ( force fx electrosurgical generator , valleylab , surgical solutions group , covidien ltd . , boulder , co ) , especially on the edge of tumor nodules .
the completeness of cytoreduction ( cc score ) 10 was evaluated before hipec , which was performed by the open colliseum technique , with 120 mg of cisplatin and 30 mg of mitomycin c each dissolved 6 l of heated saline ( drug concentration cisplatin 20 g / ml , mitomycin c 5 g / ml , as these concentration has been confirmed to be safe and effective for hipec by fujimoto et al .
the heated perfusion solution was infused into the peritoneal cavity at a rate of 500 ml / min through the inflow tube introduced from an automatic hyperthermia chemotherapy perfusion device ( es-6001 , wuhan e - sea digital engineering , wuhan , china ) .
the temperature of the perfusion solution in peritoneal space was kept at 43.0 0.5c and monitored with a thermometer on real time .
the total hipec time was 90 min , after which the perfusion solution in the abdominal cavity was removed through the suction tube .
when the conditions stabilized , usually 2448 hr later , the patient was transferred to the surgical oncology ward 9 .
adjuvant chemotherapy was delivered within 4 weeks after surgery , including systemic chemotherapy mainly with folfox ( oxaliplatin , leucovorin and 5-fu ) or folfiri ( irinotecan , leucovorin and 5-fu ) regimens , and perioperative intraperitoneal chemotherapy ( pic ) through the intraperitoneal chemotherapy port mainly using docetaxel ( 75 mg / m , on day 1 , every 3 weeks ) and carboplatin ( at calvert formula : area under the curve , auc 5 ; on day 1 , every 3 weeks ) , all dosed on the base of body surface area calculation 12 .
the following study parameters were defined : ( 1 ) perioperative period : from the day of surgery to days 30 postoperation ; ( 2 ) pci 10 : 20 was defined as low pci ( lpci ) , and > 20 as high pci ( hpci ) ; ( 3 ) cc 10 : the present study set cc0 - 1 as complete cytoreduction , and cc2 - 3 as incomplete cytoreduction ; ( 4 ) synchronous pc : pc was detected synchronously at first treatment ; ( 5 ) metachronous pc : after the primary crc had been treated , patients developed pc during follow - up ; ( 6 ) overall survival ( os ) : the period from first treatment to death due to the disease for synchronous pc , and from crs to death due to the disease for metachronous pc ; ( 7 ) adverse events : complications occurred during the perioperative period directly attributable to the treatment , including sae and other side effects ; the former referred to life - threatening complications , consisting of hemorrhage , intestinal leakage , intestinal obstruction , septicemia and death directly related to the therapy ; the latter consisting of hypoalbuminemia , respiratory infections , liver and kidney toxicities , and delayed incision healing ; all based on nci common terminology criteria ( ctc ) for adverse events version 4.0 14 ; and ( 8) the survival prolong rate ( spr ) : worked out by os difference of the better os minus worse os and divided by worse os , calculated as : all patients received regular follow - up once every 3 months for the first 2 years , and once every 6 months thereafter .
the last follow - up was on june 11 , 2013 , by which 1 patient in crs group was lost for follow - up 12 months after operation , and the overall follow - up rate was 98.4% .
the crc pc database included major clinic - pathological information such as age , gender , kps scores , histopathology , intraoperative resection area , input and output volume , pci scores , cc scores , adverse events , postoperative adjuvant chemotherapy , and follow - up information .
all data analyses were performed using the spss statistical software program , version 17.0 ( spss , inc . ,
the numerical data were directly recorded , and the category data were recorded into different categories .
differences of categorical variables between the two groups were evaluated with pearson 's chi - squared test , and those of continuous variables were evaluated with student 's t - test .
os comparisons were analyzed with kaplan meier cumulative survival curve and log rank test , and multivariate cox regression analysis was performed to delineate the independent predictors . a two - sided p < 0.05 value was considered as statistically significant .
this study included 62 consecutive patients of crc pc treated from january 2004 to december 2013 at the department of oncology , zhongnan hospital of wuhan university .
the inclusion criteria were : ( 1 ) age 2075 years old ; ( 2 ) karnofsky performance status ( kps ) score > 50 ; ( 3 ) life expectancy > 8 weeks ; ( 4 ) peripheral white blood cells count 3,500/mm and platelet count 80,000/mm ; ( 5 ) acceptable liver function , with bilirubin , aspartic aminotransferase , and alanine aminotransferase levels < 2 upper limit of normal ( uln ) ; ( 6 ) acceptable renal function , with serum creatinine level < 1.2 uln ; ( 7 ) cardiovascular pulmonary and other major organ functions could stand major operation ; and ( 8) with definite histological diagnosis .
the exclusion criteria were : ( 1 ) age < 20 or > 75 years ; ( 2 ) any lung , liver , or prominent retroperitoneal lymph node metastases during preoperative assessment ; ( 3 ) serum bilirubin or liver enzymes 2 uln ; ( 4 ) serum creatinine level 1.2 uln ; and ( 5 ) prominent mesentery contracture as revealed by medical imaging studies .
although these patients were treated in the same period at our center , they were not strictly randomized , this study was therefore defined as a case - control study , which included 29 patients receiving crs alone ( control group ) and 33 patients receiving crs + hipec treatment ( study group ) .
crs + hipec were performed by a designated team focusing on pc treatment .
after general anesthesia , a midline xiphoid - pubic incision was made , and the pci was evaluated according to sugarbaker principles 10 .
subsequently , maximal crs was performed to remove the primary tumor with acceptable margins , any involved adjacent tissue and organs , regional lymph nodes , and peritonectomy 10 .
unresectable tumors were cauterized with ball - tipped electrosurgical device at the maximal electric power ( force fx electrosurgical generator , valleylab , surgical solutions group , covidien ltd . , boulder , co ) , especially on the edge of tumor nodules .
the completeness of cytoreduction ( cc score ) 10 was evaluated before hipec , which was performed by the open colliseum technique , with 120 mg of cisplatin and 30 mg of mitomycin c each dissolved 6 l of heated saline ( drug concentration cisplatin 20 g / ml , mitomycin c 5 g / ml , as these concentration has been confirmed to be safe and effective for hipec by fujimoto et al . 11 , and
the heated perfusion solution was infused into the peritoneal cavity at a rate of 500 ml / min through the inflow tube introduced from an automatic hyperthermia chemotherapy perfusion device ( es-6001 , wuhan e - sea digital engineering , wuhan , china ) .
the temperature of the perfusion solution in peritoneal space was kept at 43.0 0.5c and monitored with a thermometer on real time .
the total hipec time was 90 min , after which the perfusion solution in the abdominal cavity was removed through the suction tube .
when the conditions stabilized , usually 2448 hr later , the patient was transferred to the surgical oncology ward 9 .
adjuvant chemotherapy was delivered within 4 weeks after surgery , including systemic chemotherapy mainly with folfox ( oxaliplatin , leucovorin and 5-fu ) or folfiri ( irinotecan , leucovorin and 5-fu ) regimens , and perioperative intraperitoneal chemotherapy ( pic ) through the intraperitoneal chemotherapy port mainly using docetaxel ( 75 mg / m , on day 1 , every 3 weeks ) and carboplatin ( at calvert formula : area under the curve , auc 5 ; on day 1 , every 3 weeks ) , all dosed on the base of body surface area calculation 12 .
the following study parameters were defined : ( 1 ) perioperative period : from the day of surgery to days 30 postoperation ; ( 2 ) pci 10 : 20 was defined as low pci ( lpci ) , and > 20 as high pci ( hpci ) ; ( 3 ) cc 10 : the present study set cc0 - 1 as complete cytoreduction , and cc2 - 3 as incomplete cytoreduction ; ( 4 ) synchronous pc : pc was detected synchronously at first treatment ; ( 5 ) metachronous pc : after the primary crc had been treated , patients developed pc during follow - up ; ( 6 ) overall survival ( os ) : the period from first treatment to death due to the disease for synchronous pc , and from crs to death due to the disease for metachronous pc ; ( 7 ) adverse events : complications occurred during the perioperative period directly attributable to the treatment , including sae and other side effects ; the former referred to life - threatening complications , consisting of hemorrhage , intestinal leakage , intestinal obstruction , septicemia and death directly related to the therapy ; the latter consisting of hypoalbuminemia , respiratory infections , liver and kidney toxicities , and delayed incision healing ; all based on nci common terminology criteria ( ctc ) for adverse events version 4.0 14 ; and ( 8) the survival prolong rate ( spr ) : worked out by os difference of the better os minus worse os and divided by worse os , calculated as :
all patients received regular follow - up once every 3 months for the first 2 years , and once every 6 months thereafter .
the last follow - up was on june 11 , 2013 , by which 1 patient in crs group was lost for follow - up 12 months after operation , and the overall follow - up rate was 98.4% .
the crc pc database included major clinic - pathological information such as age , gender , kps scores , histopathology , intraoperative resection area , input and output volume , pci scores , cc scores , adverse events , postoperative adjuvant chemotherapy , and follow - up information .
all data analyses were performed using the spss statistical software program , version 17.0 ( spss , inc . ,
the numerical data were directly recorded , and the category data were recorded into different categories .
differences of categorical variables between the two groups were evaluated with pearson 's chi - squared test , and those of continuous variables were evaluated with student 's t - test .
os comparisons were analyzed with kaplan meier cumulative survival curve and log rank test , and multivariate cox regression analysis was performed to delineate the independent predictors .
a two - sided p < 0.05 value was considered as statistically significant .
five patients each received two operations due to tumor recurrence in control group ( n = 3 ) and study group ( n = 2 ) .
major clinico - pathologic characteristics of the patients in this studya three patients in control group and two patients in study group each underwent two operations . according to the first surgery . including seven cases of stoma . including two cases of stoma . surgical procedures and major intraoperative parameters were recorded and analyzed ( table 2 ) .
the value of the important parameters for study group was greater than control group , including fluid intake and output , duration of operation .
comparisons of intraoperative parameters between the two groupsa values are in median ( range ) .
only one patient received 6 u of cryoprecipitation transfusion . including colloids and electrolytes solution .
after operation , all the 62 patients received systemic chemotherapy and 14 patients received pic ( five in control group and nine in study group ) .
none of the patients in both groups received any molecular targeting agents . by june 11 , 2013 , the median follow - up in control and study
groups were 41.5 ( range , 11.570.9 ) versus 36.6 ( range , 15.582.9 ) months ( p = 0.87 ) .
the primary endpoint was reached in 26 ( 89.7% ) cases in control group , and 26 ( 78.8% ) cases in study group .
ten patients are alive , 3 ( 10.3% ) in control group and 7 ( 21.2% ) in study group .
the median os was 8.5 ( 95% ci : 4.712.4 ) months for control group and 13.7 ( 95% ci : 10.016.5 ) months for study group ( p = 0.02 ; fig .
1 ) . compared with control group , the study group had survival prolong rate ( spr ) by 61.2% .
the 1- , 2- , and 3-year survival rates were 27.5% versus 63.6% , 12.0% versus 20.0% , and 0.0% versus 16.0% , respectively , for control versus study groups . the overall survival in patients with peritoneal carcinomatosis from colorectal cancer treated by crs + hipec regimen compared with control group .
the os comparisons between the two groups were stratified based on major clinico - pathological factors ( table 3 ) . compared with control group ,
the study group had os advantages across all major clinico - pathological factors studied , although male patients , age < 60 years , colon cancer pc , poorly / undifferentiated adenocarcinoma , synchronous pc , pci 20 , and cc0 - 1 could obtain greater os benefit .
os comparisons between the two groups stratified by major clinico - pathological factors na , not available ; os , overall survival ; mo , months . in the original surgery calculation . the os comparison was the further stratified by subgroup analysis ( table 4 ) , which revealed statistically greater os benefits ( p < 0.05 ) in some subgroups , such as synchronous pc in study group ( fig .
however , there was no statistical significance for os improvements in other subgroups including gender , age , primary tumor , histopathology , and ascites .
the subgroup analysis between control and study groups os , overall survival ; mo , months ; yr , years old .
a : the patients with synchronous pc in study group are superior survival to metachronous pc ; b : compared with the pci > 20 , the pci 20 have a significant survival advantage in control group .
either control group ( a ) or study group ( b ) , patients with cc0 - 1 cytoreduction had better survival advantage ; similarly , in both groups(c , d ) , postoperative chemotherapy 6 cycles provided far better survival advantage than < 6 cycles , particularly in study group ( d ) .
there were 12 patients surviving over 20 months in this cohort of patients , nine in study group and three in control group ( table 5 ) . in study group ,
three patients of synchronous pc with lpci and ccr-0 resection had a long - term os over 50 months and still free of disease ; however , three patients with hpci and ccr-2 resection also achieved a long - term os > 20 months , and one of them was still living over 30 months with tumor . in control group ,
two patients with pci < 10 and ccr-0 resection had the os over 23 months .
it was surprising that one patient ( pci = 26 , ccr = 3 ) in this group achieved long - term os of 28 months .
major treatment and follow - up features of long - term survivors in both groups m , male ; f , female ; ca , carcinomatosis ; d , died ; dfs , disease free survival ; swt , survival with tumor ; sae , serious adverse event .
sae ( grade 35 ) occurred in 13 patients , including 3 ( 9.4% ) in control group consisting of intestinal leakage ( 1 case , on day 7 postoperation ) and death ( 2 cases , on days 7 and 22 postoperation ) , and 10 ( 28.6% ) patients in study group , consisting of postoperative hemorrhage ( 1 case , 4 hr postoperation ) , septicaemia ( 1 case , on day 8 postoperation ) , diarrhea ( 1 case , on day 8 postoperation , grade 3 ) , intestinal leakage ( 2 cases , on days 16 and 17 postoperation ) , and intestinal obstruction ( 5 cases , on days 4 , 7 , 12 , 13 , and 13 postoperation ) .
no statistically significant difference was found in the frequency of sae between the two groups ( p = 0.11 ; table 6 ) .
distribution of adverse events in two groups three patients control group and two patients in study group each underwent two operations .
one patient developed abdominal hemorrhage 4 hr postoperation , and reoperation found knot slipping on branch of right gastroepiploic artery , double ligation was made and the bleeding was immediately stopped .
this patient recovered well and he is still living and active with dfs ( disease free survival ) of 52.2 months .
the second case was a 60-year - old male patient who developed septicaemia along with inflammatory diarrhea ( sae , grade 3 ) , abdominal pain and delirium on day 8 postoperation , which was confirmed to be infection by staphylococcus aureus by blood culture .
the septicaemia was controlled in 5 days after antibiotics therapy , and the patient fully recovered in about 10 days .
another two patients developed colonic stump fistula on postoperative days 16 and 17 , respectively ; the former had limited peritonitis syndrome and recovered after 7 days of conservative treatment ; but the latter deteriorated , with sepsis , generalized peritonitis , and abdominal abscess formation due to infection by escherichia coli as confirmed by bacteria culture .
this patient was treated with abdominal drainage , antibiotics , and total parenteral alimentation support , and survived 3 months after the procedure .
the other five patients developed ileus within 2 weeks after operation ; there were not electrolyte disturbance , serious infection or sepsis after treated with conservative therapy ; they had gradually recovered in about 1 week .
multivariate cox regression analysis identified three variables including therapeutic regimen , cc scores and postoperative adjuvant chemotherapy cycles as independent predictors for better survival ( table 7 ) . compared with control group ,
study group was about 2.2 times likely to improve survival ( hazard ratio = 2.15 , 95% ci 1.183.93 , p = 0.01 ) .
five patients each received two operations due to tumor recurrence in control group ( n = 3 ) and study group ( n = 2 ) .
major clinico - pathologic characteristics of the patients in this studya three patients in control group and two patients in study group each underwent two operations . according to the first surgery . including seven cases of stoma . including two cases of stoma . surgical procedures and major intraoperative parameters were recorded and analyzed ( table 2 ) .
the value of the important parameters for study group was greater than control group , including fluid intake and output , duration of operation .
comparisons of intraoperative parameters between the two groupsa values are in median ( range ) .
only one patient received 6 u of cryoprecipitation transfusion . including colloids and electrolytes solution .
after operation , all the 62 patients received systemic chemotherapy and 14 patients received pic ( five in control group and nine in study group ) .
by june 11 , 2013 , the median follow - up in control and study groups were 41.5 ( range , 11.570.9 ) versus 36.6 ( range , 15.582.9 ) months ( p = 0.87 ) .
the primary endpoint was reached in 26 ( 89.7% ) cases in control group , and 26 ( 78.8% ) cases in study group .
ten patients are alive , 3 ( 10.3% ) in control group and 7 ( 21.2% ) in study group .
the median os was 8.5 ( 95% ci : 4.712.4 ) months for control group and 13.7 ( 95% ci : 10.016.5 ) months for study group ( p = 0.02 ; fig .
1 ) . compared with control group , the study group had survival prolong rate ( spr ) by 61.2% .
the 1- , 2- , and 3-year survival rates were 27.5% versus 63.6% , 12.0% versus 20.0% , and 0.0% versus 16.0% , respectively , for control versus study groups .
the overall survival in patients with peritoneal carcinomatosis from colorectal cancer treated by crs + hipec regimen compared with control group .
the os comparisons between the two groups were stratified based on major clinico - pathological factors ( table 3 ) . compared with control group ,
the study group had os advantages across all major clinico - pathological factors studied , although male patients , age < 60 years , colon cancer pc , poorly / undifferentiated adenocarcinoma , synchronous pc , pci 20 , and cc0 - 1 could obtain greater os benefit .
os comparisons between the two groups stratified by major clinico - pathological factors na , not available ; os , overall survival ; mo , months . in the original surgery calculation . the os comparison was the further stratified by subgroup analysis ( table 4 ) , which revealed statistically greater os benefits ( p < 0.05 ) in some subgroups , such as synchronous pc in study group ( fig .
however , there was no statistical significance for os improvements in other subgroups including gender , age , primary tumor , histopathology , and ascites .
the subgroup analysis between control and study groups os , overall survival ; mo , months ; yr , years old .
a : the patients with synchronous pc in study group are superior survival to metachronous pc ; b : compared with the pci > 20 , the pci 20 have a significant survival advantage in control group .
mo , months ; spc , synchronous pc ; mpc , metachronous pc . either control group ( a ) or study group ( b ) , patients with cc0 - 1 cytoreduction had better survival advantage ; similarly , in both groups(c , d ) , postoperative chemotherapy 6 cycles provided far better survival advantage than < 6 cycles , particularly in study group ( d ) .
there were 12 patients surviving over 20 months in this cohort of patients , nine in study group and three in control group ( table 5 ) . in study group ,
three patients of synchronous pc with lpci and ccr-0 resection had a long - term os over 50 months and still free of disease ; however , three patients with hpci and ccr-2 resection also achieved a long - term os > 20 months , and one of them was still living over 30 months with tumor . in control group ,
two patients with pci < 10 and ccr-0 resection had the os over 23 months .
it was surprising that one patient ( pci = 26 , ccr = 3 ) in this group achieved long - term os of 28 months .
major treatment and follow - up features of long - term survivors in both groups m , male ; f , female ; ca , carcinomatosis ; d , died ; dfs , disease free survival ; swt , survival with tumor ; sae , serious adverse event .
sae ( grade 35 ) occurred in 13 patients , including 3 ( 9.4% ) in control group consisting of intestinal leakage ( 1 case , on day 7 postoperation ) and death ( 2 cases , on days 7 and 22 postoperation ) , and 10 ( 28.6% ) patients in study group , consisting of postoperative hemorrhage ( 1 case , 4 hr postoperation ) , septicaemia ( 1 case , on day 8 postoperation ) , diarrhea ( 1 case , on day 8 postoperation , grade 3 ) , intestinal leakage ( 2 cases , on days 16 and 17 postoperation ) , and intestinal obstruction ( 5 cases , on days 4 , 7 , 12 , 13 , and 13 postoperation ) .
no statistically significant difference was found in the frequency of sae between the two groups ( p = 0.11 ; table 6 ) .
distribution of adverse events in two groups three patients control group and two patients in study group each underwent two operations .
common terminology criteria for adverse events version 4.0 . detailed accounts of the 10 sae cases in study group were the following . one patient developed abdominal hemorrhage 4 hr postoperation , and reoperation found knot slipping on branch of right gastroepiploic artery , double ligation was made and the bleeding was immediately stopped .
this patient recovered well and he is still living and active with dfs ( disease free survival ) of 52.2 months .
the second case was a 60-year - old male patient who developed septicaemia along with inflammatory diarrhea ( sae , grade 3 ) , abdominal pain and delirium on day 8 postoperation , which was confirmed to be infection by staphylococcus aureus by blood culture .
the septicaemia was controlled in 5 days after antibiotics therapy , and the patient fully recovered in about 10 days .
another two patients developed colonic stump fistula on postoperative days 16 and 17 , respectively ; the former had limited peritonitis syndrome and recovered after 7 days of conservative treatment ; but the latter deteriorated , with sepsis , generalized peritonitis , and abdominal abscess formation due to infection by escherichia coli as confirmed by bacteria culture .
this patient was treated with abdominal drainage , antibiotics , and total parenteral alimentation support , and survived 3 months after the procedure .
the other five patients developed ileus within 2 weeks after operation ; there were not electrolyte disturbance , serious infection or sepsis after treated with conservative therapy ; they had gradually recovered in about 1 week .
multivariate cox regression analysis identified three variables including therapeutic regimen , cc scores and postoperative adjuvant chemotherapy cycles as independent predictors for better survival ( table 7 ) . compared with control group ,
study group was about 2.2 times likely to improve survival ( hazard ratio = 2.15 , 95% ci 1.183.93 , p = 0.01 ) .
since the late 1980s , crs + hipec has been gradually developed to treat crc pc , and several phase ii / iii clinical studies have demonstrated the efficacy of this strategy , with median os improved to 19.2 months from 6.0 months 13 , the 3-year survival rates from 25% to 47% 1517 , and the 5-year survival rate up to 40% 1820 .
although this new treatment strategy has gained increasing international acceptance in north american and european countries 2123 , convincing evidence is not yet available from china , where crc ranks number five in cancer mortality list . to address this problem ,
this case - control study was designed to compare the efficacy and safety of crs + hipec for chinese patients with crc pc .
the most important finding was that the median os could be extended from 8.5 months in control group to 13.7 months in study group , with survival prolong rate ( spr ) of 61.2% .
this improvement is comparable with both experimental studies ( 23 vs. 40 days , spr 60% 7 ; 43 vs. 75 days , spr 74% 24 ) and clinical studies by yang et al .
9 ( 6.5 vs. 11.0 months , spr 69% ) , verwaal et al . 6 ( 12.6 vs. 22.3 months , spr 77% ) ,
( 23.9 vs. 62.7 months , spr 162% ) , and cashin et al . 26 ( 23.9 vs. 36.5 months , spr 53% ) .
although this is not a strictly randomized study , and the two groups were different in terms of operation complexities , as the hipec group had more abdominal areas resection than the control group and thus longer operation time , there was no major selection bias in this study that could account for such big differences in os .
univariate analysis revealed 12 factors ( gender , age , primary tumor , histopathology , stage , pc timing , ascites , pci scores , cc scores , treatment , sae , postoperative chemotherapy cycles ) associated with os .
multivariate cox regression analysis identified three independent factors for improving os : study group , cc0 - 1 , and postoperative chemotherapy cycles 6 .
therefore , these factors could help make better patient selection . although the median os in this study was significantly better in study group than control group , it was shorter than most reported results 6,13,26 .
several facts could account for these differences : ( 1 ) a majority of patients ( 51.5% ) had high pci scores ( pci > 20 ) , and the median os for such patients was 13.0 months in study group ( vs. 5.0 months in control group , p = 0.002 ) .
this is comparable to most other studies 19,27,29 showing a median os of about 12 months for patients with pci > 20 .
29 also reported the 5-year survival rates of 50% , 20% , and 0% , respectively for patients with pci 10 , 1120 , and > 20 .
the patients could still benefit from hipec procedure , even if it was high - pci scores .
( 2 ) it is difficult to achieve complete cytoreduction for patients with high tumor burden , and in this study 57.6% of patients had cc2 - 3 resection . for patients with cc2 - 3 resection ,
the median os were 8.1 and 8.4 months in two studies by glehen et al .
therefore , for this subgroup of patients , our results were comparable with those reported in other studies .
it was worth noting that the os of cc2 - 3 patients were more significantly increased in study group than in control group ( 11.0 vs. 5.0 months , p = 0.003 ) . although cc2 - 3 resection was not an optimal surgical outcome , hipec still might work to some extent after unresectable or disseminated tumor scorched by high - frequency electrotome , especially on the edge of tumor tissue . as tumor aggressiveness or proliferating activity in the periphery
hipec is likely to have efficiency whatever the extent of cytoreduction , if optimal electric cauterization is delivered to the unresectable tumor .
however , more importantly , the multivariate cox regression analysis shows that cc0 - 1 resection is two times more likely to confer os advantage than cc2 - 3 resection ( hazard ratio = 2.15 , 95% ci : 1.183.93 ) .
consequently , it is still necessary every effort should be made to reduce the tumor burden as much as possible .
the analysis on 12 long - term survivors found that the patients in hpci and non - ccr0 state also benefit from hipec indeed ( os > 21 months ) , although those of lpci , ccr-0 and synchronous pc could benefited much better .
furthermore , all 12 patients have a similarity of well or intermediately differentiated adenocarcinoma in histopathology .
it has been demonstrated 33,34 that if crc pc patients received molecular targeted therapy alone , the os could reach 18.223.5 months , even could reach 54.0 months if crs + hipec plus conventional chemotherapy and molecular targeted therapy was administered 34 .
although our patients did not receive molecular targeted therapy in this study due to medical insurance issues , the study group still conferred significant survival advantage over the control group . to achieve complete cytoreduction ,
the crs + hipec procedure is often time - consuming , technically demanding and logistically complex , which could considerably increase the risk for sae 35 .
the reported perioperative morbidity rate ranged from 14.8% to 57.0% , and mortality rate from 0.0% to 12.0% 21 .
13 , the perioperative mortality rate was 4% . in our study , the 30-day sae rate was 9.4% in control group and 28.6% in study group ( p = 0.11 ) , and the mortality rates were 6.3% and 0.0% , respectively .
some of the important parameters associated with perioperative adverse events , including the fluid output / input volume , duration of anesthesia / crs and peritoneal resection rate , were more aggressive in study group than control group , however , the adverse events were not significant increased in study group .
these results suggest that the morbidity and mortality of study group are comparable with conventional gastrointestinal surgery and acceptable , if patients are treated at specialized pc centers , as demonstrated in a meta - analysis by chua et al
it is noteworthy that for patients without sae , study group confers greater os advantage than control group ( 14.5 vs. 7.0 months , p = 0.01 ) .
however , if sae developed the os could be considerably compromised , no matter what treatment modalities were delivered . therefore , careful attention should be paid to minimizing sae , including improved selection criteria , the open coliseum technique , optimal crs and hipec procedure , intensified perioperative management .
in summary , this study from china has provided new evidence that crs + hipec bring significant survival benefit and acceptable safety for patients with crc pc .
more knowledgeable patient selection at specialized treatment centers could ensure the value of this strategy for patients with crc pc . | backgroundadvanced colorectal cancer ( crc ) is prone to developing peritoneal carcinomatosis ( pc ) .
this case - control study was to compare the efficacy and safety of cytoreductive surgery ( crs ) versus crs plus hyperthermic intraperitoneal chemotherapy ( hipec ) in chinese patients with crc pc.methodsthe 62 consecutive pc patients were treated with crs ( control group , n = 29 ) or crs + hipec ( study group , n = 33 ) .
the primary end point was overall survival ( os ) , the secondary end points were perioperative safety profiles.resultsfor the comparison of control versus study groups , the peritoneal cancer index ( pci ) 20 was 13 ( 44.8% ) versus 16 ( 48.5% ) patients ( p = 0.78 ) , complete cytoreduction ( cc0 - 1 ) was achieved in 9 ( 31.0% ) versus 14 ( 42.4% ) cases ( p = 0.36 ) . at the median os was 8.5 ( 95% confidence interval [ ci ] 4.712.4 ) versus 13.7 ( 95% ci 10.016.5 ) months ( p = 0.02 ) , the 1- , 2- , and 3-year survival rates were 27.5% versus 63.6% , 12.0% versus 20.0% , and 0.0% versus 16.0% , respectively . serious adverse events in postoperative 30 days were 9.4% versus 28.6% ( p = 0.11 ) .
multivariate analysis revealed that crs + hipec , cc0 - 1 , adjuvant chemotherapy 6 cycles were independent factors for os benefit.conclusioncrs + hipec could improve os for crc pc patients , with acceptable perioperative safety .
j. surg .
oncol 2014 ; 109:730739 . |
traumatic brain injury ( tbi ) is known to cause several secondary effects , which lead to peripheral organs dysfunction .
the close relationship between brain injury and subsequent intestinal mucosal injury has been recognized [ 1 , 2 ] .
this pathologic course may not only influence the intestinal mucosa itself but also impair the remote organs , leading to systemic inflammatory response syndrome ( sirs ) and multiple - organ dysfunction syndrome ( mods ) .
previous studies of our laboratory demonstrated that traumatic brain injury could lead to intestinal inflammatory response mediated by increased nuclear factor kappa b ( nf-b ) and proinflammatory cytokines , which played a critical role in the pathogenesis of acute gut mucosal injury following tbi [ 4 , 5 ] .
afterwards , using nuclear factor erythroid 2-related factor 2 ( nrf2 ) knockout mice , we found that nrf2 , which is the key transcription factor that mediates the induction of cellular antioxidant defense mechanisms , played an important protective role in limiting tbi - induced intestinal inflammatory response and gut mucosal injury [ 6 , 7 ] .
however , it is not known whether activation of the factor nrf2 has a protective effect in the intestinal inflammation associated with tbi . a wide range of natural and synthetic small molecules with diverse chemical backgrounds
have been shown to induce the nrf2 activity ; tert - butylhydroquione ( tbhq ) is the major one that exerts powerful antioxidant potential [ 810 ] .
it has been documented that tbhq protects the animals and cell lines against acute toxicity and oxidative insult , presumably through the induction of many cytoprotective and detoxifying enzymes such as epoxide hydrolase , glutathione - s - transferase , and glucoronyltransferase .
this activation is dependent on the translocation of nrf2 from the cytoplasm to the nucleus through inducer interaction with keap1 , the cytoplasmic repressor of nrf2 .
nevertheless till now , no study was found in the literature to address the therapeutic effect of the activation of nrf2 by tbhq on the tbi - induced intestinal inflammatory response .
the purpose of the current study was therefore to determine the influence of tbhq administration on intestinal nf-b activity , proinflammatory cytokines expression , and apoptosis after tbi .
all procedures were approved by the institutional animal care committee and were in accordance with the guidelines of the national institutes of health on the care and use of animals .
age- and weight - matched adult male icr mice ( 68 weeks , 2832 g ) were purchased from animal center of chinese academy of sciences , shanghai , china .
mice were housed at 23 1c in humidity - controlled animal quarters with 12-hour light / dark cycle .
the experimental groups consisted of sham + vehicle group , tbi + vehicle group , and tbi + tbhq group ( n = 12 per group ) .
mice of tbi + tbhq group were fed the tbhq - supplemented diet for one week prior to injury , but mice of sham + vehicle group and tbi + vehicle group were given the control food .
for tbhq feeding , food pellets were powdered in a coffee grinder and dry mixed with 1% tbhq ( w / w ) .
distilled water was added to the powder ( equal v / w ) , thoroughly mixed , and reshaped into food pellets .
the pellets were then baked at 60c for 3 h. control food was processed in the same manner without the addition of tbhq .
this dose and medication of tbhq administration have been widely used in analogous animal models described elsewhere [ 15 , 16 ] .
the mouse model of tbi was employed as described being previously developed in our laboratory .
mice were anesthetized by intraperitoneal injection with sodium pentobarbital ( 50 mg / kg ) .
a round , flat , and 6 mm diameter teflon impounder was centered between the ears and eyes .
tbi was induced by a 100 g weight dropped from a 12 cm height along a stainless steel string , which translated into 1200 g / cm .
this model is generally associated with 20% mortality within the first 5 min after injury , and no delayer mortality was observed thereafter .
sham mice were subjected to identical treatment with the exception that no injury was performed .
six mice in each group were sacrificed for electrophoretic mobility shift assay ( emsa ) and enzyme - linked immunosorbent assay ( elisa ) , the others were for immunohistochemistry and terminal deoxynucleotidyl transferase - mediated dutp nick end labeling ( tunel ) study . for emsa and elisa analysis , mice were exsanguinated by cardiac puncture , and a 3-cm segment of the jejunum 8 cm distal to treitz ligament was rapidly removed and stored at liquid nitrogen immediately .
for immunohistochemistry and tunel study , mice were perfused via left ventricular puncture with cold saline ( 4c ) , followed by 4% neutral buffered formalin .
the 3-cm segment of the jejunum was taken , stored overnight in 4% neutral buffered formalin , and then embedded in paraffin .
emsa was performed using a commercial kit ( gel shift assay system ; promega , madison , wi ) following the methods in our laboratory . a consensus oligonucleotide probe for nf-b (
5-agt tga ggg gac ttt ccc agg c-3 ) was end - labeled with [ -p]-atp ( free biotech . ,
the intestinal levels of inflammatory cytokines such as tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and interleukin-6 ( il-6 ) were quantified using elisa kits specific for mouse according to the manufacturers ' instructions ( tnf- from diaclone research , france ; il-1 , il-6 from biosource europe sa , belgium ) and our previous study .
the paraffin - embedded sections ( 4 m ) were used for immunohistochemical assay , which was performed with a goat antimouse intercellular adhesion molecule-1 ( icam-1 ) antibody ( diluted 1 : 200 , r&d systems , inc . ,
microscopy of the immunohistochemically stained tissue sections was performed by an experienced pathologist blinded to the experimental condition .
evaluation of sections was undertaken by assessing the intensity of staining ( 5 grades ) . 0 indicates no detectable positive cell ; 1 indicates very low density of positive cells , 2 indicates a moderate density of positive cells ,
3 indicates the higher , but not maximal density of positive cells , and 4 indicates the highest density of positive cells .
the procedures were according to instruction of the kit ( iscdd , boehringer mannheim , germany ) and our laboratory methods .
ten villi and crypts of each section were observed under a light microscope , and the average number of tunel - positive cells in 100 counted cells was assigned as the apoptotic index .
small clusters of dead cell fragments were assessed as originating from one cell and given a single count , and any doubtful cells were disregarded .
as shown in figure 1 , low intestinal nf-b binding activity ( weak emsa autoradiography ) was detected in the sham + vehicle group . compared with sham + vehicle group , nf-b binding activity in the intestinal tissue
the results showed that tbhq treatment could markedly suppress the activation of nf-b in the gut following tbi .
concentrations of inflammatory cytokines including tnf- , il-1 , and il-6 were low in the mice gut of sham + vehicle group , as shown in figure 2 . compared with sham + vehicle group , intestinal levels of tnf- , il-1 , and il-6
tbhq administration after tbi could lead to significantly decreased tnf- , il-1 , and il-6 concentrations in mice intestinal tissue .
the immunohistochemical assay showed low icam-1 immunoreactivity in the villous interstitium and lamina propria in sham + vehicle group ; see figure 3 .
compared to the sham + vehicle group , icam-1 in the intestinal tissue was significantly upregulated in tbi + vehicle group . in tbi
the results suggested that tbhq administration could significantly down - regulate the icam-1 expression in mice gut following tbi .
low apoptotic index was found in the sham + vehicle group mice intestinal tissue , as shown in figure 4 . in tbi
+ vehicle group , the apoptotic index in the studied mice gut was found to be significantly increased compared with that in sham + vehicle group animals . in tbi
the results suggested that tbhq treatment could inhibit apoptotic cell death in the intestine and could potentially reduce the mucosal injury following tbi .
this study revealed that the inflammatory - related factors including nf-b , proinflammatory cytokines , and icam-1 in the intestine were significantly upregulated following tbi and could be suppressed when treated with tbhq . at the same time , treatment with tbhq markedly reduced tbi - induced apoptotic cell death in the intestine .
these findings reported here suggest for the first time that tbhq administration can suppress the intestinal inflammation and reduce the mucosal injury following tbi . to defend against exogenous toxins and injury ,
cells possess a large number of cytoprotective and detoxifying enzymes whose expression is rapidly increased in response to insults .
many of these genes contain a common promoter element called the antioxidant response element ( are ) .
several transcription factors can bind to are ; however , nrf2 is the major one that binds to and activates the expression of these are - mediated gene products [ 1921 ] . in recent researches , the central role of nrf2 in cell survival
importantly , numerous lines evidence generated in the recent years have implied the anti - inflammatory effect of nrf2 in a variety of experimental models [ 2427 ] .
our previous studies showed that in response to tbi , mice lacking nrf2 exhibited increased intestinal inflammatory response and mucosal injury [ 6 , 7 ] .
these provide the evidence that nrf2 plays an important role in the gut injury after tbi .
upregulation of nrf2 may bring great benefit to the tbi - induced intestinal inflammatory damage .
synthetic phenolic antioxidant tbhq used widely as a food - additive antioxidant for humans has long been of interest as effective inducer of nrf2 [ 14 , 28 , 29 ] .
there is abundant evidence that tbhq treatment increases nrf2 activation and confers protection [ 14 , 30 ] . in vitro studies
have shown that , as a result of its action on nrf2 , tbhq is effective in inducing are - dependent gene expression in cultured astrogytes and protecting neurons from oxidative injury .
other in vivo studies have shown that the neuroprotective effect of tbhq on oxidative stress and ischemic injury is lost in nrf2-deficient mice [ 14 , 15 ] .
additionally , the majority of tbhq - induced genes in glia and neurons have been shown to be dependent on nrf2 by microarray analyses . in light of the findings described above
, we hypothesize that activation of nrf2 with tbhq may represent an attractive target for combating tbi - induced intestinal inflammatory response and mucosal injury .
the intestinal inflammatory response following trauma is characterized by intestinal recruitments of neutrophils and monocytes through activation of nf-b , releasing many inflammatory cytokines , and upregulation of adhesion molecule , which has been implicated in the pathogenesis of tbi - induced gastrointestinal dysfunction .
nf-b is one of the most important proinflammatory modulators , which can be activated by lesion - induced oxidative stress , bacterial endotoxin , or cytokines and subsequently transactivate the expression of many cytokines and adhesion molecules .
proinflammatory cytokines , including interleukins ( ils ) and tnf- released early after an inflammatory stimulus , can initiate the infiltration of inflammatory cells into the intestine by activating icam-1 and other adhesion molecules .
nf-b activation enhances the transcription of proinflammatory cytokines , and the cytokines are known to in turn activate nf-b .
the positive feedback is believed to serve to amplify inflammatory signals and exacerbate tissue injury .
our study showed that at 24 h after tbi , upregulation of nf-b , tnf- , il-1 , and il-6 was evident in the intestine and could be inhibited when treated with tbhq .
the major changes of gastrointestinal function after tbi can be summarized into four aspects : gastrointestinal mucosa ischemia , gut motility dysfunction , disruption of gut barrier , and alteration of intestinal mucosal absorptive function .
apoptosis is an important factor in gastrointestinal physiological cell renewal , which can be triggered by noxious stimuli such as trauma and ischemia [ 35 , 36 ] .
previous studies of our laboratory definitely demonstrated that marked apoptosis of the intestinal epithelial cells occurred after tbi , and it was supposed to play an important role in the gut barrier damage and increased permeability of the intestinal epithelium , leading to possible translocation of intraluminal microbes and bacterial toxins [ 5 , 6 ] .
however , there is still no effective treatment for the gut mucosa injury caused by tbi . in the present study
, we found that at 24 h after tbi , increased intestinal mucosal apoptosis was evident and could be suppressed by tbhq administration .
these results suggested that tbhq could afford protection to the tbi - induced gut mucosal damage .
although the precise mechanism regarding the anti - inflammatory ability of tbhq remains elusive , several lines of evidence indicate that nrf2 and nf-b signaling pathways contribute to the pathophysiological process .
the prevailing theory is that nrf2 interferes with inflammatory signaling pathways by inhibiting nf-b activation through the maintenance of cellular redox status .
oxidative stress from reactive oxygen species ( ros ) is believed to be involved in the progression of gastrointestinal dysfunction secondary to tbi .
activation of the nf-b signaling pathway has been shown to be responsive to excess ros and is important in the generation of inflammation .
the antioxidant transcription factor nrf2 has been shown to play an important role in limiting ros levels and thereby affect redox - sensitive nf-b signaling pathway involved in the inflammation [ 19 , 38 ] .
it is therefore implied that tbhq may play an important role in anti - inflammation by a mechanism of the augmentation of cellular antioxidative system via upregulation of nrf2 signaling pathway resulting in decreased pro - inflammatory cytokines production and adhesion molecules expression via inactivation of nf-b signaling pathway .
it is assured that further ingenious researches are needed and will be conducted in our laboratory . in summary
, the results showed that oral treatment with tbhq prior to tbi markedly decreased nf-b activation , inflammatory cytokines production , and icam-1 expression in the gut . additionally , tbhq administration significantly attenuated tbi - induced intestinal mucosal apoptosis .
these results suggest that tbhq maybe an effective therapeutic drug for the treatment of tbi - induced gut injury with a potential mechanism of upregulation of nrf2 signaling pathway . | traumatic brain injury ( tbi ) can induce intestinal inflammatory response and mucosal injury .
antioxidant transcription factor nuclear factor erythroid 2-related factor 2 ( nrf2 ) has been shown in our previous studies to prevent oxidative stress and inflammatory response in gut after tbi .
the objective of this study was to test whether tert - butylhydroquinone ( tbhq ) , an nrf2 inducer , can protect against tbi - induced intestinal inflammatory response and mucosal injury in mice .
adult male icr mice were randomly divided into three groups : ( 1 ) sham + vehicle group , ( 2 ) tbi + vehicle group , and ( 3 ) tbi + tbhq group ( n = 12 per group ) .
closed head injury was adopted using hall 's weight - dropping method .
intestinal mucosa apoptosis and inflammatory - related factors , such as nuclear factor kappa b ( nf-b ) , tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) and intercellular adhesion molecule-1 ( icam-1 ) , were investigated at 24 h after tbi . as a result
, we found that oral treatment with 1% tbhq prior to tbi for one week markedly decreased nf-b activation , inflammatory cytokines production , and icam-1 expression in the gut .
administration of tbhq also significantly attenuated tbi - induced intestinal mucosal apoptosis .
the results of the present study suggest that tbhq administration could suppress the intestinal inflammation and reduce the mucosal damage following tbi . |
microencapsulation has gained importance in the fields of cell and tissue engineering , as well as in the development of drug formulations and oral delivery systems .
the term microencapsulation , in this work , encompasses the terms microcapsules , microparticles , microspheres , and microemulsions .
generally , the term microsphere is employed for a homogeneous structure made of one continuous phase , and the term microcapsule is used for a reservoir - like structure with a well - defined core and envelope / coat .
there exist a variety of microcapsules which can differ in size , composition , and function .
the characteristics of the microcapsules ultimately depend on the final goal of the encapsulated product , as they can be used to entrap all sorts of substances : solids , liquids , drugs , proteins , bacterial cells , stem cells , and so forth .
with such a range of substances that can be entrapped , one can conclude that microcapsules can have an assortment of objectives and applications , whether for drug delivery , enzyme retrieval , artificial cell and artificial tissue delivery , and delivery of microorganisms .
this paper provides an up - to - date review of microencapsulation and its latest developments .
it provides a comprehensive overview of microencapsulation technology , the primary goals of microencapsulation , and the processes and techniques involved .
specifically , this paper comprehensively discusses the use of microencapsulated microorganisms in renal diseases , cardiovascular diseases , colorectal cancer , inflammatory bowel disease , and others .
microencapsulation for mammalian cells is described for diabetes , hepatic diseases , parathyroid insufficiency , anemia , cancer , and neurodegenerative diseases .
the use of microencapsulated drugs and other pharmaceutics focuses on hormone therapy , gastrointestinal disorders , diabetes , pulmonary diseases , periodontitis , and hypertension .
some goals of microencapsulation include material structuration , protection of the enclosed product , and controlled release of the encapsulated contents , as shown in figure 1 .
microcapsules can provide structuration to compounds that are normally difficult to administer due to factors such as the material 's insolubility , volatility , reactivity , hygroscopicity , and physical state .
microcapsules may also serve the role of protecting the encapsulated contents to prevent the degradation of the product due to external environmental factors such as oxygen , light , heat , and humidity which could destroy any labile compound .
protection by microcapsules may also be required when orally administering a therapeutic , due to exposure to the harsh conditions of the upper gastrointestinal tract ( git ) .
in addition , the host 's immune system would quickly lead to the implanted cells ' rejection and undesired side effects if the cells are recognized as foreign .
immunoprotection and immunoisolation may be achieved by a microcapsule , important for the in vivo delivery and implantation of mammalian cells , such as stem cells , for tissue and cell engineering applications .
the capability of microcapsules to serve the purpose of immunoprotection has been well demonstrated in a number of disease contexts , including type 1 diabetes , parkinson 's disease , alzheimer 's disease , cancers , and other disorders [ 48 , 9497 ] .
microcapsules may also serve to permit the controlled release of the encapsulated contents , which can be regulated by chemical , physical , and mechanical factors .
a controlled release can permit a longer and more efficient therapeutic effect of an enzymatic by - product , which , otherwise , may have a limited half - life in vivo .
it may also regulate the release of the encapsulated product at the desired time , rate , dose , and site of action .
there are a number of techniques that can be used to fabricate microcapsules , depending on the desired characteristics and applications of the final microcapsule formulation .
these techniques can be broadly categorised into chemical , physical and physicochemical methods , as highlighted in table 1 .
chemical methods of microencapsulation include solvent evaporation , interfacial cross - linking , interfacial polycondensation , in situ polymerization , and matrix polymerization .
solvent evaporation is a technique used by many companies for the production of microcapsules , especially for drug encapsulation , as the method often requires heat .
the process necessitates that the core material be dissolved / dispersed in the coating solution followed by agitation in the liquid vehicle to obtain the desired microcapsule size .
the microencapsulation method of interfacial polycondensation , also termed interfacial condensation polymerization , was pioneered by chang .
the method involves the schotten - baumann reaction between an acid chloride and a compound containing an active hydrogen atom .
this reaction involves two polymeric reactants in a polycondensation that meet and form thin walls at the microcapsule interface .
the method of interfacial cross - linking originated from that of interfacial polycondensation and also involves the schotten - baumann reaction .
the process involves a bifunctional monomer containing active hydrogen atoms which , during encapsulation , are replaced by a biopolymer , such as a protein . at the interface of the emulsion , the membrane of the microcapsule is formed by the reaction of an acid chloride with the functional groups of the protein .
a carbohydrate or starch may also be added for an increased modulation of biodegradability and other physical properties .
in situ and
matrix polymerizations are methods used in a number of microencapsulation processes for coating of the microcapsule . in situ polymerization
is characterized by the fact that the reactants are not included in the core material , but , rather , polymerize together to form the particle surface .
matrix polymerization , on the other hand , involves the embedding of the core material in a polymeric matrix during particle formation .
this is the case in spray drying , using heat as a physical method of polymerization . in terms of a chemical method of polymerization , this can be achieved using matrices such as epoxy .
physical methods include spray drying , fluid - bed / pan coating , centrifugal extrusion , vibrating nozzle , and spinning disk microencapsulation . for spray drying ,
an emulsion is prepared by the dispersion of an oil core material or water - soluble active ingredient into a concentrated coating material .
the emulsion can then be atomized into a spray of droplets using a rotating disc and a short exposure in a heated compartment to allow water to evaporate , yielding dry microcapsules .
spray drying is an economical method that can allow for the encapsulation of labile materials such as proteins and microorganisms [ 21 , 105 ] .
fluid - bed coating is a microencapsulation technique used extensively to encapsulate pharmaceuticals into coated particles or tablets .
it is a variation of the pan coating method , one of the oldest industrial procedures , where solid particles are mixed with a dry coating material that is heated to surround the particle cores .
solid particles ( or liquids absorbed into porous solids ) are suspended on a jet of air followed by the application of a coating material using a liquid spray .
the resulting shells are solidified by cooling or solvent vaporization , and the process is repeated until the microcapsule walls are of the desired thickness .
the wurster fluid bed system , where the spray nozzle is located below the particle fluidized bed , is one commonly used system for this type of particle coating .
centrifugal extrusion is an easy - to - scale - up microencapsulation technique that involves the use of a spinning extrusion head made up of concentric nozzles .
the microcapsule core and coating materials , both immiscible with each other , are pushed through the concentric nozzles forming a flow that splits into droplets following clearing of the nozzle .
depending on the materials used during microencapsulation , solidification of the droplets can then be undertaken by cooling or gelation methods .
vibrating nozzle , also termed vibrating - jet , is a popular microencapsulation method [ 110 , 111 ] .
the liquid material to be encapsulated is extruded through a nozzle at a specific flow rate , forming a laminar jet .
unlike the centrifugal extrusion technique which relies on natural and irregular disturbances leading to irregular size and shape , this method uses a permanent sinusoidal force at determined frequencies , forming microcapsules of uniform distribution . as in centrifugal extrusion
, the solidification of the microcapsules can be undertaken using cooling or gelation . spinning disk , also termed rotational suspension separation , is another physical technique for microencapsulation .
a mixture is formed with the material for the internal core of the microcapsule and the liquid microcapsule coating material .
this dispersion is then flowed onto a turning disk , causing the microcapsules ( and coating material shells ) to be thrown off of the rim of the disk followed by solidification using cooling or gelation techniques .
physicochemical encapsulation techniques involve ionotropic gelation , polyelectrolyte complexation , coacervation , and supercritical fluid technology .
ionotropic gelation relies on the ability of polyelectrolytes to cross - link when in the presence of counterions , leading to their gelation .
this process has been extensively studied using natural polyelectrolytes such as alginate [ 114 , 115 ] , chitosan , carboxymethyl cellulose , and gellan gum .
gelated beads are produced by the addition of polymeric drops , containing the anionic therapeutic to be encapsulated , into an aqueous solution of polyvalent cations .
ionic cross - linking forms a three - dimensional lattice due to the diffusion of cations into the polymeric drops .
polyelectrolyte complexation , the addition of polycations or polyelectrolytes to the surface of the beads , can be used to further improve the mechanical strength and permeability of the gelated beads .
coacervation , also termed phase separation , is one of the oldest and most widely used methods of encapsulation that relies on polymer - polymer incompatibility .
simple coacervation involves the addition of a salt or alcohol into the polymeric mixture , promoting the liquid - liquid phase separation and the formation of coacervate polymer droplets .
complex coacervation occurs in the presence of two phases and modification of the aqueous phase ph .
this leads to the formation of a membrane at the interface of the coacervate polymer droplets .
subsequently , the membrane can be solidified and further stabilized by polymer cross - linking .
supercritical fluid technology has recently gained pharmaceutical interest for the formation of particles that are monodispersed with the capability to form nanosized particles [ 121 , 122 ] .
supercritical fluids can form particles by rapid depressurization or by exceeding the saturation point of a solute by dilution , as well as a combination of both of these processes . because these reactions occur quicker in supercritical fluids , as compared to liquids , nucleation and spinodal decomposition over crystal growth are promoted , leading to the generation of small particles . in summary
, microencapsulation has strong therapeutic potential , and using the previously methods , microcapsules can be suitably designed for a specific purpose .
the following section discusses the use of microencapsulation for the delivery of microorganisms , mammalian cells and drugs , and other pharmaceutics .
bacterial cell encapsulation is a process that can occur naturally as bacteria proliferate and produce exopolysaccharides , high - molecular - weight polymers composed of sugar residues .
the exopolysaccharide structure can act as a protective capsule and reduce the permeability and bacterial exposure to potential adverse environmental factors .
early research used microencapsulation for the immobilization of bacterial cells in the food and dairy industry , as discussed in other reviews [ 123 , 124 ] . in recent years , the microencapsulation of probiotic cells , live microorganisms , which , when administered in adequate amounts , confer a health benefit on the host , has gained interest for the treatment of a number of gastrointestinal and other health disorders .
however , orally delivered probiotic cells must be delivered and remain viable through the harsh conditions of the upper git .
hence , microencapsulation can be used as a protection for the delivery of the cells .
research focused on microencapsulation of probiotics has proven successful in the contexts of renal failure , cardiovascular diseases , and in colon disorders , as described later .
early research in the field of microencapsulated microorganisms was undertaken , by prakash and chang , using a genetically modified escherichia coli strain ( dh5 ) containing a urease gene from klebsiella aerogenes [ 2 , 126 ] .
the encapsulation was performed by gelation of alginate in calcium chloride , followed by coating steps with polylysine and alginate , to give rise to alginate - polylysine - alginate ( apa ) microcapsules containing e. coli cells . when administered orally to uremic rats
, the encapsulated e. coli successfully lowered the levels of plasma urea and ammonia back to normal levels , as well as modulating many markers of renal failure .
this was the first report that recorded the use of polymeric membrane artificial cells for the oral delivery of genetically engineered organisms .
this research also highlighted microencapsulation as a method to isolate the delivered microorganisms through the git transit until excretion , eliminating safety issues associated with the delivery of microorganisms .
research was also undertaken , in vitro , with the same e. coli but using polyvinyl alcohol microcapsules which have a significantly higher mechanical strength than apa microcapsules [ 4 , 127 ] .
supplemental research was also performed with lactobacillus delbrueckii capable of removing urea , to respond to concerns of toxicity associated with the use of the genetically engineered e. coli strain .
research by prakash et al . provided the first research investigating the use of microencapsulated yeast cells , saccharomyces cerevisiae , in renal failure .
the research group investigated the oral administration of live yeast cells in apa microcapsules in a renal failure uremic rat model .
the study demonstrated that the microencapsulated yeast cells were retained in the microcapsules through the git transit but allowed urea to diffuse through the semipermeable membrane of the microcapsule and were acted upon by yeast urease .
more importantly , a significant 18% decrease was noted for the urea levels during the 8-week treatment period , demonstrating the efficacy of the formulation as a therapeutic for eliminating the elevated levels of metabolites present in renal failure .
microencapsulation of bacterial cells has recently gained interest for the treatment and prevention of hypercholesterolemia .
early work by garofalo et al . investigated the use of pseudomonas pictorum microencapsulated with alginate - polylysine and open pore agar .
microencapsulated p. pictorum was shown to have significant cholesterol depletion activity , with the highest activity by the open pore agar microcapsule formulation . continuing the same type of work ,
investigated apa microencapsulated genetically modified bile - salt - hydrolase ( bsh- ) active lactobacillus plantarum 80 ( pcbh1 ) for its capability to break down and remove bile acids .
this research established the use of bsh - active microencapsulated organisms for lowering blood serum cholesterol . following this work , martoni et al . demonstrated that apa microencapsulated naturally bsh - active lactobacillus reuteri
can be successfully delivered to the colon and remain enzymatically active , using a simulated human gastrointestinal model .
this probiotic formulation can contribute to a significant cholesterol - lowering effect in cardiovascular diseases , by contributing to the deconjugation of bile salts in the intestine .
further research by jones et al . demonstrated the use of apa microencapsulated bsh - active l. reuteri in a human clinical study , administered as a yogurt formulation .
the formulation was shown to reduce low - density lipoprotein ( ldl ) cholesterol , total cholesterol , apolipoprotein b-100 ( apob-100 ) , and non - high - density lipoprotein ( hdl ) cholesterol in hypercholesterolemic patients more efficiently than traditional probiotic therapy and other cholesterol - lowering ingredients .
research by bhathena et al . also investigated the use of apa microencapsulated bacteria , specifically feruloyl esterase ( fae ) active l. fermentum , to lower triglyceride and cholesterol levels , major risk factors for coronary artery disease .
research was undertaken with regard to the viability and enzymatic activity of microencapsulated fae - active l. fermentum under simulated gastrointestinal conditions [ 14 , 129 ] .
it was demonstrated that , following gastrointestinal exposure , there was a significant 2.5 log difference in viability between the free and microencapsulated l. fermentum cells .
the presence of a higher probiotic viability and fae activity resulted in significant reductions in serum total cholesterol , ldl cholesterol and serum triglyceride levels in diet - induced hypercholesterolemic hamsters .
similar studies were also performed with microencapsulated l. fermentum for the treatment and prevention of metabolic syndrome .
microencapsulated microbes have also gained interest for the modulation of colonic inflammation , specifically with regard to colon cancer , but potentially for other colonic inflammatory disorders , such as inflammatory bowel syndrome ( ibs ) and inflammatory bowel disease ( ibd ) .
investigated the antitumorigenic properties of apa microencapsulated lactobacillus acidophilus in min ( multiple intestinal neoplasia ) mice that carry a germline apc mutation which spontaneously develop numerous pretumoric intestinal neoplasms .
administration of the probiotic led to a significant reduction in the number of adenomas and gastrointestinal neoplasias in the treated animals , suggesting that the microencapsulated bacteria could have a role in the development of a successful colon cancer therapeutic .
further research investigated the ability of apa microencapsulated l. acidophilus to suppress intestinal inflammation in mice , for potential applications in chronic inflammatory gut diseases such as ibs and ibd .
markers linked to colonic epithelial cell survival were also increased by the microencapsulated l. acidophilus formulation .
previously mentioned studies , with regard to fae - active microencapsulated microbes , have shown significant antioxidant properties , which could also prove beneficial for colon inflammatory disorders [ 14 , 130 ] .
research into microencapsulated microorganisms is demonstrating great potential for the treatment and prevention of a number of health disorders , and they are summarized in table 2 .
the delivery of mammalian cells has been proposed to promote the regeneration of organs such as the liver , pancreas , heart , and kidney .
unfortunately , the in vivo delivery of mammalian cells raises a number of challenges . these include ( 1 ) immune rejection by the host , ( 2 ) a loss in cell survival due to aggregation and impaired nutrition , ( 3 ) impaired cellular function due to inadequate gene expression , ( 4 ) a requirement for a large amount of readily available cells , and ( 5 ) a shortage of human cell donors [ 131 , 132 ] .
due to a shortage of human donors , research has turned to nonhuman mammalian cells , but the aforementioned impediments of immune rejection , impaired cellular function , and readily available cells remain present .
one of the earliest works in this field was by bisceglie , in the 1930s , who demonstrated the use of a polymer membrane to encase mouse tumour cells .
these were injected in a pig 's abdominal cavity and were shown to successfully survive attacks by the host immune system . since then , a lot of research has been undertaken in this field .
this section presents a synopsis of the most significant research with regard to microencapsulation in cell - based therapies , focusing on the applications of diabetes and hepatic disease .
type 1 diabetes is a growing concern , with an escalating rate of disease prevalence . with the present lack of a successful therapeutic ,
the delivery of insulin secreting pancreatic islet cells ( pics ) has proven promising for the treatment of type 1 diabetes .
unfortunately , the routine use of immunosuppressive drugs to prevent the rejection of implanted pic predisposes patients to infections and increases the risk of cancer development in the late posttransplant period [ 136 , 137 ] .
microencapsulation can act as a barrier , shielding the delivered pancreatic cells from the host 's defences , eliminating the need for immunosuppressive drugs .
the first study evaluating the morphology and function of encapsulated islet cells was performed by lim and sun in 1980 .
this research demonstrated that islet cells remained intact morphologically and functionally for 4 months , in vitro .
further investigations by lim and sun involved the intraperitoneal transplantation of encapsulated islet cells in streptozotocin - induced diabetic wistar lewis rats .
the rats transplanted with nonencapsulated cells had normoglycemia for only 68 days , demonstrating the potential of microencapsulation for the treatment of type 1 diabetes .
studies have investigated the use of microencapsulated pics to maintain normoglycemia in diabetic animal models .
kobayashi et al . investigated the therapeutic advantage of using encapsulated pic versus free pic in the diabetes animal model , nonobese diabetic mice .
pics were encapsulated in 5% ( w / w ) agarose hydrogel and injected directly into the peritoneal cavity and the omental pouch , without any immunosuppressive drug administration .
two weeks following transplantation , the control group was diabetic , as confirmed by intraperitoneal glucose tolerance tests and blood glucose levels .
it is to be noted that the free pics were no longer viable at this time . on the other hand ,
encapsulated pics were able to maintain normal blood glucose levels for over 100 days following transplantation .
omer et al . demonstrated similar results with encapsulated porcine neonatal pancreatic cell clusters ( npccs ) capable of differentiating into insulin producing cells when transplanted into streptozotocin - induced diabetic b6af1 male mice .
microcapsules , containing 1 - 2 npccs , were manufactured using highly purified alginate cross - linked by barium chloride .
the diabetic mice were intraperitoneally transplanted with 10,000 islet equivalent ( ie ) encapsulated npccs in the test group and the equivalent number of nonencapsulated npccs in the control group , with no addition of immunosuppressive therapy .
the control group ( nonencapsulated npccs ) remained hyperglycemic while the test group ( encapsulated npccs ) was normoglycemic until the completion of the trial .
the function of the transplanted npccs was confirmed by the reoccurrence of hyperglycemia following their removal at weeks 2 and 6 .
the functionality of the npccs was further demonstrated by an insulin upsurge and an improvement in the ratio of cell area to total cellular area at week 20 , confirming the differentiation of npccs into cells . like kobayashi et al .
, omer et al . confirmed that microencapsulation successfully provides the encapsulated islet cells with immune protection , without the need for immunosuppression .
moreover , omer et al . showed the differentiation of npccs into insulin producing cells , providing great therapeutic potential for the treatment of type 1 diabetes .
clinical studies are few , but research by tuch et al . investigated the transplantation of barium alginate microcapsules containing human islet cells in four type 1 diabetic patients .
this group successfully demonstrated the safety of this method , with little c - peptide detected , normal renal function , little cytokine release , and no major infection detected during the trial .
unfortunately , the research group makes the point that the efficacy of the method needs improvement for the therapy to be used clinically , although a decrease in glycemia was observed .
notably , the retrieval of the microcapsules following 16 weeks demonstrated that the encapsulated cells were no longer viable .
with respect to future human studies , there is a significant shortage of human insulin secreting cells and so the proposal for xenotransplantation .
xenotransplantation brings about concerns of host immune rejection , an obstacle that microencapsulation could potentially overcome .
type 1 diabetic dogs were transplanted with encapsulated pics and demonstrated a significant reduction ( 20%80% ) in insulin necessity after transplantation .
moreover , there was an upsurge of plasma insulin following 612 months of transplantation , along with a significant decrease in glycosylated hemoglobin .
thus , abalovich et al . demonstrated that microencapsulation may be used for xenotransplantation of pics in humans .
evaluated the function of pic apa microcapsules in a single type 1 diabetic patient . following the intraperitoneal implantation of 15,000 ie / kg bodyweight , at week 12 ,
the recovery of the microcapsules , following 9.5 years indicated that the pics were still viable and secreting small levels of insulin .
the research by elliott et al . demonstrates the potential long - term survival of microencapsulated xenogeneic pic transplanted without the need for immunosuppression .
the presented research provides optimism for the future of microencapsulated pics for the treatment of type 1 diabetes .
however , there still is a need for continuing research to demonstrate the cell viability , functionality with respect to insulin secretion , and safety associated with the xenotransplantation and allotransplantations of microencapsulated pics using long - term clinical studies .
hepatic diseases , including acute liver failure , chronic liver disease , and congenital metabolic liver disease , require the restoration of liver function .
orthotropic liver transplantation is currently the only effective treatment for end - stage liver disease [ 33 , 139142 ] .
however , the shortage of organs , the requirement for immunosuppressive therapy , and the numerous complications associated with liver transplantation limit the overall effectiveness of transplantation [ 143145 ] .
recent studies have investigated liver cell transplant ( lct ) as a potential therapeutic but , for effective lct transplantation , immunosuppression is still a requirement .
microencapsulation has been proposed as a method to address these shortcomings , with some important research presented here .
the first study evaluating the therapeutic potential of microencapsulated hepatocytes was performed by sun et al . .
rat hepatocytes , encased in apa microcapsules , were shown to secrete urea and albumin in vitro , two molecules secreted by the normal healthy liver .
the encapsulated hepatocytes were transplanted into normal wistar rats and rats with galactosamine - induced fulminant hepatic failure and still remained viable following 35 days . a recent study performed by teng et al . demonstrated the regeneration of liver cells in balb / c mice with acute liver failure ( alf ) by 70% hepatectomy , using a mixture of microencapsulated rat hepatocytes and human fetal liver stromal cells ( flscs ) supplemented with basal fibroblast growth factor ( bfgf ) .
bfgf was added to increase the metabolic activity of hepatocytes and to promote the self - renewal of human embryonic stem cells [ 33 , 147 ] .
the combined treatment of encapsulated rat hepatocytes , flscs , and bfgf enhanced the survival rate by over 86% when compared to the controls with a significant increase in liver mass following 72 hours .
furthermore , immunohistochemical inspections showed decreased levels of necrotic liver cells with increased levels of proliferating liver cells in the periportal areas .
this study concluded that a mixture of encapsulated rat hepatocytes and flscs supplemented with bfgf improved the survival of mice with alf , without the requirement for any immunosuppression .
the encapsulated cells were also protected from any host immunoreactions , demonstrating the potential for encapsulated hepatocyte xenotransplantation .
considerable research has also been performed using the microencapsulation of mammalian cells for other diseases .
an important study , performed by zhang et al . , investigated the use of microencapsulated chinese hamster ovarian ( cho ) cells that secrete vascular endothelial growth factor ( vegf ) in sprague - dawley rats , as a therapeutic for ischemic heart diseases .
the encapsulated cells were shown to be protected from immune rejection , with significantly lower levels of anti - cho in those rats as compared to the ones administered unencapsulated cells . following three weeks of transplantation , the encapsulated cho cells were found to be functionally active , secreting vegf .
there was also significant improvement in the cardiac function of the rats treated with encapsulated cho cells , as demonstrated by a decline in fractional shortening and left ventricular enlargement .
this research demonstrates great potential for the use of xenotransplantation for the treatment of ischemic heart disease .
considerable success has been achieved with microencapsulation in the treatment of other conditions such as severe anemia and neurodegenerative disease and has also found use in parathyroid replacement therapies [ 40 , 41 , 97 ] .
rinsch et al . demonstrated the increase in haematocrit value with immunosuppression for 8 weeks , with the use of encapsulated myoblasts .
likewise , rgulier et al . demonstrated the ability of encapsulated myoblasts to secrete erythropoietin which increased the haematocrit value to over 85% in anaemic mice for 80 days .
wikstrm et al . observed viable human retinal pigment epithelial in microcapsules for over 3 months , demonstrating the potential of encapsulation to maintain the viability and functionality of the encased cells .
demonstrated the reduction of daily intake of calcium and vitamin d by half , in patients suffering from hypocalcaemia by the administration of encapsulated parathyroid tissue particles .
other interesting research demonstrated that microencapsulated retinal pigment epithelial cells can be beneficial for neurodegenerative diseases like parkinson 's .
genetically engineered cells have shown great potential for the development of a cancer therapy , with microencapsulation allowing to bypass the issue of immune rejection .
table 3 provides a comprehensive list of studies where microencapsulated mammalian cells have been used for therapeutic applications .
microencapsulation technology has greatly enhanced pharmaceutics research in terms of drug delivery devices . this interdisciplinary field comprising polymer science and emulsion technology has not only covered encapsulation of drugs but also of peptides , proteins , and dna / rna therapeutic molecules for controlled release studies .
this , in turn , has improved the therapeutic efficacy of the molecule with advantages of low dosage requirements and with the ability to be delivered at the targeted site without enzyme degradation by biological fluid proteins .
the controlled release of the drug through microspheres / microcapsules occurs via four known mechanisms , namely , diffusion , dissolution , osmosis , and erosion .
the phenomenon of sustained controlled release not only protects the drug from degradation but also protects the body from potential toxic effects of the drug . most of the commonly used polymers for drug applications are poly ( lactic acid ) ( pla ) and poly ( lactic - co - glycolic acid ) ( plga ) [ 90 , 150 ] .
other polysaccharides such as chitosan [ 80 , 151 , 152 ] , alginate , and lipids [ 70 , 154 , 155 ] have also been explored .
the choice of polymer , solvents , stabilizers , and surfactants and the rigidity , integrity , and degradability are important parameters governing the formation of microparticles [ 98 , 156 ] . as aforementioned ,
solvent evaporation is accepted by many pharmaceutical industries , and has been used in products already on the market .
the microencapsulation methods used also depend on the hydrophilic or hydrophobic properties of the drug or molecule to be encapsulated . biodegradable and biocompatible polymers , such as plga , have been used to encapsulate biologically active agents such as risperidone ( antipsychotic ) and testosterone [ 60 , 158 ] to form microparticles .
microencapsulation methods have also been developed to deliver an adjuvant or an antigen , encapsulated in plga microparticles , as vaccine formulations used for immunization purposes [ 61 , 159 ] .
likewise , encapsulation of food supplements , such as vitamins and oil substances , has also been performed using emulsion technology .
a study , performed by ratnakar tandale in 2007 , demonstrated the microencapsulation of vitamin c and gallic acid , as model antioxidants , in whey protein .
the study determined the highest encapsulation ratio of vitamin c : whey protein : gallic acid , and the formulations were optimized for various storage conditions , such as humidity , temperature , and uv light and dark conditions . .
therapeutic agents such as -agonists , anticholinergics , mucolytics , and antimicrobials have also been proposed to be encapsulated for sustained respiratory drug - delivery applications .
this study compared spray drying versus emulsification method of microencapsulation to achieve highest encapsulation efficiency and determined that spray drying achieved 40% to 60% of encapsulation efficiency in comparison to emulsification , which yielded 1% to 2% encapsulation efficiency .
babtsov et al . , demonstrated microencapsulation of protein loaded chitosan nanoparticles by spray drying for pulmonary delivery of drugs .
the results showed a protein loading efficiency of 65% to 80% with its release of 75% to 80% from nanoparticles within 15 minutes .
the study also characterized the recovered nanoparticles from microspheres for size and zeta potential and found no change in the values .
other site - specific drug - delivery applications of microencapsulation include the administration of an ester prodrug , tazarotene subconjunctivally or periocularly .
in addition , a silica - lipid hybrid ( slh ) microcapsule was recently developed by tan et al .
the slh microcapsules were shown to provide physicochemical and biopharmaceutical advantages as compared to the unmodified drug , celecoxib , and the commercial celebrex product .
antibiotics such as microcycline hcl have also been administered locally in the periodontal pocket , to modulate inflammation of the periodontium , achieved by embedding the antibiotic - containing microspheres in a strip made of pectin polysaccharide [ 76 , 86 ] .
, demonstrated the successful oral delivery of clarithromycin in chitosan - alginate - ethylcellulose microspheres for the treatment of peptic ulcers caused by helicobacter pylori .
this study explored the biodegradable properties of alginate and chitosan , especially the mucoadhesive property of chitosan , which facilitates the absorption of the bioactive agent across the intestinal mucosa .
microencapsulation has also been used to deliver anticancer agents . a recent study , by patel et al . , demonstrated the advantage of ionotropic gelation to encapsulate a drug , verapamil hcl in a blend of sodium alginate , hydroxypropyl methylcellulose and hydroxymethylcellulose polymers .
other bioactive molecules such as proteins and dna / rna , that are more prone to denaturation , have also been encapsulated by solvent exchange .
likewise , dna has been encapsulated under reduced shear , to maintain its integrity , for oral delivery applications [ 62 , 82 , 163 ] .
these can be customized according to the application , to prolong the stability and controlled release of the drug .
in view of this , microcapsules have been developed using ethyl cellulose by phase separation to encapsulate highly hydrophilic drugs such as nonsteroidal anti - inflammatory drugs ( nsaids ) and diclofenac sodium , used for the treatment of rheumatoid arthritis and other diseases . a study conducted by khamanga et al .
investigated the use of eudragit rs100 ( ers ) and eudragit rl 100 ( erl ) for microsphere preparation via solvent evaporation , encapsulating losartan potassium as a model drug .
the study evaluated the effect of polymer concentration and its type on the amount of drug released .
this strategy was followed to encapsulate drugs that impose a delivery challenge due to their low - molecular weight and high hydrophilicity .
apart from drug encapsulation and release kinetic studies , microencapsulation technology has also enabled the oral delivery of high - molecular - weight proteins . due to their high - molecular weight ,
these molecules are poorly absorbed by the blood stream and are also sensitive to degradation by the acidic environment of the git .
the sustained release of peptides , proteins , and hormones , such as leutinizing hormone releasing hormone ( lhrh ) [ 164166 ] , recombinant human growth hormone , and calcitonin encapsulated in plga microcapsules , has been investigated .
, demonstrated the encapsulation of insulin using a blend of acryloyl hydroxyethyl starch ( aches ) hydrogel microparticles with plga , an interesting alternative to subcutaneous injection for the management of type 1 diabetes .
another study , involving the encapsulation of insulin , was demonstrated by caliceti et al .
they utilized microspheres prepared using an emulsion technique , comprising a blend of poly(ethylene glycol ) with pla homopolymer and plg copolymer for a 28-day sustained insulin delivery .
apart from using polymers for microencapsulation techniques , lipids have also been used to encapsulate protein , involving supercritical fluid technology .
a study demonstrated the use of this technology using dynasan 114 and gelucire 50 - 02 , for the encapsulation of a model protein , bovine serum albumin .
all of the previously mentioned applications provided a high therapeutic loading efficiency into the microparticles and provided a sustained release of the active agents [ 84 , 92 ] .
as per the literature , microencapsulation techniques , such as spray drying , phase separation , and emulsion techniques , have been extensively used for the encapsulation of drugs and proteins . however , these techniques pose a limitation in pharmaceutical applications , where the therapeutic molecule may degrade due to thermal and chemical exposure involved during the encapsulation process
. moreover , the presence of solvent residues and the polydispersity of the microcapsules account for undesired toxic effects and a lack of optimal loading of the therapeutic .
, introduced the use of a high - voltage electrostatic field to encapsulate bovine serum albumin ( bsa ) , a model protein , in sodium alginate microcapsules of < 100 m in size with 80 hours of controlled protein release .
this study brought a new dimension for the microencapsulation of protein and peptide - based pharmaceutics .
other applications of microencapsulation involve the encapsulation of nanoparticles , which can offer great advantages in biomedical applications .
for example , the microencapsulation of metals with antioxidative effects can be protected from cellular internalization while maintaining their surface - dependent biomimetic properties .
in addition , microencapsulation can prevent an initial burst release of therapeutic drugs , an important limitation of many nanoparticle formulations .
hence , microencapsulation of nanoparticles complexing therapeutic molecules has provided an approach for a controlled drug release [ 74 , 85 ] . a study by hasan et al .
elaborated the use of ibuprofen and triptorelin acetate as the model lipophilic and hydrophilic drugs encapsulated in poly--caprolactone ( pcl ) nanoparticles , entrapped in microparticles made of ethyl cellulose and ers .
this study revealed a significant reduction in the diffusion of drugs across the double - membrane polymeric wall , providing a longer and more controlled therapeutic release .
microencapsulation of nanoparticles can also serve to reduce cytoxicity effects . a recent study , by li et al .
, investigated the microencapsulation of nanoemulsions by spray drying , in which vitamin e acetate was used as a model lipophilic molecule .
this technique helped avoid the instability , the aggregation , and the hydrolysis of nanoparticles in suspension . likewise , a study performed by lee et al . demonstrated that cationic lipid nanoparticles encapsulating lipophilic drugs could be microencapsulated in an anionic polymeric membrane , forming microcapsules via ionic interactions
this method produced ph - sensitive microbeads , which proved beneficial for the oral delivery of lipophilic drugs .
microencapsulation of polymeric nanoparticles made of chitosan has also been explored for biomedical applications of pulmonary diseases due to their excellent absorption through mucosal surfaces .
protein - loaded lipid / chitosan nanoparticles were encapsulated in microspheres by spray drying , using mannitol as an aerosol excipient [ 73 , 75 ] .
carbon nanoparticles have also been widely used in biomedical research as a therapeutic vehicle for drugs and genes , specifically with respect to anticancer therapies [ 171 , 172 ] .
the microcapsule membrane has shown resistance against the varying ph of the gastrointestinal system , thereby protecting the encapsulated therapeutic payload , following a controlled release system and releasing the therapeutic at a targeted location .
it is clear from the work summarized in this paper , that microencapsulation is showing great potential for the delivery of a number of drugs and molecular pharmaceutics .
table 4 summarizes disorders where microencapsulated therapeutic drugs and other agents for the prevention and treatment of health disorders have been investigated .
as described in this paper , microencapsulation is a biomedical technology with remarkable therapeutic potential for a wide range of diseases .
the process of microencapsulation can be used in designing therapeutic formulations of microbial cells , mammalian cells , drugs , and other molecular pharmaceutics .
in addition to the presented applications , other applications are also promising [ 173175 ] .
given the importance of microencapsulation in various disease applications the technology needs to be further enhanced .
one aspect that seems critical is targeted delivery using triggered release of the encapsulated contents due to external trigger factors [ 176179 ] .
other uses of microencapsulation that seem promising are the use of this technology in developing disease models , such as models of tumors for developing pharmaceutical formulations [ 180 , 181 ] .
it is also clear that , for each microcapsule formulation , the types and physical and chemical properties of the microcapsules must be optimized .
optimization may involve a number of variables , including the type of microencapsulation process , the encapsulation materials used , and the therapeutic loading capacity . keeping these characteristics in mind
, it is also evident that the future success of microencapsulation must look at the optimization of the methods behind the fabrication of microcapsules .
specifically , characteristics such as permeability , mechanical stability , cell viability , controlled release , targeted delivery , drug stability , and shelf - life of the product , including larger - scale industrial production in therapeutically acceptable production environments , need to be optimized for each intended application . | microencapsulation is a technology that has shown significant promise in biotherapeutics , and other applications .
it has been proven useful in the immobilization of drugs , live mammalian and bacterial cells and other cells , and other biopharmaceutics molecules , as it can provide material structuration , protection of the enclosed product , and controlled release of the encapsulated contents , all of which can ensure efficient and safe therapeutic effects .
this paper is a comprehensive review of microencapsulation and its latest developments in the field .
it provides a comprehensive overview of the technology and primary goals of microencapsulation and discusses various processes and techniques involved in microencapsulation including physical , chemical , physicochemical , and other methods involved .
it also summarizes the state - of - the - art successes of microencapsulation , specifically with regard to the encapsulation of microorganisms , mammalian cells , drugs , and other biopharmaceutics in various diseases .
the limitations and future directions of microencapsulation technologies are also discussed . |
cutaneous leishmaniasis ( cl ) is a serious and increasing health problem worldwide and is endemic in nearly 90 countries , including iran ( 1 ) . in iran , the disease is mainly caused by leishmania major and l. tropica , although leishmania infantum has been infrequently isolated from patients with cutaneous leishmaniasis ( 25 ) .
the laboratorial diagnosis of cl relies mainly on the direct parasitological method that searches for the leishmania parasite in clinical samples , taken from the skin lesion of cl patients .
parasitological methods are highly specific as they demonstrate the presence of the parasite in tissues materials taken from the skin lesion , by staining the sample by giemsa s stain , or by cultivation of lesion materials , allowing for parasite growth .
although microscopic examination of lesion scrapings is specific , however its sensitivity is hampered by the fact that tissue distribution of parasites is not homogeneous and the density of leishmania is low in chronically infected cl cases ( 6 ) . on the other hand , culture of lesion biopsies and aspirates , for isolating the leishmania spp . are labor intensive , are available in reference laboratories and are positive in less than 60% of cl cases ( 7 ) . along with parasitological method ,
nevertheless , the persistent of parasite in the lesion after the treatment and the technical requirements indicate that molecular methods can not be easily incorporated routinely into diagnostic protocols for cl ( 810 ) .
serological methods have been commonly used to diagnose visceral leishmaniasis ( vl ) and considered as a routine method for diagnosis of vl ( 1114 ) .
serological methods have been considered of limited importance for the diagnosis of cl . however , in recent years such methods have been evaluated , with encouraging results , for the diagnosis of cl in few studies ( 1521 ) . among the serological tests , western blot analysis has received much attention for the diagnosis of cl .
the current study was conducted to evaluate the effectiveness of immunoblotting , aiming at the development of better tool for proper diagnosis of cl .
sera samples were collected from sixty - one parasitologically confirmed ( by microscopically examination of lesions ) cl patients ( 41 men and 20 women ) referred to university affiliated hospitals in shiraz , southern iran . from each patient , 5 ml of whole blood was collected .
most patients ( 23 cases ) had skin lesion in their hand followed by foot ( 13 cases ) , face ( 8 cases ) , and trunk ( 4 cases ) .
mean of lesion number was 3 ( range 120 ) and history of the disease was from two weeks up to 20 years .
control samples ( n=50 ) were obtained from healthy individuals who had no history of cl . moreover ,
50 sera sample were collected from non - cl patients including patients with bacterial ( n=12 ) , parasitic ( n=3 ) and autoimmune diseases ( n=35 ) .
ethical approval of the study was given by the ethics committee of shiraz university of medical sciences and consent was obtained from the participants .
promastigote form of l. major ( mrho / ir/75/er ) were cultivated at 25c in rpmi 1640 medium containing 10% heat - inactivated fetal calf serum , penicillin g ( 100 ui / ml ) , and streptomycin ( 100 mg / ml ) . moreover ,
amastigote - like form of l. major was cultured in schneider medium as previously described ( 22 ) .
soluble l. major antigen was prepared from the amastigote - like by washing the parasite , three times ( 1500g for 10 min ) with pbs .
cocktail of protease inhibitor ( sigma , p2714 ) was added and the sample was subjected to three rounds of freezing and thawing followed by five pulse of sonication .
the lysed amastigote - like was centrifuged ( 1500g for 15 min at 4 c ) and the supernatant was stored at 20 c until use .
soluble leishmania antigen ( sla ) of l. major amastigote - like ( 20 g ) was subjected to discontinuous reducing sds - page , using 5% stacking and 15% separating gels in a bio - rad apparatus at 25 ma / gel for 1 hour .
the antigen was transferred from the gel into nitrocellulose membrane ( pall gelman laboratory , p / n 66485 ) .
efficacy of transfer was checked by staining the membrane with ponceau s stain ( 0.001 g / ml in 3% trichloroacetic acid ) .
the membranes with blotted antigen were cut into strips and blocked with 5% ( w / v ) of skimmed milk in washing buffer ( 10 mm tris , 150 mm nacl , and 0.05%tween 20 ; ph 7.4 ) for 2 hours .
the strips were incubated with test sera ( 1/100 dilution in washing buffer with 1% bovine serum albumin ( bsa ) for 1.5 hours at room temperature .
after 3 washes ( each 15 min ) , the strips were incubated with horseradish peroxidase conjugated anti - human igg ( sigma , a8667 ) at a dilution of 1/4000 ( in washing buffer contained 1% bsa ) for 1 h at room temperature .
after 3 washes as before , bound antigens was developed using diaminobanzidine ( dab ) substrate ( 0.1% h2o2 , 0.5 mg / ml dab ( sigma , d5637 ) in 50 mm tris - hcl , ph 7.6 ) .
sera samples were collected from sixty - one parasitologically confirmed ( by microscopically examination of lesions ) cl patients ( 41 men and 20 women ) referred to university affiliated hospitals in shiraz , southern iran . from each patient , 5 ml of whole blood was collected .
most patients ( 23 cases ) had skin lesion in their hand followed by foot ( 13 cases ) , face ( 8 cases ) , and trunk ( 4 cases ) .
mean of lesion number was 3 ( range 120 ) and history of the disease was from two weeks up to 20 years .
control samples ( n=50 ) were obtained from healthy individuals who had no history of cl . moreover ,
50 sera sample were collected from non - cl patients including patients with bacterial ( n=12 ) , parasitic ( n=3 ) and autoimmune diseases ( n=35 ) .
ethical approval of the study was given by the ethics committee of shiraz university of medical sciences and consent was obtained from the participants .
promastigote form of l. major ( mrho / ir/75/er ) were cultivated at 25c in rpmi 1640 medium containing 10% heat - inactivated fetal calf serum , penicillin g ( 100 ui / ml ) , and streptomycin ( 100 mg / ml ) . moreover ,
amastigote - like form of l. major was cultured in schneider medium as previously described ( 22 ) .
soluble l. major antigen was prepared from the amastigote - like by washing the parasite , three times ( 1500g for 10 min ) with pbs .
cocktail of protease inhibitor ( sigma , p2714 ) was added and the sample was subjected to three rounds of freezing and thawing followed by five pulse of sonication .
the lysed amastigote - like was centrifuged ( 1500g for 15 min at 4 c ) and the supernatant was stored at 20 c until use .
soluble leishmania antigen ( sla ) of l. major amastigote - like ( 20 g ) was subjected to discontinuous reducing sds - page , using 5% stacking and 15% separating gels in a bio - rad apparatus at 25 ma / gel for 1 hour .
the antigen was transferred from the gel into nitrocellulose membrane ( pall gelman laboratory , p / n 66485 ) .
efficacy of transfer was checked by staining the membrane with ponceau s stain ( 0.001 g / ml in 3% trichloroacetic acid ) .
the membranes with blotted antigen were cut into strips and blocked with 5% ( w / v ) of skimmed milk in washing buffer ( 10 mm tris , 150 mm nacl , and 0.05%tween 20 ; ph 7.4 ) for 2 hours .
the strips were incubated with test sera ( 1/100 dilution in washing buffer with 1% bovine serum albumin ( bsa ) for 1.5 hours at room temperature .
after 3 washes ( each 15 min ) , the strips were incubated with horseradish peroxidase conjugated anti -
human igg ( sigma , a8667 ) at a dilution of 1/4000 ( in washing buffer contained 1% bsa ) for 1 h at room temperature .
after 3 washes as before , bound antigens was developed using diaminobanzidine ( dab ) substrate ( 0.1% h2o2 , 0.5 mg / ml dab ( sigma , d5637 ) in 50 mm tris - hcl , ph 7.6 ) .
all the sera samples were evaluated by the immunoblotting system . using antigens prepared from amastigotes , components of 14 to 135 kda ( 14 , 18 , 2029 , 3035 ,
3842 , 45 , 48 , 5156 , 62 , 68 , 70 , 75 , 95 , and 135 kda ) were detectable by the sera of cl patients
. from 61 sera of cl patients , 59 cases ( 96.7% ) detected a 63 kda subunit and 51 cases ( 83.6% ) recognized a 3235 kda component ( fig . 1 , 2 ) .
these components were detected by the sera of just a few of healthy subjects or non - cl patients . the highest sensitivity ( 96.7% ) of the system
was achieved with the 63-kda subunit while the highest specificity was found with a 75-kda subunit . the best negative predictive value ( 97.2% )
was achieved with the 63-kda subunit while the highest positive predictive value of the system ( 89.4% ) was obtained with the 75-kda subunit .
table 1 shows the performance of different subunits of soluble antigen of l. major amastigote - like in diagnosis of cl in immunoblotting system .
detection of leishmania parasites in lesion of cl patients remains as a gold standard for diagnosis of cl ( 910 ) .
the sensitivity of direct microscopic identification of the parasite is less that 60% ( 7 ) .
furthermore , the direct microscopic identification methods are much less sensitive in detecting the parasites in chronic cases , where parasitemia is low .
cultivation of tissue samples , taken from the skin lesions , might help to some extent for detecting the leishmania parasite , but it is time - consuming and parasite cultivation can not be easily performed in any diagnostic centers .
it is mainly because robust activation of humoral immunity occurs in vl and hyper gammaglobulinemia is a feature of this form of leishmaniasis .
therefore , antibodies against different antigens of leishmania are present in the sera of vl patients and these antibodies can be detected by serological approaches .
serological assays have a limited application in the diagnosis of cl in comparison with vl .
nevertheless , the use of the serological tools in diagnosis of cl has been evaluated in few studies with encouraging results ( 1519 , 23 , 24 ) .
ifa and elisa for serodiagnosis of cutaneous leishmaniasis on 61 confirmed cl patients revealed sensitivity of 91.6% and 83.6% and specificity of 81% and 62.7% respectively ( 25 ) .
findings of the study demonstrated an appropriate performance for immunoblotting in the diagnosis of cl where a sensitivity of more than 95% and specificity of 70% were found for a 63-kda subunit of l. major for diagnosis of cl .
moreover , the diagnostic performance of the 3235 kda and a 75 kda antigens of amastigotes of l. major in this study were satisfactory .
the detected 63 kda subunit , might be the well - known gp63 ( glycoproteins 63 ) of leishmania surface protein which is the most important surface protein of leishmania spp .
it belongs to metalloprotease family and has major role in entrance of parasite into macrophages ( 26 ) . in pomares
serum samples from 45 patients with proven cl were all positive by immunoblotting with specific bands against 14 kda and/or 18 kda leishmania antigens ( 27 ) . in our study ,
the low molecular weight antigens of l. major had a relatively high sensitivity but their specificity was low .
assessed the antigens of l. infantum for the diagnosis of anthroponotic cutaneous leishmaniasis ( acl ) caused by l. tropica and found subunits of antigens ranging from 15 to 118 kda , recognized by patients sera ( 19 ) . among all bands ,
this is consistent with our findings about the performance of the 63-kda subunit of l. major in diagnosis of cl .
the 63-kda subunit of leishmania amastigote antigen , which is detected by the sera of most of cl patients , might be the gp 63 , a well - known glycoprotein that is present in leishmania spp .
a protein of 60 kda can be recognized by the sera of american cutaneous leishmaniasis ( acl ) patients , and suggested that this subunit can be used for the diagnosis of acl ( 28 ) .
a few of samples from healthy controls or non - cl patients reacted with the 63 kda in our study .
thus , a positive immunoblotting test should be interpreted in light of the patient s clinical status .
immunoblotting has a satisfactory performance in diagnosis of cl , and this test can be used for serological diagnosis of cl along with the parasitological methods . | background : cutaneous leishmaniasis ( cl ) is a parasitic disease with a relatively wide distribution in different areas of the world , including iran .
the parasite is mainly diagnosed microscopically , but serological approaches might be useful for diagnosis as well .
this study aimed to assess the efficacy of an immunoblotting system for serodiagnosis of cutaneous leishmaniasis in iran.methods:sixty-one sera samples from parasitologically confirmed cl patients and 50 sera samples from healthy controls along with 50 sera sample from non - cl patients were collected .
native strain of leishmania major was cultured in schneider medium and soluble leishmania antigens were prepared from amastigotes - like parasites .
all of sera samples were evaluated by an immunoblotting system.results:components of 14 to 135 kda were detectable by the sera of cl patients . from 61 sera of cl patients , 59 cases ( 96.7% ) detected a 63 kda subunit and 51 cases ( 83.6% ) recognized a 3235 kda component . among all subunits ,
the 63 kda band showed the highest sensitivity ( 96.7% ) and a 75 kda band had the highest ( 98% ) specificity.conclusion:immunoblotting has a satisfactory performance in diagnosis of cl and this test can be used , as an aid , for proper diagnosis of cl . |
every year , more than one million cases of scorpion stings are reported in the world ( 1 ) .
most of these cases are reported in mexico , tunisia , and brazil ( 2 ) .
the hemiscorpius genus of scorpions is distributed throughout asia ( iran , iraq , oman , pakistan , saudi arabia , yemen , and the united arab emirates ) and africa ( eritrea , somalia , and egypt ) ( 37 ) . in iran ,
about 40,000 to 50,000 scorpion sting cases are reported annually , resulting in about 19 deaths ( 8) . to date , 1,500 species of scorpions have been identified throughout the world , and about 50 of these species can be dangerous to humans .
these 50 species include androctonus , buthus , mesobuthus , tityus , leiurus , and hemiscorpius ( 9 ) .
the scorpion s venom contains various toxins , including neurotoxin , cardiotoxin , nephrotoxin , and hemolytic toxin ( 14 ) .
children and elderly patients are at increased risk of complications from scorpion envenomation ( 15 ) .
it is well known that the potent cytotoxin , venom in h. lepturus , causes cutaneous necrosis , necrotic ulcers , psychological problems , ankylosis of the joints , hemoglubinuria , fatal haemolysis , hematuria , renal failure , and even death ( 9 , 11 , 12 , 1618 ) . in southern
this paper presents and discusses the first report of death as a result of h. acanthocercus envenomation in a 15-year - old male in southern iran .
a 15-year - old male resident of a rural area in the bandar abbas district in southern iran was stung by an h. acanthocercus scorpion on his right arm ( figure 1 ) , and he was admitted to the hospital 12 hours later due to weakness , fever , chills , and pain at the site of the sting .
the patient was conscious at the time of admission , and he was not ill , icteric . on the first day ,
the clinical signs included fever , lethargy , abdominal pain , chills , active bleeding , and hematuria .
the patient s vital signs were bp ( 115/75 mm hg ) , pr 68/min , rr ( 18/min ) , and body temperature ( 39.5 c ) ; his heart and lung functions were normal .
erythema and local necrosis were observed at the site of the sting . on the second day after admission
, the patient s fever increased to 40 c , and active bleeding and hematuria continued .
on the third day , there was no change in the clinical status of the patient .
table 1 provides the results of the patient s laboratory tests . on the second day , his data showed increases in ptt ( 56 sec ) , wbc ( 2410/l ) , urea ( 86 mg / dl ) , creatinine ( 2.1 mg / dl ) , sgot ( 240
u / l ) , sgpt ( 180 u / l ) , total and direct bilirubin ( 9 and 2 mg / dl , respectively ) , and there were decreases in his rbc ( 2.8310/l ) , hgb ( 8.8 g / dl ) , and plt ( 2010/l ) .
the patient also received 400 mg of ciprofloxacin twice a day and 600 mg of clindamycin three times a day for treatment of cellulitis .
also , intravenous paracetamol was used to control the patient s fever , and he received fresh frozen plasma ( ffp ) , platelets , and cryoprecipitate for correction of coagulation factors . in spite of these treatments , the patients died due to his impaired coagulation status and hemodynamic status .
a 15-year - old male resident of a rural area in the bandar abbas district in southern iran was stung by an h. acanthocercus scorpion on his right arm ( figure 1 ) , and he was admitted to the hospital 12 hours later due to weakness , fever , chills , and pain at the site of the sting .
the patient was conscious at the time of admission , and he was not ill , icteric . on the first day ,
the clinical signs included fever , lethargy , abdominal pain , chills , active bleeding , and hematuria .
the patient s vital signs were bp ( 115/75 mm hg ) , pr 68/min , rr ( 18/min ) , and body temperature ( 39.5 c ) ; his heart and lung functions were normal .
erythema and local necrosis were observed at the site of the sting . on the second day after admission
, the patient s fever increased to 40 c , and active bleeding and hematuria continued .
on the third day , there was no change in the clinical status of the patient .
table 1 provides the results of the patient s laboratory tests . on the second day , his data showed increases in ptt ( 56 sec ) , wbc ( 2410/l ) , urea ( 86 mg / dl ) , creatinine ( 2.1 mg / dl ) , sgot ( 240 u / l ) , sgpt ( 180 u / l ) , total and direct bilirubin ( 9 and 2 mg / dl , respectively ) , and there were decreases in his rbc ( 2.8310/l ) , hgb ( 8.8 g / dl ) , and plt ( 2010/l ) .
the patient also received 400 mg of ciprofloxacin twice a day and 600 mg of clindamycin three times a day for treatment of cellulitis .
also , intravenous paracetamol was used to control the patient s fever , and he received fresh frozen plasma ( ffp ) , platelets , and cryoprecipitate for correction of coagulation factors . in spite of these treatments , the patients died due to his impaired coagulation status and hemodynamic status .
h. lepturus envenomation has resulted in a significant number of mortalities in iran ( 19 , 20 ) .
the hemiscorpius scorpion s sting does not cause immediate or severe pain ( 1 , 19 ) , so most people who are stung do not get medical care . the delay in seeking medical care may be due to the fact that there are few early symptoms associated with h. lepturus envenomation , and those that do occur are mild ( 18 ) .
the patient did not seek medical care until 12 days after envenomation , and by that time he had developed severe complications .
the severe symptoms of envenomation caused by h. lepturus venom in children are attributable to their low body mass and their limited physiological reserves ( 19 ) .
very severe renal complications have been observed in children who had not sought medical care ( 21 ) . in rahmani & jalali s study ,
all patients who died also had been stung by h. lepturus scorpions and sought medical care days later ( 18 ) , as was the case for the patient in our study .
h. lepturus venom causes various symptoms , such as macular erythema , necrosis , ulcers , fever , and others ( 22 ) .
the clinical symptoms caused by h. acanthocercus envenomation were similar to those of h. lepturus .
studies have shown that hemiscorpius venom is highly cytotoxic and can cause cutaneous necrosis , severe hemolysis , hematuria , renal failure , and death ( 18 , 23 ) , and our observations in the case in question were similar to the findings of those studies .
table 1 shows laboratory findings that indicate the changes in a patient s hematology and biochemistry parametrics as the result of being stung by an h. acanthocercus scorpion .
these data were in good agreement with the findings of other similar studies in iran ( 10 , 16 , 18 , 2426 ) .
the most severe hemoglobinuria , i.e. , + 4 , that has been observed in people stung by h. lepturus scorpions was reported in mohseni s study ( 27 ) .
other studies has shown a rapid reduction in the level of hct with acute hemolysis among patients who were referred to hospital emergency departments ( 16 , 28 ) .
in addition , emam s report indicated that the measurement of hematologic parameters , such as ptt , hb , rbc , and plt , was important because they are important indicators in patients who have been stung by h. lepturus scorpions ( 29 ) .
in contrast to the above results , the results of mohseni s study indicated that the ptt and pt factors were not important indices in cases involving scorpion stings ( 27 ) .
dehghani showed that the venom from the h. lepturus scorpion causes an increase in wbc count ( 30 ) .
the results of hematology tests and urine analyses in this case were similar to the above results .
the results of this report showed that , when people are stung by hemiscorpius scorpions , their laboratory test results change , including the results associated with hematology , biochemistry , and urine analysis . therefore these tests can be useful in the early detection and treatment of victims .
the specific anti - venin made by the razi vaccine and serum research institute is a polyvalent antivenom ( 5-ml ampoule ) against six species of scorpions , i.e. , h. lepturus , androctonus crassicauda , mesobuthus eupeus , odonthobothus doriae , hottentotta saulcyi and hottentotta zagrozensis ( 31 ) . in iran ,
the current treatment of patients who have been stung by h. lepturus is the intravenous ( iv ) or intramuscular ( i m ) injection of anti - venom ( 12 ) .
the specific polyvalent anti - venom injection as early as possible after the sting is fundamentally important with respect to the effectiveness of the anti - venom ( 19 ) . in severe cases following envenomation ,
death can occur as a result of delaying the administration of the anti - venom , especially in children and elderly patients ( 32 ) .
the venom of h. acanthocercus such as h. lepturus venom represents serious clinical symptom such as hemoglobinuria , proteinuria , hematuria , hemolysis of blood cells and increased creatinine excretion .
urine analysis , hematology and biochemistry data are the most important factors in the follow - up of scorpion victims .
this report showed that the anemia and hemoglobinuria in patients who stung by hemiscorpius scorpions should be considered .
finally , the results of this report indicate that h. acanthocercus is one of the most dangerous scorpions in south of iran .
further studies are recommended due to lack of sufficient information on the toxicology of these scorpions done . | scorpion stings are significant causes of death in the western and southern regions of iran . to date
, reports have indicated that the h. lepturus species is the main cause of mortality due to scorpion stings .
one of the species that belongs to this genus is hemiscorpius acanthocercus ( h. acanthocercus ) .
this scorpion s venom is cytotoxic , and it causes pathological changes in the blood and can cause severe damage to the kidneys .
the pain of hemiscorpius sting is mild and asymptomatic in the early hours .
delays in the treatment of these victims can cause hemolysis , hematuria , kidney failure , and even death . in this paper , we report the first known death due to an h. acanthocercus sting in iran . |
the study population consisted of overweight ( bmi 25 kg / m ) adults without diabetes who were abdominally obese ( waist circumference > 40 inches in males and > 35 inches in females ) and who lived in eight rural underserved communities in southwestern pennsylvania .
we excluded individuals who reported a diagnosis of diabetes or had a current prescription for glucose - lowering medication , were pregnant , could not walk one - quarter mile without stopping , had bariatric surgery , were currently using weight loss medications , or could not provide informed consent . in general , the eligibility criteria for the study were more flexible than those typically used in efficacy trials ( 3,1820 ) .
the study was a prospective , multisite , quasi - experimental study with four parallel lifestyle - intervention groups ( fig .
1 ) . the community in which the participant lived determined the lifestyle - intervention group to which they were allocated .
allocation of communities to the intervention groups was based on community characteristics ( population size , ethnic majority , education level , median household income , persons below poverty , and the age - adjusted prevalence of obesity ) and the community s capacity to carry out the respective interventions .
for example , it was not practical for a community that did not have adequate access to high - speed internet to be allocated to the int group .
the study was implemented in three phases : phase 1 , training and certification in the group lifestyle balance ( glb ) program and in standardized measurement techniques ; phase 2 , community - based screening to determine eligibility of individuals to take part in the intervention ; and phase 3 , provision of the intervention with 3- and 6-month follow - up including clinical assessment .
three - month assessment results carried forward to 6-month assessment if data were missing .
phase 1 consisted of glb training provided by the university of pittsburgh diabetes prevention support center ( 22 ) .
trained community preventionists ( registered nurses and dietitians from each community site ) delivered all glb content for the intervention , while lay health coaches ( also from the communities ) were used in a support role to aid preventionists and to provide informational and emotional support to study participants .
lay health coaches were trained by the preventionists and research staff on glb content , active listening skills , research fundamentals , and the health insurance portability and accountability act requirements . for maintenance of a high level of treatment fidelity in the community , four research study coordinators were implemented to work closely with the preventionists and lay health coaches at each community site .
the research team received training in standardized measurement consistent with the diabetes prevention program ( dpp ) by the university of pittsburgh diabetes prevention support center staff or their designees .
individuals who met the inclusion criteria were targeted for screening between october 2009 and june 2010 through recruitment flyers and local newspaper and radio station advertisements .
screenings were offered at no cost at hospitals , churches , work sites , and other locations throughout the eight study communities and were facilitated by study staff ( study coordinators , preventionists , and lay health coaches ) .
five hundred and fifty - five individuals were screened for bmi 25 kg / m and abdominal obesity at 1 of 44 screenings held in the study communities .
four hundred and ninety - three individuals were eligible and were invited to participate in phase iii of the study ( lifestyle interventions ) . those who were not eligible were referred to other exercise and healthy - lifestyle programs in their communities ( fig .
the intervention visits were held at the same site where they were screened for study eligibility . in instances when the screening sites were not available or could not facilitate a group class , intervention sites in the same community as the screening sites were chosen .
change program adapted from the lifestyle intervention used in the dpp that focuses on healthy food choices , fat and calorie intake , and physical activity .
members from the original dpp lifestyle team collaborated to adapt and update the individual intervention to a 12-week group - based program ( 22 ) .
the glb was delivered via three modalities ( ff , dvd , and int ) for the purposes of this study . despite the modality ,
the theoretical framework for all modalities was based on social cognitive theory and incorporated behavioral self - management approaches designed to help participants set goals , problem solve , make behavior changes , and increase their self - efficacy .
the weight loss goal for the glb is for participants to lose at least 5% of their body weight and/or decrease at least one cardiovascular risk factor after the lifestyle intervention .
all participants , in all groups , received a copy of the glb handouts , a fat and calorie counter , self - monitoring books for keeping track of food and physical activity , a pedometer , measuring cups and spoons , and a chart for recording weekly weights .
participants were asked to self - monitor food intake and physical activity throughout the 12-week intervention and were given feedback concerning progress .
one hundred and nineteen participants from two communities participated in ff , which consisted of 12 group - education sessions that took place over the course of 1214 weeks . participants met as a group for up to 90 min / session each week .
one lay health coach per community communicated with participants and identified barriers and solutions to promote program engagement and retention and provided informational and emotional support to participants upon their request .
in addition , the lay health coaches aided in the logistics of the study and shared relevant experiences to initiate class discussion .
the dvd series was based on the glb program , covering all 12 weekly sessions .
they were provided with a set of dvds that contained four weekly sessions at a time .
it was recommended that participants watch one session per week , perhaps setting aside a specific day and time each week to view each session .
participants then met as a group at four time points throughout the 12-week period to debrief the previous sessions they watched and receive a new 4-week set of dvds .
all activities were completed as if they were attending an ff group session , including keeping track of what they ate and their physical activity levels .
preventionists and lay health coaches called participants weekly to offer information , support , and reminders as needed .
int was developed specifically for results of the rethinking eating and activity ( react ) and consisted of the use of the dvds as described above but through internet access .
in addition to viewing of the dvds online , the int incorporated behavioral tools such as e - mail prompts for online self - monitoring of eating patterns , physical activity , and weight and a graphing capability to visualize progress made toward stated goals .
participants met as a group at baseline and again after finishing the 12- to 14-week intervention .
if staff found that a participant did not log onto the react website for > 1 week and did not respond to an e - mail inquiry , a phone call was made .
one hundred and one participants from two communities in this study arm were able to self - select the intervention modality ( as described above ) of their choosing .
participants were limited to one modality to avoid contamination and bias in the results . of participants ,
eligibility , baseline , and follow - up data were collected through in - person visits .
participants were asked to make in - person visits at 3 and 6 months after enrollment into the study . at each visit , anthropometric measures ( height , weight , blood pressure , and waist circumference ) were collected .
weight was measured on a high - quality calibrated digital scale with the participant wearing clothes but no shoes .
blood pressure was measured using the auscultatory method ( 23 ) , and waist circumference was measured at the umbilical waist ( 24 ) .
questionnaires that assessed sociodemographic characteristics , quality of life , and other comorbidities were completed .
physical and mental functioning was measured with the medical outcomes study short form 12 ( 25 ) .
cvd risk factors were defined using the national cholesterol education program s adult treatment panel iii definition of metabolic syndrome ( 28 ) .
more specifically , hypertension was defined as systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg .
hyperlipidemia was defined as triglyceride levels 150 mg / dl . during the in - person data collection visits
, participants were provided with a laboratory requisition to have their blood drawn at baseline and again at 3 and 6 months after study enrollment .
blood samples were collected after an 8-h fast to determine glucose , triglycerides , and hdl cholesterol levels .
the overall 3-month response rate was 60% ( 260 of 434 ) and 6-month response rate was 46.8% ( 203 of 434 ) .
three - month response rates by group were as follows : ff 80.7% , dvd 56.7% , int 43.6% , and ss 55.4% .
six - month response rates by intervention group were ff 72.2% , dvd 38.1% , int 35.6% , and ss 37.6% ) ( fig .
the primary outcome of this study was change in weight from baseline to 3- and 6-month follow - up .
secondary outcomes included changes in cvd risk factors , including glucose , triglyceride levels , waist circumference , blood pressure values , and hdl cholesterol .
imputation analyses that carried baseline values forward and the mean values forward provided no change in interpretation of results .
data are presented using descriptive statistics ( e.g. , proportions , means , sds ) by lifestyle group and by reassessment time .
the analyses examined whether there were within - group differences from baseline to 3-month follow - up and whether improvements could be maintained at 6-month follow - up . for examination of differences between study groups , a combined between- and within - group repeated - measures anova was performed for each outcome of interest .
statistical modeling was then used to investigate whether there were differential effects on outcomes due to process or demographic differences . for investigation of the possible effect of the clustering of individuals within communities , mixed
models were adjusted for the baseline value of the dependent variable , age , sex , community , and the clustering of participants within communities . the study was designed to have 80% power to detect a between - group difference of 5% change in weight , with a two - sided significance level of 0.05 .
the study population consisted of overweight ( bmi 25 kg / m ) adults without diabetes who were abdominally obese ( waist circumference > 40 inches in males and > 35 inches in females ) and who lived in eight rural underserved communities in southwestern pennsylvania .
we excluded individuals who reported a diagnosis of diabetes or had a current prescription for glucose - lowering medication , were pregnant , could not walk one - quarter mile without stopping , had bariatric surgery , were currently using weight loss medications , or could not provide informed consent . in general , the eligibility criteria for the study were more flexible than those typically used in efficacy trials ( 3,1820 ) .
the study was a prospective , multisite , quasi - experimental study with four parallel lifestyle - intervention groups ( fig .
1 ) . the community in which the participant lived determined the lifestyle - intervention group to which they were allocated .
allocation of communities to the intervention groups was based on community characteristics ( population size , ethnic majority , education level , median household income , persons below poverty , and the age - adjusted prevalence of obesity ) and the community s capacity to carry out the respective interventions .
for example , it was not practical for a community that did not have adequate access to high - speed internet to be allocated to the int group .
the study was implemented in three phases : phase 1 , training and certification in the group lifestyle balance ( glb ) program and in standardized measurement techniques ; phase 2 , community - based screening to determine eligibility of individuals to take part in the intervention ; and phase 3 , provision of the intervention with 3- and 6-month follow - up including clinical assessment .
three - month assessment results carried forward to 6-month assessment if data were missing .
phase 1 consisted of glb training provided by the university of pittsburgh diabetes prevention support center ( 22 ) .
trained community preventionists ( registered nurses and dietitians from each community site ) delivered all glb content for the intervention , while lay health coaches ( also from the communities ) were used in a support role to aid preventionists and to provide informational and emotional support to study participants .
lay health coaches were trained by the preventionists and research staff on glb content , active listening skills , research fundamentals , and the health insurance portability and accountability act requirements . for maintenance of a high level of treatment fidelity in the community , four research study coordinators were implemented to work closely with the preventionists and lay health coaches at each community site .
the research team received training in standardized measurement consistent with the diabetes prevention program ( dpp ) by the university of pittsburgh diabetes prevention support center staff or their designees .
individuals who met the inclusion criteria were targeted for screening between october 2009 and june 2010 through recruitment flyers and local newspaper and radio station advertisements .
screenings were offered at no cost at hospitals , churches , work sites , and other locations throughout the eight study communities and were facilitated by study staff ( study coordinators , preventionists , and lay health coaches ) .
five hundred and fifty - five individuals were screened for bmi 25 kg / m and abdominal obesity at 1 of 44 screenings held in the study communities .
four hundred and ninety - three individuals were eligible and were invited to participate in phase iii of the study ( lifestyle interventions ) .
those who were not eligible were referred to other exercise and healthy - lifestyle programs in their communities ( fig .
the intervention visits were held at the same site where they were screened for study eligibility . in instances
when the screening sites were not available or could not facilitate a group class , intervention sites in the same community as the screening sites were chosen .
change program adapted from the lifestyle intervention used in the dpp that focuses on healthy food choices , fat and calorie intake , and physical activity .
members from the original dpp lifestyle team collaborated to adapt and update the individual intervention to a 12-week group - based program ( 22 ) .
the glb was delivered via three modalities ( ff , dvd , and int ) for the purposes of this study . despite the modality ,
the theoretical framework for all modalities was based on social cognitive theory and incorporated behavioral self - management approaches designed to help participants set goals , problem solve , make behavior changes , and increase their self - efficacy .
the weight loss goal for the glb is for participants to lose at least 5% of their body weight and/or decrease at least one cardiovascular risk factor after the lifestyle intervention .
all participants , in all groups , received a copy of the glb handouts , a fat and calorie counter , self - monitoring books for keeping track of food and physical activity , a pedometer , measuring cups and spoons , and a chart for recording weekly weights .
participants were asked to self - monitor food intake and physical activity throughout the 12-week intervention and were given feedback concerning progress .
one hundred and nineteen participants from two communities participated in ff , which consisted of 12 group - education sessions that took place over the course of 1214 weeks .
one lay health coach per community communicated with participants and identified barriers and solutions to promote program engagement and retention and provided informational and emotional support to participants upon their request .
in addition , the lay health coaches aided in the logistics of the study and shared relevant experiences to initiate class discussion .
the dvd series was based on the glb program , covering all 12 weekly sessions .
they were provided with a set of dvds that contained four weekly sessions at a time .
it was recommended that participants watch one session per week , perhaps setting aside a specific day and time each week to view each session .
participants then met as a group at four time points throughout the 12-week period to debrief the previous sessions they watched and receive a new 4-week set of dvds .
all activities were completed as if they were attending an ff group session , including keeping track of what they ate and their physical activity levels .
preventionists and lay health coaches called participants weekly to offer information , support , and reminders as needed .
int was developed specifically for results of the rethinking eating and activity ( react ) and consisted of the use of the dvds as described above but through internet access .
in addition to viewing of the dvds online , the int incorporated behavioral tools such as e - mail prompts for online self - monitoring of eating patterns , physical activity , and weight and a graphing capability to visualize progress made toward stated goals .
participants met as a group at baseline and again after finishing the 12- to 14-week intervention .
if staff found that a participant did not log onto the react website for > 1 week and did not respond to an e - mail inquiry , a phone call was made .
one hundred and one participants from two communities in this study arm were able to self - select the intervention modality ( as described above ) of their choosing .
of participants , 60% chose ff , 0% chose dvd , and 40% chose int .
eligibility , baseline , and follow - up data were collected through in - person visits .
participants were asked to make in - person visits at 3 and 6 months after enrollment into the study . at each visit ,
anthropometric measures ( height , weight , blood pressure , and waist circumference ) were collected .
weight was measured on a high - quality calibrated digital scale with the participant wearing clothes but no shoes .
blood pressure was measured using the auscultatory method ( 23 ) , and waist circumference was measured at the umbilical waist ( 24 ) .
questionnaires that assessed sociodemographic characteristics , quality of life , and other comorbidities were completed .
physical and mental functioning was measured with the medical outcomes study short form 12 ( 25 ) .
cvd risk factors were defined using the national cholesterol education program s adult treatment panel iii definition of metabolic syndrome ( 28 ) .
more specifically , hypertension was defined as systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg .
hyperlipidemia was defined as triglyceride levels 150 mg / dl . during the in - person data collection visits
, participants were provided with a laboratory requisition to have their blood drawn at baseline and again at 3 and 6 months after study enrollment .
blood samples were collected after an 8-h fast to determine glucose , triglycerides , and hdl cholesterol levels .
the overall 3-month response rate was 60% ( 260 of 434 ) and 6-month response rate was 46.8% ( 203 of 434 ) .
three - month response rates by group were as follows : ff 80.7% , dvd 56.7% , int 43.6% , and ss 55.4% .
six - month response rates by intervention group were ff 72.2% , dvd 38.1% , int 35.6% , and ss 37.6% ) ( fig .
the primary outcome of this study was change in weight from baseline to 3- and 6-month follow - up .
secondary outcomes included changes in cvd risk factors , including glucose , triglyceride levels , waist circumference , blood pressure values , and hdl cholesterol .
imputation analyses that carried baseline values forward and the mean values forward provided no change in interpretation of results .
data are presented using descriptive statistics ( e.g. , proportions , means , sds ) by lifestyle group and by reassessment time .
the analyses examined whether there were within - group differences from baseline to 3-month follow - up and whether improvements could be maintained at 6-month follow - up . for examination of differences between study groups , a combined between- and within - group
statistical modeling was then used to investigate whether there were differential effects on outcomes due to process or demographic differences . for investigation of the possible effect of the clustering of individuals within communities , mixed
models were adjusted for the baseline value of the dependent variable , age , sex , community , and the clustering of participants within communities .
the study was designed to have 80% power to detect a between - group difference of 5% change in weight , with a two - sided significance level of 0.05 .
phase 1 consisted of glb training provided by the university of pittsburgh diabetes prevention support center ( 22 ) .
trained community preventionists ( registered nurses and dietitians from each community site ) delivered all glb content for the intervention , while lay health coaches ( also from the communities ) were used in a support role to aid preventionists and to provide informational and emotional support to study participants .
lay health coaches were trained by the preventionists and research staff on glb content , active listening skills , research fundamentals , and the health insurance portability and accountability act requirements . for maintenance of a high level of treatment fidelity in the community , four research study coordinators were implemented to work closely with the preventionists and lay health coaches at each community site .
the research team received training in standardized measurement consistent with the diabetes prevention program ( dpp ) by the university of pittsburgh diabetes prevention support center staff or their designees .
individuals who met the inclusion criteria were targeted for screening between october 2009 and june 2010 through recruitment flyers and local newspaper and radio station advertisements .
screenings were offered at no cost at hospitals , churches , work sites , and other locations throughout the eight study communities and were facilitated by study staff ( study coordinators , preventionists , and lay health coaches ) .
five hundred and fifty - five individuals were screened for bmi 25 kg / m and abdominal obesity at 1 of 44 screenings held in the study communities .
four hundred and ninety - three individuals were eligible and were invited to participate in phase iii of the study ( lifestyle interventions ) .
those who were not eligible were referred to other exercise and healthy - lifestyle programs in their communities ( fig .
the intervention visits were held at the same site where they were screened for study eligibility . in instances when the screening sites were not available or could not facilitate a group class , intervention sites in the same community as the screening sites were chosen .
change program adapted from the lifestyle intervention used in the dpp that focuses on healthy food choices , fat and calorie intake , and physical activity .
members from the original dpp lifestyle team collaborated to adapt and update the individual intervention to a 12-week group - based program ( 22 ) .
the glb was delivered via three modalities ( ff , dvd , and int ) for the purposes of this study . despite the modality ,
the theoretical framework for all modalities was based on social cognitive theory and incorporated behavioral self - management approaches designed to help participants set goals , problem solve , make behavior changes , and increase their self - efficacy .
the weight loss goal for the glb is for participants to lose at least 5% of their body weight and/or decrease at least one cardiovascular risk factor after the lifestyle intervention .
all participants , in all groups , received a copy of the glb handouts , a fat and calorie counter , self - monitoring books for keeping track of food and physical activity , a pedometer , measuring cups and spoons , and a chart for recording weekly weights .
participants were asked to self - monitor food intake and physical activity throughout the 12-week intervention and were given feedback concerning progress .
one hundred and nineteen participants from two communities participated in ff , which consisted of 12 group - education sessions that took place over the course of 1214 weeks . participants met as a group for up to 90 min / session each week .
one lay health coach per community communicated with participants and identified barriers and solutions to promote program engagement and retention and provided informational and emotional support to participants upon their request .
in addition , the lay health coaches aided in the logistics of the study and shared relevant experiences to initiate class discussion .
the dvd series was based on the glb program , covering all 12 weekly sessions .
they were provided with a set of dvds that contained four weekly sessions at a time .
it was recommended that participants watch one session per week , perhaps setting aside a specific day and time each week to view each session .
participants then met as a group at four time points throughout the 12-week period to debrief the previous sessions they watched and receive a new 4-week set of dvds .
all activities were completed as if they were attending an ff group session , including keeping track of what they ate and their physical activity levels .
preventionists and lay health coaches called participants weekly to offer information , support , and reminders as needed .
int was developed specifically for results of the rethinking eating and activity ( react ) and consisted of the use of the dvds as described above but through internet access .
in addition to viewing of the dvds online , the int incorporated behavioral tools such as e - mail prompts for online self - monitoring of eating patterns , physical activity , and weight and a graphing capability to visualize progress made toward stated goals .
participants met as a group at baseline and again after finishing the 12- to 14-week intervention .
if staff found that a participant did not log onto the react website for > 1 week and did not respond to an e - mail inquiry , a phone call was made .
one hundred and one participants from two communities in this study arm were able to self - select the intervention modality ( as described above ) of their choosing .
participants were limited to one modality to avoid contamination and bias in the results . of participants ,
eligibility , baseline , and follow - up data were collected through in - person visits .
participants were asked to make in - person visits at 3 and 6 months after enrollment into the study . at each visit , anthropometric measures ( height , weight , blood pressure , and waist circumference ) were collected .
weight was measured on a high - quality calibrated digital scale with the participant wearing clothes but no shoes .
blood pressure was measured using the auscultatory method ( 23 ) , and waist circumference was measured at the umbilical waist ( 24 ) .
questionnaires that assessed sociodemographic characteristics , quality of life , and other comorbidities were completed .
physical and mental functioning was measured with the medical outcomes study short form 12 ( 25 ) .
cvd risk factors were defined using the national cholesterol education program s adult treatment panel iii definition of metabolic syndrome ( 28 ) .
more specifically , hypertension was defined as systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg .
hyperlipidemia was defined as triglyceride levels 150 mg / dl . during the in - person data collection visits
, participants were provided with a laboratory requisition to have their blood drawn at baseline and again at 3 and 6 months after study enrollment .
blood samples were collected after an 8-h fast to determine glucose , triglycerides , and hdl cholesterol levels .
the overall 3-month response rate was 60% ( 260 of 434 ) and 6-month response rate was 46.8% ( 203 of 434 ) .
three - month response rates by group were as follows : ff 80.7% , dvd 56.7% , int 43.6% , and ss 55.4% .
six - month response rates by intervention group were ff 72.2% , dvd 38.1% , int 35.6% , and ss 37.6% ) ( fig .
the primary outcome of this study was change in weight from baseline to 3- and 6-month follow - up .
secondary outcomes included changes in cvd risk factors , including glucose , triglyceride levels , waist circumference , blood pressure values , and hdl cholesterol .
imputation analyses that carried baseline values forward and the mean values forward provided no change in interpretation of results .
data are presented using descriptive statistics ( e.g. , proportions , means , sds ) by lifestyle group and by reassessment time .
the analyses examined whether there were within - group differences from baseline to 3-month follow - up and whether improvements could be maintained at 6-month follow - up .
for examination of differences between study groups , a combined between- and within - group repeated - measures anova was performed for each outcome of interest .
statistical modeling was then used to investigate whether there were differential effects on outcomes due to process or demographic differences . for investigation of the possible effect of the clustering of individuals within communities , mixed
models were adjusted for the baseline value of the dependent variable , age , sex , community , and the clustering of participants within communities . the study was designed to have 80% power to detect a between - group difference of 5% change in weight , with a two - sided significance level of 0.05 .
five hundred and fifty - five individuals were screened for bmi 25 kg / m and abdominal obesity . of screened individuals ,
86.1% were female and 95.1% were non - hispanic white , reflecting the racial makeup of the study area .
mean weight and waist circumference were 209.5 45.6 lbs and 43.1 18.8 inches , respectively . of those who were screened ,
493 ( 88.8% ) were eligible to participate in the intervention and 434 ( 88% ) enrolled ( fig .
1 ) . when the screening population ( n = 555 ) was compared with the intervention population ( n = 434 ) to determine generalizability , there were no statistical differences in mean weight ( 209.5 45.6 vs. 215.3 44.4 lbs ) , waist circumference ( 43.1 18.8 vs. 44.2 12.4 inches ) , sex distribution ( 86.1% female vs. 86.2% female ) , or race ( 95.1 vs. 96.8% non - hispanic white ) .
mean age of intervention participants ( n = 434 ) was 51.1 years ; 86.2% were female and 96.8% non - hispanic white .
mean weight , bmi , and waist circumference at baseline was 215.3 44.4 lbs , 36.9 9.2 kg / m , and 44.2 12.4 inches , respectively ( table 1 ) .
baseline sociodemographic characteristics of the intervention participants are presented in table 1 overall and by intervention group .
participants in int viewed an average of 6.8 sessions online , and participants in dvd attended an average of 2.9 dvd debriefing sessions out of 4 possible .
ss participants who chose ff attended an average of 8.4 classes , while those who chose int viewed an average of 9.2 sessions online ( table 1 ) .
there were no differences between groups in sex distribution , education level , income , smoking status , family history of diabetes , or whether participants attempted weight loss during the 12 months preceding the study ( table 1 ) .
baseline characteristics of react intervention participants there was a marked decline in weight from baseline to 3 months in all intervention groups ( ff 12.5 lbs , p = 0.01 ; dvd 12.2 lbs , p < 0.0001 ; int 13.7 lbs , p < 0.0001 ; and ss 14 lbs , p < 0.0001 ) , with participants in ss experiencing the largest average weight loss . at 6 months ,
the mean weight loss from baseline was 10.8 lbs in ff ( p < 0.0001 ) , 7.5 lbs in dvd ( p < 0.0001 ) , 6.8 lbs in int ( p < 0.0001 ) , and 8.7 lbs in ss ( p < 0.0001 ) ( fig .
significant differences in mean weight loss between groups at 3 and 6 months were observed , with ss having the greatest average weight loss at 3 months ( p = 0.03 ) and ff having the greatest average loss at 6 months ( p = 0.05 ) compared with other groups . when the effect of group was adjusted for the clustering of participants within communities , baseline weight , age , and sex , the magnitude of the association remained strong at 3 ( p = 0.004 ) and 6 ( p = 0.004 ) months in favor of ff .
when weight loss goals were examined at 6 months , the proportion of participants with a weight that was lower than their baseline weight was 71.4% in ff compared with 53.1 , 42.6 , and 55.5% in dvd , int , and ss , respectively . of the participants who achieved the 5% weight loss goal at 3 months ( ff 57.2% , dvd 56.7% , int 62% , and ss 66.7% ) , > 90% in each group sustained the weight loss at 6 months ( ff 90.7% , dvd 90.9% , int 92.1% , and ss 100% ) ( data not shown ) .
all groups experienced significant improvements in the proportion of participants with cvd risk factors after the intervention .
the proportion of participants with abdominal obesity significantly decreased in all groups from baseline to 3 and 6 months ( fig .
3 ) . similarly , the proportion of participants with hypertension decreased in all groups and significantly in ff ( p < 0.01 ) , dvd ( p < 0.05 ) , and int ( p < 0.0001 ) . triglyceride and hdl cholesterol levels also improved but not in int . indeed , int experienced significant increases ( p < 0.01 ) in the proportion of participants with abnormal triglyceride levels over time ( fig .
additionally , the proportion of participants with impaired fasting glucose ( ifg ) decreased in all groups from baseline to 3 and 6 months except for int .
in fact , of the 41 individuals who had ifg at baseline in ss , 26 no longer had ifg at 3 months ( data not shown ) .
all associations remained after adjustment for the effect of clustering of participants within communities , age , sex , and baseline cvd risk factors .
the proportion of individuals with cvd risk factors remained steady between 3 and 6 months in all groups and never returned back to baseline ( fig .
3 ) . when weight loss and cvd risk factors were examined within the ss group and stratified by intervention modality ( ff versus int ) , no differences in associations were found .
changes in proportion of participants with cardiovascular risk factors across study groups after lifestyle intervention ( baseline to 6-month follow - up ) .
p values represent within - group differences from baseline to 6-month follow - up .
* p < 0.05 , * * p < 0.01 , * * * p < 0.0001 .
associations remained after adjustment for the effect of clustering of participants within communities , age , sex , and baseline values .
there was a marked decline in weight from baseline to 3 months in all intervention groups ( ff 12.5 lbs , p = 0.01 ; dvd 12.2 lbs , p < 0.0001 ; int 13.7 lbs , p < 0.0001 ; and ss 14 lbs , p < 0.0001 ) , with participants in ss experiencing the largest average weight loss . at 6 months ,
the mean weight loss from baseline was 10.8 lbs in ff ( p < 0.0001 ) , 7.5 lbs in dvd ( p < 0.0001 ) , 6.8 lbs in int ( p < 0.0001 ) , and 8.7 lbs in ss ( p < 0.0001 ) ( fig .
significant differences in mean weight loss between groups at 3 and 6 months were observed , with ss having the greatest average weight loss at 3 months ( p = 0.03 ) and ff having the greatest average loss at 6 months ( p = 0.05 ) compared with other groups . when the effect of group was adjusted for the clustering of participants within communities , baseline weight , age , and sex , the magnitude of the association remained strong at 3 ( p = 0.004 ) and 6 ( p = 0.004 ) months in favor of ff .
when weight loss goals were examined at 6 months , the proportion of participants with a weight that was lower than their baseline weight was 71.4% in ff compared with 53.1 , 42.6 , and 55.5% in dvd , int , and ss , respectively . of the participants who achieved the 5% weight loss goal at 3 months (
ff 57.2% , dvd 56.7% , int 62% , and ss 66.7% ) , > 90% in each group sustained the weight loss at 6 months ( ff 90.7% , dvd 90.9% , int 92.1% , and ss 100% ) ( data not shown ) .
all groups experienced significant improvements in the proportion of participants with cvd risk factors after the intervention .
the proportion of participants with abdominal obesity significantly decreased in all groups from baseline to 3 and 6 months ( fig .
3 ) . similarly , the proportion of participants with hypertension decreased in all groups and significantly in ff ( p < 0.01 ) , dvd ( p < 0.05 ) , and int ( p < 0.0001 ) . triglyceride and hdl cholesterol levels also improved but not in int . indeed , int experienced significant increases ( p < 0.01 ) in the proportion of participants with abnormal triglyceride levels over time ( fig .
3 ) . additionally , the proportion of participants with impaired fasting glucose ( ifg ) decreased in all groups from baseline to 3 and 6 months except for int .
in fact , of the 41 individuals who had ifg at baseline in ss , 26 no longer had ifg at 3 months ( data not shown ) .
all associations remained after adjustment for the effect of clustering of participants within communities , age , sex , and baseline cvd risk factors .
the proportion of individuals with cvd risk factors remained steady between 3 and 6 months in all groups and never returned back to baseline ( fig .
3 ) . when weight loss and cvd risk factors were examined within the ss group and stratified by intervention modality ( ff versus int ) , no differences in associations were found .
changes in proportion of participants with cardiovascular risk factors across study groups after lifestyle intervention ( baseline to 6-month follow - up ) .
p values represent within - group differences from baseline to 6-month follow - up . * p < 0.05 , * * p < 0.01 , * * * p < 0.0001 .
associations remained after adjustment for the effect of clustering of participants within communities , age , sex , and baseline values .
in this comparative effectiveness study , in which overweight and abdominally obese participants from eight underserved rural communities were enrolled , four modalities ( ff , dvd , int , and ss ) of lifestyle intervention achieved statistically and clinically significant weight loss , despite the modality .
the observed weight loss was similar to weight loss observed in efficacy studies ( 38 ) , with ss participants experiencing the largest average weight loss at 3 months and ff participants experiencing the largest average weight loss at 6 months .
more than one - half of all participants lost at least 5% of their total body weight after the intervention , weighing on average 20 lbs less than they did at baseline .
evidence demonstrates that 5% weight loss is clinically meaningful and has documented health benefits , including improved diabetes and cvd risk factors ( 29 ) .
all groups experienced reductions in the proportion of individuals with impaired fasting glucose , abdominal obesity , and hypertension after the intervention .
the data presented in this study support the work of others who designed and implemented lifestyle interventions based on the dpp in community settings .
numerous studies have reported initial weight loss and cvd risk reduction after a lifestyle intervention ( 9,13,14,30 ) and demonstrated that these types of interventions are feasible and can be effectively implemented in a number of settings and populations .
however , few studies have made head - to - head comparisons of modalities of lifestyle intervention ( 31,32 ) .
we believe that the react study is the first scalable public health attempt at examining the comparative effectiveness of previously developed lifestyle intervention modalities so that informed decisions can be made regarding primary prevention of diabetes and cvd . additionally , it is one of the first studies to examine the effectiveness of empowering a group of participants to choose the intervention modality that best suited their lifestyle . in conducting community interventions ,
although we implemented standardized interventions and data collection across all communities and study groups , we had to tailor our approach to reflect the setting ( e.g. , community - based hospitals , community gathering places , community work sites , etc . ) .
additionally , because of the real - world setting of our study , we had to increase our flexibility at times .
for example , we encouraged all participants to attend the group sessions and the data - collection reassessments that were part of their study group ; however , we could not mandate it .
although attendance was highly encouraged , average attendance / curriculum viewing was ~9 of 12 sessions .
as attrition may be perceived as a limitation in our data with decreasing response rates at 3 and 6 months , all analyses were carried out using intention - to - treat methodology and included the use of mixed models that addresses attrition while not overestimating the intervention effect ( 33,34 ) .
we were powered at 80% to detect significant differences in the primary and secondary outcomes .
therefore , it is unlikely that the reported findings are subject to type ii error .
the time frame of this react report also poses a limitation . a 6-month time frame limits generalizability of the results , as many improvements in clinical outcomes can not be determined in this short period .
although all react study groups achieved notable improvements in outcomes , the largest and most significant improvements were observed in the ff and ss groups . to that end , the react study provides a sort of template for what may be needed for successful community - based primary prevention efforts .
our results suggest that individuals who are attempting to lose weight and decrease their risk for diabetes and cvd benefit from accountability to their peers and health care providers in a group - based format and the option to choose the type of intervention that best suits their lifestyle .
future reports will focus on the long - term maintenance of the observed improvements and the comparative cost - effectiveness of the lifestyle - intervention modalities .
health professionals , payers , and policy makers worldwide increasingly recognize the need for cost - effective , scalable , community - based primary prevention efforts .
the react study provides the first opportunity to compare the effectiveness of several lifestyle interventions in multiple underserved rural communities . for a meaningful impact on public health policy and clinical care , understanding the comparative effectiveness of each primary prevention modality in multiple community settings is critical .
efforts to make primary prevention a billable and reimbursable service are ongoing at the local and national levels . | objectiveto determine the comparative effectiveness of three lifestyle intervention modalities in decreasing risk for diabetes.research design and methodsfive hundred and fifty - five individuals ( 86.1% female , 95.1% white , and 55.8% obese ) from eight rural communities were screened for bmi 25 kg / m2 and waist circumference > 40 inches in men and > 35 inches in women .
communities with their eligible participants ( n = 493 ; mean age 51 years , 87.6% female , 94.1% caucasian ) were assigned to four group lifestyle balance ( glb ) intervention groups : face to face ( ff ) ( n = 119 ) , dvd ( n = 113 ) , internet ( int ) ( n = 101 ) , and self - selection ( ss ) ( n = 101 ) .
ss participants chose the glb modality .
glb is a comprehensive lifestyle behavior
change program.resultsa marked decline was observed in weight after the intervention in all groups ( ff 12.5 lbs , p = 0.01 ; dvd 12.2 lbs , p < 0.0001 ; int 13.7 lbs , p < 0.0001 ; and ss 14 lbs , p < 0.0001 ) .
participants in ss experienced the largest average weight loss .
weight loss was sustained in > 90% of participants in each group at 6 months ( ff 90.7% , dvd 90.9% , int 92.1% , and ss 100% ) .
all groups experienced improvements in the proportion of participants with cvd risk factors .
the proportion of individuals with cvd risk factors remained steady between 3 and 6 months in all groups and never returned back to baseline .
all associations remained after multivariate adjustment.conclusionsdespite the modality , the glb intervention was effective at decreasing weight and improving cvd risk factor control .
ss and ff participants experienced greater improvements in outcomes compared with other groups , establishing the importance of patient - centered decision making and a support network for successful behavior change . |
prostate cancer is the second most common cancer in male worldwide , which accounts for 14.5% of all cancer , according to globocan 2012 from world health organization . in united states and europe , it is amounting to 25% of newly diagnosed cancer . in korea ,
annual incidence of prostate cancer in korea was 8.5 per 100,000 men in 1999 and it was increased to 27.0 per 100,000 men in 2012 .
the annual increase rate of prostate cancer incidence was 11.4% , which is the highest among cancers except thyroid cancer .
the standard test for pathologic confirm of prostate cancer is transrectal ultrasonography guided prostate biopsy ( trus - bx ) , and standard 12-core biopsy is used commonly as a method of trus - bx .
the result of trus - bx provide not only an information whether prostate cancer is present or not , but also data about the location and the extent of prostate cancer . the detailed information is regarded as an important reference to decide treatment modalities such as active surveillance , radical prostatectomy and hormonal therapy . in particular , the data about location and the extent from biopsy help surgeons to determine the extent of resection , when performing radical prostatectomy . according to the biopsy result , physicians carefully decide whether to dissect closely or widely , and whether to save neurovascular bundle or not .
the decision is very crucial in radical prostatectomy , because it affects result of positive surgical margin ( psm ) .
however , trus - bx is not a perfect method for evaluation of prostate cancer , because the small amount of sampled tissue can not represent the whole prostate perfectly .
for example , 10%-18% of patients with negative result at first trus - bx were diagnosed as prostate cancer at second trus - bx .
therefore , we could not exclude a possibility that prostate cancer exists in the locations around each cores that were confirmed as benign in trus - bx .
this imperfection of trus - bx concerns physician about presence of undetected cancer which can cause psm at the location where neurovascular bundle saving is performed .
however , there are few reports that compares concordance between the location of psm and the location of positive cores in trus - bx . in this study
, we compared location of positive cores in biopsy and location of psm following radical prostatectomy .
this study was approved by the institutional review board of the konkuk university hospital ( irb no .
this retrospective study included 155 patents who were diagnosed as prostate cancer by transrectal ultrasonography guided prostate biopsy and received retropubic radical prostatectomy between august , 2005 and december , 2013 . among them , only patients with psm at final pathologic report were included .
after exclusion of lack of exact data about the location of biopsy and the location of positive cores , and the number of biopsy cores lower than 12 , 50 patients were included in final analysis .
the locations of standard 12-core biopsy were right lateral apex , right medial apex , right lateral mid , right medial mid , right lateral base , right medial base and symmetrical site of left lobe ( fig .
when suspicious hypoechoic lesion was present , 1 or 2 additional biopsies were performed and the location was regarded as the nearest core location of the 12 standard locations .
the location of psm in our pathologic results was reported routinely as 10 separate locations as followings ; right apex , right base , right lateral , right anterior , right posterior and symmetrical site of left lobe . at each location ,
presence of prostate cancer cells at margin was reported separately . when psm was present at more than 2 locations , all locations of psm were described . to compare the location of psm and the location of cancer positive cores , we defined geometrically concordant cores as fig .
2 . for example , when psm was presented at right lateral side , we could assume that prostate cancer was located at least one of right lateral apex , right lateral mid and right lateral base , therefore one of the biopsy locations must be reported as positive cores theoretically .
when we could identify at least one positive core among the locations , we regarded that the biopsy result is concordant .
when we could not identify any of positive biopsy cores at the assumed locations , we regarded that the result is discordant . in case of anterior psm and posterior surgical margin
, we could not specify assumed location of cancer exactly at coronal section of biopsy mapping .
therefore , the judgment of concordance or discordance at these psm locations was not evaluated , and 4 cases with psm exclusively at anterior or posterior location were excluded in comparative analysis . when multiple locations of psm were presented , we compared each site and regarded the result is concordant only when we could find concordant cores at every location of psm ( concordant group ) , and regarded that the result is discordant when one or more discordant result was presented at any psm locations ( discordant group ) .
all statistical analyses were performed using ibm spss statistics ver . 19.0 ( ibm co. , armonk , ny , usa ) .
a comparative analysis between the two groups was performed using the independent t - test for parametric continuous variables and the mann - whitney u test for nonparametric continuous variables .
fisher exact test was used to compare categorical variables . to evaluate factors that influence on discordant result , binary logistic regression analysis was used .
the reported p - values were two - sided , and a p - value of 0.05 was considered statistically significant .
this study was approved by the institutional review board of the konkuk university hospital ( irb no .
this retrospective study included 155 patents who were diagnosed as prostate cancer by transrectal ultrasonography guided prostate biopsy and received retropubic radical prostatectomy between august , 2005 and december , 2013 . among them , only patients with psm at final pathologic report were included .
after exclusion of lack of exact data about the location of biopsy and the location of positive cores , and the number of biopsy cores lower than 12 , 50 patients were included in final analysis .
the locations of standard 12-core biopsy were right lateral apex , right medial apex , right lateral mid , right medial mid , right lateral base , right medial base and symmetrical site of left lobe ( fig .
when suspicious hypoechoic lesion was present , 1 or 2 additional biopsies were performed and the location was regarded as the nearest core location of the 12 standard locations .
the location of psm in our pathologic results was reported routinely as 10 separate locations as followings ; right apex , right base , right lateral , right anterior , right posterior and symmetrical site of left lobe . at each location ,
presence of prostate cancer cells at margin was reported separately . when psm was present at more than 2 locations , all locations of psm were described .
to compare the location of psm and the location of cancer positive cores , we defined geometrically concordant cores as fig .
, when psm was presented at right lateral side , we could assume that prostate cancer was located at least one of right lateral apex , right lateral mid and right lateral base , therefore one of the biopsy locations must be reported as positive cores theoretically .
when we could identify at least one positive core among the locations , we regarded that the biopsy result is concordant .
when we could not identify any of positive biopsy cores at the assumed locations , we regarded that the result is discordant . in case of anterior psm and posterior surgical margin
, we could not specify assumed location of cancer exactly at coronal section of biopsy mapping .
therefore , the judgment of concordance or discordance at these psm locations was not evaluated , and 4 cases with psm exclusively at anterior or posterior location were excluded in comparative analysis . when multiple locations of psm were presented , we compared each site and regarded the result is concordant only when we could find concordant cores at every location of psm ( concordant group ) , and regarded that the result is discordant when one or more discordant result was presented at any psm locations ( discordant group ) .
all statistical analyses were performed using ibm spss statistics ver . 19.0 ( ibm co. , armonk , ny , usa ) .
a comparative analysis between the two groups was performed using the independent t - test for parametric continuous variables and the mann - whitney u test for nonparametric continuous variables .
fisher exact test was used to compare categorical variables . to evaluate factors that influence on discordant result
the reported p - values were two - sided , and a p - value of 0.05 was considered statistically significant .
mean prostate - specific antigen ( psa ) of the patients was 22.938.1 ng / ml . most of patients ( n=48 , 96% ) received 12-core trus - bx , and remained 2 patients received 12-core biopsy and 1 or 2 additional targeted biopsy .
most common location of psm in pathologic specimen was right apex ( n=21 , 42% ) followed by left apex ( n=15 , 30% ) , left base ( n=11 , 22% ) and right base ( n=10 , 20% ) ( table 2 ) . in 29 specimens ( 58% ) showed psm at only one location , however 21 specimens ( 42% ) identified to have psm at two or more locations ( table 1 ) . on comparative analysis ,
one or more concordant positive biopsy cores were identified at every psm locations in 32 specimens ( 69.6% ) with psm , but 14 specimens ( 30.4% ) had no concordant biopsy cores at one of psm locations ( table 2 ) .
when discordant rate was separated by locations of psm , right apex psm had highest rate of discordance ( 38% ) . when we compared patient characteristics and tumor characteristics between concordant group and discordant group , age , psa , biopsy gleason score sum , pathologic gleason score sum , pathologic t stage and percent tumor volume were not different between two groups . however , prostate volume was smaller , and number of cancer positive cores was lower in discordant group , compared to concordant group ( table 3 ) .
on multivariate analysis , number of cancer positive cores and prostate volume were independent factors that were related to discordant result ( table 4 ) .
this study revealed that about one fourth of psm occurred at location where prostate cancer is not detected in standard 12-core trus - bx .
this implies that prostate cancer existed at the location where cancer was not detected in trus - bx .
for the reason of missing , the undetected tumor might be so small that the cancer is located only between the biopsy cores .
in addition , out study showed that psm occurred most at apex area commonly , and inconsistent result was also most common at the apex area .
the accuracy of trus - bx was known to be only 70%-80% . as an example of inaccurate trus - bx ,
prostate cancer was detected by repeat biopsy in 25%-30% patients who showed negative result at first trus - bx .
abraham et al . reported that prostate cancer was detected in 63 patients ( 24.7% ) among 255 men who underwent repeat biopsies .
another study also reported that second , third , and fourth repeat biopsy procedures yielded a diagnosis of prostate cancer in 18% , 17% , and 14% of patients , respectively .
numao et al . also compared result of simultaneous transrectal 12-core biopsy and transperineal 14-core biopsy , and showed that 21% of cancer was missed when transrectal 12-core biopsy was performed only , and the missed lesion were frequently located anteriorly .
false negative trus - bx results also cause discordance with pathology of radical prostatectomy specimen .
compared the results of primary prostate biopsy with the radical prostatectomy , and revealed that the sensitivity for the detection of cancer in all biopsy zones was only 48% , which means that over half of cancer was missed in trus - bx .
another retrospective analysis showed that the accuracy of biopsy at apical location was lower than other site .
the positive predictive value of a positive apical core for identifying correct tumor location in the prostatectomy specimen was 71.1% , while absence of cancer in the apical biopsy had a negative predictive value of 75.5% .
our study showed that most common location of psm in pathologic specimen was right apex ( 42% ) and apex area had highest rate of discordance ( 38% ) .
these results imply that physician should be cautious to avoid psm at apex regardless biopsy result . concerning the inaccuracy of trus - bx ,
it was needed to evaluate whether decision of surgical extent based on the information from trus - bx for reducing the risk of psm was reasonable or not . to evaluate the reasonability , a few studies investigated consistency of biopsy result and surgical margin of pathologic specimen .
when biopsy result was divided to right and left , 77% of biopsy - suggested unilateral carcinoma was found to be bilateral disease on final histology following radical prostatectomy .
furthermore , 24% of unilateral disease on biopsy showed psm at contralateral biopsy - benign side .
divided biopsy location as apex and base , and investigated whether the location of preoperative biopsy positive cores could identify patients at higher risk of a psm and extraprostatic extension at radical retropubic prostatectomy . in the study ,
a positive biopsy at the apex was not predictive of an apical psm or extraprostatic extension , but a positive biopsy at the base was related to a basal psm ( 4% vs. 11% , p=0.003 ) and epe ( 32% vs. 9% , p<0.001 ) .
our results is very similar to these results that apex is more inaccurate than base .
furthermore , none of previous study subdivided the location of psm as like our study .
we subdivided the location of psm by 6 separate locations , and our result revealed that discordant result is most common in apex , following by lateral and base . furthermore
, our study showed that discordance occurred at right apex than left apex . because prostate cancer have relatively good prognosis than other malignancies , the aim of radical prostatectomy is not only control of cancers , but also preserving quality of life after treatment . in point of extended aim ,
pentafecta includes continence , potency , cancer control ( no recurrence ) , absence of postoperative complication , and negative surgical margin . among those ,
continence , potency and surgical margin are affected by the extent of resection and neurovascular bundle saving during radical prostatectomy .
especially , neurovascular bundle saving could preserve potency and improve continence results , but it increases the risk of psm .
therefore , surgeons always decides carefully whether to perform neurovascular bundle saving or not , according to the result of biopsy . however considering this study results , the pathologic locations of trus - bx are not always reliable , especially in apex .
therefore , surgeon must assumed that the cancer exist at all location of prostate , and dissect carefully even in biopsy negative site when performing neurovascular bundle saving .
there is no definite prognostic variable to predict the discordant result between biopsy and pathologic specimen . in this study , that small prostate volume and small number of cancer core are related to the discordant results .
however , discrimination of exact location in trus - bx is more difficult in small prostate . in addition
, targeting marginal area of prostate is also more difficult in small prostate . these reasons might increase the discordant rate .
in addition , relationship between small number of cancer positive biopsy cores and discordance could be explained as a result of inaccurate biopsy and neurovascular bundle saving based on the negative biopsy results .
retrospective nature of this study and small number of cases were our major limitations . because of the retrospective study design , neurovascular bundle saving and extent of dissection were not controlled during radical prostatectomy . in addition
, we could not analyze the influence of discordance on biochemical recurrence and other oncologic outcomes due to insufficient number of cases .
nonetheless , we thought this study is meaningful because this is first study that described the discordant rate of biopsy result and pathologic psm at subdivided locations .
this study showed that more than a quarter of psm occurred at location where tumor was not detected at biopsy and that apex psm had highest rate of discordant .
although tumor was not identified in specific biopsy location , careful dissection to avoid psm should be performed in every location , especially apex . | purposewe compared location of positive cores in biopsy and location of positive surgical margin ( psm ) following radical prostatectomy.materials and methodsthis retrospective analysis included patients who were diagnosed as prostate cancer by standard 12-core transrectal ultrasonography guided prostate biopsy , and who have psm after radical prostatectomy .
after exclusion of number of biopsy cores < 12 , and lack of biopsy location data , 46 patients with psm were identified .
locations of psm in pathologic specimen were reported as 6 difference sites ( apex , base and lateral in both sides ) .
discordance of biopsy result and psm was defined when no positive cores in biopsy was identified at the location of psm.resultsmost common location of psm were right apex ( n=21 ) and left apex ( n=15 ) .
multiple psm was reported in 21 specimens ( 45.7% ) . in 32 specimens ( 69.6% ) with psm , one or more concordant positive biopsy cores were identified , but 14 specimens ( 28% ) had no concordant biopsy cores at psm location .
when discordant rate was separated by locations of psm , right apex psm had highest rate of discordant ( 38% ) .
the discordant group had significantly lower prostate volume and lower number of positive cores in biopsy than concordant group.conclusionsthis study showed that one fourth of psm occurred at location where tumor was not detected at biopsy and that apex psm had highest rate of discordant .
careful dissection to avoid psm should be performed in every location , including where tumor was not identified in biopsy . |
during the past decade , organic light - emitting diodes ( oled ) have been one of the most intensively explored topics in chemistry and applied physics owing to their wide applications in full - color displays with color filters , backlighting for liquid crystal displays , and general lighting sources [ 13 ] .
while a large number of organic materials exhibit high fluorescence quantum efficiencies and bright in the visible region , there are some drawbacks such as low stability , high driving voltage associated with these materials .
therefore , they are not suitable for use in large area flat - panel displays . to overcome these problems and improve properties of electroluminescence ( el ) devices ,
it is highly desirable to design and synthesize new dipolar and multifunctional materials , which could maintain well the carrier balance .
-conjugated polymers / oligomers due to their combined desirable properties , such as fluorescence quantum yield , and good film - forming and hole - transporting properties have emerged as a very promising candidate for oleds .
color tuning in these polymers can be achieved by incorporating an electron - deficient monomer , benzothiadiazole ( bt ) , into the polymer backbone [ 4 , 5 ] . by incorporating bt into the alkylphenothiazine or alkyl phenoxazine polymer ,
an electron - rich and electron - deficient heterocycles composing the polymers possess a dual band of absorption in the visible spectrum consisting of short- and long - wavelength absorption bands .
many of desirable properties of conjugated organic polymers are dependent on the material s band gap ( egap ) , which is energetic separation between the material s filled valence and empty conduction bands . because of this , the significant effort has been applied to control the polymer egap and to produce low egap useful in technological fields novel materials .
phenothiazine , as well as phenoxazine , is well known very strong electron donor and has high homo energy level because of sulfur atom .
in addition , the homo energy level of phenothiazine copolymers is dominated by contribution of phenothiazine unit .
therefore , it is expected phenothiazine ring to be an excellent building block for lowering the ionization potential of conjugated polymers . moreover ,
efficient electrogenerated chemiluminescence was observed in co - oligomers of methylphenothiazine and interesting redox properties were found in other oligophenothiazines .
the possible consequence of the nonplanarity of these rings for the photophysics , light - emitting properties , charge transport of -conjugated polymers are intriguing and motivated .
however , the device based on poly(alkylphenothiazine ) are not enough to make efficient device since it has unbalanced charge transporting property . in order to improve electron affinity and transporting properties of material
the research presented here deals with the synthesis , characterization and optical properties of novel -conjugated oligomers based on 2,1,3-benzothiadiazole . reported here palladium promoted coupling reaction of suzuki is effective method of obtaining this class of highly luminescent -extended benzothiadiazole derivatives . it is expected that these polymers could be used as air - stable conducting materials ( through base doping for various electrical and electrochemical solid - states ) .
chemical shifts are denoted in 1 unit ( ppm ) and were referenced to internal tetramethylsilane ( 0.0 ppm ) .
the splitting patterns are designated as follows : s ( singlet ) , d ( doublet ) , t ( triplet ) , quin ( quintet ) , and m ( multiplet ) .
preparative column chromatography was carried out on glass columns of different sizes packed with silica gel merck 60 ( 4063 m ) .
ms spectra were taken on a bruker microtof - q , fwhm-17500 , 20 hz . chemical reagents were obtained from aldrich and used as received .
atomic force microscopy ( afm ) studies of langmuir - blodgett films were carried out using the afm dimension v veeco .
gel permeation chromatography ( gpc ) analysis of the polymer was performed on a waters gel permeation chromatograph with shodex gel columns and waters model 150 c refractive index detectors using thf as eluent and polystyrene standards as reference .
thermogravimetric analysis ( tga ) were conducted with a ta instrument q50 tga at a heating rate of 20 c / min under nitrogen gas flow .
vis absorption spectra were recorded on uv vis hp 8452a diode array spectrophotometer in either 1 or 4 cm quartz cuvettes depending on the sample concentration ( 1010 mol dm ) .
emission spectra were measured at 295 k in 1 cm long - neck sealed quartz cuvettes using 90 geometry on hitachi f2500 fluorescence spectrophotometer .
all emission spectra were corrected using correction data obtained with rhodamine and methylene blue as quantum counters for wavelength out to 720 nm , beyond which the response was estimated from the manufacturer s photomultiplier response data .
the luminance - current voltage characteristics were recorded by combining the spectrometer through a keithly model 2400 programmable voltage - current source .
the calculations were performed applying the dft td / b3lyp method and the standard 631 g * atomic basis set .
4,7-dibromobenzothiadiazole ( 2 ) to a 250 ml two necked round bottom flask were added 5.00 g ( 36.72 mmol ) benzothiadiazole ( 1 ) and 75 ml of hbr ( 48% ) . a solution containing 17.47 g br2 ( 109.19 mmol ) in 50 ml of hbr
after the total addition of br2 , the solution was heated at reflux for 6 h. precipitation of a dark orange solid was noted .
the mixture was cooled to room temperature , and a sufficient amount of a saturated solution of nahso3 was added to completely consume any excess of br2 .
the solid was then washed once with cold et2o and dried under vacuum for ca .
data of 4,7-dibromobenzothiadiazole 2 y = 90% ( 9.71 g , 33.03 mmol ) , orange crystals , mp
. 190 c , h nmr ( cdcl3 ) : 7.72 ( s , 2 h ) , c nmr ( cdcl3 ) : 152.9 , 132.3 , 113.9 .
3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - nonylphenoxazine ( 4a ) to a solution of 1.00 g 3,7-dibromo - n - nonylphenoxazine ( 3a ) ( 2.14 mmol ) in dry thf ( 30 ml ) at 78 c was added , with a syringe , 0.44 ml ( 4.71 mmol ) of n - butyllithium ( 2.5 m in hexane ) .
the mixture was stirred at 78 c , then warmed to 0 c for 15 min , and cooled again to 78 c for 15 min .
2-isopropoxy-4,4,5,5-tetramethyl-2,1,3-dioxoborolane 1.07 ml ( 5.25 mmol ) was added rapidly to the solution , warmed to room temperature , and stirred for 24 h. the mixture was poured into water and extracted with diethyl ether .
the solvent was removed , and the residue was purified by column chromatography with hexane : ethyl acetate ( 1:1 ) as eluent [ 10 , 11 ] .
data of 3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - nonylphenoxazine 4a y = 64% ( 0.79 g , 1.37 mmol ) , yellowish crystals , mp
. 88 c , h nmr ( cdcl3 ) : 7.267.23 ( m , 2 h , arom .
h ) , 3.453.40 ( m , 2 h , ch2 ) , 1.561.41 ( m , 8 h , ch2 ) , 1.351.27 ( m , 24 h , ch3 ) , 1.241.23 ( m , 6 h , ch2 ) , 0.88 ( t , 3 h , j = 6.71 hz , ch3 ) .
c nmr ( cdcl3 ) : 145.26 , 132.25 , 127.80 ( 2 c linked to boron , broad peak ) , 126.57 , 118.61 , 112.46 , 112.23 , 44.31 , 31.86 , 29.56 , 29.38 , 29.24 , 26.86 , 24.73 , 22.68 , 14.12 . 3,6-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - octylphenothiazine ( 4b ) to a solution of 3.02 g 3,7-dibromo - n - octylphenothiazine ( 3b ) ( 6.44 mmol ) in dry thf ( 30 ml ) at 78 c was added , with a syringe , 1.31 ml ( 14.16 mmol ) of n - butyllithium ( 2.5 m in hexane ) .
the mixture was stirred at 78 c , then warmed to 0 c for 15 min , and cooled again to 78 c for 15 min .
2-isopropoxy-4,4,5,5-tetramethyl-2,1,3-dioxoborolane 3.22 ml ( 15.77 mmol ) was added rapidly to the solution , warmed to room temperature , and stirred for 24 h. the mixture was poured into water and extracted with diethyl ether .
the solvent was removed , and the residue was purified by column chromatography with hexane : ethyl acetate ( 1:1 ) as eluent [ 10 , 11 ] .
data of 3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - octylphenothiazine 4b y = 45% ( 1.68 g , 2.90 mmol ) , yellow
white oil , h nmr ( cdcl3 ) : 6.84 ( d , j = 8.3 hz , 2 h , arom .
h ) , 6.52 ( d , j = 3.2 hz , 2 h , arom .
h ) , 6.44 ( dd , j = 3.2 hz , j = 1.7 hz , 2 h , arom .
h ) , 3.85 ( t , j = 7.1 hz , 2 h , ch2 ) , 1.811.79 ( m , 2 h , ch2 ) , 1.351.27 ( m , 24 h , ch3 ) , 1.251.23 ( m , 10 h , ch2 ) , 0.83 ( t , j = 6.3 hz , 3 h , ch3 ) .
c nmr ( cdcl3 ) : 153.2 , 143.9 , 127.80 ( 2 c linked to boron , broad peak ) , 125.6 , 124.5 , 122.9 , 122.7 , 115.3 , 111.8 . 111.5 , 103.8 , 47.6 , 31.8 , 29.5 , 28.0 , 27.6 , 26.8 , 22.6 , 14.0 .
poly[(n - nonylphenoxazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5a ) the following general suzuki polymerization procedure was used for the preparation of all the copolymers .
to a three necked flask 0.20 g 3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - nonylphenoxazine ( 4a ) ( 0.35 mmol ) , 0.10 g 4,7-dibromobenzothiadiazole ( 2 ) ( 0.35 mmol ) , 0.01 g pd(pph3)4 ( 4.80 mol ) , and 0.57 g potassium carbonate ( 0.04 mmol ) was added .
a mixture of water ( 0.21 ml ) and toluene ( 15 ml ) was added to the flask , and the reaction vessel was degassed .
the mixture was heated at 90 c for 24 h under nitrogen and then precipitated into methanol .
the polymer was filtered and washed with methanol and acetone and then dried under vacuum overnight to afford the neutral oligomer [ 12 , 13 ] .
data of poly[(n - nonylphenoxazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5a ) y = 45% ( 0.05 g , 0.16 mmol ) , dark brown crystals , h nmr ( cdcl3 ) : 7.87 ( d , 2 h , j = 7.68 hz , arom .
h ) , 6.34 ( d , 2 h , j = 8.57 hz , arom .
h ) , 3.453.40 ( m , 2 h , ch2 ) , 1.561.25 ( m , 14 h , ch2 ) , 0.88 ( t , 3 h , j = 6.71 hz , ch3 ) .
c nmr ( cdcl3 ) : 132.28 , 128.00 , 126.67 , 126.29 , 124.99 , 118.53 , 115.83 , 112.44 , 111.38 , 44.16 , 31.80 , 29.52 , 29.35 , 29.20 , 26.85 , 24.83 , 22.62 , 14.07 .
three necked flask 2.92 g 3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - octylphenothiazine ( 4b ) ( 5.45 mmol ) , 1.60 g 4,7-dibromobenzothiadiazole ( 2 ) ( 5.45 mmol ) , 0.09 g pd(pph3)4 ( 75.46 mol ) , and 9.04 g potassium carbonate ( 65.40 mmol ) was added .
a mixture of water ( 32.7 ml ) and toluene ( 15 ml ) was added to the flask , and the reaction vessel was degassed .
the mixture was heated at 90 c for 24 h under nitrogen and then precipitated into methanol .
the polymer was filtered and washed with methanol and acetone and then dried under vacuum overnight to afford the neutral oligomer [ 12 , 13 ] .
data of poly[(n - octylphenothiazine-3,7-diyl - alt-(1,2,3-benzothiadiazole ) ] ( 5b ) y = 57% ( 1.03 g , 2.96 mmol ) , brown crystals , h nmr ( cdcl3 ) : 7.64 ( s , 2 h , arom .
h ) , 6.64 ( d , 2 h , j = 8.39 hz , arom .
h ) , 3.743.69 ( m , 2 h , ch2 ) , 1.781.67 ( m , 10 h , ch2 ) , 1.401.24 ( m , 10 h , ch2 ) , 0.90 ( t , 3 h , j = 5.37 hz , ch3 ) .
c nmr ( cdcl3 ) : 144.10 , 132.34 , 132.22 , 132.09 , 132.00 , 130.13 , 129.67 , 128.63 , 128.47 , 127.40 , 127.18 , 126.43 , 122.29 , 116.66 , 115.39 , 114.76 , 113.93 , 47.65 , 31.77 , 29.44 , 29.23 , 27.03 , 26.96 , 22.78 , 14.18 .
chemical shifts are denoted in 1 unit ( ppm ) and were referenced to internal tetramethylsilane ( 0.0 ppm ) .
the splitting patterns are designated as follows : s ( singlet ) , d ( doublet ) , t ( triplet ) , quin ( quintet ) , and m ( multiplet ) .
preparative column chromatography was carried out on glass columns of different sizes packed with silica gel merck 60 ( 4063 m ) .
ms spectra were taken on a bruker microtof - q , fwhm-17500 , 20 hz . chemical reagents were obtained from aldrich and used as received .
atomic force microscopy ( afm ) studies of langmuir - blodgett films were carried out using the afm dimension v veeco .
gel permeation chromatography ( gpc ) analysis of the polymer was performed on a waters gel permeation chromatograph with shodex gel columns and waters model 150 c refractive index detectors using thf as eluent and polystyrene standards as reference .
thermogravimetric analysis ( tga ) were conducted with a ta instrument q50 tga at a heating rate of 20 c / min under nitrogen gas flow .
vis absorption spectra were recorded on uv vis hp 8452a diode array spectrophotometer in either 1 or 4 cm quartz cuvettes depending on the sample concentration ( 1010 mol dm ) .
emission spectra were measured at 295 k in 1 cm long - neck sealed quartz cuvettes using 90 geometry on hitachi f2500 fluorescence spectrophotometer .
all emission spectra were corrected using correction data obtained with rhodamine and methylene blue as quantum counters for wavelength out to 720 nm , beyond which the response was estimated from the manufacturer s photomultiplier response data .
the luminance - current voltage characteristics were recorded by combining the spectrometer through a keithly model 2400 programmable voltage - current source .
the calculations were performed applying the dft td / b3lyp method and the standard 631 g * atomic basis set .
4,7-dibromobenzothiadiazole ( 2 ) to a 250 ml two necked round bottom flask were added 5.00 g ( 36.72 mmol ) benzothiadiazole ( 1 ) and 75 ml of hbr ( 48% ) . a solution containing 17.47 g br2 ( 109.19 mmol ) in 50 ml of hbr was added dropwise very slowly ( slow addition is essential ! ) .
after the total addition of br2 , the solution was heated at reflux for 6 h. precipitation of a dark orange solid was noted .
the mixture was cooled to room temperature , and a sufficient amount of a saturated solution of nahso3 was added to completely consume any excess of br2 .
the solid was then washed once with cold et2o and dried under vacuum for ca .
data of 4,7-dibromobenzothiadiazole 2 y = 90% ( 9.71 g , 33.03 mmol ) , orange crystals , mp .
190 c , h nmr ( cdcl3 ) : 7.72 ( s , 2 h ) , c nmr ( cdcl3 ) : 152.9 , 132.3 , 113.9 .
3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - nonylphenoxazine ( 4a ) to a solution of 1.00 g 3,7-dibromo - n - nonylphenoxazine ( 3a ) ( 2.14 mmol ) in dry thf ( 30 ml ) at 78 c was added , with a syringe , 0.44 ml ( 4.71 mmol ) of n - butyllithium ( 2.5 m in hexane ) .
the mixture was stirred at 78 c , then warmed to 0 c for 15 min , and cooled again to 78 c for 15 min .
2-isopropoxy-4,4,5,5-tetramethyl-2,1,3-dioxoborolane 1.07 ml ( 5.25 mmol ) was added rapidly to the solution , warmed to room temperature , and stirred for 24 h. the mixture was poured into water and extracted with diethyl ether .
the solvent was removed , and the residue was purified by column chromatography with hexane : ethyl acetate ( 1:1 ) as eluent [ 10 , 11 ] .
data of 3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - nonylphenoxazine 4a y = 64% ( 0.79 g , 1.37 mmol ) , yellowish crystals , mp
. 88 c , h nmr ( cdcl3 ) : 7.267.23 ( m , 2 h , arom .
h ) , 3.453.40 ( m , 2 h , ch2 ) , 1.561.41 ( m , 8 h , ch2 ) , 1.351.27 ( m , 24 h , ch3 ) , 1.241.23 ( m , 6 h , ch2 ) , 0.88 ( t , 3 h , j = 6.71 hz , ch3 ) .
c nmr ( cdcl3 ) : 145.26 , 132.25 , 127.80 ( 2 c linked to boron , broad peak ) , 126.57 , 118.61 , 112.46 , 112.23 , 44.31 , 31.86 , 29.56 , 29.38 , 29.24 , 26.86 , 24.73 , 22.68 , 14.12 .
3,6-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - octylphenothiazine ( 4b ) to a solution of 3.02 g 3,7-dibromo - n - octylphenothiazine ( 3b ) ( 6.44 mmol ) in dry thf ( 30 ml ) at 78 c was added , with a syringe , 1.31 ml ( 14.16 mmol ) of n - butyllithium ( 2.5 m in hexane ) .
the mixture was stirred at 78 c , then warmed to 0 c for 15 min , and cooled again to 78 c for 15 min .
2-isopropoxy-4,4,5,5-tetramethyl-2,1,3-dioxoborolane 3.22 ml ( 15.77 mmol ) was added rapidly to the solution , warmed to room temperature , and stirred for 24 h. the mixture was poured into water and extracted with diethyl ether .
the solvent was removed , and the residue was purified by column chromatography with hexane : ethyl acetate ( 1:1 ) as eluent [ 10 , 11 ] .
data of 3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - octylphenothiazine 4b y = 45% ( 1.68 g , 2.90 mmol ) , yellow
white oil , h nmr ( cdcl3 ) : 6.84 ( d , j = 8.3 hz , 2 h , arom .
h ) , 6.52 ( d , j = 3.2 hz , 2 h , arom .
h ) , 6.44 ( dd , j = 3.2 hz , j = 1.7 hz , 2 h , arom .
h ) , 3.85 ( t , j = 7.1 hz , 2 h , ch2 ) , 1.811.79 ( m , 2 h , ch2 ) , 1.351.27 ( m , 24 h , ch3 ) , 1.251.23 ( m , 10 h , ch2 ) , 0.83 ( t , j = 6.3 hz , 3 h , ch3 ) .
c nmr ( cdcl3 ) : 153.2 , 143.9 , 127.80 ( 2 c linked to boron , broad peak ) , 125.6 , 124.5 , 122.9 , 122.7 , 115.3 , 111.8 . 111.5 , 103.8 , 47.6 , 31.8 , 29.5 , 28.0 , 27.6 , 26.8 , 22.6 , 14.0 .
poly[(n - nonylphenoxazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5a ) the following general suzuki polymerization procedure was used for the preparation of all the copolymers .
to a three necked flask 0.20 g 3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - nonylphenoxazine ( 4a ) ( 0.35 mmol ) , 0.10 g 4,7-dibromobenzothiadiazole ( 2 ) ( 0.35 mmol ) , 0.01 g pd(pph3)4 ( 4.80 mol ) , and 0.57 g potassium carbonate ( 0.04 mmol ) was added .
a mixture of water ( 0.21 ml ) and toluene ( 15 ml ) was added to the flask , and the reaction vessel was degassed .
the mixture was heated at 90 c for 24 h under nitrogen and then precipitated into methanol . the polymer was filtered and washed with methanol and acetone and then dried under vacuum overnight to afford the neutral oligomer [ 12 , 13 ] .
data of poly[(n - nonylphenoxazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5a ) y = 45% ( 0.05 g , 0.16 mmol ) , dark brown crystals , h nmr ( cdcl3 ) : 7.87 ( d , 2 h , j = 7.68 hz , arom .
h ) , 6.34 ( d , 2 h , j = 8.57 hz , arom .
h ) , 3.453.40 ( m , 2 h , ch2 ) , 1.561.25 ( m , 14 h , ch2 ) , 0.88 ( t , 3 h , j = 6.71 hz , ch3 ) .
c nmr ( cdcl3 ) : 132.28 , 128.00 , 126.67 , 126.29 , 124.99 , 118.53 , 115.83 , 112.44 , 111.38 , 44.16 , 31.80 , 29.52 , 29.35 , 29.20 , 26.85 , 24.83 , 22.62 , 14.07 .
poly[(n - octylphenothiazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5b ) to a three necked flask 2.92 g 3,7-bis(4,4,5,5-tetramethyl-2,1,3-dioxaborolan-2-yl)-n - octylphenothiazine ( 4b ) ( 5.45 mmol ) , 1.60 g 4,7-dibromobenzothiadiazole ( 2 ) ( 5.45 mmol ) , 0.09 g pd(pph3)4 ( 75.46 mol ) , and 9.04 g potassium carbonate ( 65.40 mmol ) was added .
a mixture of water ( 32.7 ml ) and toluene ( 15 ml ) was added to the flask , and the reaction vessel was degassed .
the mixture was heated at 90 c for 24 h under nitrogen and then precipitated into methanol .
the polymer was filtered and washed with methanol and acetone and then dried under vacuum overnight to afford the neutral oligomer [ 12 , 13 ] .
data of poly[(n - octylphenothiazine-3,7-diyl - alt-(1,2,3-benzothiadiazole ) ] ( 5b ) y = 57% ( 1.03 g , 2.96 mmol ) , brown crystals , h nmr ( cdcl3 ) : 7.64 ( s , 2 h , arom .
h ) , 6.64 ( d , 2 h , j = 8.39 hz , arom .
h ) , 3.743.69 ( m , 2 h , ch2 ) , 1.781.67 ( m , 10 h , ch2 ) , 1.401.24 ( m , 10 h , ch2 ) , 0.90 ( t , 3 h , j = 5.37 hz , ch3 ) .
c nmr ( cdcl3 ) : 144.10 , 132.34 , 132.22 , 132.09 , 132.00 , 130.13 , 129.67 , 128.63 , 128.47 , 127.40 , 127.18 , 126.43 , 122.29 , 116.66 , 115.39 , 114.76 , 113.93 , 47.65 , 31.77 , 29.44 , 29.23 , 27.03 , 26.96 , 22.78 , 14.18 .
the reaction of 1 with molecular bromine ( added dropwise very slowly ) in hydrobromic acid exclusively affords 4,7-dibromobenzothiadiazole ( 2 ) as a major product in 95% yield ( scheme 1 ) .
scheme 1synthesis of dibromobenzothiadiazole synthesis of dibromobenzothiadiazole the synthesis of oligomers 5a b was provided as shown in scheme 2 .
conversion of n - alkyl-3,7-dibromo - n - alkylphenoxazine ( 3a ) , -phenothiazine ( 3b ) to the boronic ester ( 4a - b ) was achieved ( according to literature general procedure ) under reaction conditions in presence 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane .
oligomers 5a b were obtained in 50% yield after purification by silica column chromatography .
the molar ratio of phenoxazine moiety in the oligomers was controlled by adjusting the molar ratio between dibromobenzothiadiazole and monomers 4a b while a 1/1 molar ratio between the dibromides and the bisborylated compounds was maintained .
scheme 2synthetic route to the co - oligomers based on benzothiadiazole synthetic route to the co - oligomers based on benzothiadiazole the structures of the obtained co - oligomers were confirmed by hnmr , cnmr as well as ms spectroscopy .
all structures were found to be highly soluble in most of organic solvents such as methylene chloride , chloroform , toluene , and o - dichlorobenzene .
both oligomers gave number - average molecular weights of about 3,500 g / mol .
this value corresponds to a degree of polymerization of seven to eight , meaning that the polymers synthesized here have an average of about 30 aromatic rings per chain .
the molecular weights obtained were adequate for processing and film formation , and thus , further optimization of the polymerization was not attempted .
the average molecular weight ( mw5a b ) of the copolymers was determined by gel permeation chromatography against polystyrene standards in thf ( polydispersity in every case near 1.6 ) .
table 1summary of gpc data ( polystyrene standard ) for benzothiadiazole co - oligomerspolymermn [ g / mol]mw[g / mol]tg [ c]pdi5a3,5005,2001301.575b3,6005,400801.62 summary of gpc data ( polystyrene standard ) for benzothiadiazole co - oligomers thin films of these compounds were also easily fabricated from their solutions in chloroform by langmuir - blodgett ( lb ) technique . synthesized oligomers 5a b dissolved in organic solvents ( chloroform ) were spread on the water , then organic layers were deposited by lb technique onto ito ( 50 ) or a set of eight interdigital , buried au electrodes ( 200 m wide and spaced ) photolithographically fixed on sio2 thermally coated silicon substrates .
etching canals in silicone substrates were then filling by mixture of gold and trace amount of wolfram .
this configuration provides flat , polished ( measured by interferometric method ) electrodes , ready to lb deposition .
all initially non measured under water films were deposited at the velocity lower than the draining rate of film of carboxylic acids i.e. 1.3 mm / min . after deposition
langmuir monomolecular films of 5a b were spread from chcl3 solution on high purity water at room temperature .
blodgett deposition was carried out with a ksv system 5000 lb through at a surface pressure of around 2025 mn / m .
the relationship between absorbance and number of layers and constant transfer ratio during the deposition indicate on constant architecture of lb film layers . the lb film morphology was examined using atomic force microscopy .
1 ) show that the lb film is compact and highly ordered . on fig . 1
, for comparison , is also shown the topography of clean substrate ( glass slide , fig
the surface roughness is rather smooth , it is due the reorganization of oligomer molecules during the lb deposition .
1surface morphology ( afm ) of lb 5a film ( 20 layers ) on glass slide ( a ) ; image of the bare glass ( b ) surface morphology ( afm ) of lb 5a film ( 20 layers ) on glass slide ( a ) ; image of the bare glass ( b ) the major result of investigations was pointed on the electrical conductivity of langmuir - blodgett ( lb ) films consisting several layers of synthesized compound ( 5a ) as an effective conducting material .
figure 2 shows typical current voltage ( i - v ) curves of fabricated lb films at room temperature .
the conductivity found for oligomer 5a lb film equals = 1.2 x 10 s / m .
2the current voltage characteristics of 20 layer lb film built of poly[(n - nonylphenoxazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5a ) and measuring system ( left ) the current voltage characteristics of 20 layer lb film built of poly[(n - nonylphenoxazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5a ) and measuring system ( left ) from current voltage characteristics obtained at higher temperatures ( 60 c , 80 c ) , it was found that the characteristics of the i - v maintain its asymmetrical shape and character .
therefore , these results indicate that obtained films behave as p - type semiconductors . moreover ,
good environmental stabilities observed for this material makes it a promising candidate for sensing elements .
the maximum luminance brightness is 6,857 cd m at 11 v. the brightness of 5a is improved largely , which denotes that compound has good hole - transporting ability and luminance property .
3the voltage - luminance curves of compound 5a as lb film the voltage - luminance curves of compound 5a as lb film figure 4 illustrated the luminance efficiency at different voltages . as the voltages increased gradually ,
the maximum luminescent efficiency increased to 3.10 cd a. this phenomenon is a consequence of the formation of excitons required to emit light .
4the voltage - efficiency curves of compound 5a as lb film the voltage - efficiency curves of compound 5a as lb film while the origin of the dual - band absorption sometimes encountered in da - type semiconducting polymers remains a source of debate , two mainstream rationales have been frequently proposed .
a first assumption attributes the lower - energy optical transition to the presence of intermolecular charge - transfer excitons occurring on the presence of covalently bound da segments along the backbone .
a second assumption considers the presence of low - lying unoccupied energy levels , strictly localized on the electron - deficient heterocycles , yet forming a discrete band of easily accessed energy states within the bandgap of the conjugated system in its ground state . in both cases ,
the higher - energy transitions appear localized on the most - electron - rich building units incorporated along the polymer backbone with a clear dependence on their relative concentration to the electron - deficient heterocycles .
this distinct contribution from the electron - rich segments on the short - wavelength absorption band in da backbones as well , the long - wavelength absorption band ( red shifted ) is illustrated in figure 5 .
5uv visible absorption spectra of polymers 5a and 5b in chloroform diluted solution ( 2 m ) , as insert absorption spectra of 5a and 5b in the solid state uv visible absorption spectra of polymers 5a and 5b in chloroform diluted solution ( 2 m ) , as insert absorption spectra of 5a and 5b in the solid state the photophysical characteristic of the copolymers ( 5a b ) were investigated by ultraviolet
visible ( uv vis ) absorption and photoluminescence ( pl ) in diluted chloroform solution and in solid state . the optical data summarized are also listed in table 2 .
a plot of extinction coefficient versus wavelength for the compound as measure in chloroform is depicted in figure 5 .
as expected , 5a b exhibit a dual band of absorption , in this case peaking at 280 , 425 nm and 280 , 400 nm for 5a b , respectively .
it should be noted that the absorption bands characteristic for phenoxazine ring are localized at about 400 nm .
however , the extent of conjugation between neighbouring benzothiadiazole units and phenoxazine ones will largely depend on the twist angle .
the above data clearly suggest that the corresponding polymer has potential as low - band - gap material .
the uv vis spectrum of 5b is very similar to spectrum of 5a , but there is very characteristic broad absorption at 490510 nm in solid state which was found also in another compounds based on benzothiadiazole and n - alkylphenothiazine ( poly(3,7-divinylene - n - octylphenothiazine - alt - benzothiadiazole ) ) .
similar donor - acceptor interaction between the electron - poor benzothiadiazole unit and the relatively electron - rich unit max at 460 nm , leading to an orange colour was found also in case of carbazole benzothiadiazole copolymers .
table 2summary of physical properties of benzothiadiazole co - oligomerspolymerin solutionin solid stateegap [ ev]egap [ ev]abs max [ nm]pl max [ nm]stokes shift [ cm]fabs max [ nm]5a28047661 7280.423552.452.615b28448068 9650.48360 , 5002.572.58polymer in chloroform solution was excited at 389 nmtheoretical datafigured out from the absorption edge from the pl spectrum summary of physical properties of benzothiadiazole co - oligomers polymer in chloroform solution was excited at 389 nm figured out from the absorption edge from the pl spectrum in general , the absorption range of solid of 5a and 5b is slightly red - shifted than those of solution because of the formation of aggregates ( fig .
however , the absorption range of polyphenothiazine and 5b are not much different , this is due to that phenothiazine impede formation of aggregates in the solid state .
the photoluminescent properties of the synthesized oligomers ( 5a b ) were investigated by fluorescence spectroscopy in dilute chloroform solution ( 2 m ) .
the fluorescence quantum yield of the oligomers 5a b were rather good ( 5a-f = 0.42 , 5b- f = 0.48 ) .
the fluorescence quantum yield of benzothiadiazole is very low ( f = 0.006 ) .
molecules 5a and 5b have rather large stokes shifts , 162 ( 61 728 cm ) and 145 nm ( 68 965 cm ) respectively .
the emission spectra of 5a and 5b solution were recorded at different excitation wavelengths in the range 310390 nm , but the emission values were almost the same .
however , when excited diluted solution of 5a at 389 nm the emission was centred at 476 nm ( 2.61 ev , fig .
the emission maximum was found at 480 nm ( 2.58 ev , fig .
the observed stokes shift according to phenoxazine emission suggests that there is a large twist between benzothiadiazole and neighbouring aromatic units .
6the pl spectra of 5a in chloroform diluted ( 2 m ) solutionfig .
7the pl spectra of 5b in chloroform diluted ( 2 m ) solution the pl spectra of 5a in chloroform diluted ( 2 m ) solution the pl spectra of 5b in chloroform diluted ( 2 m ) solution the luminescence emission spectrum of 5a has well resolved vibronic structrures in dilute chloroform solution .
the emission spectrum of 5a has a vibronic progression with the 00 transition at 476 nm .
incorporation of benzothiadiazole moieties into polyphenoxazine results in a slight red shift in the 00 in the emission band from 458 nm ( polyphenoxazine ) to 476 nm , both polyphenoxazine and polyphenoxazine with incorporated benzothiadiazole emits blue light in diluted solutions .
2,1,3-benzothiadiazole based oligomers and polymers have been widely studied in recent years as active materials in various optoelectronic devices because of the heterocyclic group and the observed low - band - gap in polymers containing it .
copolymerization of benzothiadiazole with phenoxazine , phenothiazine , fluorene and carbazole or other suitable arylenes can be used as a means to tune the homo
the homo and lumo energy level of -conjugated polymer are important for understanding charge injection processes in the luminescent devices . by the theoretical study the luminescence of benzothiadiazole
phenoxazine oligomer ( 5a ) was found as 506 nm ( table 2 ) , in case of 5b the value is 482 nm . the above data , similar to experimental ( 476 nm5a , 480 nm5b ) , clearly suggests that the corresponding copolymers has potential as low - band - gap materials . the low optical band gaps of the oligomers should result from the alternating between the electron - rich unit of alkylphenothiazine / alkylphenoxazine and the strong electron - deficient unit of benzothiadiazole .
thin films of these compounds were also easily fabricated from their solutions in chloroform by langmuir - blodgett ( lb ) technique . synthesized oligomers 5a b dissolved in organic solvents ( chloroform ) were spread on the water , then organic layers were deposited by lb technique onto ito ( 50 ) or a set of eight interdigital , buried au electrodes ( 200 m wide and spaced ) photolithographically fixed on sio2 thermally coated silicon substrates .
etching canals in silicone substrates were then filling by mixture of gold and trace amount of wolfram .
this configuration provides flat , polished ( measured by interferometric method ) electrodes , ready to lb deposition .
all initially non measured under water films were deposited at the velocity lower than the draining rate of film of carboxylic acids i.e. 1.3 mm / min . after deposition
langmuir monomolecular films of 5a b were spread from chcl3 solution on high purity water at room temperature .
blodgett deposition was carried out with a ksv system 5000 lb through at a surface pressure of around 2025 mn / m .
22 c as well as conductivity measurements . the transference of lb film was y type in first deposition .
the relationship between absorbance and number of layers and constant transfer ratio during the deposition indicate on constant architecture of lb film layers . the lb film morphology was examined using atomic force microscopy .
1 ) show that the lb film is compact and highly ordered . on fig . 1 , for comparison
, is also shown the topography of clean substrate ( glass slide , fig .
the surface roughness is rather smooth , it is due the reorganization of oligomer molecules during the lb deposition .
1surface morphology ( afm ) of lb 5a film ( 20 layers ) on glass slide ( a ) ; image of the bare glass ( b ) surface morphology ( afm ) of lb 5a film ( 20 layers ) on glass slide ( a ) ; image of the bare glass ( b ) the major result of investigations was pointed on the electrical conductivity of langmuir - blodgett ( lb ) films consisting several layers of synthesized compound ( 5a ) as an effective conducting material .
figure 2 shows typical current voltage ( i - v ) curves of fabricated lb films at room temperature .
the conductivity found for oligomer 5a lb film equals = 1.2 x 10 s / m .
2the current voltage characteristics of 20 layer lb film built of poly[(n - nonylphenoxazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5a ) and measuring system ( left ) the current voltage characteristics of 20 layer lb film built of poly[(n - nonylphenoxazine-3,7-diyl - alt-(2,1,3-benzothiadiazole ) ] ( 5a ) and measuring system ( left ) from current voltage characteristics obtained at higher temperatures ( 60 c , 80 c ) , it was found that the characteristics of the i - v maintain its asymmetrical shape and character .
moreover , good environmental stabilities observed for this material makes it a promising candidate for sensing elements .
the maximum luminance brightness is 6,857 cd m at 11 v. the brightness of 5a is improved largely , which denotes that compound has good hole - transporting ability and luminance property .
3the voltage - luminance curves of compound 5a as lb film the voltage - luminance curves of compound 5a as lb film figure 4 illustrated the luminance efficiency at different voltages . as the voltages increased gradually ,
the maximum luminescent efficiency increased to 3.10 cd a. this phenomenon is a consequence of the formation of excitons required to emit light .
4the voltage - efficiency curves of compound 5a as lb film the voltage - efficiency curves of compound 5a as lb film
while the origin of the dual - band absorption sometimes encountered in da - type semiconducting polymers remains a source of debate , two mainstream rationales have been frequently proposed .
a first assumption attributes the lower - energy optical transition to the presence of intermolecular charge - transfer excitons occurring on the presence of covalently bound da segments along the backbone .
a second assumption considers the presence of low - lying unoccupied energy levels , strictly localized on the electron - deficient heterocycles , yet forming a discrete band of easily accessed energy states within the bandgap of the conjugated system in its ground state . in both cases ,
the higher - energy transitions appear localized on the most - electron - rich building units incorporated along the polymer backbone with a clear dependence on their relative concentration to the electron - deficient heterocycles .
this distinct contribution from the electron - rich segments on the short - wavelength absorption band in da backbones as well , the long - wavelength absorption band ( red shifted ) is illustrated in figure 5 .
5uv visible absorption spectra of polymers 5a and 5b in chloroform diluted solution ( 2 m ) , as insert absorption spectra of 5a and 5b in the solid state uv visible absorption spectra of polymers 5a and 5b in chloroform diluted solution ( 2 m ) , as insert absorption spectra of 5a and 5b in the solid state the photophysical characteristic of the copolymers ( 5a b ) were investigated by ultraviolet
visible ( uv vis ) absorption and photoluminescence ( pl ) in diluted chloroform solution and in solid state . the optical data summarized are also listed in table 2 .
a plot of extinction coefficient versus wavelength for the compound as measure in chloroform is depicted in figure 5 .
as expected , 5a b exhibit a dual band of absorption , in this case peaking at 280 , 425 nm and 280 , 400 nm for 5a b , respectively .
it should be noted that the absorption bands characteristic for phenoxazine ring are localized at about 400 nm .
however , the extent of conjugation between neighbouring benzothiadiazole units and phenoxazine ones will largely depend on the twist angle .
the above data clearly suggest that the corresponding polymer has potential as low - band - gap material .
the uv vis spectrum of 5b is very similar to spectrum of 5a , but there is very characteristic broad absorption at 490510 nm in solid state which was found also in another compounds based on benzothiadiazole and n - alkylphenothiazine ( poly(3,7-divinylene - n - octylphenothiazine - alt - benzothiadiazole ) ) .
similar donor - acceptor interaction between the electron - poor benzothiadiazole unit and the relatively electron - rich unit max at 460 nm , leading to an orange colour was found also in case of carbazole
table 2summary of physical properties of benzothiadiazole co - oligomerspolymerin solutionin solid stateegap [ ev]egap [ ev]abs max [ nm]pl max [ nm]stokes shift [ cm]fabs max [ nm]5a28047661 7280.423552.452.615b28448068 9650.48360 , 5002.572.58polymer in chloroform solution was excited at 389 nmtheoretical datafigured out from the absorption edge from the pl spectrum summary of physical properties of benzothiadiazole co - oligomers polymer in chloroform solution was excited at 389 nm figured out from the absorption edge from the pl spectrum in general , the absorption range of solid of 5a and 5b is slightly red - shifted than those of solution because of the formation of aggregates ( fig .
however , the absorption range of polyphenothiazine and 5b are not much different , this is due to that phenothiazine impede formation of aggregates in the solid state .
the photoluminescent properties of the synthesized oligomers ( 5a b ) were investigated by fluorescence spectroscopy in dilute chloroform solution ( 2 m ) .
the fluorescence quantum yield of the oligomers 5a b were rather good ( 5a-f = 0.42 , 5b- f = 0.48 ) .
the fluorescence quantum yield of benzothiadiazole is very low ( f = 0.006 )
. molecules 5a and 5b have rather large stokes shifts , 162 ( 61 728 cm ) and 145 nm ( 68 965 cm ) respectively .
the emission spectra of 5a and 5b solution were recorded at different excitation wavelengths in the range 310390 nm , but the emission values were almost the same .
however , when excited diluted solution of 5a at 389 nm the emission was centred at 476 nm ( 2.61 ev , fig .
the emission maximum was found at 480 nm ( 2.58 ev , fig . 7 ) .
the observed stokes shift according to phenoxazine emission suggests that there is a large twist between benzothiadiazole and neighbouring aromatic units .
6the pl spectra of 5a in chloroform diluted ( 2 m ) solutionfig .
7the pl spectra of 5b in chloroform diluted ( 2 m ) solution the pl spectra of 5a in chloroform diluted ( 2 m ) solution the pl spectra of 5b in chloroform diluted ( 2 m ) solution the luminescence emission spectrum of 5a has well resolved vibronic structrures in dilute chloroform solution . the emission spectrum of 5a has a vibronic progression with the 00 transition at 476 nm .
incorporation of benzothiadiazole moieties into polyphenoxazine results in a slight red shift in the 00 in the emission band from 458 nm ( polyphenoxazine ) to 476 nm , both polyphenoxazine and polyphenoxazine with incorporated benzothiadiazole emits blue light in diluted solutions .
2,1,3-benzothiadiazole based oligomers and polymers have been widely studied in recent years as active materials in various optoelectronic devices because of the heterocyclic group and the observed low - band - gap in polymers containing it .
copolymerization of benzothiadiazole with phenoxazine , phenothiazine , fluorene and carbazole or other suitable arylenes can be used as a means to tune the homo
the homo and lumo energy level of -conjugated polymer are important for understanding charge injection processes in the luminescent devices . by the theoretical study the luminescence of benzothiadiazole
phenoxazine oligomer ( 5a ) was found as 506 nm ( table 2 ) , in case of 5b the value is 482 nm .
the above data , similar to experimental ( 476 nm5a , 480 nm5b ) , clearly suggests that the corresponding copolymers has potential as low - band - gap materials .
the low optical band gaps of the oligomers should result from the alternating between the electron - rich unit of alkylphenothiazine / alkylphenoxazine and the strong electron - deficient unit of benzothiadiazole .
in summary , according to the donor - acceptor - alternating strategy , this paper described the synthesis of novel -conjugated co - oligomers based on benzothiadiazole structure through well - known palladium catalyzed suzuki polycondensation .
the ability of connection of electron - donating and electron - withdrawing groups in benzothiadiazole co - oligomers , has been applied to developing methods for controlling the polymer egap and producing technologically useful low - band - gap conjugated materials .
the use of electron - withdrawing groups , however , provides reduced homo - lumo energies and lower egap values .
the application of such alternating co - oligomers employing electron - withdrawing groups appears to be a promising new approach for the production of low egap materials .
the obtained semiconducting oligomers - bifunctional materials as viable luminance and high hole - transporting one , exhibit excellent solubility in common organic solvents , thermal stability and luminescence in blue region and can be cast into uniform films .
the band gap values of synthesized compounds are in the adequate range ( 2.542.75 ev ) for testing as oleds .
thin , ordered film fabricated of these compounds were found as semiconducting material for diode application .
based copolymers have not only good processability due to the backbone , in which alkyl groups are incorporated , but also bright and uniform luminescence in the visible region .
since stable , bright blue emission is still a limiting factor in high performance led devices
. polymers with these unique backbones will be able to be introduced in many applications for low cost electronics , which needs good reproducibility of device performance and moderate field - effect mobility . | the benzothiadiazole arylene alternating conjugated oligomers have been designed and synthesized via suzuki coupling reaction . the structures and properties of the conjugated oligomers were characterized by 1hnmr , 13cnmr , uv vis absorption spectroscopy , photoluminescence ( pl ) spectroscopy .
the luminescent measurements demonstrate that polybenzothiadiazoles are good chromophores able to form thin films by langmuir - blodgett ( lb ) technique , making them suitable for further applications .
also the electrical properties of obtained films confirm the good potential of these novel aryl - based -conjugated polymers for the development of various electrical and electrochemical solid - state devices . |
ami and scd are among the most serious and catastrophic of acute cardiac disorders , accounting for hundreds of thousands of deaths each year worldwide .
although the incidence of ami has been decreasing in the us according to the american heart association , heart disease is still the leading cause of mortality in adults . in most cases of ami and in a majority of cases of scd ,
the underlying pathology is acute intraluminal coronary thrombus formation within an epicardial coronary artery leading to total or near total acute coronary occlusion .
this article summarizes our current understanding of the pathophysiology of these acute coronary syndromes and briefly discusses new approaches currently being researched in an attempt to define and ultimately reduce their incidence .
ami is usually defined as myocardial necrosis in the setting of clinical evidence consistent with its diagnosis .
this clinical evidence includes the patient 's symptoms , acute electrocardiograph ( ecg ) findings , or other evidence indicating a new wall motion abnormality in a segment of the myocardium .
myocardial infarction is caused by an acute imbalance in the ratio of myocardial blood supply to myocardial oxygen demand in the heart . in the case of an acute coronary thrombosis
, there is an acute drop in blood flow , leading to myocardial necrosis in the myocardial segment supplied by the coronary artery in question .
sudden cardiac death describes the unexpected natural death from a cardiac cause within a short time period , generally not more than 1 hour from the onset of symptoms , in a person without any prior condition that would appear fatal .
scd is usually secondary to a fatal arrhythmia such as ventricular fibrillation , which is a direct result of the coronary thrombosis lowering the threshold for the onset of this arrhythmia .
the underlying pathophysiologic mechanisms for these syndromes begin with the process of atherosclerosis , which develops and progresses for decades prior to the acute event .
atherosclerosis can be described as a low - grade inflammatory state of the intima ( inner lining ) of medium - sized arteries that is accelerated by the well - known risk factors such as high blood pressure , high cholesterol , smoking , diabetes , and genetics . in the case of coronary atherosclerosis
, this slow progression leads to the gradual thickening of the inner layer of the coronary arteries , which may over time narrow the lumen of the artery to various degrees .
atherosclerosis leading to the acute syndromes of ami and scd has a predilection for the proximal segments of the major coronary arteries often at arterial bifurcation points that alter flow in the artery .
this slow atherosclerotic progression may be interrupted by one or more cycles of rapid progression related to one of two processes : either asymptomatic plaque disruption with formation of a non - occlusive intraluminal thrombus or plaque hemorrhage ( figure 1 ) .
an atherosclerotic plaque is composed of inflammatory cells , cellular debris , smooth muscle cells ( smcs ) , and varied amounts of cholesterol and cholesterol ester , some of which is in the form of cholesterol crystals .
this lipid core forms in some plaques under a fibrous cap composed of collagen , smcs , and elastin . the cap is covered on its luminal side by a single layer of endothelial cells as is the inner layer of all arteries within the body .
an abundance of inflammatory cells derived foam cells originating from circulating monocytes migrate into the arterial wall and may weaken and thin out the fibrous cap .
these plaques are termed thin - capped fibroatheromas ( or tcfas for short ) ( figure 2 ) .
these processes can ultimately result in a tear in the cap , exposing the thrombogenic lipid core under the cap to the flowing blood , leading to the formation of an intraluminal coronary thrombus .
depending on several factors , including the plaque composition , plaque volume and the degree of luminal narrowing , size of the cap tear , and the thrombotic milieu ( a complicated interaction of different forces that ultimately determine how prothrombotic the blood is ) , the thrombus that forms may either lyse spontaneously , remain , and subsequently be incorporated into the wall of the artery ( further narrowing the lumen ) or grow and progress to total or near coronary occlusion and a symptomatic acute coronary event .
based on pathologic evaluation of patients with scd , there are often several bouts of asymptomatic coronary thrombus that have been incorporated into the arterial wall prior to the final fatal event .
plaque erosion is another cause for intraluminal coronary thrombus formation . in this condition , the thrombus forms on a defect in the endothelial layer covering a plaque .
these plaques may or may not be inflamed , and the cap is not usually thin . here , the prothrombotic milieu is felt to be very important in the process , and plaque erosions are common in smokers and in women less than 50 years of age . at present , it is not known how frequently this process occurs asymptomatically .
most of the data on plaque erosion are derived from patients with a symptomatic acute coronary event .
as the intima of an artery thickens , it must be nourished by an adequate blood supply . this blood supply or vasa vasorum usually grows from the adventitia or outer layer of the artery into the media and intima supplying needed nutrients .
these blood vessels are thin - walled , and their endothelial integrity is not always completely intact .
rupture of these vessels into the intima may acutely enlarge the size of the plaque by the deposition of blood .
furthermore , the red cell membrane is lipid - rich , and hemorrhage can also increase the lipid and inflammatory cell content within the plaque .
however , as the plaque grows , the lumen of the artery does not necessarily narrow .
the arterial wall remodels , and the lumen does not begin to narrow until the plaque volume approaches 40% .
this phenomenon , called glagovian or positive remodeling , is in part responsible for the angiographic finding that plaques ultimately responsible for acute coronary events are often non - obstructive ( < 50% diameter stenosis ) in the weeks to month prior to the event .
the angiogram visualizes the inside of the artery but can not evaluate the arterial wall .
whereas the plaque ultimately responsible for the acute event ( usually termed vulnerable or high - risk plaques ) may be large and bulky , the lumen may look normal or only mildly narrowed on the angiogram because of this remodeling process .
based on post - mortem pathologic evaluation of patients with a fatal coronary thrombosis , plaque rupture is present in about 66% to 75% of cases .
angiograms performed within 4 hours of the onset of ami symptoms in patients presenting with st segment elevation on their ecg have shown total occlusion of the coronary artery in 84% of cases , and the remaining cases demonstrated a near total occlusion but with some flow to the distal vessel .
the thrombus that forms in many cases is older than the clinical presentation of the ami .
when thrombus was extracted from the responsible coronary at the acute presentation during percutaneous coronary intervention ( < 12 hours after the onset of symptoms ) , in about 50% of cases the thrombus that was removed was partially organized , indicating that it formed prior to the onset of symptoms .
the underlying pathophysiology of ami appears to be slightly different when analyzed in the living patient and also depends on the type of ami . from a clinical standpoint ,
one divides ami based on the ecg into st elevation myocardial infarction ( stemi ) and non - st elevation myocardial infarction ( nstemi ) .
stemi in general nearly always presents with total coronary occlusion ( see above ) , and the degree of myocardial necrosis ( or the myocardium at risk of necrosis ) is larger than in nstemi .
nstemi has a lower incidence of total coronary occlusion , but if the artery lumen is not totally obstructed , it usually demonstrates a severe blockage ( > 70% diameter stenosis ) in one or more arteries most often with an intraluminal coronary thrombus . during evaluations by intracoronary devices at the time of acute presentation to the catheterization laboratory for coronary intervention , differences have been reported between stemi and nstemi in pathophysiology . when ocular coherence tomography was used to evaluate intraluminal pathology , plaque rupture
was seen in 72% of stemi and 32% of nstemi and plaque erosion in 28% of stemi and 48% of nstemi . in the remaining nstemi cases ,
calcified nodules are found infrequently ( < 10% ) as the underlying mechanism in fatal coronary thrombus at post - mortem examination . another intraluminal catheter device that can be used during the acute presentation of ami that detects
the presence of lipid - rich plaque has also been studied in patients presenting with stemi . in 20 patients with an acute coronary occlusion ,
this catheter found intense yellow plaques ( a signal for plaque lipid ) in 19 of 20 cases .
if the thrombosed plaque is the immediate cause of most acute coronary events , can one find that plaque that is likely to progress in the future and cause the event ?
as most are non - obstructive and asymptomatic in the weeks prior to the event , relying on symptomatology alone is not useful .
however , there is a difference of opinion in the literature as to the best and most cost - effective methods to prevent future acute coronary events .
should one attempt to locate the responsible plaque or concentrate on the high - risk or vulnerable patient likely to develop the acute clinical event ?
for vulnerable plaque detection , one would need to find a device , either invasive or ( preferably ) non - invasive , that could detect the vulnerable plaque .
this would necessitate knowing the natural history of such plaques and being able to show that intervening on a presumed vulnerable plaque by some approach such as a stent was safer and more cost - effective than the best medical therapy alone . at present , this is unknown .
we still do not know the natural history of a presumed vulnerable plaque , and a plaque that looks vulnerable on an initial evaluation may change at some later point and appear stabilized .
one concern with an invasive methodology is the fact that all of the detectors are concentrating on finding the responsible tcfa but do not consider the plaque erosion which is not an infrequent cause of ami or scd .
furthermore , if the detector is invasive , what about individuals in whom the first presentation of symptomatic coronary disease is ami or scd ? in the framingham study , 53% of men and 36% of women presented with either ami or scd .
non - invasive detectors of a vulnerable plaque are , in our estimation , inadequate and will require additional prospective evaluation .
if vulnerable plaque detection and therapy are challenging , should we concentrate our efforts on the high - risk or vulnerable patient ?
the answer is probably yes , but similar to the above , this can be difficult , particularly in primary prevention .
while patients with known coronary artery disease ( cad ) or a cad equivalent such as diabetes or peripheral artery disease should be treated with guideline - directed medical therapies , how do we identify most of the patients who will ultimately develop ami or scd .
traditional risk factor scores are inadequate because , while those at highest risk are more likely to develop adverse events , most adverse events on follow - up do not occur in these patients but in the middle- or lower - risk populations , which are much larger in numbers than the high - risk . various studies assessing different methods of risk evaluation in primary prevention , such as the presence and extent of coronary calcium by computed tomography scanning
, intimal - medial thickness of the carotid artery , and iliofemoral atherosclerosis by ultrasound , are attempting to identify that subgroup who might require intensive medical management to reduce subsequent risk .
these are ongoing , and results are not yet available . in conclusion , our understanding of these acute coronary syndromes has matured over the last 30 years as outlined above .
nevertheless , our methods for detecting and identifying most patients or plaques likely to progress to these syndromes in the future are still inadequate and require further study .
it is exciting to speculate , given our past successes , what the next 30 years will reveal .
| acute myocardial infarction ( ami ) and sudden cardiac death ( scd ) are among the most serious and catastrophic of acute cardiac disorders , accounting for hundreds of thousands of deaths each year worldwide .
although the incidence of ami has been decreasing in the us according to the american heart association , heart disease is still the leading cause of mortality in adults . in most cases of ami and in a majority of cases of scd ,
the underlying pathology is acute intraluminal coronary thrombus formation within an epicardial coronary artery leading to total or near - total acute coronary occlusion .
this article summarizes our current understanding of the pathophysiology of these acute coronary syndromes and briefly discusses new approaches currently being researched in an attempt to define and ultimately reduce their incidence . |
although considerable evidences have demonstrated that percutaneous coronary intervention ( pci ) is the most advantageous and rewarding reperfusion strategy available in patients with acute st - segment - elevation myocardial infarction ( stemi ) , but they fail to restore optimal myocardial reperfusion in a sizeable portion of patients , mostly because of no - reflow phenomenon .
no - reflow is defined as reduced coronary reperfusion ( failure to restore thrombolysis in myocardial infarction [ timi ] flow grade 3 ) without any arterial obstruction , dissection , or spasm in angiography .
the incidence of this phenomenon is present in 10 - 54% of the procedures depending on the characteristics of the studied population and the method used for its diagnosis , and it is associated with an adverse clinical outcome with greater short and long - term progression to heart failure and increased mortality .
several authors have established a significant association between this phenomenon , and plasma levels of different markers or with certain features of atherosclerotic plaque visualized by intra vascular ultra sonography .
other studies have shown that the combination of clinical variables and procedural findings is useful to identify this group of patients .
tobacco smoking is a well - established preventable risk factor for the development and progression of coronary artery disease ( cad ) and is strongly related to cardiovascular causes morbidity and mortality ; but several recent studies showed that smokers have less no - reflow phenomenon after pci in the setting of acute stemi .
this phenomenon , often termed the smoker 's paradox , presumably exists because smokers are usually younger and they have fewer cardiovascular risk factors and their infarct lesions have a greater thrombotic component with relatively less atherosclerotic plaque .
due to increasing number of smokers , especially among women and young adults in iran and lack of sufficient studies about the effect of smoking on no - reflow phenomenon , we undertook this study to assess the relationship between cigarette smoking and postprocedural timi flow grade , no - reflow phenomenon and short - term outcomes .
a total of 141 patients who were admitted to chamran hospital ( isfahan , iran ) between march and september of 2012 with a diagnosis of stemi , enrolled into our cohort study .
the diagnosis of stemi was established in the presence of chest pain lasting for 20 min associated with electrocardiographic changes ( new st elevation at the j point in at least 2 contiguous leads of 2 mm [ 0.2 mv ] in men or 1.5 mm [ 0.15 mv ] in women in leads v2-v3 and/or of 1 mm [ 0.1 mv ] in other contiguous chest leads or the limb leads or new or presumably new left bundle branch block ) .
we excluded patients with restenosis after stenting , coronary artery bypass graft failure , severe heart failure or cardiogenic shock , important systemic disease , or serum creatinine > 2.5 mg / dl .
the ethical committee of the isfahan university of medical sciences reviewed and approved this study ( ethical code : 392317 ) . according to self - reported smoking status
any patient who had smoked 10 cigarettes / day within the year preceding the index procedure was considered a smoker .
thus , nonsmokers included those patients who had never smoked and those who had quit smoking 1-year before the index procedure ( former smokers ) . in the beginning
then , a questionnaire containing demographic data ( age and gender ) , history of diseases ( diabetes mellitus , stroke , hypertension , cad , and hyperlipidemia ) , and sk prescription was completed .
blood samples were obtained before angiography . within the first 12-h of chest pain , the subjects underwent angiography and then primary pci or rescue pci if they had chest pain and/or persistent st - segment elevation despite receiving sk . in order to perform angiography , seldinger method
the results of angiography including the involved artery , position and extent of stenosis were recorded for each patient . after receiving a 70 iu / kg dose of stat heparin
pci was done in an infarct - related artery ( ira ) and the interventionist selected the stent number and size based on the involved artery and plaque length and diameter .
the size of catheter balloon was determined by a skilled operator who simultaneously viewed a cine angiogram .
angiography was performed after pci to assess timi flow , and no - reflow phenomenon , and the results were recorded for all patients .
no - reflow was defined as post - pci timi grade 0 , 1 , or 2 flow in the absence of mechanical obstruction .
all patients received 600 mg of clopidogrel and 325 mg of aspirin stat prior to pci .
they were also prescribed a daily 325 mg of aspirin and 75 - 150 mg of clopidogrel following pci .
the complications , including death , reinfarction , bleeding , arrhythmia and cerebrovascular accident , during the first 24-h after pci were recorded . in order to analyze the collected data , descriptive statistics ( percentages and mean standard deviation ) was applied .
differences between groups stratified by smoking status were tested by the chi - square tests ( fisher 's exact test if needed ) for dichotomous variables and the independent t - test for continuous variables .
we also used this test to compare platelet count between groups because of abnormal distribution .
then , differences in no - reflow phenomenon and 24-h outcomes between smokers and nonsmokers were assessed .
results were adjusted for age , gender , diabetes , hypertension , history of cad and the extent of stenosis by logistic regression analysis .
all analyses were performed using spss for windows 16.0 ( ibm corporation , chicago ) .
a total of 141 patients who were admitted to chamran hospital ( isfahan , iran ) between march and september of 2012 with a diagnosis of stemi , enrolled into our cohort study .
the diagnosis of stemi was established in the presence of chest pain lasting for 20 min associated with electrocardiographic changes ( new st elevation at the j point in at least 2 contiguous leads of 2 mm [ 0.2 mv ] in men or 1.5 mm [ 0.15 mv ] in women in leads v2-v3 and/or of 1 mm [ 0.1 mv ] in other contiguous chest leads or the limb leads or new or presumably new left bundle branch block ) .
we excluded patients with restenosis after stenting , coronary artery bypass graft failure , severe heart failure or cardiogenic shock , important systemic disease , or serum creatinine > 2.5 mg / dl .
the ethical committee of the isfahan university of medical sciences reviewed and approved this study ( ethical code : 392317 ) .
according to self - reported smoking status , participants were categorized as smokers and nonsmokers .
any patient who had smoked 10 cigarettes / day within the year preceding the index procedure was considered a smoker .
thus , nonsmokers included those patients who had never smoked and those who had quit smoking 1-year before the index procedure ( former smokers ) .
in the beginning , study procedure was explained to subjects and informed consents were obtained from them .
then , a questionnaire containing demographic data ( age and gender ) , history of diseases ( diabetes mellitus , stroke , hypertension , cad , and hyperlipidemia ) , and sk prescription was completed .
blood samples were obtained before angiography . within the first 12-h of chest pain , the subjects underwent angiography and then primary pci or rescue pci if they had chest pain and/or persistent st - segment elevation despite receiving sk . in order to perform angiography , seldinger method
the results of angiography including the involved artery , position and extent of stenosis were recorded for each patient . after receiving a 70 iu / kg dose of stat heparin
pci was done in an infarct - related artery ( ira ) and the interventionist selected the stent number and size based on the involved artery and plaque length and diameter .
the size of catheter balloon was determined by a skilled operator who simultaneously viewed a cine angiogram .
angiography was performed after pci to assess timi flow , and no - reflow phenomenon , and the results were recorded for all patients .
no - reflow was defined as post - pci timi grade 0 , 1 , or 2 flow in the absence of mechanical obstruction .
all patients received 600 mg of clopidogrel and 325 mg of aspirin stat prior to pci .
they were also prescribed a daily 325 mg of aspirin and 75 - 150 mg of clopidogrel following pci .
the complications , including death , reinfarction , bleeding , arrhythmia and cerebrovascular accident , during the first 24-h after pci were recorded .
in order to analyze the collected data , descriptive statistics ( percentages and mean standard deviation ) was applied .
differences between groups stratified by smoking status were tested by the chi - square tests ( fisher 's exact test if needed ) for dichotomous variables and the independent t - test for continuous variables .
we also used this test to compare platelet count between groups because of abnormal distribution .
then , differences in no - reflow phenomenon and 24-h outcomes between smokers and nonsmokers were assessed .
results were adjusted for age , gender , diabetes , hypertension , history of cad and the extent of stenosis by logistic regression analysis .
all analyses were performed using spss for windows 16.0 ( ibm corporation , chicago ) .
the baseline characteristics of both groups are summarized in table 1 . among the total number of 141 patients , 47 patients ( 32% ) were smokers , and 94 ( 65% ) were nonsmokers .
all patients in the smoker group were a heavy smoker ( 10 pack / year ) .
nonsmokers were 8 years older ( p < 0.001 ) and a larger proportion of nonsmokers were women ( p < 0.01 ) .
in addition , major co - existing conditions such as hypertension ( p < 0.001 ) , and diabetes mellitus ( p < 0.05 ) were more prevalent among nonsmokers .
baseline demographics and clinical status onadmission there were no significant differences in hematocrit ( hct ) , platelet ( plt ) count and left ventricle ejection fraction in both groups .
there were modest differences in angiographic features between smokers and nonsmokers [ table 2 ] .
primary pci was performed nearly at the same rate between smokers and nonsmokers ( p = 0.34 ) .
one stent was implanted in most of the patients ( 80.85% of smokers and 81.91% of nonsmokers , p = 0.59 ) but in a small subset of patients , according to the interventionist opinion two or three stent were implanted in ira .
the angiographic efficacy of the intervention measured as a proportion of patients with final timi 3 flow in the culprit vessel was high in both groups , without any significant differences ( p = 0.32 ) .
angiographic and procedural characteristics of the analyzed groups table 3 shows major adverse cardiac events ( mace ) during the first 24-h after pci .
mace such as death , arrhythmia and reinfarction during the first 24-h after pci were slightly lower in the smoker group , but did not show any statistical significance .
mace during the first 24-h after pci multivariable analysis was performed to identify independent relationship between smoking status and no - reflow phenomenon .
age , gender , diabetes , hypertension , history of cad and extent of stenosis were tested for multivariable analysis .
smoking status , when adjusted for these variables by logistic regression analysis , did not possess any predictive value in terms of postpci timi flow and no - reflow phenomenon ( odds ratio [ or ] = 1.68 ; 95% confidence interval [ ci ] : 0.68 - 4.10 , p = 0.25 ) .
despite widespread awareness of smoking deleterious effect , it remains the single largest preventable cause of cardiovascular morbidity and premature death in developed countries . in the interheart trial investigating 27089 participants from 52 countries ,
current smoking was associated with an almost 3-fold greater risk of nonfatal acute myocardial infarction ( mi ) compared with never smoking . in the present study , we assessed the impact of smoking on no - reflow phenomenon after pci in stemi patients .
smokers , despite having lower incidence of diabetes mellitus and hypertension ( p = 0.019 and 0.01 respectively ) , require pci 8 years earlier than nonsmokers ( p = 0.001 ) .
however , once smokers underwent successful pci , the first 24-h in - hospital outcomes were similar to nonsmokers ( p = 0.6 ) .
sherif et al . studied 4660 patients for 1-year after pci , and showed that short - term complications were similar in both groups of patients .
in contrast to our results , sukiennik et al . found higher unadjusted mortality rates after pci among nonsmokers during hospitalization ( 2.4% for smokers compared with 4.6% for nonsmokers , p = 0.0532 ) .
weisz et al . analyzing the data obtained in the randomized cadillac trial found the lowest mortality in current smokers , intermediate in former smokers , and highest in nonsmokers after 30 days and 1-year .
however , after a multivariate correction for differences in baseline variables , current smoking status was no longer protective against mortality .
furthermore , part of these apparently contradicting results might be explained by differences in the definition of smoking status and the concomitant therapies .
probably because our patient had lesser risk factors for bleeding such as female sex , chronic renal failure , systolic blood pressure < 100 and old age . also , because heparin was not prescribed after pci .
no - reflow is one of the major problems in patients with stemi who undergo primary pci and may limit the benefits of recanalization of the ira .
it is known that angiographic no - reflow is strongly correlated with morbidity and mortality in acute mi .
the rate of no - reflow phenomenon in our study was 30.6% , which was consistent with previously published no - reflow rates .
we found no relationship between smoking and angiographic no - reflow ( p = 0.77 ) .
study on plaque component on 190 acute coronary syndromes ( acs ) patients demonstrated no difference in no - reflow between smokers and nonsmokers ( p = 0.7 ) .
data from albertal et al . showed that postprocedural timi flow grade and timi frame were better in smokers , whereas myocardial blush grade was similar between both groups .
percentage of complete ( > 70% ) st - segment resolution ( str ) at 60 min was higher in active smokers than nonsmokers ( 76.4% vs. 50% , p = 0.002 ) .
multivariate logistic regression analysis identified active smoking as an independent predictor of complete str at 60 min ( or = 3.47 ; 95% ci : 1.48 - 8.14 ; p = 0.004 ) .
showed that among 1140 patients with stemi undergoing primary pci , smokers had less no - reflow phenomenon ( p = 0.04 ) .
univariable and multivariable logistic regression models were used to identify the correlates of the no - reflow after primary pci . this could be explained by healthier coronary artery risk factor profile , less extensive atherosclerotic disease and hence , smaller plaque burden in smokers rather than nonsmokers . elevated plt , fibrinogen , and hct levels are also encountered in active smokers , indicating hypercoagulable state promoting vascular thrombosis . therefore , coronary obstructions in patients who smoke may be more of a thrombogenic origin than atherosclerotic , so they are more susceptible to thrombolysis , either pharmacological or mechanical , compared to nonsmokers . on the other hand ,
when there is a significantly greater thrombus burden , thrombi tend to fragment with balloon dilatation , which can lead to distal embolization and no - reflow phenomenon .
so maybe better result in previous studies is because of widespread use of anti - plt agents .
desai et al . found that clopidogrel reduced the rate of closed ira or death / mi before angiography in the clarity - timi 28 trial , which was especially marked among those who smoked 10 cigarettes / day .
similarly , clopidogrel was significantly more effective in reducing the rate of cardiovascular death , mi , or urgent revascularization through 30 days among those who smoked 10 cigarettes / day compared to those who did not .
probably , enhanced plt activation induced by smoking makes the effect of clopidogrel more pronounced in smokers , in addition to improved pharmacokinetics of clopidogrel in smokers .
first , the potential bias from misclassification of smoking status may exist because of the possible inaccurateness of a patient 's reporting of smoking status .
second , all participants were from a single high - volume coronary treatment center , which would restrict the generalization of our findings .
third , the diagnosis of no - reflow was made considering only the epicardial flow .
it is better to consider complementary diagnostic methods such as str 3-h after reperfusion and the evaluation of myocardial blush grade to increase sensitivity .
but , the quality of our coronary angiogram did not allow adequate assessment of myocardial blush score in some patients , so we used conventional timi flow grading system .
finally , we followed patients for a short period , so the impact of smoking on mace is not estimated precisely
. however , within these limitations , we believe that our work will stimulate more research to verify our results and explore the pathophysiology of adverse outcomes in different smoking status of patients undergoing pci .
first , the potential bias from misclassification of smoking status may exist because of the possible inaccurateness of a patient 's reporting of smoking status .
second , all participants were from a single high - volume coronary treatment center , which would restrict the generalization of our findings .
third , the diagnosis of no - reflow was made considering only the epicardial flow .
it is better to consider complementary diagnostic methods such as str 3-h after reperfusion and the evaluation of myocardial blush grade to increase sensitivity .
but , the quality of our coronary angiogram did not allow adequate assessment of myocardial blush score in some patients , so we used conventional timi flow grading system .
finally , we followed patients for a short period , so the impact of smoking on mace is not estimated precisely .
however , within these limitations , we believe that our work will stimulate more research to verify our results and explore the pathophysiology of adverse outcomes in different smoking status of patients undergoing pci .
there was no significant difference in no - reflow phenomenon and mace during the first 24-h after pci among smokers and nonsmokers .
the survival advantage in some other studies is not a consequence of smoking status per se making the commonly used term
this may be fully explained by the younger age of smokers , their more favorable clinical characteristics and the less extensive coronary atherosclerosis .
in fact , smoking contributes to the occurrence of acs at a younger age and no convincing evidence concerning a protective role of smoking in the interventional setting exists . finally , bearing in mind the results of primary and secondary prevention trials clearly indicating beneficial effects of a nonsmoking lifestyle and smoking cessation , any form of smoking
hs contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
fdt contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
hz contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
aa contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . | background : no - reflow phenomenon after percutaneous coronary intervention ( pci ) in patients with acute st - segment - elevation myocardial infarction ( stemi ) is relatively common and has therapeutic and prognostic implications .
cigarette smoking is known as deleterious in patients with coronary artery disease ( cad ) , but the effect of smoking on no - reflow phenomenon is less investigated .
the aim of this study was to compare no - reflow phenomenon after percutneous coronary intervention for acute myocardial infarction , between smokers and non smokers.materials and methods : a total of 141 patients who were admitted to chamran hospital ( isfahan , iran ) between march and september , 2012 with a diagnosis of stemi , enrolled into our cohort study .
patients were divided into current smoker and nonsmoker groups ( based on patient 's information ) .
all patients underwent primary pci or rescue pci within the first 12-h of chest pain .
no - reflow phenomenon , thrombolysis in myocardial infarction ( mi ) flow , and 24-h complications were assessed in both groups.results:a total of 47 current smoker cases ( 32.9% ) and 94 ( 65.7% ) nonsmoker cases were evaluated .
smokers in comparison to nonsmokers were younger ( 53.47 10.59 vs. 61.46 10.55 , p < 0.001 ) and they were less likely to be hypertensive ( 15.2% vs. 44.7% , p < 0.001 ) , diabetic ( 17% vs. 36.2% , p < 0.05 ) , and female gender ( 4.3% vs. 25.5% , p < 0.01 ) . angiographic and procedural characteristics of both groups were similar .
9 patients died during the first 24-h after pci ( 4.3% of smokers and 6.4% of nonsmokers , p : 0.72 ) .
no - reflow phenomenon was observed in 29.8% of current smokers and 31.5% of nonsmokers (
p = 0.77).conclusion : no - reflow phenomenon or short - term complications were not significantly different between current smokers and non smokers . |
fibromatoses / desmoid tumors are fibroblastic proliferations with an intermediate dignity between benign fibroblastic tumors and fibrosarcoma . they are characterized by an infiltrative growth pattern , high rate of local recurrence , and the inability to metastasize [ 13 ] . referring to weiss and goldblum
there are two main groups of fibromatoses- superficial ( fascial ) and deep ( musculoaponeurotic ) fibromatosis .
furthermore , these tumors are divided into several subcategories according to their anatomic location ( abdominal , extra - abdominal , intra - abdominal ) . wide or radical resection with negative microscopic surgical margins is treatment of choice and a significant prognostic factor in primary case as well as in recurrence cases ( p < 0.001 ) .
mri represents the gold - standard imaging modality for the purpose of planning management , especially surgery , and performing surveillance of desmoid patients in order to detect disease progression and postoperative recurrence [ 511 ] .
peptide receptor imaging ( pri ) enables visualization of receptor expressing tissues noninvasively [ 12 , 13 ] .
furthermore , peptides labeled with -radiation emitters can be used to eradicate receptor expressing tissues ( peptide receptor radionuclide therapy prrt )
. however , the best examples of targets for radiopeptide - based imaging are somatostatin receptors ( sstrs ) , since they are frequently expressed in neuroendocrine tumors as well as in a wide variety of other malignancies , even soft tissue tumors [ 13 , 14 ] .
studies on somatostatin receptor scintigraphy ( srs ) have started in 1988 and have initially concerned with the use of 123i - tyr3-octreotide , which has already been abandoned for practical reasons and cost concerns . in 1994 , imaging with the radiopharmaceutical 111in - pentetreotide ( commercially available as octreoscan ) was approved for patients with neuroendocrine tumors ( nets ) .
positive scans reflect the presence of an increased density of sstrs , in particular sstr 2 and sstr 5 .
nevertheless , the density of sstrs varies among different tumors and therefore , the sensitivity of 111in - pentetreotide also varies .
therefore , new somatostatin analogues with similar binding profile and imaging quality but labeled with 99 mtc have frequently been studied and are growing in importance [ 12 , 13 , 17 ] , due to the cost effectiveness and wide availability of this radioisotope [ 13 , 18 ] .
studies of various authors revealed several advantages of 99 mtc - toc compared to 111in - oct , hence 99mtc - toc is considered as a promising radiopharmaceutical to replace 111in - oct in diagnostic nuclear medicine [ 18 , 20 ] .
although advanced imaging modalities can provide clues to the correct diagnosis of aggressive fibromatosis , histological examination of tumour tissue remains the golden diagnostic standard [ 5 , 7 , 9 , 21 ] . based on the report of de pas et al .
, the aim of this retrospective study was to investigate the percentage of desmoid tumors that express sstr 2 and show adequate tracer uptake in srs .
the hypotheses were that there is a low percentage of sstr 2 positive desmoid tumors and that only few cases show adequate uptake in srs .
thirteen patients who have been treated for desmoid tumors which have been histologically verified and who have been examined with srs since 1999 were included in the current ( table 1 ) .
mean age at diagnosis was 38 years ( range , 18 to 52 ) and the average followup was 54 months ( range , 6 to 153 ) .
although deep fibromatoses are rare on hands and feet , one patient developed a metacarpal tumour .
another patient , who initially presented with a proliferation of the lower leg , developed a plantar recurrence .
the extra - abdominal tumors were located at the following anatomical sites trunk : n = 1 ; upper and lower extremities : n = 10 , whereas six occurred in the upper extremity and four in the lower one .
all patients were analyzed either for uptake of 111in - dtpa - octreotide or for uptake of 99mtc - edda / hynic - toc .
furthermore , scintigraphic findings were correlated with corresponding mr images as well as with immunohistochemical expression of sstr 2a .
somatostatin receptor scintigraphy was performed either preoperatively or at suspicion of disease relapse , by using established radiopharmaceuticals , namely , either 111in - dtpa - octreotide or 99mtc - edda / hynic - toc ( figure 1(a ) ) .
the tracers were prepared with commercially available kits using standard techniques as described by the producers .
planar and spect investigations of the tumour regions were performed 4 or 5 and 24 hours after application of the radioactive tracer . since
2009 srs has been performed with 99mtc - edda / hynic - toc , due to cost effectiveness and a simplifying one - day protocol .
the scintigraphic procedure includes 1 and 4 hours postinjection planar and spect images of the tumour regions .
after methodological setting by testing the sstr 2a antibody ( code ss-800 , biotrend chemikalien , cologne , germany ) , all cases of desmoid tumors with available pathological material were retrospectively analyzed for immunohistochemical expression of sstr 2a ( figure 1(b ) ) .
immunohistochemical studies were performed using an established specific rabbit polyclonal antibody against sstr 2a receptors ( code ss-800 , biotrend chemikalien ) , more precisely against its carboxyl - terminus ( amino acid sequence 355369 ( etqrtllngdlqtsi ) ) .
appropriate positive and negative controls were included : specimens of neuroendocrine tumours ( nets ) were stained as positive control , while normal human soft tissue was used as negative control .
the staining pattern was described as absent ( score 0 : no immunoreactivity ) , moderate ( score 1 : focal to multifocal immunoreactivity in < 50% of cells ) , and distinct ( score 2 : immunoreactivity in > 50% of cells ) .
all slides were evaluated independently by light microscopy by two of the authors , including a senior pathologist ( b. atzwanger ) .
ten patients were examined with srs at least once and three were investigated twice . in all three cases , the findings of the first scintigraphy correlated with the findings of the second investigation ( table 1 ) .
altogether , five cases showed nonpathological tracer uptake ( 38% ) , while eight cases ( 62% ) revealed increased tracer uptake at tumour site and were therefore considered as sstr expressing tumors ( figure 1(a ) ) .
tissue samples from seven patients were available and were exclusively specimens from extra - abdominal locations . in total ,
nine specimens were tested for the expression of sstr 2a , seven tissue samples were from one location of the tumour and in two cases , specimens from two various locations of the tumour were investigated .
staining was moderate in four cases and absent in five cases ( figure 1(b ) ) .
the other sample did not stain in the first location , but revealed moderate staining in the second location .
the desmoids showed an adequate tracer uptake in srs and their tumour samples revealed positive staining by immunohistochemistry ( figures 1(a ) and 1(b ) ) .
three of these had adequate tracer uptake in srs , while the tissue samples revealed absent staining .
on the other hand , in one patient the tumour showed non - pathological tracer uptake in srs , while the tissue specimen stained moderately .
another patient showed non - pathological tracer uptake in srs and the tissue sample revealed absent staining of one location and moderate staining of the other location . in conclusion ,
a positive correlation between srs and immunohistochemistry could be found in two patients ( 29% ) .
in the present investigation , 62% of the examined proliferations showed an enhanced tracer uptake at the site of the desmoid lesion in srs , hence these tumors were regarded as sstr expressing proliferations .
desmoids of five cases ( 38% ) showed nonpathological scintigraphic findings and , consequently , were considered as sstr negative lesions .
furthermore , three patients , who have been investigated twice , revealed correlating findings of the first and the second scintigraphy which is in agreement with the generally admitted fact that recurrences usually express the same receptors as the primary tumors and , additionally , show the same tracer uptake pattern .
somatostatin receptors are frequently expressed in a wide variety of neoplastic proliferations with soft tissue tumors and desmoids being no exception .
although the amount of sstrs as well as the expressed subtypes are varying from tumour to tumour and within one tumour , sstr 2 is the most frequently expressed subtype in the vast majority of sstr positive tumors and , moreover , its splice variant sstr 2a has been shown to be the actually expressed isoform [ 24 , 25 ] .
this is of diagnostic importance for srs , since commercially available radiopharmaceuticals contain the somatostatin analogue octreotide or derivates of the same and have distinct affinity to sstr 2 and only moderate affinity to subtypes 3 and 5 . in 2003
data was published leading to the assumption that desmoid tumors may express sstrs as well .
florio et al . reported on four samples of aggressive fibromatosis and analyzed them for the expression of sstrs by using rt - pcr ( rna isolation and reverse transcription - polymerase 44 chain reaction ) .
they found that two samples where negative for all sstr subtypes , while one abdominal fibromatosis expressed sstr 14 and one extra - abdominal fibromatosis located on a limb expressed sstr 1 and sstr 5 . in 2003 , de pas et al .
examined six patients with fast growing relapses of desmoid tumors using srs and found that two of them were sstr positive .
subsequently , they were apparently successfully treated ( partial response or disease stabilization ) with 90y - dotatoc .
immunohistochemical evaluation of sstr 2a in human tumors is well established and has been shown to be a fast and reliable method . de pas et al .
reported on ten patients with aggressive fibromatosis , among whom two showed immunopositivity to a rabbit polyclonal antibody sstr 2a .
krner et al . found that one antibody , namely the commercially available sstr 2a antibody ( code ss-800 , biotrend chemikalien , cologne , germany ) , which has also been used in the current study , is suitable for routine immunohistochemical assessment of the sstr 2a status of neoplastic tissues .
the main disadvantage of immunohistochemical investigations of sstrs is the general lack of standardization concerning interpretation of staining patterns , which results in problematic reproducibility of this method . in this series
, nine specimens have been tested for the expression of sstr 2a . in our study
only four cases revealed a moderate staining by immunohistochemistry whereas five cases were completely negative .
several studies have directly compared the results of srs with corresponding immunohistochemical sstr 2a investigations and none of these concerned desmoid tumors [ 23 , 27 , 28 ] .
nevertheless , reasons for discrepant findings among these two techniques are presumably independent from the examined tumour type . as with this study ,
two various constellations of discrepant srs and immunohistochemistry findings can occur : discrepancies between enhanced tracer uptake in srs and absent immunohistochemical staining for sstr 2a or nonpathological tracer uptake in srs and positive immunohistochemical staining .
first , the somatostatin analogues used for srs binding , apart from sstr 2 , also the subtypes sstr 3 and 5 with moderate affinity .
although sstr 2 is the most frequent expressed receptor subtype in most tumors , it is possible that scintigraphy results are positive because of the other two receptor subtypes predominating within the lesion .
however , since this survey investigated immunohistochemical expression of sstr 2a only , no assertion can be stated , though some authors [ 23 , 27 ] found that the analysis of the other two receptors did not increase the congruency of the two techniques .
second , nonpathological tracer uptake in srs can be based upon a poor sstr density and may be another explanation for inconsistent results , since asnacios et al .
reported that nonpathological scintigraphy combined with positive immunohistochemical staining implicate an overall lower amount of positive tumour cells as compared with correlative positive results of both techniques . in the current study ,
the amount of immunohistochemical stained cells in positive histological sections was lower than 50% in all samples and regardless of enhanced or non - pathological srs .
degradation of sstrs in tissue specimens with time may another reason for the inconsistent results between srs and immunohistochemistry .
another important factor might be the used antibody for immunohistochemical staining as well as the area of its binding . in an earlier series ,
our study group found positive staining for somatostatin in only six of 46 extra - abdominal , two of 21 abdominal , and one of 13 intra - abdominal fibromatoses ( overall 11% ) using a polyclonal , cytoplasmatic somatostatin antibody ( code a0566 , dako , vienna , austria ) . in the current series , a surface antibody for sstr 2a ( code ss-800 , biotrend chemikalien ) was used being positive in four of nine cases ( 44% ) , indicating higher specificity .
several authors [ 23 , 27 ] agree that the frequently found heterogeneous distribution of sstr subtypes in neoplasms may be the most plausible explanation for both , discrepant positive and discrepant negative results in immunohistochemistry as compared with srs . in concordance with this assumption ,
the tissue sample of one patient with discrepant results had been obtained by biopsy , thus it is not representative for the sstr expression pattern of the whole tumour .
moreover , except for two tissue samples , only one histological section per specimen has been examined for the expression of sstr 2a , which again seems to be unrepresentative for the whole tumour .
the tumors ' frequently found heterogeneity is also emphasized by the tissue sample of one case that has been investigated on two various locations and showed opposed results .
the greatest problem seems to be the general lack of standardization of immunohistochemical sstr analysis and , therefore , the development of standardized scoring systems is of prime importance .
furthermore , immunohistochemistry fails to provide information on the whole tumour and one histological section per tissue sample seems diagnostically inconclusive .
consequently , immunohistochemistry seems to provide the more convincing results , the more histological sections of different locations of one tumour have been investigated .
however , the value of immunohistochemical sstr 2a examinations of desmoid tumors remains unclear and further investigations are needed .
overall , eight out of 13 desmoids ( 62% ) showed an enhanced tracer uptake . on the other hand , immunohistochemical investigations for sstr 2a did not correlate with findings of somatostatin receptor scintigraphy .
this may likely be due to the lack of standardization of this technique as well as the heterogeneous receptor distribution within the tumors .
nevertheless , further investigations are needed to determine the value of somatostatin receptor scintigraphy and immunohistochemistry for desmoid tumors . | background .
magnetic resonance imaging is considered as imaging modality of choice in diagnosis of desmoid tumors , though even this technique can lack the ability to distinguish aggressive fibromatosis from other benign or malignant soft tissue tumors .
the aim of this study was to investigate if desmoid tumors would show an adequate tracer uptake in somatostatin receptor scintigraphy and moreover to correlate these results with immunohistochemical staining .
patients and methods .
thirteen patients with desmoid tumors were examined with somatostatin receptor scintigraphy . additionally , seven of these patients have been tested for the immunohistochemical expression of somatostatin receptor subtype 2a .
the results of somatostatin receptor scintigraphy and the results of immunohistochemical staining ( somatostatin receptor subtype 2a ) were evaluated and correlated .
results .
somatostatin receptor scintigraphy revealed that eight of 13 affected patients ( 62% ) showed an enhanced tracer uptake . on the other hand ,
the correlation between the results of somatostatin receptor scintigraphy and immunohistochemical investigations was poor ( two out of seven cases ) . conclusion .
the current study demonstrated that desmoid tumors frequently express somatostatin receptor subtype 2 , while immunohistochemical investigations did not correlate with these findings .
this may likely be due to lack of standardization of this technique and also due to heterogeneous receptor distribution within the tumors . |
osseointegrated implants have provided alternative treatments to conventional prostheses for patients who lost their teeth and achieved predictable long - term results.1,2 an accurate and passively fitting prosthesis as well as successful surgical operation is suggested as one of the critical requirements for long term implant success.3 - 8 since the uneven distribution of occlusal loads and torquing stresses on the various elements due to problems related to poor fit of frameworks connected to implant may lead to marginal bone loss and failure of implants as well as in relation to mechanical problems as loosening of screws and fatigue fractures of implant components.4 - 10 it will probably not be possible to connect a multi - unit implant prostheses with a completely passive fit in clinical situation because there are many potential inaccuracies with current materials and techniques , which include dimensional changes in impression materials , expansion of gypsum die product , dimensional changes in wax and acrylic pattern , dimensional changes in investment materials and volumetric shrinkage of metal casting on solidification.11 among these variables , the precise transfer of the spatial relationships of implants from the mouth to the master cast with an impression is the first and critical step to ensure passive fit of implant framework .
therefore , clinicians should strive for improving the transfer accuracy of the impression copings.12 - 14 various techniques have been suggested to achieve an accurate master cast .
squared impression coping15 - 18 and custommade , open - top impression tray19 - 21 were recommended instead of tapered impression coping and stock impression tray . in regard to splint the impression copings , there are many controversies exist since brnemark et al . emphasized the importance of splinting impression copings together before registration of impression.22 humphries et al.,12 hsu et al.,23 philips et al.,24 and herbst et al.25 found no significant differences between the values obtained with acrylic - splinted versus unsplinted groups in impression techniques .
spector et al.26 also investigated the accuracy of three varied impression procedures using the direct and indirect transfer copings .
though the study involved multiple variables of techniques and materials , the consistent findings was one of distortion resulting from the transfer manipulations .
the common practice of joining the direct transfer copings with acrylic resin is an attempt to stabilize the copings against rotation during fixture or abutment analog fastening , control the relationship between implants in a rigid fashion . in their study ,
the same objective could be partially accomplished with a rigid impression material or an elastic material with a low flexibility , both of which do not introduce the polymerization shrinkage variables inherent in the use of acrylic resin .
interregui et al.27 and burawi et al.28 showed better result with unsplinted group using polyether or additional silicone impression material alone and presumed the main reason of distortion with resin splinted group possibly occurred by the residual polymerization shrinkage .
however , assif et al.29 and naconecy et al.30 showed that splinting technique was significantly more accurate than unsplinted techniques .
vigolo et al.31 suggested that the impression technique involved square impression copings joined together with autopolymerizing acrylic resin or square impression copings , previously airborne particle - abraded and adhesivecoated could improve accuracy of the master cast than non - modified squared transfer coping without splinting .
cabral et al.32 compared 4 impression techniques and direct impression technique with square impression coping with acrylic resin splints sectioned 17 minutes after setting and welded with the same resin before impression making showed better results than other techniques studied .
also rhyu et al.33 suggested vinyl polysiloxane ( vps ) bite registration material as a splinting material and found impressions made with square impression coping splinted with vps bite registration material were better than acrylic resin splinted group and unsplinted group .
the purpose of this in vitro study was to evaluate the effect of dimensional stability of splinting material on the accuracy of master casts .
a stainless steel metal model ( ss 316 , seoul , korea ) with six 3.75 10 mm ad modum brnemark external hex implant ( 29108 : noble biocare , gteborg , sweden ) was fabricated .
the fixtures were widely distributed throughout the stainless steel model to simulate fully edentulous condition .
1 ) . to make accurate impressions for fabrication of sample casts , custom impression tray incorporating 6 squared transfer copings was fabricated using light - polymerizing tray material according to manufacturer 's instruction ( triad tru - tray ; dentsply international inc , york , pa , usa)(fig .
the 6 squared transfer copings were attached to each implant fixtures and were covered by 2 layers of baseplate wax ( kims international , seoul , korea ) .
the impression tray was designed to have superior openings for the access of guide pins .
six squared pick - up type transfer copings ( 29072 ; nobel biocare , gteborg , sweden ) connected on each fixture with guide pins ( fig .
impressions were made after 6 impression copings were splinted with each other using five different methods as follows .
group 1 , squared transfer copings splinted with autopolymerizing acrylic resin ( gc pattern resin ; gc corp , tokyo , japan ) for 24hours and sectioned , reconnected just before impression procedure ( fig .
4 ) . group 2 , squared transfer copings splinted with autopolymerizing acrylic resin ( gc pattern resin ; gc corp , tokyo , japan ) 17 minutes before impression procedure ( fig . 5 ) .
group 3 , primary impression was made around transfer copings with impression plaster ( snow - white plaster no.2 ; kerr , romulus , mi , usa ) following manufacturer 's instruction and then secondary impression was made with polyether impression material ( fig .
group 4 , squared transfer copings splinted with impression plaster ( snow - white plaster no.2 ; kerr , romulus , mi , usa ) over dental floss ( fig .
group 5 , squared transfer copings splinted with vps bite registration material ( blu - mousse ; parkell bio - materials , farmingdale , ny , usa)(fig .
the impression material was machine - mixed ( pentamix;3 m espe , seefeld , germany ) and the mixed material was both syringed around impression coping and loaded in the impression tray .
the impression tray was hand - pushed until its periphery meets the epoxy resin base and maintained in position with hand pressure .
polyether impression materials ( impregum penta , 3 m espe , seefeld , germany ) were used for impression procedure except group 5 impressions ( fig .
they were made using additional type polyvinyl siloxane impression material ( dimension penta h ; 3 m espe , seefeld , germany ) .
after impressions were made , fixture analogs ( 29108 ; nobel biocare , gteborg , sweden ) were screwed into squared transfer copings ( 29072 ; nobel biocare , gteborg , sweden ) in the impressions .
each impression was poured with vacuum - mixed improved dental stone ( resinrock ; whip - mix , louisville , ky , usa ) .
the casts were retrieved from the impressions after 24 hours . from master model , 5 impressions and experimental models
were made for each of 5 splinting methods represented by group 1 to 5 ( fig .
a computerized numerical control ( cnc ) , coordinate measuring machine ( cmm ) ( strato bright 710 ; mitutoyo corporation , tokyo , japan ) was used for all coordinate measurements ( fig .
the accuracy of this cmm was less than 0.0001 mm for the x , y , and z axes .
all measurements were performed by the same operator using probe head ( ph10 m ; mitutoyo corporation , tokyo , japan ) and signal probe ( tp7 m ; mitutoyo corporation , tokyo , japan ) .
geopak - win software ( mitutoyo corporation , tokyo , japan ) was used for geometric transformation and data processing ( fig .
the centroid of fixture or analog 1 which is on the right side of model was designated as the origin of coordinated system .
an imaginary line was laid on the zx plane between centroid of cylinder 1 and the centroid of cylinder 6 .
thus the centroid of fixture or analog 1 lay on the origin ( 0,0,0 ) and the centroid of fixture or analog 6 lay on the zx plane ( x,0,z ) ( fig .
this coordinate system marks several dots on the elliptical plane created by platforms of fixtures or analogs and imaginary z plane .
the software convert ellipse to circle and determine centroids of each platform . to evaluate the accuracy of each impression methods , coordinates of the centroids on the master model
were located in three dimensions and compared with the coordinates of the centroids on the experimental casts obtained from 5 different impression methods .
means and standard deviations were calculated for each item measured ( i.e. , each type of impression methods ) .
a one - way analysis of variance ( anova ) at a confidence level of 95% was used to evaluate the data and tukey 's studentized range test was used to determine significant differences between the groups .
six squared pick - up type transfer copings ( 29072 ; nobel biocare , gteborg , sweden ) connected on each fixture with guide pins ( fig .
impressions were made after 6 impression copings were splinted with each other using five different methods as follows .
group 1 , squared transfer copings splinted with autopolymerizing acrylic resin ( gc pattern resin ; gc corp , tokyo , japan ) for 24hours and sectioned , reconnected just before impression procedure ( fig .
4 ) . group 2 , squared transfer copings splinted with autopolymerizing acrylic resin ( gc pattern resin ; gc corp , tokyo , japan ) 17 minutes before impression procedure ( fig . 5 ) .
group 3 , primary impression was made around transfer copings with impression plaster ( snow - white plaster no.2 ; kerr , romulus , mi , usa ) following manufacturer 's instruction and then secondary impression was made with polyether impression material ( fig .
group 4 , squared transfer copings splinted with impression plaster ( snow - white plaster no.2 ; kerr , romulus , mi , usa ) over dental floss ( fig
group 5 , squared transfer copings splinted with vps bite registration material ( blu - mousse ; parkell bio - materials , farmingdale , ny , usa)(fig .
the impression material was machine - mixed ( pentamix;3 m espe , seefeld , germany ) and the mixed material was both syringed around impression coping and loaded in the impression tray .
the impression tray was hand - pushed until its periphery meets the epoxy resin base and maintained in position with hand pressure .
polyether impression materials ( impregum penta , 3 m espe , seefeld , germany ) were used for impression procedure except group 5 impressions ( fig .
they were made using additional type polyvinyl siloxane impression material ( dimension penta h ; 3 m espe , seefeld , germany ) .
after impressions were made , fixture analogs ( 29108 ; nobel biocare , gteborg , sweden ) were screwed into squared transfer copings ( 29072 ; nobel biocare , gteborg , sweden ) in the impressions .
each impression was poured with vacuum - mixed improved dental stone ( resinrock ; whip - mix , louisville , ky , usa ) .
the casts were retrieved from the impressions after 24 hours . from master model , 5 impressions and experimental models
were made for each of 5 splinting methods represented by group 1 to 5 ( fig .
a computerized numerical control ( cnc ) , coordinate measuring machine ( cmm ) ( strato bright 710 ; mitutoyo corporation , tokyo , japan ) was used for all coordinate measurements ( fig .
the accuracy of this cmm was less than 0.0001 mm for the x , y , and z axes .
all measurements were performed by the same operator using probe head ( ph10 m ; mitutoyo corporation , tokyo , japan ) and signal probe ( tp7 m ; mitutoyo corporation , tokyo , japan ) .
geopak - win software ( mitutoyo corporation , tokyo , japan ) was used for geometric transformation and data processing ( fig .
the centroid of fixture or analog 1 which is on the right side of model was designated as the origin of coordinated system .
an imaginary line was laid on the zx plane between centroid of cylinder 1 and the centroid of cylinder 6 .
thus the centroid of fixture or analog 1 lay on the origin ( 0,0,0 ) and the centroid of fixture or analog 6 lay on the zx plane ( x,0,z ) ( fig .
this coordinate system marks several dots on the elliptical plane created by platforms of fixtures or analogs and imaginary z plane .
the software convert ellipse to circle and determine centroids of each platform . to evaluate the accuracy of each impression methods , coordinates of the centroids on the master model
were located in three dimensions and compared with the coordinates of the centroids on the experimental casts obtained from 5 different impression methods .
means and standard deviations were calculated for each item measured ( i.e. , each type of impression methods ) .
a one - way analysis of variance ( anova ) at a confidence level of 95% was used to evaluate the data and tukey 's studentized range test was used to determine significant differences between the groups .
table 1 shows means of actual distortion amount on each fixture analogs of 5 experimental models resulted from 5 different impression methods .
the x , y , and z values are the amounts of displacement of each fixture analogs in the direction of axis from standard position ( circle 1 ) and they were compared with x , y , z points of master model . the difference of means for each of 3 distortion variables between master model and each splinting method are given as each entry in table 2 .
the result of one sample t - test shows discrepancies in x , y and z axes between master model and each of 5 splinting method ( p<.0001 ) .
not any impression method perfectly duplicated the master model , but some extent of distortion values was clinically acceptable .
table 3 shows comparison between the global means in each splinting methods . in x - axis ,
the mean distortion value of group 1 model was 14 11.3 m which was significantly less than other groups .
the mean distortion values of group 3 : 19.6 10.5 m ; group 4 : 28.9 20.5 m ; group 5 : 31.2 29.7 m were found to have no significant difference .
the maximum mean distortion value was measured in group 2 : 32.1 11.2 m . in the same manner
the mean distortion value of group 1 in y - axis was 7.4 6.9 m which was also significantly less than other groups .
the mean distortion values of group 2 : 18.4 15 m , group3 : 19.5 14.9 m , group 4 : 18.2
the mean distortion value of group 5 in y - axis was 28.4 23.6 m and it was the largest among all groups .
likewise , similar result was observed from z - axis as well , group 1 had the statistically smallest mean distortion value which was 8.6 7.1 m .
the mean distortion values of group 3 , group 4 , group 5 model were 10.2 7.6 m , 25.7 17 m , 23.4 17.9 m , respectively and found to have statistically no significant difference between group 1 , 3 , 4 , and 5 .
the mean distortion value of group 2 was 108 57.7 m that gave the worst results within all distortion data of experimental models ( table 3 ) . in summary ,
group 1 showed the smallest distortion and the mean distortion values of group 3 and 4 were shown to be the similar and next to group 1 . on the other hand , group 2 showed relatively larger distortion than any other group .
passive fit was described by brnemark to be ideally in the 10 m range.34 the definition has evolved to describe a clinically acceptable fit in which stress / strain conditions are within the physiologic range that enables the immature bone to mature or remodel in response to occlusal loads following prosthesis connection .
horizontal fit discrepancy leads to binding of the screws and bending stresses in implant system and when vertical fit discrepancy is present , the preload is used to bring the mating surface closer together , which makes the screw vulnerable to fatigue fractures and loosening.35 horizontal ( x- and y - axes ) displacement in relation to the master cast replicas contributes to a major part of the distortion of prostheses .
however , the vertical dimension of distortion ( z - axis ) seems to increase more than the increase in the horizontal axis when tested in the mouth , which could be critical because this direction of distortion may be more related to introduce preload in the implant.36 it is known that there are some built in machining tolerances in each implant system but any discrepancy above the machining tolerance in the x , y or z axis may not allow a proper clamping effect of the components .
furthermore the amount of introduced stress may vary , depending on the stiffness of the framework for exactly the same degree of misfit .
thus , the biomechanical impact of fit between osseointegrated implants and superstructures seems to be complex , and there is at present time no answer to the question of what acceptable clinical fit should be.37 also there are many variables with current materials and techniques that can influence clinical acceptable fit of implant framework but clinician must strive to overcome these variables .
the precise transfer of the spatial relationships of implants from the mouth to the master cast with an impression is the first and critical step to ensure passive fit of implant framework .
various techniques had been introduced to get accurate impression and splinting of impression coping is one of those even if there are still controversies .
some studies found no difference between splinted and nonsplinted technique.12,23 - 26 however , other studies showed that splinting may provide stabilization of transfer copings against torque from analog tightening and reduce rotational freedom within resilient impression material.29 - 33 and it is advocated splinting is the determining factor for the most accurate cast fabrication , regardless of impression material even though the polyether impression material is very rigid after setting.16,29 in the present study , authors compared autopolymerizing acrylic resin , impression plaster and vps bite registration paste as splinting material to evaluate their effect of polymerization shrinkage of splinting material .
it has been reported that the total shrinkage of acrylic resin is between 6.5% and 7.9% in the first 24 hours , with 80% of shrinkage occurring in the first 17 minutes after mixing,38 whereas the setting expansion of impression plaster is between 0.01% and 0.12%.39 perfect duplication of master model was impossible in all groups .
group 1 , 3 , and 4 showed clinically acceptable mean distortion value and the largest distortion were measured in group 2.27 minimal distortion were found out in group 1 impression method using resin splinting for more than 24 hours , then sectioned and reconnected just before the impression procedure .
adequate polymerization time and the process of compensation seemed like the reason of best accuracy .
thus , dumbrigue et al.40 and naconecy et al.30 presented simple and less time consuming procedure that can rigidly connect transfer coping and minimize the effect of resin polymerization shrinkage such as using prefabricating resin bar or carbon steel bar .
they stated that impression plaster sets rapidly , is quite accurate and rigid , and does not bend or distort ; it is also easy to manipulate , less time consuming and less expensive to use .
and eid also described how to use impression plaster to make implant impression in their clinical studies and stated about the accuracy , easy of manipulation and decreased working time.42,43 on the other hand , the proper use of vps bite registration material could be a doubt as a splinting material because of its short working time and low flowability although their dimensional stabilities are excellent .
however , this study shows that the bite registration material was found out the similar accuracy compared to the impression plaster except the distortion values in y - axis .
within the limitations of this in vitro study , 1 . splinting square impression coping with autopolymerizing resin , adequate polymerization time and compensation procedure before impression ( group 1 impression method )
was found to be statistically the most accurate method of splinting ( mean distortion values < 20 m ) .
clinically acceptable accuracy could be obtained from the splinting methods used with the impression plaster .
statistically significant difference was not found between the group 3 and 4 ( table 3 ) .
the splinting method used with the vps bite registration material showed statistically more distortion than impression plaster in y - axis .
however , there was no statistically significant difference of accuracy in the x- and z - axes .
the impressions made with direct autopolymerizing resin splint method without compensation procedure ( group 2 ) resulted in more distortion than other methods as a whole .
especially , there was a significant loss of accuracy in the z - axis with the high mean distortion value over 100 m . | purposethe aim of this study was to evaluate the effect of dimensional stability of splinting material on the accuracy of master casts.materials and methodsa stainless steel metal model with 6 implants embedded was used as a master model .
implant level impressions were made after square impression copings were splinted using 5 different techniques as follows .
( 1 ) splinted with autopolymerizing resin and sectioned , reconnected to compensate polymerization shrinkage before the impression procedure .
( 2 ) splinted with autopolymerizing resin just before impression procedure . ( 3 ) primary impression made with impression plaster and secondary impression were made over with polyether impression material .
( 4 ) splinted with impression plaster . ( 5 ) splinted with vps bite registration material . from master model , 5 impressions and 5 experimental casts ,
total 25 casts were made for each of 5 splinting methods .
the distortion values of each splinting methods were measured using coordinate measuring machine , capable of recordings in the x- , y- , z - axes .
a one - way analysis of variance ( anova ) at a confidence level of 95% was used to evaluate the data and tukey 's studentized range test was used to determine significant differences between the groups.resultsgroup 1 showed best accuracy followed by group 3 & 4 .
group 2 and 5 showed relatively larger distortion value than other groups .
no significant difference was found between group 3 , 4 , 5 in x - axis , group 2 , 3 , 4 in y - axis and group 1 , 3 , 4 , 5 in z - axis ( p<.0001).conclusionboth splinting impression copings with autopolymerizing resin following compensation of polymerization shrinkage and splinting method with impression plaster can enhance the accuracy of master cast and impression plaster can be used simple and effective splinting material for implant impression procedure . |
schizophrenia is a chronic and often debilitating illness affecting approximately 1% of the world population.1 it is a complex disorder characterized by profound disturbance of perception , cognition , emotion , and social function .
the onset of schizophrenia is typically in late adolescence or early adulthood , and includes distinctive symptoms , commonly referred to as positive , negative , and cognitive . to date
, effective treatments for schizophrenia have been limited to medications with antidopaminergic activity , which alleviate symptoms by augmenting dysfunctional neurotransmitter systems.2 while antipsychotics are most effective for positive symptoms , negative and cognitive symptoms are less well addressed.3 treatment of patients with minimal or no response to adequate doses of antipsychotics represents an enormous challenge for clinicians .
these antipsychotic - resistant patients constitute up to 25%30% of all patients suffering from schizophrenia.2,4,5 one of the main reasons for this is our lack of understanding of the etiology of schizophrenia .
although the exact mechanism of schizophrenia remains to be elucidated , several hypotheses have been proposed , including disruption of neurotransmitter systems,610 genetic factors,11 and neurodevelopmental and neurotoxic mechanisms.12,13 in recent years , however , growing evidence has supported the idea that neuroinflammation , in particular that focused on the microglia , plays an important role in the etiology of schizophrenia , so appropriate control of microglial activation may be a promising strategy in the treatment of the disease.1,1416 minocycline , a second - generation tetracycline , has a distinct neuroprotective profile independent of its antibacterial activity.17 minocycline is almost completely absorbed when taken orally and shows excellent penetration of brain tissue . these properties , as well as its beneficial effect in animal models of neurologic disorders ,
has led to investigation of its potential use in the treatment of schizophrenia.18,19 recent reports have demonstrated a possible antipsychotic effect for minocycline , which is a potent inhibitor of microglial activation . in these studies ,
use of minocycline as an adjuvant to antipsychotics was reported to be beneficial in patients with schizophrenia.2023 here we review the existing literature focusing on the preclinical and clinical potential of minocycline in the treatment of schizophrenia and propose directions for future research .
although dopaminergic neurotransmission is involved in the pathophysiology of schizophrenia , the exact mechanism leading to dopaminergic dysfunction remains unclear .
accumulating evidence indicates the significance of neuroinflammation involving microglia in schizophrenia.16,24 maternal inflammation during critical stages of gestation has been shown to form the basis of the link between prenatal infection and schizophrenia.25,26 longitudinal studies in animal models further indicate that infection - induced developmental neuroinflammation may be pathologically relevant beyond the antenatal and neonatal periods , and may contribute to progression of disease , associated with gradual development of full - blown schizophrenia.27 arion et al carried out a dna microarray study that showed increased expression of genes related to immune and chaperone function in the prefrontal cortex in schizophrenia.28 narayan et al profiled genome - wide expression patterns in the prefrontal cortex in subjects with schizophrenia at different stages of the illness .
their study demonstrated that the molecular basis for schizophrenia changes between the early to chronic stages , providing evidence that the nature of schizophrenia changes with disease progression , with the long - term illness possibly being associated with inflammation , stimulus response , and immune function.29 microglial cells are the primary reservoirs of proinflammatory cytokines , such as interleukin-6 , tumor necrosis factor - alpha ( tnf- ) , and interferon - gamma ( ifn- ) , and act as the main antigen - presenting cells in the central nervous system ( cns).30 microglia are important for the cross - talk between the immune system and glutamatergic neurotransmission.31 the cells gradually become present in the brain and participate in various aspects of brain development , including cell death , axonal remodeling , synaptogenesis , and synaptic pruning.3236 prolonged microglial hyperactivity may lead to neuronal apoptosis and brain damage , which are commonly seen in neurodegenerative disorders such as parkinson s disease and alzheimer s disease.37,38 a neurodegenerative and neurodevelopmental process is indicated in the course of schizophrenia and may be associated with microglial activation.39,40 animal studies , autopsy studies , and positron emission tomography ( pet ) studies have demonstrated that the neuropathology of schizophrenia is associated with microglial activation.4149 bayer et al found that there were more activated microglia in the frontal cortex and hippocampus in patients with schizophrenia post mortem than in controls.45 their findings were strengthened further by positive results from other autopsy studies in patients with schizophrenia.43,44,46,47 the evidence from autopsy studies suggests that schizophrenia is associated with an increased number of activated microglia cells .
pet , a noninvasive brain imaging technique , provides the opportunity to study the presence of microglial activation in vivo .
van berckel et al found overactive microglia in patients with schizophrenia of recent onset.48 doorduin et al also reported finding more activated microglia in the hippocampus of patients with schizophrenia than in healthy volunteers , and suggested that focal neuroinflammation may play an important role in schizophrenia.49 as such , there has been growing evidence of a microglial hypothesis of schizophrenia ( figure 1 ) whereby stressful events , damage - associated molecular patterns , and pathogen - associated molecular patterns activate microglia in the cns via immunologic or inflammatory activators .
proinflammatory cytokines and free radicals released by overactivated microglia can cause neuronal degeneration , abnormalities in white matter , and decreased neurogenesis .
the damage done to the neuron by overactivated microglia may thus lead eventually to the development of schizophrenia.1,16 if microglial pathology proves to be an important causative factor in schizophrenia , the processes involving modulators of microglial activation may represent a novel therapeutic strategy.50,51 inflammation of the cns is detrimental to neurogenesis in the adult hippocampus.52,53 the negative effects of inflammation on differentiation and survival of neuronal cells are due to microglia - derived proinflammatory cytokines.52,54 interleukin-1 and tnf- have been reported to inhibit neurogenesis in vivo.55,56 however , neurogenesis can be restored by anti - inflammatory compounds such as minocycline , indomethacin , and cyclo - oxygenase-2 inhibitors ( eg , celecoxib ) which inhibit microglial activation.52,53,57,58 other reports have shown that minocycline is able to inhibit microglial activation via anti - inflammatory and antioxidant properties which are independent of its antimicrobial action.5967 yrjanheikki et al showed that minocycline is able to provide neuroprotection against global ischemia in gerbils and focal brain ischemia in rats.59,60 this neuroprotection was associated with reduced activation of microglia and inhibition of interleukin-1-converting enzyme , suggesting that minocycline may function by reducing the cytotoxic effects of the microglia .
minocycline can also reduce the proliferation and activation of resting microglia.61 tikka et al investigated whether minocycline was able to reduce excitotoxicity in a primary neuronal culture , and found that glutamate - induced microglial activation occurred via the p38 mitogen - activated protein kinase pathway and that minocycline inhibited activation of this pathway in microglia , providing neuroprotection against excitotoxicity.62 their findings were strengthened further by positive results in other studies using animal models of ischemic injury to the brain and neurodegenerative diseases.6365 in addition , it was found that interleukin-1 levels following traumatic brain injury can be reduced in mice by a high dose of minocycline , and that increased tnf- and nitric oxide levels in rats treated with 3-nitropropioic acid ( an inhibitor of succinate dehydrogenase , an electron transport enzyme ) can be attenuated by treatment with minocycline.66,67 minocycline has also been reported to reduce superoxide production by granular neurons in the rat following treatment with n - methyl - d - aspartate , a glutamate receptor agonist.68 overall , the above studies suggest that minocycline targets overactive microglia and removes proinflammatory cytokines and oxygen radicals.5967 increasing evidence indicates that the neurodegenerative course of schizophrenia involves an increased susceptibility to apoptotic death , and activation of the apoptotic process results in rapid neuronal death.6973 the mechanisms of cell death in neurodegenerative diseases include oxidative stress , excitotoxicity , mitochondrial dysfunction , and inflammation.74 proinflammatory cytokines , including tnf- , have been well characterized as mediators of oxidative stress and induce apoptosis in human cortical neurons as well as oligodendrocytes.75,76 apoptosis is indirectly suppressed by minocycline by inhibition of proinflammatory cytokines and nitric oxide.77 moreover , minocycline has been shown to be an inhibitor of poly-(adp - ribose ) , polymerase-1 , and matrix metalloproteinases.78,79 the antiapoptotic profile of minocycline has been attributed to upregulation of the anti - apoptotic protein bcl-2 , reduced expression of caspases , and inhibition of release of proapoptotic proteins from the mitochondria.8084 animal studies have shown that minocycline is able to increase expression of antiapoptotic proteins and decrease expression of these proteins after treatment with transforming growth factor beta-2 .
hydrogen peroxide can also be reversed by minocycline , which leads to inhibition of apoptosis.85,86 minocycline may directly activate the kinase g pathway by activation of kinase g1 , leading to increased expression of bcl-2.19,87 minocycline is able to block the mitochondrial permeability transition pore ( a large - conductance mega channel in the inner mitochondrial membrane ) , inhibiting release of proapoptotic proteins from the mitochondria.8891 minocycline has also been shown to reduce glutamate excitotoxicity in the glutamatergic pathways.9299 it has been reported that minocycline - treated rat cortical neurons show increased cell viability following glutamate administration , which indicates that minocycline is able to reduce glutamate - induced neurotoxicity.92,93 glur1 receptors , which are critical for the cognitive processes that are impaired in schizophrenia , has been shown to be involved in the pathobiology of schizophrenia.94,95 minocycline affects a subtype of glutamate receptors known as glur1 alpha - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa ) receptors in neurons in vitro and in the cns in vivo . increased membrane localization of the glur1 ampa receptor improves glutamatergic activity and modulates neuroplasticity.96 overall , these findings suggest that minocycline has an antiapoptotic profile and is involved in several pathways known to be disturbed in schizophrenia .
although dopaminergic neurotransmission is involved in the pathophysiology of schizophrenia , the exact mechanism leading to dopaminergic dysfunction remains unclear .
accumulating evidence indicates the significance of neuroinflammation involving microglia in schizophrenia.16,24 maternal inflammation during critical stages of gestation has been shown to form the basis of the link between prenatal infection and schizophrenia.25,26 longitudinal studies in animal models further indicate that infection - induced developmental neuroinflammation may be pathologically relevant beyond the antenatal and neonatal periods , and may contribute to progression of disease , associated with gradual development of full - blown schizophrenia.27 arion et al carried out a dna microarray study that showed increased expression of genes related to immune and chaperone function in the prefrontal cortex in schizophrenia.28 narayan et al profiled genome - wide expression patterns in the prefrontal cortex in subjects with schizophrenia at different stages of the illness .
their study demonstrated that the molecular basis for schizophrenia changes between the early to chronic stages , providing evidence that the nature of schizophrenia changes with disease progression , with the long - term illness possibly being associated with inflammation , stimulus response , and immune function.29 microglial cells are the primary reservoirs of proinflammatory cytokines , such as interleukin-6 , tumor necrosis factor - alpha ( tnf- ) , and interferon - gamma ( ifn- ) , and act as the main antigen - presenting cells in the central nervous system ( cns).30 microglia are important for the cross - talk between the immune system and glutamatergic neurotransmission.31 the cells gradually become present in the brain and participate in various aspects of brain development , including cell death , axonal remodeling , synaptogenesis , and synaptic pruning.3236 prolonged microglial hyperactivity may lead to neuronal apoptosis and brain damage , which are commonly seen in neurodegenerative disorders such as parkinson s disease and alzheimer s disease.37,38 a neurodegenerative and neurodevelopmental process is indicated in the course of schizophrenia and may be associated with microglial activation.39,40 animal studies , autopsy studies , and positron emission tomography ( pet ) studies have demonstrated that the neuropathology of schizophrenia is associated with microglial activation.4149 bayer et al found that there were more activated microglia in the frontal cortex and hippocampus in patients with schizophrenia post mortem than in controls.45 their findings were strengthened further by positive results from other autopsy studies in patients with schizophrenia.43,44,46,47 the evidence from autopsy studies suggests that schizophrenia is associated with an increased number of activated microglia cells .
pet , a noninvasive brain imaging technique , provides the opportunity to study the presence of microglial activation in vivo .
van berckel et al found overactive microglia in patients with schizophrenia of recent onset.48 doorduin et al also reported finding more activated microglia in the hippocampus of patients with schizophrenia than in healthy volunteers , and suggested that focal neuroinflammation may play an important role in schizophrenia.49 as such , there has been growing evidence of a microglial hypothesis of schizophrenia ( figure 1 ) whereby stressful events , damage - associated molecular patterns , and pathogen - associated molecular patterns activate microglia in the cns via immunologic or inflammatory activators .
proinflammatory cytokines and free radicals released by overactivated microglia can cause neuronal degeneration , abnormalities in white matter , and decreased neurogenesis .
the damage done to the neuron by overactivated microglia may thus lead eventually to the development of schizophrenia.1,16 if microglial pathology proves to be an important causative factor in schizophrenia , the processes involving modulators of microglial activation may represent a novel therapeutic strategy.50,51
inflammation of the cns is detrimental to neurogenesis in the adult hippocampus.52,53 the negative effects of inflammation on differentiation and survival of neuronal cells are due to microglia - derived proinflammatory cytokines.52,54 interleukin-1 and tnf- have been reported to inhibit neurogenesis in vivo.55,56 however , neurogenesis can be restored by anti - inflammatory compounds such as minocycline , indomethacin , and cyclo - oxygenase-2 inhibitors ( eg , celecoxib ) which inhibit microglial activation.52,53,57,58 other reports have shown that minocycline is able to inhibit microglial activation via anti - inflammatory and antioxidant properties which are independent of its antimicrobial action.5967 yrjanheikki et al showed that minocycline is able to provide neuroprotection against global ischemia in gerbils and focal brain ischemia in rats.59,60 this neuroprotection was associated with reduced activation of microglia and inhibition of interleukin-1-converting enzyme , suggesting that minocycline may function by reducing the cytotoxic effects of the microglia .
minocycline can also reduce the proliferation and activation of resting microglia.61 tikka et al investigated whether minocycline was able to reduce excitotoxicity in a primary neuronal culture , and found that glutamate - induced microglial activation occurred via the p38 mitogen - activated protein kinase pathway and that minocycline inhibited activation of this pathway in microglia , providing neuroprotection against excitotoxicity.62 their findings were strengthened further by positive results in other studies using animal models of ischemic injury to the brain and neurodegenerative diseases.6365 in addition , it was found that interleukin-1 levels following traumatic brain injury can be reduced in mice by a high dose of minocycline , and that increased tnf- and nitric oxide levels in rats treated with 3-nitropropioic acid ( an inhibitor of succinate dehydrogenase , an electron transport enzyme ) can be attenuated by treatment with minocycline.66,67 minocycline has also been reported to reduce superoxide production by granular neurons in the rat following treatment with n - methyl - d - aspartate , a glutamate receptor agonist.68 overall , the above studies suggest that minocycline targets overactive microglia and removes proinflammatory cytokines and oxygen radicals.5967
increasing evidence indicates that the neurodegenerative course of schizophrenia involves an increased susceptibility to apoptotic death , and activation of the apoptotic process results in rapid neuronal death.6973 the mechanisms of cell death in neurodegenerative diseases include oxidative stress , excitotoxicity , mitochondrial dysfunction , and inflammation.74 proinflammatory cytokines , including tnf- , have been well characterized as mediators of oxidative stress and induce apoptosis in human cortical neurons as well as oligodendrocytes.75,76 apoptosis is indirectly suppressed by minocycline by inhibition of proinflammatory cytokines and nitric oxide.77 moreover , minocycline has been shown to be an inhibitor of poly-(adp - ribose ) , polymerase-1 , and matrix metalloproteinases.78,79 the antiapoptotic profile of minocycline has been attributed to upregulation of the anti - apoptotic protein bcl-2 , reduced expression of caspases , and inhibition of release of proapoptotic proteins from the mitochondria.8084 animal studies have shown that minocycline is able to increase expression of antiapoptotic proteins and decrease expression of these proteins after treatment with transforming growth factor beta-2 .
hydrogen peroxide can also be reversed by minocycline , which leads to inhibition of apoptosis.85,86 minocycline may directly activate the kinase g pathway by activation of kinase g1 , leading to increased expression of bcl-2.19,87 minocycline is able to block the mitochondrial permeability transition pore ( a large - conductance mega channel in the inner mitochondrial membrane ) , inhibiting release of proapoptotic proteins from the mitochondria.8891 minocycline has also been shown to reduce glutamate excitotoxicity in the glutamatergic pathways.9299 it has been reported that minocycline - treated rat cortical neurons show increased cell viability following glutamate administration , which indicates that minocycline is able to reduce glutamate - induced neurotoxicity.92,93 glur1 receptors , which are critical for the cognitive processes that are impaired in schizophrenia , has been shown to be involved in the pathobiology of schizophrenia.94,95 minocycline affects a subtype of glutamate receptors known as glur1 alpha - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa ) receptors in neurons in vitro and in the cns in vivo . increased membrane localization of the glur1 ampa receptor improves glutamatergic activity and modulates neuroplasticity.96 overall
, these findings suggest that minocycline has an antiapoptotic profile and is involved in several pathways known to be disturbed in schizophrenia .
minocycline has been reported to reduce inflammation and provide neuroprotection in a variety of experimental models , including ischemic stroke , brain and spinal cord injury , multiple sclerosis , and parkinson s disease , alzheimer s disease , and huntington s disease.17 du et al have reported on the neuroprotective effects of minocycline in animal models of stroke / ischemic injury and huntington s disease , and shown that minocycline can prevent nigrostriatal dopaminergic neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) mouse model of parkinson s disease.97 minocycline has been shown to play an important role in the treatment of disorders involving neuronal damage.17,97 however , in a rat model of intracerebral hemorrhage , wasserman et al found that minocycline did not reduce neuronal loss outside the hematoma or striatal tissue , despite reducing the number of neutrophils and activated microglia .
these authors suggested that minocycline did not appear to target the mechanisms responsible for cell death in this model of intracerebral hemorrhage.98 however , the complex behavioral tests needed to assess functional recovery in this model of intracerebral hemorrhage were not used , and it was not known whether rescuing the band of damaged neurons at the edge of the hematoma would have lasting benefits .
the neurotherapeutic potential of minocycline in the treatment of mental disorders has also been explored.19,99101 it has been reported that minocycline is able to ameliorate behavioral changes as well as neurotoxicity at dopaminergic terminals after administration of methamphetamine , a compound that causes long - term cognitive deficits and psychiatric signs such as hallucination and delusions.99 hashimoto et al did a pet study in conscious monkeys , and found that minocycline attenuated the reduction of dopamine transporters in monkeys treated with methamphetamine .
they suggested that minocycline protects against methamphetamine - induced neurotoxicity in the monkey brain.102 levkovitz et al100 compared the effects of minocycline and haloperidol in an animal model of schizophrenia ( n - methyl - d - aspartate antagonist , dizocilpine maleate , mk801 ) .
after 3 days of treatment with minocycline 35 mg / kg / day , the rats were injected with mk801 and assessed using behavioral tests .
the findings showed that mk801 caused cognitive visuospatial memory deficits and changes in sensorimotor gating , similar to those evident in schizophrenia .
minocycline reversed the cognitive effects of mk801 , and the effect was similar to that of haloperidol .
levkovitz et al suggested that minocycline may be able to protect against the disturbed cognitive processes seen in the mk801 animal model of schizophrenia.100 zhang et al also indicated that minocycline attenuates the behavioral changes ( such as acute hyperlocomotion and prepulse inhibition deficits ) occurring in mice after administration of dizocilpine , and suggested that minocycline could be a potential therapeutic compound for schizophrenia.103 monte et al reported the prevention and reversal of ketamine - induced schizophrenia - related behavior by minocycline.101 moreover , minocycline has been shown to have beneficial effects on cognition in animal models of schizophrenia . in a study by fujita et al saline 10 ml / kg / day or phencyclidine 10 mg / kg / day
three days after the final dose of saline or phencyclidine , vehicle ( 10 ml / kg / day , physiologic saline ) or minocycline ( 4.0 or 40 mg / kg / day ) was administered intraperitoneally for 14 consecutive days .
one day after the final injection , a novel object recognition test was performed , and it was found that phencyclidine - induced cognitive deficits in mice were significantly improved by subsequent subchronic administration of minocycline ( 40 mg / kg ) , suggesting minocycline could be a potential therapeutic compound for cognitive deficits in schizophrenic patients.104 improvement by minocycline of impaired recognition memory in methamphetamine - treated mice was also reported by mizoguchi et al.105 in summary , these findings indicate that minocycline may be useful in the treatment in schizophrenia .
however , regardless of how encouraging the preclinical studies have been , clinical trials are required to verify these effects of minocycline .
miyaoka et al reported two cases of minocycline being used as adjunctive treatment . in these cases , minocycline produced a remarkable improvement in individuals with acute schizophrenia who had predominantly catatonic symptoms.106 these observations are supported by a case report of a patient with schizophrenia given minocycline 200 mg / day for 8 weeks in addition to a stable dose of olanzapine 20 mg / day.107 the combination of minocycline and antipsychotic treatment significantly reduced positive symptoms , with no negative symptoms seen in the baseline assessment .
significantly decreased hyperperfusion in the posterior cingulate gyrus along with a simultaneous improvement of positive symptoms was observed after treatment with minocycline .
based on these case reports , miyaoka et al conducted an open - label study in 22 patients with schizophrenia , in whom minocycline was administered for 4 weeks as an adjunct to antipsychotic medication .
minocycline was initiated as 100 mg orally twice daily for the first week , and 150 mg orally three times daily from weeks 2 through 4 .
the patients showed statistically significant and robust clinical improvements on the positive and negative syndrome scale , and these improvements were maintained at the follow - up evaluation 4 weeks after the end of treatment with minocycline , with few adverse events.108 however , this was an open - label study without a control group , and the effects of the adjunctive medication can not be ruled out as an explanation for the observed improvement .
interestingly , some studies indicate that minocycline may have specific effects on negative symptoms and impaired cognitive function in patients with schizophrenia , which contribute to their poor social and occupational functioning .
case reports indicate the effective role of add on minocycline in treating negative symptoms in schizophrenia.109,110 recently , levkovitz et al carried out a double - blind , randomized , placebo - controlled study demonstrating that add on treatment with minocycline 200 mg / day for 6 months had a beneficial effect on negative symptoms and general outcomes in patients with early - phase schizophrenia .
a similar pattern was seen for cognitive functioning , mainly of the executive type , ie , working memory , cognitive shifting , and cognitive planning.111 seventy patients with early - phase schizophrenia were recruited for the study ; however , of 54 patients randomly allocated in a 2:1 ratio to the minocycline group , only 13 completed the 6-month trial .
another randomized , double - blind , placebo - controlled clinical trial also suggested that addition of minocycline ( 200 mg / day for one year ) to treatment as usual in patients with early psychosis could reduce negative symptoms without a detectable effect on cognition.112 the investigators recruited 144 patients with psychosis ( including schizophrenia , schizoaffective disorder , psychosis not otherwise specified , or schizophreniform disorder ) within 5 years of first onset in brazil and pakistan , and 94 completed the trial .
although the dropout rates were not high when compared with other trials , the validity and reliability of cognitive measures in two different racial groups and cultures could have increased the variability of the results .
in addition , neither the type nor dose of antipsychotic medication was controlled , which may have further increased the variability and obscured the effects of the medication .
sofuoglu et al showed that minocycline attenuated dextroamphetamine - induced subjective reward effects and improved reaction times on a go no - go task in healthy volunteers.113 although case reports should to be interpreted with caution and large - scale studies are needed to determine the effectiveness of minocycline and related compounds in clinical use , the results of the above studies suggest that minocycline may be a safe and effective adjunct to antipsychotic medication for the treatment of schizophrenia .
although the etiology of schizophrenia remains unclear , there has been growing evidence suggesting that neuroinflammation , characterized by overactive microglia , plays an important role in the neuropathology of schizophrenia .
the microglial hypothesis of schizophrenia may shed new light on a therapeutic strategy for the disease.1,16 according to this hypothesis and the results of previous related studies , we suggest that overactivation of microglia can lead to neuronal degeneration and abnormal development in the brain , and that this neuropathologic process may be more likely to result in negative symptoms and cognitive impairment in schizophrenia.1,16,2123,48,49 neuroinflammation could constitute the basic framework for further study in the neuropathology of negative schizophrenia . as an inhibitor of microglial activation
, minocycline has anti - inflammatory , antioxidant , and antiapoptotic properties , and can modulate glutamate - induced excitotoxicity .
the therapeutic potential of minocycline has been demonstrated in studies using animal models of schizophrenia.19,99105 in addition to the evidence presented here , recent clinical trials have demonstrated that minocycline can be used as an add on therapy in schizophrenia , especially for amelioration of negative symptoms.106112 because of the limited and preliminary nature of the studies mentioned above , more detailed studies are required to determine whether minocycline is effective as an adjunctive treatment for symptoms in patients with schizophrenia who derive limited benefit from standard antipsychotic treatment .
moreover , considering the complicated etiology and heterogeneity of schizophrenia , it will be necessary to categorize the disease phenotype in further studies , including patient status and the severity of negative symptoms , which possibly need to be used as inclusion / exclusion criteria or stratifying variables .
finally , given the mechanism of action of minocycline and the data on the role of overactivated microglia in schizophrenia , future clinical studies should include inflammatory markers in blood or cerebrospinal fluid , and functional imaging such as pet to verify the hypothesis that subgroups of schizophrenic patients with neuroinflammation are the most appropriate candidates for augmentation therapy with minocycline . | accumulating evidence suggests that neuroinflammation affecting microglia plays an important role in the etiology of schizophrenia , and appropriate control of microglial activation may be a promising therapeutic strategy for schizophrenia .
minocycline , a second - generation tetracycline that inhibits microglial activation , has been shown to have a neuroprotective effect in various models of neurodegenerative disease , including anti - inflammatory , antioxidant , and antiapoptotic properties , and an ability to modulate glutamate - induced excitotoxicity .
given that these mechanisms overlap with neuropathologic pathways , minocycline may have a potential role in the adjuvant treatment of schizophrenia , and improve its negative symptoms . here , we review the relevant studies of minocycline , ranging from preclinical research to human clinical trials . |
endodontic treatment of teeth is a routine therapy to treat inflammation or necrosis of the pulp .
it is established that the success rate of conventional endodontic therapy is 85% to 95% .
inspite of this high success rate , root canal therapy occasionally fails for which retreatment is needed .
however , some cases can not be retreated conservatively because of various limitations and therefore an endodontic surgery is required to save the affected tooth .
the management of the root during endodontic surgery includes root end resection , root - end preparation , and placement of a root end filling to seal the root canal . during root end cavity preparation with ultrasonic retro - tips ,
the presence of smear layer has been postulated as an avenue for leakage and source for bacterial growth and ingress , particularly following root end preparation .
the debris created during root - canal instrumentation should be removed from the dentine surface of the canal wall and the dentine tubules .
various gels and solutions such as citric acid , tannic acid , polyacrylic acid , phosphoric acid , chelating agents such as ethylenediaminetetraacetic acid ( edta ) or redta , doxycycline , and tetracycline isomers have been used to remove the smear layer formed on the walls of the retrocavity following preparation .
hence the objective of this study was to evaluate the efficacy of 35% orthophosphoric acid gel , 24% edta gel and 10% citric acid gel in removal of smear layer from retrocavities prepared with ultrasonic retro - tips using scanning electron microscopy .
fifty human single - rooted teeth , extracted for orthodontic / periodontal reasons , were employed in the study . to facilitate instrumentation , the crowns of all teeth were decoronated at the level of the cementoenamel junction using diamond disc ( horico , germany ) .
the working length was determined by inserting a # 10 k - file ( mani , japan ) into the canal until it just exits the apical foramen and then subtracting 1 mm from the obtained length .
the root canals were prepared with the protaper ( dentsply , maillefer , switzerland ) rotary file system according to manufacturer 's instructions in a crown down manner .
the final apical size at the working length was f2 having an apical diameter 0.25 mm and taper of 0.08 . during biomechanical preparation
the canals were irrigated with 2 ml of 3% sodium hypochlorite ( naocl ) after each instrumental change . at the end of the instrumentation , the root canals were dried and irrigated with 17% edta .
preliminary control group ( n = 10 ) and experimental group ( n = 40 ) . the preliminary control group did not receive any obturation , apicoectomy or retrograde cavity preparation .
the experimental groups were obturated with gutta - percha ( dentsply - maillefer , ballaigues , switzerland ) ah plus sealer ( dentsply/ de trey , konstanz , germany ) using lateral condensation technique .
after obturation , the apical 3 mm of each root was resected at an angle of 90 with respect to the long axis of the root with a diamond disc .
retrograde cavities were prepared with kis # 1 diamond - coated surgical ultrasonic retrotips ( dentsply , maillefer , switzerland ) with ultrasonic handpiece ( mini piezo ; ems , nyon , switzerland ) set at full power to a depth of 3 mm into root canal . in this group , specimens were split longitudinally in the buccolingual plane . to facilitate separation of the root into two halves ,
all roots were grooved longitudinally on the external surface with a diamond disc ( horico , germany ) and split into two halves with a chisel .
the half containing the most visible portion of the apex was selected for sem observation ( cambridge s60 , germany ) .
retro cavities in group i were treated with a gel of 35% orthophosphoric acid for 15 seconds , followed by 1 minute rinsing with distilled water .
group ii retrocavities were treated with a gel of 24% edta at a neutral ph for 2 minutes , followed by 1 minute rinsing with distilled water .
group iii retrocavities were treated with gel of 10% citric acid for 2 minutes , followed by 1 minute rinsing with distilled water .
group iv ( negative control group ) did not receive any treatment of the reretrocavity .
all roots were grooved longitudinally on the external surface with a diamond disc ( horico , germany ) , then split into two halves with a chisel .
the half containing the most visible part of the retrocavity was mounted on a brass stub and gold sputtered to examine under scanning electron microscopy ( cambridge s60 , germany ) .
an evaluation was performed to record the presence of smear layer on the surface of the retrocavity walls based on the following score described by hulsmann et al.,(1997 ) : score 1 : dentinal tubules completely open score 2 : more than 50% of dentinal tubules open score 3 : less than 50% of dentinal tubules
the data were then analyzed statistically using a non - parametric test ( kruskall - wallis test ) and post - hoc analysis , using statistical package for the social sciences software ( spss 17 ) data analyzing software .
fifty human single - rooted teeth , extracted for orthodontic / periodontal reasons , were employed in the study .
to facilitate instrumentation , the crowns of all teeth were decoronated at the level of the cementoenamel junction using diamond disc ( horico , germany ) .
the working length was determined by inserting a # 10 k - file ( mani , japan ) into the canal until it just exits the apical foramen and then subtracting 1 mm from the obtained length .
the root canals were prepared with the protaper ( dentsply , maillefer , switzerland ) rotary file system according to manufacturer 's instructions in a crown down manner .
the final apical size at the working length was f2 having an apical diameter 0.25 mm and taper of 0.08 . during biomechanical preparation
the canals were irrigated with 2 ml of 3% sodium hypochlorite ( naocl ) after each instrumental change . at the end of the instrumentation , the root canals were dried and irrigated with 17% edta .
the specimens were then divided into two groups . preliminary control group ( n = 10 ) and experimental group ( n = 40 ) .
the preliminary control group did not receive any obturation , apicoectomy or retrograde cavity preparation .
the experimental groups were obturated with gutta - percha ( dentsply - maillefer , ballaigues , switzerland ) ah plus sealer ( dentsply/ de trey , konstanz , germany ) using lateral condensation technique .
after obturation , the apical 3 mm of each root was resected at an angle of 90 with respect to the long axis of the root with a diamond disc .
retrograde cavities were prepared with kis # 1 diamond - coated surgical ultrasonic retrotips ( dentsply , maillefer , switzerland ) with ultrasonic handpiece ( mini piezo ; ems , nyon , switzerland ) set at full power to a depth of 3 mm into root canal .
in this group , specimens were split longitudinally in the buccolingual plane . to facilitate separation of the root into two halves ,
all roots were grooved longitudinally on the external surface with a diamond disc ( horico , germany ) and split into two halves with a chisel .
the half containing the most visible portion of the apex was selected for sem observation ( cambridge s60 , germany ) .
retro cavities in group i were treated with a gel of 35% orthophosphoric acid for 15 seconds , followed by 1 minute rinsing with distilled water .
group ii retrocavities were treated with a gel of 24% edta at a neutral ph for 2 minutes , followed by 1 minute rinsing with distilled water .
group iii retrocavities were treated with gel of 10% citric acid for 2 minutes , followed by 1 minute rinsing with distilled water .
group iv ( negative control group ) did not receive any treatment of the reretrocavity .
all roots were grooved longitudinally on the external surface with a diamond disc ( horico , germany ) , then split into two halves with a chisel .
the half containing the most visible part of the retrocavity was mounted on a brass stub and gold sputtered to examine under scanning electron microscopy ( cambridge s60 , germany ) .
an evaluation was performed to record the presence of smear layer on the surface of the retrocavity walls based on the following score described by hulsmann et al.,(1997 ) : score 1 : dentinal tubules completely open score 2 : more than 50% of dentinal tubules open score 3 : less than 50% of dentinal tubules open score 4 : almost all dentinal tubules covered with smear layer .
the data were then analyzed statistically using a non - parametric test ( kruskall - wallis test ) and post - hoc analysis , using statistical package for the social sciences software ( spss 17 ) data analyzing software .
the analyzed samples that showed a cleanliness of the walls with dentinal tubules completely open ( score 1 ) were all from group i. in this group , 70% of the observed samples had this score [ figure 1a ] .
the majority of analyzed samples in group ii ( 5 samples of 10 [ 50% ] ) showed dentinal tubules covered with the smear layer ( score 4 ) [ figure 1b ] and in the group iii , 40% of samples , all dentinal tubules were completely covered with smear layer [ figure 1c ] .
eighty percent of samples in preliminary control group showed root canal walls covered with smear layer [ figure 1d ] .
all the analyzed samples of the negative control group showed dentinal tubules covered with smear layer [ figure 1e ] [ table 1 ] .
sem images of ( a ) orthophosphoric acid group , ( b ) edta group , ( c ) citric acid group ( d ) preliminary control group and ( e ) negative control group percentage distribution of scores for smear layer removal and inter - group comparison statistical inter - group comparison was done between orthophosphoric acid group and citric acid group .
orthophosphoric acid ( group i ) was significantly superior in removing smear layer when compared with citric acid ( p value-0.001 ) .
also , phosphoric acid proved to be more effective than edta in removal of smear layer .
however , there was no significant difference in smear layer removal between edta and citric acid ( p value-0.59 ) [ table 1 ] .
analysis of the results reveal that ( group i ) orthophosphoric acid was more efficacious ( statistically significant ) than ( group ii ) citric acid and ( group iii ) edta in removing smear layer from retrocavities prepared with ultrasonic retro - tips .
currently , ultrasonic retro - tips are more popular for this purpose . ultrasonic technique of root end preparation allows optimal root end preparation in terms of dimension and direction .
an evaluation of the technique of root end preparation by wuchenich et al . , ( 1993 ) showed that ultrasonically created cavities had more parallel walls , deeper depths for retention , and preparation which followed the line of the root canal , and cleaner surfaces than those created with burs .
the use of ultrasonic retro - tips during root end cavity preparation produces a smear layer on the walls of the retrocavity .
smear layer is an amorphous , irregular entity containing organic ( pulp tissue , bacteria ) and inorganic ( dentin ) materials with microorganisms and necrotic pulpal debris .
pashley et al , 1997 believed that the smear layer contains bacteria and bacterial by - products and thus it must be completely removed from the root canal system . while the removal of smear layer subsequent to coronal cavity preparation and restoration has been advocated in some clinical situations ( brannstrom 1984 ) , the removal of smear layer from apical retrocavity preparations has not been clinically addressed .
however , because smear layer may serve as either an avenue for leakage or as a bacterial substrate ( pitt ford and roberts 1990 ) , removal of the smear layer prior to filling the retrocavity may be clinically appropriate .
effective removal of smear layer has been reported using a combination of 5.25% naocl and 17% edta solution ( baumgartner et al , 1987 ) .
although naocl remains gold standard as a result of its anti - microbial effect and tissue dissolution properties , it has no effect on inorganic portion of smear layer .
therefore , naocl has been used in association with edta , which acts on inorganic debris formed in instrumented root canals .
other agents such as citric acid , tannic acid , polyacrylic acid , phosphoric acid and redta are also used for the removal of smear layer .
since most ramifications are within the apical 3 mm of the root , this is the least amount of root end that should be removed .
they recommended resection of the apical 3 mm , followed by the preparation of a root end cavity 3 mm deep , thus making the therapeutic length to 6 mm . during root end resection
, the apical 3 mm of roots are resected at a 90 angle with respect to the long axis of the tooth .
this minimizes exposure of dentinal tubules on the resected surface when compared to the 45 angle root end resection .
there are no comparative studies on the different methodologies of studying smear layer removal from the walls of retrograde cavities during surgical endodontics , for this reason , we used the scoring criteria developed by hulsmann et al ( 1997 ) to evaluate the endodontic smear layer after instrumentation of the canal .
seventy percent of the teeth in othophosphoric acid group showed cleanliness of the retrocavity walls with dentinal tubules completely open . majority of analyzed samples of edta and citric acid groups showed dentinal tubules covered with smear layer .
eighty percent of samples in the preliminary control group showed root canal walls covered with smear layer .
all the analyzed samples of negative control group showed dentinal tubules covered with smear layer .
the results of this study were consistent with those of cristiano fabiani et al ( 2011 ) who compared removal of smear layer from retrocavities using 35% orthophosphoric acid and 24% edta gel .
they found that orthophosphoric acid was more effective in removing smear layer than edta . also findings of this study were in accordance with maira prado et al ( 1995 ) who reported 37% phosphoric acid solution to be effective in removing smear layer when compared with that of 17% edta and 10% citric acid at 30 seconds , 1 and 3 minutes experimental time period .
overall , the results of our study suggested that orthophosphoric acid gel is more effective in removing smear layer when compared with citric acid and edta with lesser application time .
removal of smear layer enhances adhesion by intimate adaptation of the retrofilling material , making the root surface biocompatible and optimizing periodontal healing .
the following conclusions can be drawn from this study :
application of 35% orthophosphoric acid gel for 15 seconds on retrocavities prepared with ultrasonic retro - tips is the most effective means for removal of smear layer.35% orthophosphoric acid is more effective than 10% citric acid gel and 24% edta gel .
its action requires less time ; hence , may be preferred for removal of smear layer.there is no significant difference with the application of 24% edta and 10% citric acid in removal of smear layer on retrocavities prepared with ultrasonic retro - tips .
application of 35% orthophosphoric acid gel for 15 seconds on retrocavities prepared with ultrasonic retro - tips is the most effective means for removal of smear layer .
35% orthophosphoric acid is more effective than 10% citric acid gel and 24% edta gel .
its action requires less time ; hence , may be preferred for removal of smear layer .
there is no significant difference with the application of 24% edta and 10% citric acid in removal of smear layer on retrocavities prepared with ultrasonic retro - tips . | objectives : to evaluate the efficacy of 35% orthophosphoric acid , 24% ethylenediaminetetraacetic acid ( edta ) and 10% citric acid in the removal of smear layer from retrocavities prepared with ultrasonic retro - tips using scanning electron microscopy.materials and methods : root canals of fifty single - rooted teeth were cleaned , shaped , and obturated with gutta - percha and ah plus sealer .
the apical 3 mm of each root was sectioned with a diamond disc and retrograde cavities were prepared with kis # 1 ultrasonic retro - tips to a depth of 3 mm .
retro cavities in group i were treated with a gel of 35% orthophosphoric acid for 15 seconds , group ii were treated with a gel of 24% edta at neutral ph for 2 minutes , and group iii were treated with a gel of 10% citric acid for 2 minutes , followed by 1 minute rinsing with distilled water for all groups .
the samples were prepared for scanning electron microscopic observation .
scoring was performed for the presence of the smear layer on the walls of the retrocavity.results:in the orthophosphoric acid group , it was observed that all dentinal tubules were open in 70% of the samples .
the majority of analyzed samples in edta and citric acid group showed dentinal tubules covered with the smear layer.conclusions:application of 35% orthophosphoric acid gel for 15 seconds on retrocavities prepared with ultrasonic retro - tips is the most effective means for removal of smear layer . |
vascular calcification is often observed in advanced vascular lesions and is a common consequence of aging , diabetes mellitus , and chronic kidney disease .
patients with diabetes mellitus or chronic kidney disease often exhibit more severe atherosclerosis and a higher prevalence of vascular calcification .
vascular calcification is closely associated with arterial stiffness and ultimately contributes to increased cardiovascular mortality .
therefore , developing therapeutic strategies to prevent and treat vascular calcification has a great clinical importance .
vascular calcification was long considered to be a passive degenerative process . however , accumulating evidence shows that bone - associated proteins , including osteocalcin , osteopontin , and alkaline phosphatase , are preferentially expressed in calcified atherosclerotic plaques .
in addition , bone - associated structures such as matrix vesicles , which are the initial sites of primary nucleation during the mineralization of bone , were found in calcified atherosclerotic plaques .
these findings suggest that vascular calcification is an active and highly regulated process that is similar to bone formation . however , although a number of studies explored the mechanism of vascular calcification , it remains poorly understood .
metabolic flexibility is the capacity of the body to adjust fuel oxidation on the basis of nutrient availability .
competition between glucose and fatty acids for fuel oxidation is primarily controlled by the pyruvate dehydrogenase complex ( pdc ) .
pdc is a mitochondrial enzyme complex that regulates the entry of glycolytic products into the tricarboxylic acid ( tca ) cycle by converting pyruvate into acetyl coenzyme a ( acetyl - coa ) in mammalian cells .
pdc is relatively active in a fed state and stimulates glucose oxidation to produce energy from glucose or convert it to fat for energy storage in peripheral tissues .
however , inhibition of pdc activity by pyruvate dehydrogenase kinase ( pdk)-dependent phosphorylation reduces glucose oxidation and provides three - carbon substrates such as pyruvate , lactate , and alanine for gluconeogenesis in a fasted state . to date , four pdk isozymes ( pdk1 , 2 , 3 , and 4 ) have been identified in humans and rodents and are expressed in a tissue - specific manner . among them , pdk4 expression levels were found to be dramatically increased in several peripheral tissues , including skeletal muscle , heart , mammary glands , adipose tissue , kidneys , and liver in fasting or diabetic rodents . in addition , pdk4 knockout mice had lower blood glucose levels in a fasted state than wild - type mice , consistent with an important role for pdk4 in maintaining glucose levels during fasting , and , after feeding a high - fat diet , pdk4 knockout mice exhibited improved glucose tolerance and insulin sensitivity compared with wild - type mice .
in diabetic mice lacking hepatic insulin receptor substrates 1 and 2 , deletion of the pdk4 gene resulted in improvement in hyperglycemia and glucose tolerance .
furthermore , pdk4 deficiency attenuated fat accumulation in the livers of mice fed a high - fat diet .
these results suggest that pdk4 plays an important role in the development of metabolic diseases , including hyperglycemia , insulin resistance , and hepatic steatosis .
a more comprehensive overview of recent research findings regarding the roles of pdks in metabolic diseases is provided by other excellent reviews . in this review , we will summarize recent studies on the mechanism of vascular calcification and discuss our recent findings regarding the role of pdk4 in the development of vascular calcification .
accumulating evidence suggests that a phenotypic change in vascular smooth muscle cells ( vsmcs ) plays a critical role in the development of vascular calcification . before the initiation of vascular calcification , vsmcs undergo a phenotypic change from a contractile to a synthetic and osteochondrogenic phenotype .
this phenotype change is accompanied by downregulated expression of vsmc contractile markers such as smooth muscle -actin and smooth muscle 22 and upregulated expression of osteochondrogenic markers such as osteocalcin , osteopontin , and alkaline phosphatase .
bone morphogenetic proteins ( bmps ) provide essential signals for determining cell fate , embryonic patterning , organogenesis , and the postnatal remodeling of diverse tissues .
bmps form the largest group of proteins within the transforming growth factor superfamily , and more than 20 subtypes of bmps have been identified . among them , bmp2 is well known to play a role in the development of vascular calcification .
bmp2 was found to be expressed in human calcified atherosclerotic plaques , and smooth muscle - specific overexpression of bmp2 in apolipoprotein e ( apoe)-deficient mice accelerated vascular calcification .
in addition , pharmacological inhibition of bmp signaling ameliorated vascular calcification in low density lipoprotein receptor ( ldlr)-deficient mice , suggesting that bmp signaling plays an important role in the development of vascular calcification .
interestingly , a number of studies provide evidence that bmp2 signaling contributes significantly to the transdifferentiation of vsmcs into osteochondrogenic cells . under atherosclerotic calcifying conditions , bmp2 binds
after activation by the type ii receptors , the type i receptors phosphorylate small mothers against decapentaplegic ( smad ) 1/5/8 to propagate the signal into the cell . smad1/5/8
form heteromeric complexes with smad4 and move into the nucleus , where they assemble into transcriptional machinery that regulates the expression of osteogenic genes .
recently , we found that expression of estrogen - related receptor ( err ) , a member of the orphan nuclear receptor superfamily , is upregulated during in vitro osteogenic differentiation of vsmcs .
adenovirus - mediated overexpression of err in vsmcs induced bmp2 expression , leading to increased phosphorylation of smad1/5/8 .
in addition , inhibition of endogenous err expression or activity using specific sirnas or the selective inverse agonist ameliorated vascular calcification both in vitro and in vivo .
our findings suggest that err plays an important role in the development of vascular calcification by upregulating bmp2 signaling , and that inhibition of err may be a promising therapeutic strategy for preventing vascular calcification .
the best - studied transcription factors regulated by bmp2 signaling are runt - related transcription factor 2 ( runx2 ) and muscle segment homeobox 2 ( msx2 ) .
runx2 is a member of the runt - related transcription factor family and plays an essential role in osteoblast differentiation and bone formation .
multiple signaling pathways , including the bmp2 pathway , converge on runx2 to induce osteoblast differentiation .
runx2 regulates the expression of osteochondrogenic markers , including osteocalcin , osteopontin , and alkaline phosphatase .
although runx2 is not expressed in normal vessels , it is abundantly expressed in calcified human vessels and calcified vsmcs in mice .
previous studies demonstrated that functional inactivation of runx2 by dominant - negative mutations or knockdown prevents calcification in vsmcs , while its overexpression stimulates calcification , suggesting that runx2 is essential for the osteochondrogenic phenotype change in vsmcs .
furthermore , smooth muscle - specific deficiency of runx2 markedly inhibited vascular calcification in mice .
msx2 is also a key transcription factor involved in vascular calcification induced by bmp2 signaling .
msx2 is a member of the homeodomain transcription factor family and plays an important role in osteoblast differentiation and bone formation .
previous studies show that bmp2-dependent activation of msx2 promotes the osteogenic differentiation of vsmcs and vascular myofibroblasts . in ldlr - deficient mice ,
a high - fat diet stimulated vascular calcification , and this was accompanied by upregulation of msx2 expression in vessel walls . in addition , transgenic overexpression of msx2 in the vessel wall promoted vascular calcification via activation of canonical wnt signaling .
furthermore , smooth muscle - specific deficiency of msx1 and msx2 attenuated vascular calcification and aortic stiffness in ldlr - deficient mice fed high - fat diets .
the main pathological stimuli that induce the osteochondrogenic phenotype change in vsmcs are oxidative stress , oxylipids , and phosphates . among them
, oxidative stress plays a critical role in the pathogenesis of atherosclerosis and other cardiovascular diseases .
in addition , increased oxidative stress is closely associated with several medical conditions that are linked to an elevated prevalence of vascular calcification , including diabetes mellitus and chronic kidney disease .
several in vitro studies show that oxidative stress can induce an osteochondrogenic phenotype change in vsmcs .
expression of runx2 was found to be involved in oxidative stress - induced osteogenic differentiation and calcification of vsmcs .
furthermore , a recent study showed that antioxidant treatment inhibited osteogenic differentiation of vsmcs and vascular calcification in uremic rats , supporting the idea that antioxidants may represent promising therapeutic agents for the treatment and prevention of vascular calcification .
the transcription factor nuclear factor e2-related factor 2 ( nrf2 ) plays a critical role in cellular antioxidant defenses by activating a wide range of antioxidant genes .
a recent in vitro study demonstrated that nrf2 inhibits osteoblast differentiation through the inhibition of runx2-dependent transcriptional activity .
recently , we found that dimethyl fumarate , a potent synthetic nrf2 activator , inhibits in vitro osteogenic differentiation and calcification of vsmcs , ex vivo calcification of vessel rings , and vitamin d - induced in vivo vascular calcification , suggesting that nrf2 is a potential therapeutic target for the treatment of vascular calcification . several anticalcific molecules , including matrix gla protein ( mgp ) , fetuin - a , and osteoprotegerin ( opg ) , have been identified and these anticalcific molecules play an important role in suppressing vascular calcification under normal conditions . in patients with chronic kidney disease , dysregulation of anticalcific molecules
mgp is an extracellular matrix protein that binds to calcium ions with high affinity and acts as an inhibitor of vascular mineralization .
mgp - deficient mice exhibited vascular calcification , while overexpression of mgp in apoe - deficient mice reduced the amount of vascular calcification .
vitamin k - dependent -carboxylation of glutamate residues is required to convert mgp into its active form .
recent studies show that , in animal models , treatment with therapeutic doses of warfarin , a vitamin k antagonist , stimulates the development of vascular calcification , while treatment with high dietary vitamin k1 inhibits it .
in addition , treatment with warfarin is associated with coronary artery plaque calcification in patients with suspected coronary artery disease .
fetuin - a is a glycoprotein that is secreted from the liver and adipose tissue and is present at high concentrations in human blood , where it binds calcium ions and hydroxyapatite .
fetuin - a inhibited vsmc calcification in vitro , and fetuin - a deficiency in apoe - deficient mice promoted vascular calcification .
low serum levels of fetuin - a are associated with increased vascular calcification and cardiovascular mortality in patients on dialysis .
opg is a phosphoprotein that regulates bone formation by inhibiting apatite crystal growth and osteoclast differentiation .
opg was found to inhibit osteogenic differentiation and calcification in vsmcs , and opg - deficient mice developed osteoporosis and vascular calcification .
in addition , treatment with opg ameliorated warfarin or vitamin d - induced vascular calcification in animal models .
clinical studies show that serum opg levels are significantly higher in patients with chronic kidney disease than in controls , indicating that this increase might be a compensatory response to the disease , rather than a risk factor .
accumulating evidence suggests that matrix vesicles play an important role in the development of vascular calcification .
vascular calcification is initiated both by matrix vesicles released from viable vsmcs and by apoptotic bodies from dying cells .
in addition , several studies show that the growth arrest - specific gene 6 ( gas6)-mediated survival pathway plays a central role in preventing phosphate - induced vsmc apoptosis and calcification
. we also found that -lipoic acid , a naturally occurring antioxidant with anti - apoptotic properties , reduced phosphate - induced vsmc apoptosis and calcification through inhibiting phosphate - induced downregulation of cell survival signals via the binding of gas6 to its cognate receptor axl and subsequent akt activation .
there is increasing evidence suggesting that mitochondrial dysfunction can be an important contributor to the development of atherosclerosis .
mitochondrial dna damage results in decreased mitochondrial function , including impaired respiratory chain function and reduced adenosine triphosphate ( atp ) production , and eventually compromises cellular function . in animal studies , it was shown that mitochondrial dna damage and mitochondrial dysfunction are early events in the development of atherosclerotic lesions and promote progression of atherosclerosis .
furthermore , mitochondrial dna damage was observed in blood cells and atherosclerotic lesions of patients with coronary artery disease .
these findings raise the prospect that mitochondrial dysfunction may induce vascular calcification , because atherosclerosis is a progressive disease that can lead to vascular calcification , which is often found in advanced atherosclerotic lesions .
interestingly , we observed that functional and structural mitochondrial defects , as evidenced by reduced mitochondrial membrane potential , decreased intracellular atp content , increased production of mitochondrial reactive oxygen species , and disruption of mitochondrial structural integrity , in calcifying vsmcs treated with inorganic phosphate .
these results suggest a potential role for mitochondrial dysfunction in vsmc apoptosis and calcification . indeed , mitochondria play an essential role in the regulation of intrinsic apoptotic pathways .
mitochondria - dependent intrinsic apoptosis involves the release of cytochrome c from the inner membrane space to the cytosol , which in turn triggers the activation of caspase-9 and effector caspases , leading to nuclear dna fragmentation and other changes that culminate in apoptotic death . in line with this
, we showed that the protective effect of -lipoic acid against phosphate - induced vsmc apoptosis and calcification can be attributed to the restoration of mitochondrial function as well as to the activation of the gas6/axl / akt survival pathway .
finally , administration of -lipoic acid ameliorated vitamin d - induced vascular calcification and mitochondrial dysfunction in mice .
normal resting cells generate energy by converting glucose into pyruvate via the glycolysis pathway , which does not require oxygen , followed by oxidation reactions in the mitochondria . under normoxic conditions ,
pyruvate produced by glycolysis is transported primarily into the mitochondria and then decarboxylated by pdc into acetyl - coa , which enters the tca cycle . however , under hypoxic conditions , inhibition of pdc prevents the conversion of pyruvate into acetyl - coa , leading to decreased tca cycle activity in the mitochondria and increased conversion of pyruvate into lactate in the cytosol .
interestingly , cancer cells depend largely on glycolysis for energy , even though sufficient oxygen is available .
cancer cells meet the requirement for energy and biosynthetic precursors for proliferation and metastasis through aerobic glycolysis .
dichloroacetate , an inhibitor of pdk , blocks aerobic glycolysis and causes cancer cell apoptosis and tumor regression , suggesting that pdk is a novel therapeutic target for cancer treatment .
recently , accumulating evidence suggests that aerobic glycolysis also plays a critical role in meeting the demand for energy and biosynthetic precursors during proliferation and differentiation of other types of cells , including immune cells .
given that osteogenic differentiation of vsmcs is critical for the development of vascular calcification , and that this process may require glucose metabolism similar to the warburg effect to produce energy and the necessary biosynthetic precursors , it is reasonable to hypothesize that pdk plays an important role in metabolic regulation of the osteogenic switch in vsmcs .
indeed , our unpublished observations indicate that glucose consumption and lactate production are increased in phosphate - induced vsmc calcification . furthermore , because it is suggested that mitochondrial dysfunction is a metabolic feature that controls the vsmc phenotype , pdk may regulate the osteogenic switch in vsmcs by controlling mitochondrial function .
recently , we observed that expression of pdk4 and phosphorylation of pdc are increased in calcifying vsmcs and calcified vessels of patients with atherosclerosis .
interestingly , the mrna and protein levels of pdk4 were markedly increased in a time- and dose - dependent manner , whereas expression of other pdk isozymes was not significantly changed in phosphate - treated human vsmcs , suggesting that pdk4 plays a specific role in phosphate - induced osteogenic differentiation and calcification of vsmcs .
we also demonstrated that both genetic and pharmacological inhibition of pdk4 ameliorated in vitro calcification of vsmcs , ex vivo calcification of aortic rings , and vitamin d - induced in vivo calcification . to gain mechanistic insight into how pdk4 modulates vascular calcification , we examined the role of pdk4 in regulation of the osteogenic switch in vsmcs .
adenovirus - mediated overexpression of pdk4 increased , while pdk4 deficiency decreased , the expression of osteogenic genes in human vsmcs .
we previously reported that -lipoic acid , which also inhibits pdk4 activity , ameliorates vascular calcification by improving mitochondrial function .
consistent with this , overexpression of pdk4 induced mitochondrial dysfunction in vitro , as evidenced by decreased atp content , oxygen consumption rate , and maximal respiration capacity , and this was reversed by treatment with an inhibitor of pdk .
these results suggest that pdk4 induces mitochondrial dysfunction , which may contribute to the development of vascular calcification .
since pdk4 increased the expression of several osteogenic genes that are induced by bmp2 with no change in bmp2 expression , we explored the possibility that pdk4 may regulate the signaling pathway downstream of bmp2 without affecting the expression of bmp2 itself .
interestingly , we found that , under calcifying conditions , pdk4 phosphorylates and activates smad1/5/8 , which leads to translocation of phosphorylated smads into the nucleus for the transcriptional regulation of osteogenic markers , thus enhancing bmp2 signaling pathway activity ( fig .
a direct interaction between pdk4 and smad seems unlikely , because pdk4 is located in the mitochondrial matrix .
however , using various methods , including a binding prediction model , confocal imaging analysis , immunoblots of subcellular fractions , co - immunoprecipitation , glutathione s - transferase pull - down assay , and in vitro kinase assay , we demonstrated that , after being transported from mitochondria into cytosol in response to calcifying stimuli , pdk4 can directly interact with and phosphorylate smad1/5/8 , .
this finding is consistent with a previous report showing that the mammalian pdk4 ortholog in caenorhabditis elegans is located in the cytosol as well as the mitochondria . finally , we evaluated whether the inhibitory effect of vascular calcification adversely affects bone remodeling , since vascular calcification shares many similarities with physiological bone formation .
bone remodeling and osteoblastic differentiation in pre - osteoblasts were found to not be adversely affected by pdk4 deficiency , even though pdk4 promotes osteogenic differentiation of vsmcs in response to calcifying stimuli .
these results indicate that deletion of pdk4 effectively attenuates vascular calcification without adverse effects on bone remodeling .
accumulating evidence suggests that a phenotypic change in vascular smooth muscle cells ( vsmcs ) plays a critical role in the development of vascular calcification . before the initiation of vascular calcification , vsmcs undergo a phenotypic change from a contractile to a synthetic and osteochondrogenic phenotype .
this phenotype change is accompanied by downregulated expression of vsmc contractile markers such as smooth muscle -actin and smooth muscle 22 and upregulated expression of osteochondrogenic markers such as osteocalcin , osteopontin , and alkaline phosphatase .
bone morphogenetic proteins ( bmps ) provide essential signals for determining cell fate , embryonic patterning , organogenesis , and the postnatal remodeling of diverse tissues .
bmps form the largest group of proteins within the transforming growth factor superfamily , and more than 20 subtypes of bmps have been identified . among them , bmp2 is well known to play a role in the development of vascular calcification .
bmp2 was found to be expressed in human calcified atherosclerotic plaques , and smooth muscle - specific overexpression of bmp2 in apolipoprotein e ( apoe)-deficient mice accelerated vascular calcification .
in addition , pharmacological inhibition of bmp signaling ameliorated vascular calcification in low density lipoprotein receptor ( ldlr)-deficient mice , suggesting that bmp signaling plays an important role in the development of vascular calcification .
interestingly , a number of studies provide evidence that bmp2 signaling contributes significantly to the transdifferentiation of vsmcs into osteochondrogenic cells . under atherosclerotic calcifying conditions , bmp2 binds
after activation by the type ii receptors , the type i receptors phosphorylate small mothers against decapentaplegic ( smad ) 1/5/8 to propagate the signal into the cell . smad1/5/8
form heteromeric complexes with smad4 and move into the nucleus , where they assemble into transcriptional machinery that regulates the expression of osteogenic genes .
recently , we found that expression of estrogen - related receptor ( err ) , a member of the orphan nuclear receptor superfamily , is upregulated during in vitro osteogenic differentiation of vsmcs .
adenovirus - mediated overexpression of err in vsmcs induced bmp2 expression , leading to increased phosphorylation of smad1/5/8 .
in addition , inhibition of endogenous err expression or activity using specific sirnas or the selective inverse agonist ameliorated vascular calcification both in vitro and in vivo .
our findings suggest that err plays an important role in the development of vascular calcification by upregulating bmp2 signaling , and that inhibition of err may be a promising therapeutic strategy for preventing vascular calcification .
the best - studied transcription factors regulated by bmp2 signaling are runt - related transcription factor 2 ( runx2 ) and muscle segment homeobox 2 ( msx2 ) .
runx2 is a member of the runt - related transcription factor family and plays an essential role in osteoblast differentiation and bone formation .
multiple signaling pathways , including the bmp2 pathway , converge on runx2 to induce osteoblast differentiation .
runx2 regulates the expression of osteochondrogenic markers , including osteocalcin , osteopontin , and alkaline phosphatase .
although runx2 is not expressed in normal vessels , it is abundantly expressed in calcified human vessels and calcified vsmcs in mice .
previous studies demonstrated that functional inactivation of runx2 by dominant - negative mutations or knockdown prevents calcification in vsmcs , while its overexpression stimulates calcification , suggesting that runx2 is essential for the osteochondrogenic phenotype change in vsmcs .
furthermore , smooth muscle - specific deficiency of runx2 markedly inhibited vascular calcification in mice .
msx2 is also a key transcription factor involved in vascular calcification induced by bmp2 signaling .
msx2 is a member of the homeodomain transcription factor family and plays an important role in osteoblast differentiation and bone formation .
previous studies show that bmp2-dependent activation of msx2 promotes the osteogenic differentiation of vsmcs and vascular myofibroblasts . in ldlr - deficient mice ,
a high - fat diet stimulated vascular calcification , and this was accompanied by upregulation of msx2 expression in vessel walls . in addition , transgenic overexpression of msx2 in the vessel wall promoted vascular calcification via activation of canonical wnt signaling .
furthermore , smooth muscle - specific deficiency of msx1 and msx2 attenuated vascular calcification and aortic stiffness in ldlr - deficient mice fed high - fat diets .
the main pathological stimuli that induce the osteochondrogenic phenotype change in vsmcs are oxidative stress , oxylipids , and phosphates . among them
, oxidative stress plays a critical role in the pathogenesis of atherosclerosis and other cardiovascular diseases .
in addition , increased oxidative stress is closely associated with several medical conditions that are linked to an elevated prevalence of vascular calcification , including diabetes mellitus and chronic kidney disease .
several in vitro studies show that oxidative stress can induce an osteochondrogenic phenotype change in vsmcs .
expression of runx2 was found to be involved in oxidative stress - induced osteogenic differentiation and calcification of vsmcs .
furthermore , a recent study showed that antioxidant treatment inhibited osteogenic differentiation of vsmcs and vascular calcification in uremic rats , supporting the idea that antioxidants may represent promising therapeutic agents for the treatment and prevention of vascular calcification .
the transcription factor nuclear factor e2-related factor 2 ( nrf2 ) plays a critical role in cellular antioxidant defenses by activating a wide range of antioxidant genes .
a recent in vitro study demonstrated that nrf2 inhibits osteoblast differentiation through the inhibition of runx2-dependent transcriptional activity .
recently , we found that dimethyl fumarate , a potent synthetic nrf2 activator , inhibits in vitro osteogenic differentiation and calcification of vsmcs , ex vivo calcification of vessel rings , and vitamin d - induced in vivo vascular calcification , suggesting that nrf2 is a potential therapeutic target for the treatment of vascular calcification .
several anticalcific molecules , including matrix gla protein ( mgp ) , fetuin - a , and osteoprotegerin ( opg ) , have been identified and these anticalcific molecules play an important role in suppressing vascular calcification under normal conditions . in patients with chronic kidney disease , dysregulation of anticalcific molecules may contribute to the development and progression of vascular calcification .
mgp is an extracellular matrix protein that binds to calcium ions with high affinity and acts as an inhibitor of vascular mineralization .
mgp - deficient mice exhibited vascular calcification , while overexpression of mgp in apoe - deficient mice reduced the amount of vascular calcification .
vitamin k - dependent -carboxylation of glutamate residues is required to convert mgp into its active form .
recent studies show that , in animal models , treatment with therapeutic doses of warfarin , a vitamin k antagonist , stimulates the development of vascular calcification , while treatment with high dietary vitamin k1 inhibits it .
in addition , treatment with warfarin is associated with coronary artery plaque calcification in patients with suspected coronary artery disease .
fetuin - a is a glycoprotein that is secreted from the liver and adipose tissue and is present at high concentrations in human blood , where it binds calcium ions and hydroxyapatite .
fetuin - a inhibited vsmc calcification in vitro , and fetuin - a deficiency in apoe - deficient mice promoted vascular calcification .
low serum levels of fetuin - a are associated with increased vascular calcification and cardiovascular mortality in patients on dialysis .
opg is a phosphoprotein that regulates bone formation by inhibiting apatite crystal growth and osteoclast differentiation .
opg was found to inhibit osteogenic differentiation and calcification in vsmcs , and opg - deficient mice developed osteoporosis and vascular calcification .
in addition , treatment with opg ameliorated warfarin or vitamin d - induced vascular calcification in animal models .
clinical studies show that serum opg levels are significantly higher in patients with chronic kidney disease than in controls , indicating that this increase might be a compensatory response to the disease , rather than a risk factor .
accumulating evidence suggests that matrix vesicles play an important role in the development of vascular calcification .
vascular calcification is initiated both by matrix vesicles released from viable vsmcs and by apoptotic bodies from dying cells .
in addition , several studies show that the growth arrest - specific gene 6 ( gas6)-mediated survival pathway plays a central role in preventing phosphate - induced vsmc apoptosis and calcification
. we also found that -lipoic acid , a naturally occurring antioxidant with anti - apoptotic properties , reduced phosphate - induced vsmc apoptosis and calcification through inhibiting phosphate - induced downregulation of cell survival signals via the binding of gas6 to its cognate receptor axl and subsequent akt activation .
there is increasing evidence suggesting that mitochondrial dysfunction can be an important contributor to the development of atherosclerosis .
mitochondrial dna damage results in decreased mitochondrial function , including impaired respiratory chain function and reduced adenosine triphosphate ( atp ) production , and eventually compromises cellular function . in animal studies , it was shown that mitochondrial dna damage and mitochondrial dysfunction are early events in the development of atherosclerotic lesions and promote progression of atherosclerosis .
furthermore , mitochondrial dna damage was observed in blood cells and atherosclerotic lesions of patients with coronary artery disease .
these findings raise the prospect that mitochondrial dysfunction may induce vascular calcification , because atherosclerosis is a progressive disease that can lead to vascular calcification , which is often found in advanced atherosclerotic lesions .
interestingly , we observed that functional and structural mitochondrial defects , as evidenced by reduced mitochondrial membrane potential , decreased intracellular atp content , increased production of mitochondrial reactive oxygen species , and disruption of mitochondrial structural integrity , in calcifying vsmcs treated with inorganic phosphate .
mitochondria - dependent intrinsic apoptosis involves the release of cytochrome c from the inner membrane space to the cytosol , which in turn triggers the activation of caspase-9 and effector caspases , leading to nuclear dna fragmentation and other changes that culminate in apoptotic death . in line with this
, we showed that the protective effect of -lipoic acid against phosphate - induced vsmc apoptosis and calcification can be attributed to the restoration of mitochondrial function as well as to the activation of the gas6/axl / akt survival pathway .
finally , administration of -lipoic acid ameliorated vitamin d - induced vascular calcification and mitochondrial dysfunction in mice .
normal resting cells generate energy by converting glucose into pyruvate via the glycolysis pathway , which does not require oxygen , followed by oxidation reactions in the mitochondria . under normoxic conditions ,
pyruvate produced by glycolysis is transported primarily into the mitochondria and then decarboxylated by pdc into acetyl - coa , which enters the tca cycle . however
, under hypoxic conditions , inhibition of pdc prevents the conversion of pyruvate into acetyl - coa , leading to decreased tca cycle activity in the mitochondria and increased conversion of pyruvate into lactate in the cytosol .
interestingly , cancer cells depend largely on glycolysis for energy , even though sufficient oxygen is available .
cancer cells meet the requirement for energy and biosynthetic precursors for proliferation and metastasis through aerobic glycolysis .
dichloroacetate , an inhibitor of pdk , blocks aerobic glycolysis and causes cancer cell apoptosis and tumor regression , suggesting that pdk is a novel therapeutic target for cancer treatment .
recently , accumulating evidence suggests that aerobic glycolysis also plays a critical role in meeting the demand for energy and biosynthetic precursors during proliferation and differentiation of other types of cells , including immune cells .
given that osteogenic differentiation of vsmcs is critical for the development of vascular calcification , and that this process may require glucose metabolism similar to the warburg effect to produce energy and the necessary biosynthetic precursors , it is reasonable to hypothesize that pdk plays an important role in metabolic regulation of the osteogenic switch in vsmcs .
indeed , our unpublished observations indicate that glucose consumption and lactate production are increased in phosphate - induced vsmc calcification . furthermore , because it is suggested that mitochondrial dysfunction is a metabolic feature that controls the vsmc phenotype , pdk may regulate the osteogenic switch in vsmcs by controlling mitochondrial function .
recently , we observed that expression of pdk4 and phosphorylation of pdc are increased in calcifying vsmcs and calcified vessels of patients with atherosclerosis .
interestingly , the mrna and protein levels of pdk4 were markedly increased in a time- and dose - dependent manner , whereas expression of other pdk isozymes was not significantly changed in phosphate - treated human vsmcs , suggesting that pdk4 plays a specific role in phosphate - induced osteogenic differentiation and calcification of vsmcs .
we also demonstrated that both genetic and pharmacological inhibition of pdk4 ameliorated in vitro calcification of vsmcs , ex vivo calcification of aortic rings , and vitamin d - induced in vivo calcification . to gain mechanistic insight into how pdk4 modulates vascular calcification
, we examined the role of pdk4 in regulation of the osteogenic switch in vsmcs .
adenovirus - mediated overexpression of pdk4 increased , while pdk4 deficiency decreased , the expression of osteogenic genes in human vsmcs .
we previously reported that -lipoic acid , which also inhibits pdk4 activity , ameliorates vascular calcification by improving mitochondrial function .
consistent with this , overexpression of pdk4 induced mitochondrial dysfunction in vitro , as evidenced by decreased atp content , oxygen consumption rate , and maximal respiration capacity , and this was reversed by treatment with an inhibitor of pdk .
these results suggest that pdk4 induces mitochondrial dysfunction , which may contribute to the development of vascular calcification .
since pdk4 increased the expression of several osteogenic genes that are induced by bmp2 with no change in bmp2 expression , we explored the possibility that pdk4 may regulate the signaling pathway downstream of bmp2 without affecting the expression of bmp2 itself .
interestingly , we found that , under calcifying conditions , pdk4 phosphorylates and activates smad1/5/8 , which leads to translocation of phosphorylated smads into the nucleus for the transcriptional regulation of osteogenic markers , thus enhancing bmp2 signaling pathway activity ( fig .
a direct interaction between pdk4 and smad seems unlikely , because pdk4 is located in the mitochondrial matrix .
however , using various methods , including a binding prediction model , confocal imaging analysis , immunoblots of subcellular fractions , co - immunoprecipitation , glutathione s - transferase pull - down assay , and in vitro kinase assay , we demonstrated that , after being transported from mitochondria into cytosol in response to calcifying stimuli , pdk4 can directly interact with and phosphorylate smad1/5/8 , .
this finding is consistent with a previous report showing that the mammalian pdk4 ortholog in caenorhabditis elegans is located in the cytosol as well as the mitochondria .
finally , we evaluated whether the inhibitory effect of vascular calcification adversely affects bone remodeling , since vascular calcification shares many similarities with physiological bone formation .
bone remodeling and osteoblastic differentiation in pre - osteoblasts were found to not be adversely affected by pdk4 deficiency , even though pdk4 promotes osteogenic differentiation of vsmcs in response to calcifying stimuli .
these results indicate that deletion of pdk4 effectively attenuates vascular calcification without adverse effects on bone remodeling .
the osteogenic phenotypic change in vsmcs plays a key role in the development of vascular calcification .
accumulating evidence suggests that mitochondrial dysfunction is a characteristic feature of osteogenic differentiation in vsmcs . in this review
, we summarized our knowledge of the main mechanisms underlying vascular calcification , and discussed the role of pdk4 in the molecular and metabolic processes that contribute to the osteogenic switch in vsmcs during the development of vascular calcification .
although further studies are required to ascertain the metabolic changes that occur in vsmcs during osteogenic differentiation and vascular calcification , and their relationship with pdk4 , the current evidence indicates that pdk4 may be a promising therapeutic target for the treatment of vascular calcification .
understanding the mechanisms of vascular calcification will be crucial for the development of novel therapeutic strategies against vascular calcification . | vascular calcification , abnormal mineralization of the vessel wall , is frequently associated with aging , atherosclerosis , diabetes mellitus , and chronic kidney disease .
vascular calcification is a key risk factor for many adverse clinical outcomes , including ischemic cardiac events and subsequent cardiovascular mortality .
vascular calcification was long considered to be a passive degenerative process , but it is now recognized as an active and highly regulated process similar to bone formation . however , despite numerous studies on the pathogenesis of vascular calcification , the mechanisms driving this process remain poorly understood .
pyruvate dehydrogenase kinases ( pdks ) play an important role in the regulation of cellular metabolism and mitochondrial function .
recent studies show that pdk4 is an attractive therapeutic target for the treatment of various metabolic diseases . in this review ,
we summarize our current knowledge regarding the mechanisms of vascular calcification and describe the role of pdk4 in the osteogenic differentiation of vascular smooth muscle cells and development of vascular calcification .
further studies aimed at understanding the molecular mechanisms of vascular calcification will be critical for the development of novel therapeutic strategies . |
outpatients with mucocele of the glands of blandin nuhn treated with absolute ethanol at the department of oral and maxillofacial surgery , tianjin hospital for stomatology , totaled 14 patients from 2013 to 2015 . of them ,
the age range was from 6 to 30 years , with an average age of 14.4 years .
lesion dimensions ranged from 0.2 0.3 to 1.5 1 cm , with an average dimension of 0.9 0.6 cm .
mucoceles were observed on the midline ventral surface of the tongue tip in 10 patients , and the lesions were located on the left and the right of the midline in 3 and 1 patients , respectively .
after blood investigation , the lesion and its adjacent mucosa were disinfected , followed by performing superficial anesthesia with 2% lidocaine .
various doses of absolute ethanol ( from 0.1 to 0.5 ml ) , according to the volume of mucocele , were injected into the mucous cavities until the superficial mucosae of the lesions turned pale .
all patients were informed return visit after a week and continued with out - patient follow - up for 3 months to 2 years .
mucoceles disappeared a week after injection therapy in 9 patients , the lesion size diminished in 5 patients , 2 of whom and the other 3 received injection once and twice later at once a week , respectively .
the chief complaint was slight swelling and pain occurred on the same day when the patients were treated .
all patients were without clinical evidence of recurrence during the follow - up period ( fig .
1 ) . the effect of treatment in a patient with mucocele of the glands of blandin nuhn before and after injection of absolute ethanol . ( a ) clinical presentations ( the black arrow ) before injection of absolute ethanol .
( b ) the mucocele was disappeared a week after the patient was given an injection .
the glands of blandin nuhn resembling a horseshoe , namely the anterior lingual glands , are located near the tip of the tongue and embedded within muscle beneath the tongue ventrum .
so , mucocele will be prone to occur when the glands are injured , and an incomplete removement of the lesion results in recurrence more easily as well as very soon . surgical excision of the lesion including blandin
it is sometimes important of a clear demarcation of the lesion to perform a complete and easier surgery , because collapse of the lesion resulting from an extravasation of mucos or its recurrence may lead to an inconvenient exploration during the operation . to improve the visual access , an alternative method is injection of ultraflow rubber base impression material or alginate impression material into the lesion presurgically , but the method is suggested to not be used because of a foreign body reaction by some scholars . if the lesion is on the midline ventral surface of the tongue , it will be necessary to excise the bilateral glands for preventing the recurrence
. meanwhile , the younger patients must be hospitalized and undergo the surgery under general anesthesia , which may not be accepted by some parents .
absolute ethanol as a sclerosant proved to be safe and effective , has been applied in treatment of renal tumors , cysts , and vascular malformations . to the best of our knowledge , there is no report about absolute ethanol injection for treating the mucocele .
the new method was attempted for observing the effect of therapy . in the study , although some patients , whose lesions were larger in size , received more than 1 intralesional injection , a satisfactory result was achieved without severe comorbidity .
the patients or their parents could actively cooperate for the evident effect and the simple process . according to the mucous ingredients and the histological characterization that a torn end of a main duct of a minor salivary gland is found to communicate with the mucus pool surrounded by connective tissue
, it is our speculation , for the functionary mechanism of absolute ethanol , that it denature the ingredients to deposit and make the epithelial cells near the gap of a mucous tubule dehydration and necrosis , which may lead to an inflammatory reaction followed by development of scars that can close the gap to prevent the mucus from overbrimming . in conclusion ,
injection of absolute ethanol is a simple , mininvasive , effective , and economic method with few complications for therapy of mucocele compared with the surgery . | abstractmucocele of the anterior lingual salivary glands is a more common cystic lesion , especially in patients aged less than 20 years .
the study is aimed to observe the effect of treatment by injection of absolute ethanol instead of surgery .
fourteen outpatients diagnosed as mucocele of the glands of blandin nuhn were selected .
these patients , after blood investigation , were treated by injection of absolute ethanol into a mucous cavity of lesion under superficial anesthesia with 2% lidocaine once a week and followed up from 3 months to 2 years .
mucocele of the glands of blandin
nuhn was extirpated in all patients after 1 to 3 injections .
there were no other complications except slight distending pain occurred on the same day when the patients were treated .
the recurrence was not observed during the follow - up period . in conclusion
, the study suggests that injection of absolute ethanol may be an alternative means for treating mucocele of the glands of blandin nuhn , because it is mininvasive , safe , effective , economic , and simply manipulated compared with surgical treatment . |
a 73-year - old man was admitted to our intensive care unit ( icu ) via the emergency department with severe diarrhea , oliguria , and generalized edema .
the patient 's vital signs were blood pressure ( bp ) 85/50 mmhg , heart rate 120 beats / min , and drowsy mental status .
his medical history included diabetes mellitus for 5 years with the operation of coil embolization in the right posterior communicating artery aneurysm 3 years previously .
on icu admission , urinalysis revealed protein 4 + and serum albumin 2.1 g / dl .
also , he was diagnosed with prerenal acute kidney injury due to severe dehydration with laboratory test results of blood urea nitrogen 63 and creatinine ( cr ) 3.0 .
brain computed tomography ( ct ) showed no remarkable findings and mental status recovered with adequate fluid therapy .
after the kidney biopsy , he was diagnosed with nephrotic syndrome with minimal change disease and prednisolone was administered .
the physician in charge of the patient judged that he had a relatively slight nephrotic syndrome with minimal change .
therefore , further evaluation , such as echocardiography was not considered as a treatment plan except for laboratory test and renal dialysis .
he was determined to take dialysis and a 12 french , 2 lumen hemodialysis catheter ( niagara , slimcath catheter ; bard , usa ) was inserted in the right internal jugular vein .
vital signs were stable and the patient was transferred to the general ward . because his renal function was improving , the physician decided to remove the hemodialysis catheter . with the patient in the supine position holding his breath ,
the catheter was removed slowly from the right internal jugular vein while the exit site was pressed with gauze for 5 minutes .
after about 10 minutes , the spouse of another patient informed staff that the patient had suddenly slumped onto the floor of the ward .
the attending physician and nurses entered the room and found the patient lying unconscious on the floor .
bp and pulse was not checked . mental status was coma and glasgow coma scale score was 3 .
the electrocardiogram recordings showed increase in st segment of ii , iii , and avf leads ( fig .
, air embolism due to catheter removal was not diagnosed . with a diagnosis of acute myocardial infarction at the inferior wall , coronary angiography ( cag )
the angiography showed slow blood flow pattern which was compatible with thrombolysis in myocardial infarction ( timi ) grade 1 with multiple air bubbles in the mid segment of the right coronary artery ( fig .
after the air passed , blood flow in the artery totally recovered to timi grade 3 .
the final diagnosis was acute myocardial infarction due to coronary air embolism because any narrowing site or thrombus was not found in coronary arteries as the cause of obstruction except for air bubbles .
however , the patient was still pulseless . to assist the patient 's impaired cardiopulmonary function ,
coronary air embolism is a rare complication of coronary catheterization , with a reported incidence of about 0.1% . .
also , it can occur as a result of venous air embolism by many factors other than coronary catheterization . in one report , venous air embolism that occurred from uterus exteriorization during a cesarean section caused coronary air embolism through patent foramen ovale ( pfo ) .
the mechanism by which venous air penetrate into the arterial circulation is responsible for the paradoxical embolism by pfo . in the case
presented , echocardiography was not performed because air embolism was not suspected to be the cause of cardiac arrest at first . and after the cag , no diagnostic procedure for further evaluation could be performed due to low possibility of resuscitation .
it was an imperfective point of our case that we were unable to demonstrate the presence of a pfo .
it is possible that the patient in our case had a pfo . if not , the filtrating property of the lungs is limited to the amount of air into the vein .
therefore , air in the vein can be transported to the artery by physiologic shunting of systemic circulation via pulmonary capillaries . in one report ,
air embolism in the left ventricle occurred by the removal of a central venous catheter with echocardiographic finding which showed no pfo .
it is especially risky to have air bubbles in arteries supplying the brain or the heart because these organs are most vulnerable to hypoxia .
signs and symptoms of coronary air embolism include chest pain , hypotension , transient electrocardiography changes consistent with myocardial ischemia , arrhythmias ( bradycardia , heart block , ventricular tachycardia , and ventricular fibrillation ) , and even cardiac arrest
. the severity of cardiac impairment caused by coronary air embolism can be different from the amount of air injected into coronary artery and the patient 's cardiac function state .
a study in dogs showed that injection of 0.02 ml / kg air into the coronary artery resulted in a 28% rate of mortality . also , khan et al
. classified the magnitude of air emboli based on the angiographic findings and severity of symptoms .
they defined " massive emboli " as the injection of air bubbles greater than 35 ml , with the condition associated with serious complications .
the present case had 3.5 ml of air bubbles in the right coronary artery , as estimated by the amount of air bubbles in the length of the right coronary mid segment observed in the cag , the artery diameter , and the time of air passage .
considering the amount of air into the flow and fatal complications like acute coronary syndrome and severe pulmonary edema , the present case is consistent with the definition of massive air emboli .
coronary air embolism can be conclusively diagnosed by the observation of air passing through the coronary artery in fluoroscopy or angiography . in general , air emboli passing distal to coronary arterial circulation results in slow flow phenomenon in epicardial vessels .
the treatment of coronary air embolism is most often the forceful injection of the patient 's blood or saline to propel the air bubbles to the terminal portion of the coronary artery during angiography .
other treatments are breaking the air bubbles using guide wires or aspirating the air using a thrombectomy catheter .
if the amount of air is minimal , administration of 100% oxygen for 30 minutes can displace nitrogen from the air emboli , which causes shrinkage of the air bubbles .
venous air embolism by cvc removal occurs when a fistula is formed between the skin and the vena cava due to a long - standing catheter .
fibrin tract may be formed around the catheter , usually after a long stay , but sometimes even within 24 hours .
fibrin tract can be visible more than 24 hours after catheter removal and air occlusive dressing .
therefore , the time of cvc should be as short as possible to avoid fistula formation between the central vein and the skin .
pressure should be firmly applied on the exit site for more than 10 minutes and dressing maintained for at least 24 hours .
even if an air embolism occurs , the symptoms ( from asymptomatic to fatal ) depend on the volume and speed of the air .
the factors determining the volume and speed of the air bubbles are the size of catheter lumen and the pressure gradient between the atmosphere and the patient 's central vein . in the case of humans
, death can occur when 200300 ml of air is delivered into the right ventricle .
a 14 gauge catheter enables gas flow at the rate of 100 ml / s and a pressure of 5 cmh2o .
a double lumen hemodialysis catheter ( 12 french , 8.5 gauge ) was used in the present case .
supposing that the pressure difference is 5 cmh2o , the volume flow rate of air capable passing through the catheter is calculated to be about 1280.99 ml / s by a modification of poiseuille 's law ( q = r(p1-p2)/8 nl , r = radius of tube , p1-p2 = pressure difference , n = viscosity of fluid , l = length of tube ) .
when the air penetrates the vascular system some extent , air inflow will be blocked due to reversal of the pressure difference between the central vein and the atmosphere .
however , it should be kept in mind that the velocity of air influx will be extremely fast even though the diameter of the catheter increases a little bit according to poiseuille 's law , in which the fluid speed is proportional to the four - square of the radius .
it can be helpful to place the patient in the trendelenburg position with the head pointing down at an angle of 1030 during catheter removal . because the venous pressure increases and therefore , the pressure difference decreases , less air will enter the blood . using the same principle , in the case of co - operational patients ,
it is much safer to have the patient hold their breath or expire air during catheter removal .
when air embolism is detected , it is important to discover abnormal air in the heart , vena cava , or brain through echocardiography or ct scan images . in our case ,
ct or echocardiography was not performed because air embolism was not suspected at first . in general ,
when venous air embolism occurs , an electrocardiogram shows right ventricular strain pattern ( t wave inversion , st segment depression ) as right ventricular outflow tract is obstructed .
but the electrocardiogram at cardiac arrest in our case showed st segment elevation , which was suggestive of acute myocardial infarction and the patient had a nephrotic syndrome accompanied by thrombosis .
therefore , he was first diagnosed with acute myocardial infarction due to thrombus and an emergent cag was performed . if he were diagnosed with venous air embolism at first , ct or echocardiography would have been done , which would likely have discovered any air bubbles in the cardiac cavities . a chest x - ray taken after the cag shows severe pulmonary edema as the distinctive " butterfly " pattern .
this is thought to be the result of non - cardiogenic pulmonary edema from secretion of vasoactive mediators by pulmonary vascular obstruction and the cardiogenic pulmonary edema from acute myocardial infarction by coronary air embolism .
first , stop the procedure that is creating the air embolism and turn the patient to the left lateral trendelenburg position , to maintain air in the right atrium and prevent further air entry to the right ventricle .
it decreases the size of air bubbles by denitrification and improves the arterial oxygen gas saturation treating organ from ischemic damage .
when cvc is inserted , special care and attention is necessary from a highly qualified specialist .
however , there are many cases in which a relatively inexperienced physician removes cvc and neglects prevention . in the present case ,
a hemodialysis double lumen catheter was inserted to treat acute kidney injury , and it was well treated .
but , air embolism was not prevented during catheter removal , resulting in the patient 's death .
the physician should have thought about the possibility about any fibrin tract due to a long - standing catheter and taken more attention to use air occlusive dressing , ensure the best position for the patient , and keep pressure on the exit site for the required length of time .
video : coronary angiography showing air bubbles passing the mid segment of right coronary artery . | coronary air embolism is a rare event .
we report a case in which an acute myocardial infarction occurred in the region supplied by the right coronary artery after the removal of a double - lumen hemodialysis catheter .
emergent coronary angiography revealed air bubbles obstructing the mid - segment of the right coronary artery with slow flow phenomenon distally .
the patient expired due to myocardial infarction . |
although differential diagnosis of deep t - wave inversion includes life - threatening conditions , such as myocardial infarction and stroke , even a thorough workup may not reveal its etiology 2 .
deep t - wave inversion is commonly associated with takotsubo cardiomyopathy or takotsubo - like myocardial dysfunction 3 , but this can not be diagnosed without wall motion abnormalities 4 .
we experienced an otherwise healthy elderly woman in whom deep t - wave inversion of unknown origin was incidentally found and who developed typical takotsubo cardiomyopathy 10 years later .
our findings provide evidence that isolated deep t - wave inversion is an after - effect of takotsubo cardiomyopathy .
in 2003 , 10 years before the present admission , the electrocardiogram ( ecg ) of a 73-year - old healthy woman showed deep t - wave inversion during an annual health check .
she recalled no chest symptoms , and her ecg at a health check 6 months previously was negative .
her vital signs were normal , except for a blood pressure of 178/80 mmhg .
the ecg showed negative t waves in leads ii , iii , avf , and v26 ( fig.1a ) .
days after discharge , the t - wave inversion was much shallower ( fig.1b ) , and 3 months later , it had disappeared ( fig.1c ) .
we treated the hypertension with candesartan cilexetil , and 6 months later , her blood pressure was under control . in 2013 , 10 years later , after hearing of the death of her grandchild , she became short of breath and experienced chest pain . on admission 7 h after the onset of symptoms , she was still in acute distress , although she appeared alert and well oriented .
( a ) ecg on day 1 shows negative t waves in leads ii , iii , avf , and v26 .
the creatine kinase level was 499 u / l ( reference : 40150 u / l ) , creatine kinase mb isoenzymes were 51.2 ng / ml ( reference : 0.06.9 ng / ml ) , and the creatine kinase isoenzyme index was 10.7% ( reference : 03.5% ) .
ecg on admission showed a normal sinus rhythm with st - segment elevation in leads ii , iii , avf , and v26 ( fig.2a ) .
transthoracic echocardiography indicated severe segmental left ventricular dysfunction involving the septum , anteroseptal territory , and apex , with grade two mitral regurgitation .
left ventriculography revealed akinesis of the apical wall and compensatory hyperkinesis of the basal walls ( fig.3a and b ) .
( a ) ecg on day 1 shows a normal sinus rhythm with st - segment elevation in leads ii , iii , avf , and v26 .
( b ) ecg on day 11 shows deeply inverted t waves in leads ii , iii , avf , and v36 .
( c ) ecg 3 months after the onset shows normalization of st - t changes .
diastolic ( a ) and systolic ( b ) phases show akinesis of the apical wall and compensatory hyperkinesis of the basal walls .
however , 24-h urine tests showed no elevation in metanephrine or normetanephrine levels , making a diagnosis of pheochromocytoma less probable .
blood tests , echocardiography , and left ventriculography results excluded myocarditis and hypertrophic cardiomyopathy . finally , we diagnosed the patient with takotsubo cardiomyopathy .
a transthoracic echocardiogram on day 10 showed mild improvement of global wall motion . on day 11 , she had become asymptomatic , although the ecg showed deeply inverted t waves in leads ii , iii , avf , and v36 ( fig.2b ) , similar to those observed at the first admission ( fig.1a ) .
the ecg obtained 3 months after the onset of symptoms was nearly normal ( fig.2c ) .
in 1998 , well before takotsubo cardiomyopathy was generally recognized , hansoti and dharani 2 reported 10 previously healthy patients ( nine women ) with idiopathic isolated global t - wave inversion , similar to that observed in the present case . aside from negative echocardiography and left ventriculography , the clinical features of female predominance , 4060 years of age , acute chest pain or discomfort , normal creatine kinase mb isoenzymes , a negative coronary angiogram , and a good prognosis are compatible with the diagnosis of takotsubo cardiomyopathy .
therefore , the isolated deep t - wave inversion observed during the first episode in our patient was most likely an after - effect of takotsubo cardiomyopathy .
this conclusion is based on the fact that no other cause was identified , and the ecg findings at the second admission to hospital were similar to those associated with typical recurrent takotsubo cardiomyopathy 5 . in typical cases of takotsubo cardiomyopathy , st - segment elevation on the ecg and abnormal wall motion on echocardiography are observed at the onset .
these events are followed by t - wave inversion , which persists after normalization of echocardiographic images 6 , as observed during the first episode in our patient .
most patients with takotsubo cardiomyopathy complain of chest pain and dyspnea , although some are asymptomatic 7 . in our patient
, asymptomatic takotsubo cardiomyopathy may have been present before the periodic health examination . in conclusion , when clinicians encounter isolated deep t - wave inversion , they should suspect that it is an after - effect of takotsubo cardiomyopathy because the generally favorable prognosis does not warrant an invasive workup , such as coronary angiography .
written informed consent was obtained from the patient for publication of this case report and any accompanying images .
a copy of the written consent is available for review by the editor of this journal .
| key clinical messagethe electrocardiogram ( ecg ) of a 73-year - old , asymptomatic woman showed deep t - wave inversion .
the complete workup was negative .
ten years later , she developed takotsubo cardiomyopathy with abnormal ecg again .
isolated deep t - wave inversion might be an aftereffect of takotsubo cardiomyopathy that does not warrant an invasive workup . |
cells of the immune system continually monitor a host for invading pathogens and participate in clearing those pathogens so the body remains free of infection .
this active response is an immunogenic response . under non - infectious conditions , cells of the immune system
are also continually exposed to proteins of the host and any immune responses which develop are suppressed so the host maintains a state of self - tolerance .
the cells that direct the outcome of pathogen exposure are the antigen - presenting cells ( apc ) , and these include dendritic cells ( dc ) , macrophages and some b lymphocytes .
the potential for control of the uptake and presentation of environmental antigens by apc is important for immunotherapy aimed at both the tolerogenic and immunogenic capacity of apc .
achieving therapeutic control over dc could have impact on immunization for pathogens and cancers on the one hand , and immunosuppression for autoimmune diseases and organ transplant rejection on the other .
to date , the difficulty has been to determine the conditions under which dc behave in an immunogenic versusa regulatory or suppressive manner .
spleen has been the organ of focus in many dc studies due to its unique role as a site of dc development [ 13 ] .
it is also a site for dc exposure to blood - borne pathogens and apoptotic or dying cells .
the main function of dc is to endocytose and process antigen from pathogens or apoptotic cells , and to present antigen as peptide in the context of major histocompatibility complex ( mhc ) class i and ii molecules for t - cell recognition and activation
. the outcome of t - cell activation can be either immunity or tolerance depending on the signals that accompany t - cell receptor ( tcr ) recognition of antigen .
cell surface markers like cd11c , cd11b , cd8a , cd4 , mhc - ii and cd45ra are most widely used for phenotypic characterization of dc subsets .
different subsets of dc are present across lymphoid organs like spleen , bone marrow ( bm ) , lymph nodes and thymus , with different subsets distinguishable by function and marker expression .
the murine thymus , for example , contains mainly cd8a conventional ( c)dc that also express cd11c and mhc - ii , which develop within this organ , while cd8ot cdc resident in murine thymus are thought to be immigrants .
spleen is distinct in that it contains multiple dc subsets including plasmacytoid ( p ) dc , and the cd8ot and cd8a subsets of cdc .
the cd8ot cdc in murine spleen are cd11ccd11bmhc - iib220 cells , and can be further distinguished on the basis of cd4 expression .
the cd8 cdc are also distinguishable as cd11bcd4 cells , while pdc can be characterized by marker expression as cd11ccd11bcd8tmhc - ipb220 cells , with cd8 and mhc - ii expression dependent on the state of maturation .
pdc are also marked by secretion of interferon- ( ifn- ) , which is effective in mediating viral clearance [ 9 , 10 ] .
both cdc subsets are derived from precursors originating in bm , which migrate to spleen where they develop into immature , steady - state dc . as well as differing in marker expression , cdc differ in their location within organs , with cd8 cdc localizing to the t - cell areas of spleen and cd8 cdc to the marginal zone .
distinct monocyte - derived dc with the functional and pheno - typic characteristics of cdc are also recruited into sites of inflammation .
langerhans cells ( lc ) also appear to be inflammatory dc and represent a distinct monocyte - derived cell type expressing langerin ( cd207 ) that resides in the epidermis and the intestinal epithelium .
lc migrate to lymph nodes in response to contact with antigen in the presence of inflammation or danger signals . inflammatory dc derive from circulating monocytes , which differentiate under the influence of inflammatory factors like granulo - cyte - macrophage colony stimulating factor ( gm - csf ) , tumour necrosis factor ( tnf)-a and il-4 [ 1214 ] .
they are distinct from the dc subsets described in spleen in the resting state , although they have phenotypic and functional similarity to cd8ot cdc .
the developmental relationship between cdc and inflammatory dc is not yet clear , although recent evidence for a common dendritic progenitor in bm for cdc and pdc [ 2 , 15 ] , now implicates inflammatory dc as a different lineage with a distinct myeloid progenitor and developmental pathway more closely aligned with mono - cyte development .
recent studies have shown that by 6 weeks of age , mouse spleen contains 2.4% dc which express mhc - ii on their cell surface and are immunocompetent .
a primary function of dc is uptake of antigen from a site of infection with subsequent migration to lymph nodes and presentation of antigen to cd4 t cells in the context of mhc - ii molecules .
if dc also become activated via pathogen - associated danger signals , which signal through toll - like receptors ( tlr ) on the dc , then there is up - regulation of t - cell co - stimulatory molecules like cd86 and cd40 , leading to t - cell activation and an immunogenic response .
however , capacity to present antigen to t cells and to initiate an immune response differs with their cd8a phenotype .
cd8ot cdc endocytose foreign antigen , and induce a cd4 t - cell response both in the presence and absence of bacterial lipopolysaccharide ( lps ) as a
some cdc subsets can endocytose exogenous antigen and present it to cd8 t cells in the context of mhc - i molecules in a process termed
cross - presentation , whereby foreign antigen is endocytosed and degraded within endosomes to be presented as peptide in the context of mhc - i molecules .
this pathway is important for clearance of pathogens like viruses or parasites that do not infect apc , and for removal of apoptotic normal and cancerous cells . in terms of cdc in spleen ,
cd8a cdc are able to cross - present antigen most efficiently [ 17 , 18 ] .
functional studies on dc are difficult due to the problem of isolating small subsets of cells and of maintaining their functional potential .
removal of cells from the host , injection into recipient animals , or staining cells with antibody for facs analysis , can lead to activation .
it is difficult to obtain an ex vivo population in the resting or steady - state for use in comparative studies .
a wealth of evidence now indicates that dc maturation or activation status rather than dc lineage per se determines the immuno - genicity of dc .
dc tolerogenicity is not therefore a characteristic of a specific subset , or lineage of dc , but a feature of the environmental niche surrounding the developing cell .
indeed , an important characteristic of dc is their functional plasticity and their ability to adopt different apc characteristics depending on the cytokine milieu or inflammation site in which they are located .
several environmental factors are well known to support the development of tolerogenic / suppressive dc including the immunosuppressive cytokines il-10 and transforming growth factor- ( tgf- ) , as well as other factors like hepatocyte growth factor , granulocyte colony stimulating factor ( g - csf ) , prostaglandin e2 and histamine [ 21 , 22 ] .
dc that are tolerogenic or suppressive contribute to peripheral tolerance mechanisms essential in supporting the state of central or thymic tolerance established at birth .
peripheral tolerance is maintained by multiple mechanisms including anergy or deletion of self - reactive t cells , and the induction of regulatory t cells ( tregs ) , which suppress the function of self - reactive t cells .
it is well established that immature cdc resident in spleen are tolerogenic in the steady - state , taking up antigen from the environment as soluble molecules or apoptotic cells , and presenting these to t cells in the absence of a co - stimulatory signal , often with low mhc - ii expression , so inducing to a tolerogenic outcome [ 2326 ] .
in contrast , these same cells also become highly immunogenic when exposed to inflammatory or danger signals , inducing strong immunogenic responses in nave t cells . activating proin - flammatory cytokines can include tnf- , ifn- and cd40-ligand ( cd40-l ) , while danger signals can include lps , cpg motifs and double stranded ( ds ) rna .
for example , cd8 cdc have been shown to be highly immunogenic and able to induce cd8 cytotoxic t cells in response to viral infection [ 18 , 27 ] .
dc of the same type can also function in cross - presentation of apoptotic cells to cd8 t cells , mediating deletion of reactive t cells , although the mechanism of deletion is unclear .
much of the work on immature , tolerogenic dc has involved in vitro cultured cells , and the identity of the in vivo cell equivalent is unclear .
many studies have involved dc produced by culture of bm cells with gm - csf and il-4 , which yields a subset of dc very similar to cd8ot cdc .
several papers now report the ability of these cells to induce both regulatory and anergic t cells [ 30 , 31 ] .
indeed , in the collagen - induced arthritis model in mice , orally induced tolerance due to collagen administration has been associated with pdc and their production of induction of 2,3 indoleamine dioxygenase ( ido ) with subsequent formation of tregs .
tregs then have an inhibitory effect on t - cell proliferation and their production of inflammatory cytokines .
soluble factors produced by tolerogenic dc could represent valuable immunotherapeutic agents for control of t - cell proliferation and activation in autoimmune diseases .
immature pdc induce anergy in cd4 t cells , and mature pdc can induce regulatory t - cell function in cd8 t cells . while the induction of cd4 t - cell anergy by immature pdc is thought to occur through dc - t - cell contact in the absence of costimulatory molecules , the induction of regulatory function in cd8 t cells by mature pdc may be mediated by the activation of the pdc by cd40l .
while a number of different examples exist in the literature , this group of cells is not well defined [ 20 , 3234 ] .
several reports have described the induction of dcregs with suppressive or regulatory function for cd4 t cells after in vitro co - culture of haematopoietic progenitors , precursors or even dc above splenic stroma [ 3335 ] . indeed ,
evidence that splenic stromal cells support dc haematopoiesis is consistent with a central role for microenvironments in spleen in dc development [ 1 , 36 , 37 ] .
subsets of murine monocytes and dc with regulatory function some authors describe dcregs that can induce the development of treg cells [ 20 , 35 ] .
immature dc expressing cd45rb , isolated directly from bm or induced by culture of bm progenitors with stroma comprising a combination of fibroblastic and endothelial cells .
others have described dcregs , which do not induce treg formation , but are immunosuppressive due to their production of inhibitory factors .
tang et al . reported dcregs as cd11ccd11bmhc - ii dc developing when bm - derived haematopoietic stem cells ( hsc ) were cultured over a spleen endothelial stroma .
these cells were derived in vitro from mature cd11cmhc - ii dc induced after culture of bm cells with gm - csf and il-4 . when cultured above a splenic stroma , they proliferated and differentiated further dependent on stroma - produced soluble tgf- and contact with fibronectin .
these two dc subsets are quite distinct in terms of lineage origin , although both are regulatory in that they directly inhibit cd4 t - cell proliferation through release of nitric oxide , which interferes in the il-2 signalling pathways important in t - cell proliferation [ 33 , 34 ] .
dcregs described by tang et al . also activate cd4 t cells in terms of cytokine production , but are unable to induce their proliferation .
wong and rodriguez showed expansion of a cd11clocd8mhc - iiocd45rb subset of dcreg in response to infection with plasmodium yoelii , p. berghei and lps treatment .
the expansion of dcreg lasted until 10 days after infection and corresponded with a drop in numbers of cdc .
the dcreg population was shown to stimulate tregs , which secrete il-10 and suppress cd4 t - cell function and appear to resemble the dcreg described previously by svensson et al .. indeed , descriptions and mode of action of dcreg differ , and the challenge for immu - nologists now is to reconcile cells identified by in vitro studies , with in vivo subsets , and to determine the in vivo conditions that favour one cell type over another .
recently , myeloid suppressor cells ( msc ) were described that have a cd11bcd11cgr-1 phenotype , and so bear some phenotypic relationship with monocytes and myeloid dc .
this cell type has been described in several mouse models and in association with a variety of disease states .
the exact phenotype and mechanism of action of msc appears to vary with cell location and the disease state of the host , so it is possible that different subsets of this cell type exist , although they generally reflect inflammatory monocytes .
, describe a cd11bgr-1 cell type , which is expanded in tumour - bearing mice .
these cells up - regulate arginase ( arg1 ) and nitric oxide synthase ( nos2 ) activity in response to ifn- and il-13 , leading to an environment containing reactive nitric oxide species and depleted levels of l - argi - nine .
this results in t - cell apoptosis , impaired proliferation and failure of t cells to express functional antigen - specific tcr .
other groups report cells that act through either arg1 or nos2 [ 40 , 41 ] .
msc reported in mice that have undergone traumatic stress like surgery , are cd11bgr-1 myeloid cells with up - regulated arg1 activity , and are found in spleen in close proximity to t - cell zones .
, , develop from bm cells cultured with gm - csf and resemble cd11bly-6cly-6 g monocytes .
these become suppressive in the presence of ifn- and appear to require cell - cell contact and nos2 activity to achieve this effect .
a subset of cd11bly-6cly-6 g msc that resemble monocytes have also been described by zhu et al .. these were observed in the neural tissue of experimental autoimmune encephalitis ( eae)-immu - nized mice .
they can up - regulate both arg1 and nos2 in response to a variety of cytokines , including ifn- and il-4 , so suppressing t cells in a mechanism initiated by cell - cell contact .
recently weber et al . described type ii monocytes and their suppressive effect upon adoptive transfer into mice with induced experimental autoimmune encephalomye - litis ( eae ) .
ameliorated disease and reduced t - cell infiltration into the central nervous system , due to production of tregs and t helper type 2 cells through increased il-10 and tgf- expression , which led to an anti - inflammatory response . differences between the myeloid cell types described by these groups could be explained by the different disease states of the mice from which they were isolated .
msc differ by comparison with described dcregs in that they are associated with disease states and often appear to contribute to pathology as opposed to playing a role in immunosuppression .
dendritic cells are emerging as a highly versatile cell type that displays plasticity in response to environmental factors .
several theories have been proposed regarding the subsets of cells involved in these two processes .
most of the well - described steady - state dc have tolerogenic capacity as immature cells , but can become immunogenic upon maturation / activation in the presence of a danger signal .
tolerance mediated by these cells most likely involves ubiquitous self - antigens , like those released by apoptotic cells .
other subsets of cells , including dcregs and msc , function to turn down an immune response under inflammatory conditions .
cells like these would be important in ensuring that tolerance is maintained even when pathogens are present .
increasing evidence now suggests that dc can emerge from a number of precursors and can acquire a range of immunosup - pressive or regulatory functions under different environmental triggers .
msc may also have a developmental origin common with some dc and particularly diffdc , but appear to be distinct from other dcregs .
our conclusion is that dcregs represent a broad class of cells , which participate in a number of ways to turn down an immune response .
their role in homeostasis and switching off immune responses therefore reflects yet another immune capacity for dc , which now extends to immunostimula - tion , induction of tolerance , maintenance of peripheral tolerance and immunosuppression . | dendritic cells ( dc ) are central to the immune system because of their role in antigen presentation leading to either tolerance or immunity among cells of the adaptive immune response .
it is becoming increasingly evident that dc show extensive plasticity in terms of their origin and function , giving rise to a number of subsets represented differentially in all lymphoid organs .
this article considers the tolerogenic capacity of murine dc and draws a distinction between dc that induce tolerance in the immature state and immunity in an inflammatory context , and those that act as regulatory cells inducing immunosuppression in the presence of inflammation . |
lung cancer can be mainly classified into squmous cell carcinoma , adenocarcinoma , small cell carcinoma and the other subtypes .
it is very important to diagnose the subtype of lung cancer fast and accurately as the therapy is different .
endobronchial ultrasound - guided transbronchial needle aspiration ( ebus - tbna ) , a new clinical technology , has been applied in the clinical diagnosis of advanced cancer and tuberculosis . because of direct smears from needle aspiration and brushing specimens , ebus - tbna can be used for on - site evaluation .
however , few studies have investigated the sensitivity and specificity of cytology compared to histology using ebus - tbna .
however , few researches are focused on sensitivity and specificity of the cytopathology based on the ebus - tbna . through a retrospective study of ebus - tbna cases
, we provided the sensitivity and specificity of lung cancer subtype based on the ebus - tbna and found that ebus - tbna was a simple , fast , safe and effective technology which can be routinely applied in the clinical practice .
the cytological specimens were collected using an ultrasound bronchofiber videoscope with a 22-gauge needle and was followed by cytology and histological diagnosis .
the average number of slides of each case were 24 , with a range of 17 .
all cases diagnosed as thoracic disease at shanghai pulmonary hospital from april 2010 to may 2011 were reviewed . during this period ,
379 cytological samples were obtained by ebus - tbna during this period are originally diagnosed as inadequate , suspicious , benign or malignant . of all 379 cases
159 histological - control - paired cases underwent a bronchofiber biopsy , percutaneous transthoracic biopsy , or postsurgical pathology .
immunocytochemistry ( icc ) was requested on the cytological material if there were enough cells left .
based on the diagnosis by two cytopathologists , we classify the cases into the positive group and the negative group .
the positive group referred to the cases of malignant diagnosis such as squamous cell carcinoma , adenocarcinoma , small cell carcinoma and adenosqamous carcinoma etc .
and the negative group contained the cases diagnosed as benign or no evidence of malignancy .
samples obtained by ebus - tbna were centrifuged for 5 minutes in the 50ml centrifuge tube at speed of 2000r / s .
supernatant was collected in the centrifuge tube and the cleaning solution was added ( tct ) .
when the sediment contained more blood cells , 10% acetic acid wash solution was used after shaking centrifuge ( at 2000 r / s , 5 min ) .
95% ethanol was added into the supernatant along the wall ( gentle movements ) and fixed for 1 h. the amount of alcohol added into the precipitate was at least five times that of the supernatant volume . after discarding the alcohol ,
10% neutral formalin was added along the wall to separate the natural sediment from the wall and fixed for 1 h. the sediment was poured from the centrifuge tube with a gauze wrapping and pressed down gently from the side into a color - embedded basket .
ihc was performed on 4-m thick formalin - fixed and paraffin - embedded ( ffpe ) sections .
slides were deparaffinized and pretreated with heat - mediated antigen retrieval solution in a microwave oven .
p63 ( 1:50 , dako ) , ck5/6 ( 1:50 , dako ) , ttf-1 ( 1:100 , dako ) , ck7 ( 1:100 , dako ) , cea ( 1:100 , dako ) , and cd56 ( 1:100 , dako ) , syn ( 1:100 dako ) were applied .
the slides were then washed in tris - hcl and detected with horseradish peroxidase - conjugated anti - rabbit envision+kit ( dako ) .
all slides were counterstained with hematoxylin . sensitivity and specificity of the cytological diagnosis is calculated as below equation shows : % sensitivity = true positive/(true positive + false negative ) 100% .
the cytological specimens were collected using an ultrasound bronchofiber videoscope with a 22-gauge needle and was followed by cytology and histological diagnosis .
the average number of slides of each case were 24 , with a range of 17 .
all cases diagnosed as thoracic disease at shanghai pulmonary hospital from april 2010 to may 2011 were reviewed . during this period ,
379 cytological samples were obtained by ebus - tbna during this period are originally diagnosed as inadequate , suspicious , benign or malignant . of all 379 cases
159 histological - control - paired cases underwent a bronchofiber biopsy , percutaneous transthoracic biopsy , or postsurgical pathology .
immunocytochemistry ( icc ) was requested on the cytological material if there were enough cells left .
based on the diagnosis by two cytopathologists , we classify the cases into the positive group and the negative group .
the positive group referred to the cases of malignant diagnosis such as squamous cell carcinoma , adenocarcinoma , small cell carcinoma and adenosqamous carcinoma etc .
and the negative group contained the cases diagnosed as benign or no evidence of malignancy .
samples obtained by ebus - tbna were centrifuged for 5 minutes in the 50ml centrifuge tube at speed of 2000r / s .
supernatant was collected in the centrifuge tube and the cleaning solution was added ( tct ) .
when the sediment contained more blood cells , 10% acetic acid wash solution was used after shaking centrifuge ( at 2000 r / s , 5 min ) .
95% ethanol was added into the supernatant along the wall ( gentle movements ) and fixed for 1 h. the amount of alcohol added into the precipitate was at least five times that of the supernatant volume . after discarding the alcohol ,
10% neutral formalin was added along the wall to separate the natural sediment from the wall and fixed for 1 h. the sediment was poured from the centrifuge tube with a gauze wrapping and pressed down gently from the side into a color - embedded basket .
ihc was performed on 4-m thick formalin - fixed and paraffin - embedded ( ffpe ) sections .
slides were deparaffinized and pretreated with heat - mediated antigen retrieval solution in a microwave oven .
p63 ( 1:50 , dako ) , ck5/6 ( 1:50 , dako ) , ttf-1 ( 1:100 , dako ) , ck7 ( 1:100 , dako ) , cea ( 1:100 , dako ) , and cd56 ( 1:100 , dako ) , syn ( 1:100 dako ) were applied .
the slides were then washed in tris - hcl and detected with horseradish peroxidase - conjugated anti - rabbit envision+kit ( dako ) .
sensitivity and specificity of the cytological diagnosis is calculated as below equation shows : % sensitivity = true positive/(true positive + false negative ) 100% .
of the all 379 cases , 277 ( 73% ) patients were males and 102 ( 26.9% ) were females .
the median age was 59 years ( range : 1583 years ) . in this study , there was no association between the histological subtype of thoracic disease and neither patient 's age ( = 2.7 , p = 0.412 ) or sex ( = 0.687 , p = 0.812 ) .
the number of slides in these cases ( excluding icc slides ) ranged from 2 to 6 slides ( median : 4 slides ) .
the rest 240 cases that had enough cytological materials on the smear comprised the objects for this study . among 240 cases ,
135(56.25% ) cases were negative while the left 105 ( 43.75% ) cases were positive .
further , the 37.92% ( 91/240 ) of the adequate cytological sample had been made the cell block [ table 1 ] .
correlation between cytologic diagnoses and the routine reports to compare the differences in sensitivity and specificity between the two methods , we analyzed the 159 cases which we analyze 159 cases which were diagnosed by cytology and histology at the same time .
the sensitivity rate of the cytology reached to 94.52% and the specificity rate was as high as 95.12% [ table 2 ] .
correlation between cytologic outcome ( original report ) and the final histologic diagnosis of lung cancer ( =0.213 , p=0.645 ) there are 69 cases diagnosed as positive by both cytology and histology . to further analysis these cases , the subtype diagnosis by cytology was inconsistent with histology .
when adding cell block into cytology diagnosis , it did not improve the consistency of diagnosis by cytology and histology [ table 3 ] .
comparison of smear , cell block , and histology icc was dedicated to the cases which were hard to distinguish between benign and malignant or hard to subtype malignant diseases based on the smear . in our research
, the following icc biomarkers were routinely used to differentiate between benign cells ( such as lymphocytes ) and malignant cells : ttf-1 , ck7 , and cea for adenocarcinoma [ figure 1a and b ] ; p63 and ck5/6 for squamous cell carcinoma [ figure 1c ] ; and ttf-1 , cd56 , and syn for small cell carcinoma [ table 4 , figure 1d ] .
there was an association between histological subtypes of ebus - tbna and the sensitivity of the cytological diagnosis was showed in table 3 .
further , the icc helped to improve the sensitivity rate of the cytology based on the ebus .
p63 positive was significant ( squamous cell carcinoma vs. adenocarcinoma or sclc , p < 0.001 ) . ttf-1 positive of adenocarcinoma was similar with p63 for squamous cell carcinoma ( adenocarcinoma vs. squamous cell carcinoma , p < 0.001 ) .
however , ttf-1 positive between adenocarcinoma and small cell carcinoma was no difference ( adenocarcinoma vs. small cell carcinoma , = 0.003 , p > 0.05 ) .
cd56 and nse positive of small cell lung carcinoma was showed significantly ( small cell carcinoma vs. squamous cell carcinoma or adenocarcinoma , p < 0.05 ) [ table 4 ] .
( a ) the immunostain of cea in adenocarcinoma . it showed membrane positive ( ihc : cea 400 ) .
( d ) the immunostain of syn in small cell carcinoma ( ihc : syn 400 ) the expression of ihc - marker in sqcc , adcc , and sclc the sensitivity rate of icc diagnosis was 88.24% in squamous cell carcinoma , 100.00% in adenocarcinoma , 96.00% in small cell carcinoma compared to the conventional cytology of 82.35% in squamous cell carcinoma , 81.48% in adenocarcinoma , 72.00% in small cell carcinoma , and the specificity rate of icc diagnosis was 100.00% in squamous cell carcinoma , 100.00% in adenocarcinoma , 99.25% in small cell carcinoma compared to the cytology of 98.59% in squamous cell carcinoma , 98.51% in adenocarcinoma , 72.00% in small cell carcinoma .
they always appear in lung cancer , as well as in tuberculosis , sarcoidosis , etc .
, lung cancer is the second most common cancer and is the leading cause of cancer mortality worldwide for both men and women .
most patients present with incurable disease and face a 5-year relative survival rate of 17% with current standard therapies .
the treatment of lung cancer depends on the tumor type , clinical stage , and the patients overall health condition .
earlier effective treatment based on histology is much important for patient to extend their life expectancy . because the advanced patients have lost the chance of surgery , it is difficult to get pathological samples from them , except body fluid , hyperlink http://dict.baidu.com/s?wd=sputum
ebus - tbna , a new way to obtain the samples of the lung cancer patients , is helpful for medical treatment .
ebus - tbna is proved to be a safe , sensitive , and inexpensive diagnostic procedure .
clinical studies have shown that ebus - tbna has the sensitivity , specificity , and positive and negative predictive values of 84100% , 100% , and 100% and 6797% , respectively .
the rate of diagnosis of thoracic disease is improved by appling ebus - tbna applied , while patients get little hurt during the diagnosis .
therefore , cytological diagnosis in thoracic disease is important for the detection and management to improve patients survival .
however , the sensitivity of cytological examination for its diagnosis varies widely in literature . in this study
, we have reviewed a large series of histological diagnostics combining ebus - tbna technology to provide an estimate of the sensitivity and specificity of the cytological diagnosis .
our results showed that cytological diagnosis provided a definite or suspected diagnosis of ebus - tbna in 63.32% of all the cases ( 240/379 ) .
the sensitivity and specificity of the squamous cell carcinoma , adenocarcinoma , and small cell carcinoma were high based on the characteristic of the cell .
these data were not improved on revision of the cytological preparations , which were carried out by two experienced cytopathologists .
the data was obtained either in the presence of a relevant clinical history with accompanying positive endobronchial biopsy or with a background knowledge of the reviewing . the rest of the 139 cases had insufficient diagnostic value cells on the smears .
if the lymph node phagocytic cell had been seen , we considered it was a valid puncture , otherwise the smear was not proper .
the sensitivity and specificity was improved in small cell carcinoma when the cell block was applied in the cytological diagnosis .
the size of the small cell carcinoma is as large as that of the lymphocyte when the cell on the smear was swelling .
the icc would label the tumor cells of the small cell carcinoma , syn and cd56 was showed in the cell plasma , while ttf-1 was stained in the nucleus .
the sensitivity of the squamous cell carcinoma was not high because the probability that squamous cell carcinoma metastasizes to lymph nodes was less than adenocarcinoma and small cell carcinoma .
because the transbronchial puncture would bring the bronchial epithelium onto the smear , we could not differ it from tumor cells when squamous metaplasia occurred and the ck5/6 and p63 were both expressed in the bronchial epithelium and squamous cell carcinoma .
however , it would be more difficult to do this in case of the solid adenocarcinoma , which was without any glandular cavity structure .
we should use cell block and icc in the diagnosis of solid adenocarcinoma . both the specificity and the sensitivity reached 100% when cell block and icc were used in the diagnosis .
the primary reason was that no tumor cells were taken from the puncture ; in other words , the puncture just got a sample of lymphocyte .
therefore , the technique of the operating doctor and cytopathologist is very important and influences the results of the ebus - tbna . here
, we reported that cytological diagnosis had a high sensitivity and specificity of thoracic disease detection based on sampling from ebus - tbna .
the data would be more satisfactory if we make the cytology diagnosis according to the results of icc .
in conclusion , combined with newly developed ebus - tbna , cytological diagnosis is sensitive .
the value of cytological examination of ebus - tbna should be acknowledged as a simple , fast , and safe procedure that provides a reasonable degree of sensitivity and specificity . in case of the high skilled puncture doctors and cytopathologists ,
. it will give the patient a choice for diagnosis , which is painless and inexpensive .
this work was supported in part by grants from the projects of the shanghai municipal commission of health and family planning ( no .
this work was supported in part by grants from the projects of the shanghai municipal commission of health and family planning ( no .
| aims : endobronchial ultrasound ( ebus ) is a relatively new modality that can be used to guide transbronchial needle aspiration ( tbna ) of mediastinal lymph nodes . at present , researches on the sensitivity and specificity of cytopathology based on the ebus - tbna are deficient ; therefore , we want to evaluate the value of cytology based on the ebus - tbna in this article.materials and methods : we reviewed the 379 cases that underwent the ebus - tbna in shanghai pulmonary hospital from april 2010 to may 2011 . discarding the 139 cases with insufficient cells , we analyzed the remaining 240 cases that had enough cells on the smears.statistical analysis used : the statistical package for the social sciences version 15.0 ( spss inc .
, chicago , il ) was used for data analysis . a p value of < 0.05 was considered significant.results:we found that the cytologic diagnosis of sensitivity and specificity reached 94.52% and 95.12% , respectively .
the sensitivity of squamous cell carcinoma , adenocarcinoma , and small cell carcinoma was up to 88.24% , 100.00% , and 96.00% , respectively .
the specificity of squamous cell carcinoma , adenocarcinoma , and small cell carcinoma reached to 100.00% , 100.00% , and 99.25% , respectively.conclusion:here , we report that the cytological examination of ebus - tbna should be acknowledged as a simple , fast , and safe procedure that provides a reasonable sensitivity and specificity of diagnosis in lung cancer . |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.