article stringlengths 0 826k | abstract stringlengths 174 4.34k |
|---|---|
renal transplantation ( rtx ) is best therapeutic modality for end - stage renal disease ( esrd ) . compared with dialysis
, a transplant leads to a longer life , enhances quality of life , and is cost - effective for the health care system . in india ,
175,000 new patients develop esrd annually , and < 10% are able to gain access to renal replacement therapy .
the rate of renal transplantations performed yearly in india translates to 3.25 per million population ; the deceased - donation rate is 0.08 per million population per year .
this discrepancy between the number of waiting patients and transplantations performed can be reduced by developing deceased donor organ transplantation ( ddot ) program .
the reasons for such a low rate are many , ranging from lack of awareness to socioeconomic reasons .
apart from the medical issues , legal , social , and ethical issues are the key factors in obtaining consent from the relatives of potential deceased donors .
this was a retrospective study of 160 ddot carried out in our institute from january 2006 to december 2009 .
demographics and post - transplant follow up including investigations , immunosuppression requirement , rejection episodes , and survival were evaluated .
all patients received induction with rabbit - anti - thymocyte globulin ( r - atg ) ( 1.5 mg/ kg ) , and methylprednisolone ( mp ) 500 mg intravenously and mp was continued for 3 days postoperatively .
maintenance of immunosuppression consisted of prednisolone ( 30 mg / day tapered to 10 mg / day at 3 months post - transplant and continued thereafter ) , mycophenolate mofetil ( mmf ) ( 2 g / day ) , and calcineurin inhibitors ( cni ) [ cyclosporine { csa } ( 5 mg / kgbw / day or tacrolimus , 0.08 mg / kgbw / day ) ] .
doses of cni were adjusted as per trough levels ( c0 ) by hplc method in initial 23 months thereafter it was done in event of graft dysfunction due to economic constraints.cyclosporine dosing was adjusted to achieve target c0 concentrations of 200300 ng / ml during the first 23 months after transplantation , 100250 ng / ml up to 6 months after transplantation , and ~100 ng / ml thereafter .
tacrolimus dosing was adjusted to achieve target c0 concentrations of 1015 ng / ml during the first 23 months after transplantation , and 48 ng / ml thereafter .
all patients received prophylaxis against cytomegalovirus ( cmv ) infection ( gancyclovir 1 g thrice a day3month ) , fungal infections ( fluconazole 100 mg once a day6 months ) , and pneumocystis carinii pneumonia ( trimethoprim / sulfamethaxazole ( tmp / smx 160/800 mg ) once a day9 months ) .
all patients were followed at weekly intervals for the first 3 months , fortnightly for the next 3 months , monthly for the next 6 months , and 3 monthly intervals thereafter . on every visit , renal and liver function status was monitored ; complete blood counts and ultrasound doppler studies were performed .
recipients underwent renal graft biopsy for clinical suspicion of acute rejection based on a decline in renal function . an acute rejection episode diagnosed by an allograft biopsy as per the modified banff classification , was treated with standard anti - rejection therapy ( art ) .
t - cell rejections were treated with mp 500 mg3 dosesr - atg 1.5 mg / kg single dose .
b - cell rejections were treated with mp 500 mg 3 dosesplasmapharesis ( 40 ml / kg per session48 sessions)+ivig 5 g / day510 dosesrituximab 375 mg / m bsa single dose .
all patients received induction with rabbit - anti - thymocyte globulin ( r - atg ) ( 1.5 mg/ kg ) , and methylprednisolone ( mp ) 500 mg intravenously and mp was continued for 3 days postoperatively .
maintenance of immunosuppression consisted of prednisolone ( 30 mg / day tapered to 10 mg / day at 3 months post - transplant and continued thereafter ) , mycophenolate mofetil ( mmf ) ( 2 g / day ) , and calcineurin inhibitors ( cni ) [ cyclosporine { csa } ( 5 mg / kgbw / day or tacrolimus , 0.08 mg / kgbw / day ) ] .
doses of cni were adjusted as per trough levels ( c0 ) by hplc method in initial 23 months thereafter it was done in event of graft dysfunction due to economic constraints.cyclosporine dosing was adjusted to achieve target c0 concentrations of 200300 ng / ml during the first 23 months after transplantation , 100250 ng / ml up to 6 months after transplantation , and ~100 ng / ml thereafter .
tacrolimus dosing was adjusted to achieve target c0 concentrations of 1015 ng / ml during the first 23 months after transplantation , and 48 ng / ml thereafter .
all patients received prophylaxis against cytomegalovirus ( cmv ) infection ( gancyclovir 1 g thrice a day3month ) , fungal infections ( fluconazole 100 mg once a day6 months ) , and pneumocystis carinii pneumonia ( trimethoprim / sulfamethaxazole ( tmp / smx 160/800 mg ) once a day9 months ) .
all patients were followed at weekly intervals for the first 3 months , fortnightly for the next 3 months , monthly for the next 6 months , and 3 monthly intervals thereafter . on every visit ,
renal and liver function status was monitored ; complete blood counts and ultrasound doppler studies were performed .
recipients underwent renal graft biopsy for clinical suspicion of acute rejection based on a decline in renal function . an acute rejection episode diagnosed by an allograft biopsy as per the modified banff classification , was treated with standard anti - rejection therapy ( art ) .
t - cell rejections were treated with mp 500 mg3 dosesr - atg 1.5 mg / kg single dose .
b - cell rejections were treated with mp 500 mg 3 dosesplasmapharesis ( 40 ml / kg per session48 sessions)+ivig 5 g / day510 dosesrituximab 375 mg / m bsa single dose .
out of 1109 rtxs performed , 160 ( 14.42% ) were ddot , out of which 68.8% ( n=110 ) were males and 31.2% ( n=50 ) were females with mean age , 35.614.68 years .
the underlying diseases for development of esrd were chronic glomerulonephritis ( cgn ) ( n=79 ) , diabetes mellitus ( n=16 ) , benign nephrosclerosis ( n=16 ) , autosomal dominant polycystic kidney disease ( n=9 ) , focal and segmental glomerulosclerosis ( n=3 ) , iga nephropathy ( n=4 ) , obstructive uropathy ( n=8 ) , tubulointerstitial nephritis ( n=7 ) , lupus nephritis ( n=6 ) , single unit kidney with cgn ( n=5 ) , mpgn ( n=2 ) , chronic pyelonepritis(n=2 ) , and alport syndrome ( n=3 ) .
kidneys were retrieved from 89 donors , including 14.3% non - heart - beating donors and 25.2% expanded criteria donors ( ecd ) .
out of 89 donors , 24.7% ( n=22 ) were from ahmedabad , 55.1% ( n=49 ) from surat ( about 250 km away ) , 16.9% ( n=15 ) from rajkot ( about 200 km away ) , 2.2% ( n=2 ) from bhuj ( about 600 km away ) , and 1.1% ( n=1 ) was from bhavnagar ( about 250 km away ) .
mean cold ischaemia time was 5.562.04 h and a total of 30.6% ( n=49 ) patients developed delayed graft function .
patient survival was 79.58% , 76.7% , 74.8% , and graft survival was 92.4% , 87.9%% , and 87.9% at 1 , 2 and 3 years respectively [ figure 1 ] . mean scr ( mg / dl ) at 1 year , 2 years and 3 years was 1.340.34 , 1.430.41 , and 1.400.36 , respectively .
kaplan curve showing patient and graft survival total of 36 ( 22.5% ) patients died , 16 due to gram - negative sepsis , 4 due to fungal infection , 4 due to cmv disease , 3 due to staphylococcal pneumonia with acute respiratory distress , 4 due to acute myocardial infarction , and 5 due to cerebrovascular stroke .
all patients received triple immunosuppression , 60% ( n=96 ) received csa , 31.9% ( n=51 ) received tacrolimus , and 8.22% ( n=13 ) received sirolimus - based regimens .
14% ( 10% in 1st year and 4% in 2nd year ) had biopsy proven acute rejection ( n=22 ) .
acute b - cell mediated rejections were noted in 3.8% ( n=6 ) , acute t - cell mediated rejections in 2.5% ( n=4 ) , combined acute t- and b - cell mediated rejections in 3.8% ( n=6 ) , acute borderline t+b - cell mediated rejections were noted in 3.8% ( n=6 ) , and chronic t - cell mediated rejection was noted in 0.63% ( n=1 ) patients .
ddot in india is low despite tremendous potential . following steps which are prerequisite for successful ddot program in our set - up
have been taken by our institute.[781115 ]
( 1)increased public awareness on the need for deceased donor organs like public education campaigns on brain death especially on world kidney day.(2)encouraging participation of surgeons in general hospitals in identification of potential donors.(3)expediting in passing legislation to procure deceased donor organs.(4)reduction of cost of transplantation ( since the financial burden is borne by individuals with state support).(5)early brain death identification and certification by neurosurgeons , recognizing potential donors.(6)requesting for organ donation and obtaining consent for organ donation.(7)establishment of a committed and integrated rapid response team capable of transporting potential donor to hospitals in a timely manner.(8)improvement in transportation , communication networks , and ambulance services , successful organ retrieval.(9)efficient and trained transplant co - coordinator.(10)adequate hospital infrastructure and support logistics along with intensivists with adequate intensive care facilities and fully qualified , trained medical and paramedical staff.(11)inclusion of expanded criteria donors ( ecd).(12)positive steps have been taken by the central and state governments , and few non - government ( ngo)/nonprofit organizations in the country .
increased public awareness on the need for deceased donor organs like public education campaigns on brain death especially on world kidney day .
reduction of cost of transplantation ( since the financial burden is borne by individuals with state support ) . early brain death identification and certification by neurosurgeons , recognizing potential donors . requesting for organ donation and obtaining consent for organ donation .
establishment of a committed and integrated rapid response team capable of transporting potential donor to hospitals in a timely manner .
adequate hospital infrastructure and support logistics along with intensivists with adequate intensive care facilities and fully qualified , trained medical and paramedical staff .
positive steps have been taken by the central and state governments , and few non - government ( ngo)/nonprofit organizations in the country .
ddot with elderly donors or donors with suboptimal donation criteria ( marginal or ecd ) are increasing in number . in the united states ,
15%20% of donors were ecd in 2002 . in europe , according to eurotransplant data in 2007 , 20% of donors were 65 years old . in india , where ddot account for around 4% of total transplants , discarding the marginal kidneys would hamper the program . in this study , ecd comprised 25.2% of ddot . in the circumstances of organ shortage , ddot with expanded criteria donor is a feasible option . individual centers in the india have reported their outcomes : in a study by mani , 1 year and 4 year graft survivals of 88 ddot in chennai were 72% and 63% , respectively , and patient survival was hardly different from graft survival .
the inability of most of the patients to afford monoclonal antibodies for immunosuppression might be the reason for inferior 1-year results in this study .
five - year patient and graft survivals of 68 ddot in chennai were 61.7% and 58.8% , respectively , with biopsy proven acute rejection ( bpar ) , 26.4% , delayed graft function ( dgf ) in 50% and cold ischemia time ( cit ) was 5.63.2 h , respectively . short - term graft survival was reduced because of a high 1-year post - transplantation mortality , with most of these deaths caused by sepsis or multiple organ failure .
risk factors for early graft loss were retransplantation , longer cold ischemia time , and acute rejection episode .
the 1-year allograft and patient survivals , of 100 ddot from four major centers in chennai , were 82% and 86% , respectively , with 2-year allograft and patient survivals of 74% and 80% , respectively . in a study by feroz et al .
, from our centre , 1-year patient and graft survival rate for 38 ddot were 90% and 85% , respectively , with bpar in 17% , dgf in 68% and cit of 6.93.8 h , respectively .
in indian , graft survival rates in good centers are 80% in cadaver donor transplants . in our study , over mean follow - up of 2.351.24 years , patient and graft survival rates were 77.5% and 89.3% respectively with high 1-year post - transplantation mortality , with most of these deaths caused by sepsis .
it is possible that triple immunosuppressive regimens with atg induction , unhygienic living conditions , delayed presentation and diagnosis , tropical climate , limited availability , and the expense of diagnostic tools and economic constraints for treatment in majority of patients , may have contributed to high infection rate , similar to experience by other studies.[131618 ] infection , long duration of hd before transplant , ecd , increased dgf , socioeconomic factors pervasively influenced access to health care , may have contributed to high 1-year post - transplantation mortality , with most of these deaths caused by sepsis.[1921 ] recipients of renal allograft in developing countries may be more prone to infections , which are the most common cause of post - transplant mortality .
in the circumstances of organ shortage ddot have a potential to expand the donor pool and shorten the waiting lists for rtx . graft function along with patient and graft survival rates are also acceptable . | renal transplantation ( rtx ) is the best therapeutic modality for patient suffering from end - stage renal disease ( esrd ) .
deceased donor organ transplantation ( ddot ) accounts for < 4% of rtx in india .
we report 4 years single centre experience on ddot vis - - vis patient / graft survival , graft function in terms of serum creatinine ( scr ) , rejection episodes , and delayed graft function in 160 ddot . between january 2006 to december 2009 , 160 rtx from 89 donors were performed , of which 25.2% were expanded criteria donors .
majority of the donors were brain dead due to road traffic / cerebrovascular accidents .
the commonest recipient diseases leading to esrd were chronic glomerulonephritis ( 49% ) , diabetes mellitus ( 10% ) , and benign nephrosclerosis ( 10% ) .
mean recipient / donor age was 35.614.68 and 44.0318.19 years .
mean dialysis duration pretransplantation was 15.372.82 months .
mean cold ischemia time was 5.562.04 hours .
all recipients received single dose rabbit - anti - thymocyte globulin induction and steroids , mycophenolate mofetil / calcinueurin inhibitor for maintenance of immunosuppression .
delayed graft function was observed in 30.6% patients and 14% had biopsy proven acute rejection . over mean follow - up of 2.351.24 years , patient and graft survival rates were 77.5% and 89.3% with mean scr of 1.400.36 mg / dl .
ddot has acceptable graft / patient survival over 4 years follow - up and should be encouraged in view of organ shortage . |
herbal medicines , with their effective pharmacological functions , low toxicity , and less side effects , have been widely used for thousands of years all over the world , which are becoming more and more popular and promising type of medicines [ 15 ] .
however , the same species of herbal medicines with different growing conditions can have some different chemical constituents and pharmacological activities , and what is more , even different parts of the same herbal may considerably differ in types and quantities of chemical components [ 68 ] .
thus , the quality assessment and control of herbal medicines are an important concern for both the health authorities and the public [ 911 ] . the traditional methods such as visible inspection and thin - layer chromatography either are subjective in the aspect of nature or require experienced users , resulting in scarcely meeting the criteria needed to support their use widely .
therefore , a faster , more accurate , and sensitive identification method is required to determine the herbal medicines .
the large majority of investigations only focused on several pharmacologically active constituents to assess the quality and potency of the herbal medicine , which barely represent the whole quality of herbal medicines [ 12 , 13 ] .
the fingerprint technique , which emphasized the whole compositions of samples and focuses on identifying and assessing the quality of the samples , was admitted by who , state food and drug administration ( sfda ) of china ( 2000 ) , and other authorities as a strategy for quality assessment of herbal medicines . due to its reliability , versatility , and precision ,
high - performance liquid chromatography ( hplc ) played an important role among all the fingerprint techniques [ 1517 ] . in recent years , hplc method with various detectors has been developed for qualitative and quantitative analysis of various herbal medicines , such as angelica dahurica , panax ginseng , and berberidis radix .
if the peaks are well separated , a herbal sample can be authenticated by chemical fingerprinting based on the presence or absence of characteristic peaks . however , when coming to identifying a large number of similar samples , numerous variables ( peaks ) may not be efficiently discriminated . in addition
, the traditional hplc fingerprints can not meet the requirements of high throughput analysis due to the low column efficiency and long analysis time with generally more than 1 hr . in recent years ,
near - infrared ( nir ) as having characteristics of high efficiency , low cost , simple measurement , little sample preparation , quick data analysis , and nondestructive analytical technique has been widely used in several scientific fields , especially in the identification of herbal medicines [ 2124 ] .
nonetheless , just as hplc , the fingerprint information provided by nir spectra may be difficult to interpret .
for example , woo 's team used mahalanobis distance and discriminant pls2 combined with nir spectroscopy to discriminate herbal medicines according to geographical origin , frizon used the principal component analysis ( pca ) and the partial least squares ( pls ) regression to determine the total phenolic compounds in yerba mate by nir , and segtnan 's research studied the structure of water using two - dimensional near - infrared correlation spectroscopy . unfortunately , these researches all failed to reach the completely correct recognition . considering that wavelengths of near - infrared reflectance spectra set as the explanatory variable can be hundreds or even thousands , which often had further quantity than the number of samples , multiple correlation was caused by this ;
ordinary pattern recognition methods like pca or lda are difficult to use to create an effective mathematical model . for another , by using plsda , the spectral data can be compressed to a lower - dimensional spatial data . through reasonable selection of the main components and removing interfering components and main ingredients of interference factors ,
only useful principal components can be involved in linear regression , which can perfectly solve the problem of prediction results being confused .
thus , plsda model can be much better to relate the dummy code for the full nir original and preprocessing spectral variables of herbal medicines [ 29 , 30 ] . in this study
, traditional fingerprint of chromatograms combined with plsda and three classical chemometrics methods , as pca , lda , and plsda model with different fingerprint preprocessing of nir spectra variables , are adopted to extract and discriminate otherness of different collection parts , collection time , and different origins of hibiscus mutabilis l. and different species of berberidis radix .
other than pca and lda that can merely have good learning performance and do well in the training sets , plsda model shows good performance in the area of identification of herbal medicines and can be perfectly employed in the analysis of the influence of different collection parts , collection time , and different origins or various species belonging to the same genera of herbal medicines by both chromatograms and nir spectra .
what is more , this is the first time for a supervised pattern recognition method based on plsda algorithm by hplc or nir to identify the sheer amount of information of herbal medicines at one time .
hibiscus mutabilis l. leaves and stems samples were purchased in anhui , collected in nanning , guangxi , or south - central university for nationalities while berberis soulieana schneid ( bss ) , berberis henryana schneid ( bhs ) , berberis gagnepainii schneid ( bgs ) , and berberis triacanthophora fedde ( btf ) samples were collected from jianshi , wuhan , between may and june in 2012 .
antaris ii ft - nir spectrometer , result 3.0 spectral collecting software ( thermo electron co. , usa ) , ultimate 3000 analytical hplc ( dionex co. , usa ) , dzf-6021 vacuum oven ( shanghai yiheng technical co. , ltd . ) , and fw135 herbal grinder ( tianjin taisite instrument co. , ltd . ) were used .
hibiscus mutabilis l. samples used in hplc were crushed with herbal medicine grinder , sifted by 80 mesh sieve , and set aside while berberidis radix samples used in hplc were dried in vacuum at 60c for 24 hours , crushed with the grinder , and sifted by 40 mesh sieve for further use .
all the samples used in nir were crushed with the grinder and sifted into fine powders by 200 mesh sieve , then vacuum - dried at 60c for 24 hours , and stored in a dryer spare .
an ecosil 120 - 5-c18 sh ( 250 mm 4.6 mm , 5 m ) column was used in the fingerprint analysis of hibiscus mutabilis l. while a hyperchrome - hplc - c18 ( 250 mm 4.6 mm , 5 m ) column was used in the analysis of berberidis radix .
all analyses of hibiscus mutabilis l. were performed at a column temperature of 30c , with a mobile phase of methanol ( a)-0.1% acetate aqueous solution ( b ) ( see table s1 of the supplementary material available online at http://dx.doi.org/10.1155/2015/727589 ) , an injection volume of 10 l , and a flow rate of 1.0 ml / min . to gain the most suitable measurement wavelength ,
the test solution will be full - wavelength- ( 200400 nm ) scanned by uv .
taking into account the end absorbance of the mobile phase of methanol and maximum absorption wave of hibiscus mutabilis l. test solution at 269 nm , several wavelengths with large absorbance were added to select one with more peaks and better separation .
ultimately , the uv absorbance of the eluent was set at 269 nm . on the other hand ,
analyses of berberidis radix were performed in the same situation except with a mobile phase of methanol ( a)-0.25% phosphoric acid ( b ) ( table s2 ) and the uv absorbance of the eluent was measured at 230 nm . in order to make it possible for fingerprint peaks to show what ingredients hibiscus mutabilis l. really contains and avoid byproducts ( such as hydrolysis products ) and nonmedicinal ingredients interferences , petroleum ether was used to remove chlorophyll or other fat - soluble ingredients and then dried in a rotary vacuum evaporator and volume was set with methanol . by comparing the transfer rate of products using ultrasound methanol , ethanol , ultrasound , methanol water mixture reflux , ethanol water mixture reflux , and so forth , extraction methods , ethanol water mixture reflux 2 h ( ethanol concentration of 60% ) was taken for the highest transfer rate . accurately weighed 2 g hibiscus mutabilis l. leaves or stems sample was dissolved in 30 ml of 60% ethanol , refluxed for 2 h , and then filtered after it cooled down .
the petroleum ether layer was discarded while the aqueous alcohol layer was evaporated on a rotary instrument rotation evaporated and the residue was dissolved in methanol . after filtering , the sample was set to a volume of 25 ml in a volumetric flask and filtered by 0.22 m microporous membrane and the resulting solution was used as the test solution 1 .
compared with methanol , ethanol , and reflux in water bath and ultrasonic extraction method , hydrochloric acid - methanol ( volume ratio = 1 : 50 ) with ultrasonic extraction was found to be the optimal choice .
accurately weighed 0.50 g berberidis radix sample was dissolved in 50 ml of methanol and 1 ml of hydrochloric acid .
then the sample was weighed , ultrasonic extracted for 2 h , and cooled to room temperature . after being weighed again
the filtrate was set to a volume of 100 ml by methanol in a volumetric flask . at last ,
2 ml of the sample was taken and filtered by 0.22 m microporous membrane ; the resulting solution was used as the test solution 2 .
chromatographic conditions were used as above ; accurately drawn 10 l of each test solution was injected into the hplc and recorded 72 min chromatogram .
precision of the sample application and measurement of peak area were carried out using 5 replicates of the same sample and were expressed in terms of percent relative standard deviation ( % rsd ) .
stability studies : the measurement of peak area were carried out at 0 , 4 , 8 , 16 , and 24 hours , respectively , using 5 replicates of the same sample and were expressed in terms of percent relative standard deviation ( % rsd ) .
repeatability studies : repeatability of the sample application and measurement of peak area were carried out using 5 different samples which come from the same batch of herbs and were expressed in terms of percent relative standard deviation ( % rsd ) .
sample powders were directly put into the quartz sample cup smoothly , and then air was used as the reference and the background was deducted .
spectra were collected by using integrating sphere diffuse reflectance with the collecting region at 100004000 cm and a resolution of 8 cm . each sample
was randomly divided into four parts and scanned 32 times and 60 spectra were collected .
all original chromatograms and spectra were preprocessed by second - order derivative ( 2nd derivative ) and multiple scatter correction ( msc ) .
preprocessed methods , pca , lda , and plsda programs , were written and performed using a matlab 2010a ( math works , natick , ma , usa ) .
for plsda model , vector fj was used to encode category of the jth samples group , in which the element of the jth position was 1 and the other elements were 0 .
the types of unknown samples can be identified according to the dummy codes of the vectors in the classification matrix .
if the maximal element of the ith sample appeared at the jth position of the classification vector , the sample will be identified as the jth group .
the % rsd of precision , stability , and repeatability study for the samples of hibiscus mutabilis l. and berberidis radix and measurement of peak areas was found to be less than 5% , while measurement of relative retention time was less than 3% .
the % rsd indicates that the method has an acceptable level of precision . in order to develop and validate an efficient method for the analysis of the influence of different collection parts , collection time , and different origins of hibiscus mutabilis l.
samples with different collection parts numbered 1 - 1 , 1 - 2 , and 1 - 3 as shown in table 1 were made into test solutions and analyzed by the used above methods .
these three fingerprints were superimposed in figure 1(a ) . as were shown in the spectra , compared to the spectrum of sample 1 - 1 , the peaks in fingerprint of 1 - 2 were much fewer and had a greater difference in height and peak areas in parts of some common peaks which had a lower peak height and less peak area than sample 1 - 1 . on the other hand , compared to sample 1 - 1 , both the peak position and peak appearance time of 1 - 3 were roughly the same and the only differences were the peak heights and peak areas .
it reveals that big differences exist in chemical compositions and their corresponding contents of different collection parts of hibiscus mutabilis , which means that confused collection parts of herbals will generate a lot of problems , such as the impact of the quality of herbals and even medication effects , which may cause major medical incidents .
when coming to the comparison of fingerprints of different origins of hibiscus mutabilis l. , samples 1 - 1 , 1 - 4 , and 1 - 7 with different origins as listed in table 1 were made into test solutions and analyzed by the used above methods .
these three fingerprints were superimposed in figure 1(b ) . compared to the spectrum of sample 1 - 1 ,
the situations of peak appearance are quite different and worse in fingerprints of 1 - 4 , 1 - 7 which had less peak shapes and different peak heights and peak areas at the same peak appearance time .
this may be caused by the differences in chemical compositions and their corresponding contents of hibiscus mutabilis l. leaves from different origins .
as is known to all , some herbs collected in certain areas contain higher content of active ingredients which has a better quality and a more obvious treatment effect , but , instead , some herbs in this area are of poor quality .
this method , however , provides an important technical basis for the identification of authentic ingredients .
samples 1 - 5 , 1 - 6 , 1 - 7 , and 1 - 8 with different collection time as shown in table 1 were made into test solutions and analyzed by the used above methods to recognize different collection time of hibiscus mutabilis l. according to the four fingerprints superimposed in figure 1(c ) , under the same retention time , part of common peak areas and peak heights also had small differences , which proved that different collection time of hibiscus mutabilis l. can lead to the variations in chemical compositions and their contents . because the formation of chemical compositions in the plant takes place in a particular period , chemical compositions and their corresponding contents in different acquisition time also have slight differences .
either the acquisition time is earlier or later than the correct time , these active ingredients may be degraded or even transformed into other harmful substances , and the quality and safety of medicines can not be guaranteed .
thus , this method provides a reliable way to identify collection time of herbal medicines and guarantee the better safety for patients . in the recognition of berberidis radix , to study an efficient method for the identification of various species belonging to the same genera , fingerprints of four different species of berberidis radix named berberis soulieana schneid ( bss ) , berberis henryana schneid ( bhs ) , berberis gagnepainii schneid ( bgs ) , and berberis triacanthophora fedde ( btf ) by hplc
the samples correspond to the numbers in table 1 and were made into test solutions and then analyzed by above methods .
fingerprints of the four samples numbered 2 - 1 , 2 - 2 , 2 - 3 , and 2 - 4 were superimposed in figure 1(d ) .
according to these 4 fingerprints , the peak heights and the peak areas were different at 8 common peaks marked in the chromatograms , which means that small chemical composition differences but big content differences exist in these four different species of berberidis radix . compared with the other three fingerprints , some peak areas and peak heights were significantly smaller in sample 2 - 2 at the same retention time .
what is more , a common peak in the retention time between 30 min and 40 min lacked in 2 - 2 , which certified that less types of the chemical composition and lesser content of common chemical composition were in bhs . in addition , spectrograms of 2 - 1 and 2 - 4 had similar positions of peaks appearing , peak areas , and peak heights at the same retention time , showing that chemical compositions were substantially the same in bss and btf but with a few differences in the content .
this showed that , even if belonging to the same genera , different species of herbal medicines had their own character which were different from each other .
thus , fingerprints by hplc for the analysis of species difference provided an efficient and reliable method to identify herbal medicines and find similar ones to replace some herbs which were unavailable . to obtain a more rapid and effective identification results by hplc
, chemometric methods were required to extract useful information for the recognition of different types of hibiscus mutabilis l. and berberidis radix .
48 hplc chromatograms of the test solution data for 8 groups of hibiscus mutabilis l. and 24 hplc chromatogram for 4 groups of berberidis radix were randomly divided into the training set and prediction set and the optimal proportions of the training set in all the samples were selected .
detailed information about the samples from 8 groups of hibiscus mutabilis l. and 4 groups of berberidis radix was listed in table 2 .
plsda model was used to relate the dummy code for the full hplc original chromatogram variables for different sample groups .
the optimal number of latent variables was , respectively , determined as 6 to obtain the least error numbers of training and prediction sets .
figure 2 shows the assigned plots of dummy codes of the training set and prediction set for raw hplc chromatogram in plsda model .
herein , the dummy codes for the 8 groups of hibiscus mutabilis l. can be coded as f1 ( 1,0 , 0,0 , 0,0 , 0,0 ) , f2 ( 0,1 , 0,0 , 0,0 , 0,0 ) , f3 ( 0,0 , 1,0 , 0,0 , 0,0 ) , f4 ( 0,0 , 0,1 , 0,0 , 0,0 ) , f5 ( 0,0 , 0,0 , 1,0 , 0,0 ) , f6 ( 0,0 , 0,0 , 0,1 , 0,0 ) , f7 ( 0,0 , 0,0 , 0,0 , 1,0 ) , and f8 ( 0,0 , 0,0 , 0,0 , 0,1 ) while four groups of berberidis radix can be coded as f1 ( 1,0 , 0,0 ) , f2 ( 0,1 , 0,0 ) , f3 ( 0,0 , 1,0 ) , and f4 ( 0,0 , 0,1 ) .
samples from all groups were classified by the position of the maximal dummy codes of samples .
it can be clearly seen that , no matter for hibiscus mutabilis l. or berberidis radix , all training samples and prediction samples belonging to all groups were identified accurately with a perfect recognition rate of 100% in both training set and prediction set .
this result proved that the hplc chromatograms combined with partial least squares discriminant analysis can be used as a new method for a fast and accurate discrimination of herbal medicines of different collection parts , collection time , and different origins of hibiscus mutabilis l. or various species of berberidis radix .
although fingerprints by hplc provided an efficient and accurate method for the identification of herbal medicines , it was too cumbersome and time - consuming .
thus , near - infrared spectroscopy combined with chemometric methods was used to identify herbal medicines which were different in collection parts , collection time , origins , and species .
chemical components in both hibiscus mutabilis l. and berberidis radix are complex that contain many types of bioactive constituents .
the average nir spectra of each group are displayed to reflect the overlay fingerprint information in figures 3(a ) and 3(b ) . as is shown ,
the raw nir spectra were seriously overlapped , which made the accurate assignment of characteristic peaks quite difficult .
this can be attributed to the contributions of multicomponents in the samples and the shifts resulted from their interactions .
therefore , chemometric methods were required to extract useful information for the recognition of hibiscus mutabilis l. and berberidis radix of samples . in order to obtain better recognition results , classical chemical pattern recognition methods of principal component analysis ( pca ) , linear discriminant analysis ( lda ) , and partial least squares discriminant analysis ( plsda ) model were used to relate the dummy code for the full nir original and preprocessing spectral variables of different sample groups .
640-sample spectra of 8 different kinds of hibiscus mutabilis l. and 240-sample spectra of 4 different species of berberidis radix were randomly divided into the training set and the prediction set ( table 2 ) .
the training set was used to build the model while the prediction set was used to validate it , and the discrimination results were analyzed for comparison .
firstly , pca , known as a common method in the chemical pattern recognition which is mainly used for classification and clustering in the analytic processes of herbal identification , was used to do the principal component decomposition of spectral matrix of the samples in both training sets and prediction sets to make the new variables data structure characterize features of the original ones as much as possible .
the two corresponding largest eigenvalues , that is , the score vectors of the first and the second principal components , were obtained and set as the projection - axis - to - plot to describe the spatial distribution characteristics of the samples . as is shown in figure 4 , score plots of the first and the second principal components for raw nir spectra of 8 different kinds of hibiscus mutabilis l. samples in training sets ( a ) can be completely distinguished and so do msc and 2nd derivative spectra ( which is not shown here ) , which shows that pca training model does have relatively good learning performance .
but unfortunately , those in prediction sets were confused and failed to show the correct results .
on the other hand , the results of berberidis radix were quite good ( figure s1 ) .
so this method can not be used extensively to distinguish herbal medicines with a variety of mixed information .
other than looking for the vector space that can best describe the original data like pca , lda was used to find the vector space that can distinguish between various types of data completely .
this method can be used to extract the low - dimensional features with the most discriminating ability from the high - dimensional feature space which can gather all the samples in the same type and separate different ones .
thus , spectral matrix by nir of the samples in both training sets and prediction sets was decomposed with lda and the two largest eigenvalues , that is , the first and the second hidden variables , were obtained and set as the projection - axis - to - plot to describe the spatial distribution characteristics of the samples .
figure 5 shows scores of the first and the second hidden variables for raw nir spectra , msc , and 2nd derivative spectra of 8 different kinds of hibiscus mutabilis l. samples , respectively . just as the results by pca , samples in training sets
can be completely distinguished ( the results of msc and 2nd derivative spectra were not listed here ) , while those in prediction sets ( b ) were confused and failed to show the correct results and so did samples of berberidis radix ( figure s2 ) .
thus , this method was also abandoned . due to the fact that the wavelengths of near - infrared spectra set as the explanatory variable had further quantity than the number of samples , ordinary pattern recognition methods were far from creating a stable , high precision mathematical model .
thus , plsda model was used to solve the problem of prediction results confusion and relate the dummy code for the full nir original and preprocessing spectral variables of the 8 different hibiscus mutabilis l. groups or the 4 different berberidis radix groups .
table 2 showed that assigned plots of dummy codes of the training set and prediction set for raw nir spectra in plsda model .
similar figures were not shown on assigned plots of dummy codes for msc and 2nd derivative spectra in plsda model .
herein , the dummy codes for the 8 groups were coded as f1 ( 1,0 , 0,0 , 0,0 , 0,0 ) , f2 ( 0,1 , 0,0 , 0,0 , 0,0 ) , f3 ( 0,0 , 1,0 , 0,0 , 0,0 ) , f4 ( 0,0 , 0,1 , 0,0 , 0,0 ) , f5 ( 0,0 , 0,0 , 1,0 , 0,0 ) , f6 ( 0,0 , 0,0 , 0,1 , 0,0 ) , f7 ( 0,0 , 0,0 , 0,0 , 1,0 ) , f8 ( 0,0 , 0,0 , 0,0 , 0,1 ) while for the 4 groups were coded as f1 ( 1,0 , 0,0 , 0,0 ) , f2 ( 0,1 , 0,0 , 0,0 ) , f3 ( 0,0 , 1,0 , 0,0 ) , f4 ( 0,0 , 0,1 , 0,0 ) , f5 ( 0,0 , 0,0 , 1,0 ) , and f6 ( 0,0 , 0,0 , 0,1 ) .
samples from all groups were classified by the position of the maximal dummy codes of samples .
it can be clearly seen that , no matter for raw nir spectra ( figure 6 ) , msc or 2nd derivative spectra ( not shown here ) , all training samples and prediction samples belonging to all groups of hibiscus mutabilis l. were identified accurately and so did berberidis radix ( figure s3 ) .
excellent forecasted results of nir by plsda were obtained with all the recognition rate of 100% .
a perfect recognition rate for original fingerprint , msc fingerprint , and 2nd derivative fingerprint of nir spectra was achieved by the plsda models .
all the recognition rates of 100% for above three fingerprints of nir spectra were obtained by the plsda models .
this revealed that near - infrared spectroscopy combined with plsda method can be used to identify hibiscus mutabilis l. and berberidis radix , which were different in collection parts , collection time , origins , and species .
what is more , it provided a more rapid , efficient , and reliable identification method of herbal medicines than the traditional ones , which had a very broad application prospect .
a supervised pattern recognition method based on plsda algorithm by hplc and nir has been established to study and identify the information of herbal medicines . in addition , it was clarified from the results that , other than pca and lda that can merely have good learning performance and do well in the training sets , plsda model shows good performance in the area of identification of herbal medicines and can be perfectly employed in the analysis of the influence of different collection parts , collection time , different origins , and various species belonging to the same genera of herbal medicines at one time .
the results show that this recognition method is a promising approach for the identification of complex information of herbal medicines . | as an effective method , the fingerprint technique , which emphasized the whole compositions of samples , has already been used in various fields , especially in identifying and assessing the quality of herbal medicines .
high - performance liquid chromatography ( hplc ) and near - infrared ( nir ) , with their unique characteristics of reliability , versatility , precision , and simple measurement , played an important role among all the fingerprint techniques . in this paper , a supervised pattern recognition method based on plsda algorithm by hplc and
nir has been established to identify the information of hibiscus mutabilis l. and berberidis radix , two common kinds of herbal medicines . by comparing component analysis ( pca ) , linear discriminant analysis ( lda ) , and particularly partial least squares discriminant analysis ( plsda ) with different fingerprint preprocessing of nir spectra variables , plsda model showed perfect functions on the analysis of samples as well as chromatograms .
most important , this pattern recognition method by hplc and nir can be used to identify different collection parts , collection time , and different origins or various species belonging to the same genera of herbal medicines which proved to be a promising approach for the identification of complex information of herbal medicines . |
to address the need for sensitive multiplex assays in diagnostic molecular microbiology , we created a polymerase chain reaction ( pcr ) platform in which microbial gene targets are coded by a library of 64 distinct masscode tags ( qiagen masscode technology , qiagen , hilden , germany ) .
microbial nucleic acids ( rna , dna , or both ) are amplified by multiplex reverse transcription ( rt)-pcr using primers labeled by a photocleavable link to molecular tags of different molecular weight . after removing unincorporated primers ,
the identity of the microbe in the clinical sample is determined by its cognate tags .
schematic representation of mass tag polymerase chain reaction ( pcr ) . as a first test of this technology
, we focused on respiratory disease because differential diagnosis is a common clinical challenge , with implications for outbreak control and individual case management .
multiplex primer sets were designed to identify up to 22 respiratory pathogens in a single mass tag pcr reaction ; sensitivity was established by using synthetic dna and rna standards as well as titered viral stocks ; the utility of mass tag pcr was determined in blinded analysis of previously diagnosed clinical specimens .
oligonucleotide primers were designed in conserved genomic regions to detect the broadest number of members for a given pathogen species by efficiently amplifying a 50- to 300-bp product . in some instances ,
we selected established primer sets ; in others , we used a software program designed to cull sequence information from genbank , perform multiple alignments , and maximize multiplex performance by selecting primers with uniform melting temperatures and minimal cross - hybridization potential ( table a1 ) .
primers , synthesized with a 5 c6 spacer and aminohexyl modification , were covalently conjugated by a photocleavable link to masscode tags ( qiagen masscode technology ) ( 8,9 ) .
masscode tags have a modular structure , including a tetrafluorophenyl ester for tag conjugation to primary amines ; an o - nitrobenzyl photolabile linker for photoredox cleavage of the tag from the analyte ; a mass spectrometry sensitivity enhancer , which improves the efficiency of atmospheric pressure chemical ionization of the cleaved tag ; and a variable mass unit for variation of the cleaved tag mass ( 8,1012 ) .
thus , amplification of a microbial gene target produces a dual signal that allows assessment of specificity .
gene target standards were cloned by pcr into pcr2.1-topo ( invitrogen , carlsbad , ca , usa ) by using dna template ( bacterial and dna viral targets ) or cdna template ( rna viral targets ) obtained by reverse transcription of extracts from infected cultured cells or by assembly of overlapping synthetic polynucleotides .
assays were initially established by using plasmid standards diluted in 2.5-g / ml human placenta dna ( sigma , st .
louis , mo , usa ) and subjected to pcr amplification with a multiplex pcr kit ( qiagen ) , primers at 0.5 mol / l each , and the following cycling protocol : an annealing step with a temperature reduction in 1c increments from 65c to 51c during the first 15 cycles and then continuing with a cycling profile of 94c for 20 s , 50c for 20 s , and 72c for 30 s in an mj ptc200 thermal cycler ( mj research , waltham , ma , usa ) .
amplification products were separated from unused primers by using qiaquick 96 pcr purification cartridges ( qiagen , with modified binding and wash buffers ) .
masscode tags were decoupled from amplified products through uv light - induced photolysis in a flow cell and analyzed in a single quadrapole mass spectrometer using positive - mode atmospheric pressure chemical ionization ( agilent technologies , palo alto , ca , usa ) .
a detection threshold of 100 dna copies was determined for 19 of 22 cloned targets by using a 22-plex assay ( table 1 ) . *
na , not assessed ; rsv , respiratory syncytial virus ; cov , coronavirus ; sars , severe acute respiratory syndrome ; hpiv , human parainfluenza virus .
many respiratory pathogens have rna genomes ; thus , where indicated , assay sensitivity was determined by using synthetic rna standards or rna extracts of viral stocks .
after quantitation by uv spectrometry , rna was serially diluted in 2.5-g / ml yeast trna ( sigma ) , reverse transcribed with random hexamers by using superscript ii ( invitrogen , carlsbad , ca , usa ) , and used as template for mass tag pcr .
as anticipated , sensitivity was reduced by the use of rna instead of dna templates ( table 1 ) .
the sensitivity of mass tag pcr to detect live virus was tested by using rna extracted from serial dilutions of titered stocks of coronaviruses ( severe acute respiratory syndrome [ sars ] and oc43 ) and parainfluenzaviruses ( hpiv 2 and 3 ) .
rna extracted from a 1-tcid50/ml dilution , representing 0.025 tcid50 per pcr reaction , was consistently positive in mass tag pcr .
rna extracted from banked sputum , nasal swabs , and pulmonary washes of persons with respiratory infection was tested by using an assay panel comprising 30 gene targets that represented 22 respiratory pathogens .
infection in each of these persons had been previously diagnosed through virus isolation , conventional nested rt - pcr , or both .
reverse transcription was performed using random hexamers , and mass tag pcr results were consistent in all cases with the established diagnosis .
infections with respiratory syncytial virus , human parainfluenza virus , sars coronavirus , adenovirus , enterovirus , metapneumovirus , and influenza virus were correctly identified ( table 2 and figure 2 ) . a panel comprising gene targets representing 17 pathogens related to central nervous system infectious disease ( influenza a virus matrix gene ; influenza b virus ; human coronaviruses 229e , oc43 , and sars ; enterovirus ; adenovirus ; human herpesvirus-1 and -3 ; west nile virus ; st .
louis encephalitis virus ; measles virus ; hiv-1 and -2 ; and streptococcus pneumoniae , haemophilus influenzae , and nesseria meningitidis ) was applied to rna obtained from banked samples of cerebrospinal fluid and brain tissue that had been previously characterized by conventional diagnostic rt - pcr .
eleven of 12 cases of enteroviral meningitis were detected representing serotypes cv - b2 , cv - b3 , cv - b5 , e-6 , e-11 , e-13 , e-18 , and e-30 ( data not shown ) . *
rsv , respiratory syncytial virus ; hpiv , human parainfluenza virus ; cov , coronavirus ; sars , severe acute respiratory syndrome .
positive and consistent with previous diagnosis / number tested ( with respective previous diagnosis ) .
rna extracts from clinical specimens containing known pathogens were reverse transcribed into cdna ( superscript rt system , invitrogen , carlsbad , ca ; 20-l volume ) .
five microliters of the reaction were subjected to mass tag pcr by using primers coupled to masscode tags ( qiagen masscode technology , qiagen , hilden , germany ) .
detection of ( a ) influenza virus a ( h1n1 ) , ( b ) respiratory syncytial virus ( rsv ) group b , ( c ) human coronavirus sars ( hcov - sars ) , ( d ) human parainfluenza virus ( hpiv ) types 1 and ( e ) 3 , and ( f ) enterovirus ( ev ) by using a 30-plex assay , including 60 primers targeting influenza a virus matrix gene ( fluav - m ) , and for typing n1 , n2 , h1 , h2 , h3 , and h5 sequences , as well as influenza b virus ( flubv ) , rsv groups a and b , hcov-229e , -oc43 , and -sars , hpiv types 1 , 2 , 3 , and 4 ( groups a and b combined ; 4 primers ) , human metapneumovirus ( hmpv , 4 primers ) , measles virus ( mev ) , ev ( degenerate primer pair targeting all serogroups ) , human adenoviruses ( hadv , degenerate primer pair targeting all serogroups ) , human herpesvirus 1 ( hhv-1 , herpes simplex virus ) , human herpesvirus 3 ( hhv-3 ; varicella - zoster virus ) , mycoplasma pneumoniae , chlamydia pneumoniae , legionella pneumophila , streptococcus pneumoniae , haemophilus influenzae .
the bar indicates an arbitrary cut - off threshold of 2.7 ( 4 times average background determined with random human dna ) .
our results indicate that mass tag pcr is a sensitive and specific tool for molecular characterization of microflora .
the advantage of mass tag pcr is its capacity for multiplex analysis . although the use of degenerate primers ( e.g. , enteroviruses and adenoviruses , table a1 and table 1 ) may reduce sensitivity , the limit of multiplexing to detect specific targets will likely be defined by the maximal primer concentration that can be accommodated in a pcr mix .
although these steps are now performed manually , and mass spectrometers are not yet widely distributed in clinical laboratories , the increasing popularity of mass spectrometry in biomedical sciences and the advent of smaller , lower - cost instruments could facilitate wider use and integrated instrumentation .
in addition to developing additional pathogen panels , our continuing work is focused on optimizing multiplexing , sensitivity , and throughput .
potential applications include differential diagnosis of infectious diseases , blood product surveillance , forensic microbiology , and biodefense . | naturally emerging and deliberately released pathogens demand new detection strategies to allow early recognition and containment .
we describe a diagnostic system for rapid , sensitive , multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples . |
nerve sheath myxoma ( neurothekeoma ) is an uncommon benign soft tissue tumor of the peripheral nerves with fairly distinctive histological features .
a recent immunohistochemical pattern enables a differentiation between myxoid neurothekeoma , melanocytic tumors , and nervous system tumors .
we report a rare case of myxoid neurothekeoma in a three - year - old child .
a three - year - old boy presented with a nontender , left thumb swelling of four - week duration , without any history of trauma .
fine needle aspiration cytology of the mass revealed a few plump - to - oval spindle - shaped cells in a myxoid background .
gross examination of the submitted specimen revealed a firm , elastic , tan - colored smooth nodule , measuring 10 8 6 mm with a solid homogeneous gray cut surface [ figure 1 ] .
histopathologic examination revealed a nonencapsulated dermal tumor , composed of multiple , closely situated medium - sized nodules with scattered spindle - shaped cells in a myxoid - rich stroma [ figure 2 ] .
the tumor exhibited strong positivity for s-100 immunohistochemical stain [ figure 3 ] , and focal positivity for neuron - specific enolase ( nse ) .
based on the histopathologic and immunohistochemical results , the case was diagnosed as nerve sheath myxoma of classical type .
neurothekeoma : cut section shows solid homogeneous soft , yellowish gray tissue neurothekeoma : tissue section shows a nonencapsulated dermal tumor , composed of multiple , medium - sized nodules composed of few oval - to - plump spindle cells , in a myxoid - rich stroma ( h and e , 10 ) neurothekeoma : the tumor cells show strong positivity for s-100 immunohistochemical stain
neurothekeomas commonly present as small solitary erythematous nodules on the face and the upper limb .
however , other anatomic sites have been reported in the literature , such as oral cavity , cauda equina , lower limb , shoulder , and neck .
it is a tumor of the adolescent and young adult , with a female predilection , but is extremely rare in infancy .
neurothekeoma is a rare benign tumor of the nerve sheath , with a distinct histological presentation .
it was described initially by hakin and reed , in 1969 , as nerve sheath myxoma but the term neurothekeoma was given by gallager and helwing .
there are three histologic variants of neurothekeoma : myxoid ( classical or hypocellular ) , cellular , and mixed type .
the myxoid type is characterized by a high degree of myxomatous changes , low cellularity with scattered spindle cells , and is well circumscribed .
there may also be multinucleated giant cells and the lesions characteristically stain positively for s-100 , collagen type iv , and nerve growth factor receptor but are negative for epithelial membrane antigen or markers of histiocytic differentiation .
in contrast , the cellular types of neurothekeoma are not encapsulated and the mixed type shows areas of varied cellularity with focal myxoid regions . our case is a typical example of a myxoid type of neurothekeoma .
the cell of origin of neurothekeoma is controversial with theories proposing a proliferation of schwann cells or other perineural cells .
dual immunoreactivity for nse and s-100 protein in a myxoid background supports schwann cell origin , whereas the absence of s-100 protein and positive epithelial membrane antigen in the cellular type suggests perineural cell lineage .
fetsch , et al . postulated a tendency of neurothekeoma to recruit histiocytic cells and to differentiate into myofibroblasts .
smooth muscle actin ( sma ) positivity suggests some similarity to the epithelioid variant of leiomyoma or myofibroblastic proliferations .
glial fibrillary acidic protein ( gfap ) positivity is rare in neurothekeoma ; however , its positivity further supports a peripheral nerve sheath origin , as occasional peripheral nerve sheath tumors may coexpress gfap and s-100 .
nerve sheath myxoma and neurothekeoma are recently identified as different entities in gene expression profiling , although they might share some morphologic similarities .
gene expression array data strongly supports that nerve sheath myxomas are of peripheral nerve sheath origin and are distinct neoplasms from neurothekeomas .
furthermore , data shows many similarities in gene expression between neurothekeomas and cellular fibrous histiocytomas , supporting recent arguments that these entities may in fact be related .
there is an overlapping feature with other neural tissue tumors such as schwannoma , nerve sheath myxoma , and neurofibroma , leading to difficulties in diagnosis .
differential diagnosis of dermal nodule in infancy and young adulthood should include fibrous tumors , histiocytic tumors , lymphocytic tumors , melanocytic tumors , and neural tumors .
reporting such a case will increase awareness about this disease , as it should be included in the differential diagnosis of dermal lesions in young children . | neurothekeomas are rare benign neoplasms , typically occurring in young patients with a remarkable predilection for the female population . patients usually present with a small nodule in different anatomical sites , commonly involving the face and the upper limb .
we report a case of a three - year - old boy , who presented with a nontender nodule on the left thumb . surgical biopsy and
immunostaining confirmed the diagnosis as myxoid neurothekeoma .
the rarity of this unusual skin tumor in a toddler prompted the following report . |
abnormalities in their normal circulating number either in the form of thrombocytosis or thrombocytopenia are associated with many pathologic conditions .
cytokines that stimulate thrombopoiesis are often elevated in cancer , and as a result , various cancers have been associated with thrombocytosis . in addition thrombocytosis has been found to be an adverse prognostic factor in many types of common cancers . breast cancer is the most common female malignancy . when localized , it is treated with surgery and often with adjuvant therapies to decrease the risk of local or systemic recurrences .
both adjuvant therapies and therapies in the metastatic disease setting are guided by biologic characteristics such as hormone receptors and growth factor her-2 expression .
these characteristics possess also prognostic information , but additional markers are needed to further promote prognostication of breast cancer . in early stage breast cancer patients undergoing chemotherapy in five clinical trials of the austrian breast and colorectal cancer study group , pretreatment thrombocytosis was an independent prognostic factor for overall survival and breast cancer - specific survival .
we investigated if pretreatment high thrombocyte counts provide prognostic information in patients with metastatic breast cancer .
case records of women with breast cancer treated in the medical oncology department of the university hospital of lausanne over the last 12 years were retrospectively reviewed .
further information was collected from charts of patients with metastatic disease at diagnosis or at any later time of their disease history and with complete follow - up .
follow - up was considered complete if a patient was followed till her death or was seen within the last 6 months from data collection .
data on patients ' age , biologic characteristics of tumors , time from original diagnosis to metastatic disease , and site(s ) of metastases were recorded .
platelet number on diagnosis of metastatic breast cancer ( before the start of any therapy for metastatic disease ) was evaluated in 179 patients for whom there were complete follow - up data . overall survival ( os )
was defined as the interval from the date of diagnosis of metastatic disease to death .
progression free survival ( pfs ) was defined as the interval from the date of diagnosis of metastatic disease to disease progression or death , whichever happened first .
positivity was defined as 3 + by immunohistochemistry ( ihc ) or fish amplification according to standard criteria . for the purposes of the current study her-2
was considered negative if ihc was 2 + and fish had not been performed . in the normal platelet count group patients with platelet counts of 150 to 350
10/l were included . in the thrombocytosis group patients with platelet counts of > 350
survival plots of patients with normal platelet counts and thrombocytosis were constructed using the kaplan - meier method and were compared using the logrank test .
the test was used to evaluate differences in clinical and biologic characteristics in the two groups .
a cox regression proportional hazard multivariate analysis was performed to identify statistically significant factors associated with overall and progression free survival .
all p values were considered to be significant at the level of p < 0.05 .
statistical calculations were performed with online tools available from the technical university of denmark ( http://www.iscc-serv2.imm.dtu.dk/ ) and a noncommercial site ( http://www.statpages.org/ ) .
among 215 patients with metastatic breast cancer followed in our department during the period of the last 12 years , 179 patients had complete follow - up .
fourteen patients had thrombocytopenia at diagnosis and were excluded from further analysis . from the remaining 165 patients , 135 ( 81.8% )
had normal platelet counts at diagnosis of metastatic disease , and 30 ( 18.2% ) had thrombocytosis ( table 1 ) .
the median platelet count of the whole group of patients was 261 10/l ( range 154694 ) .
the median platelet count in the normal platelet group was 241 10/l ( range 154349 ) and in the thrombocytosis group was 407 10/l ( range 354694 ) .
the median age of the whole group was 62 years old ( range 3192 ) .
110 patients had died , and 55 patients were alive at last follow - up .
the median follow - up of patients alive was 25 months and the mean 30.14 months [ 95% confidence interval ( ci ) 23.5536.74 ] .
the median age of the patients with normal counts was 62 years old ( range 3192 ) and of those with thrombocytosis was 66 years old ( range 4485 ) . in the normal platelets group
92 patients had died , and 43 patients were alive with a median follow - up of 33 months ( mean 33.88 months , 95% ci 25.9541.82 ) . in the thrombocytosis group 18 patients had died and 12 patients were alive with a median follow - up of 12.5 months ( mean 16.75 months , 95% ci 9.3324.17 ) .
there were no statistically significant differences in the proportion of patients in the two groups regarding the hormone receptors status , the her-2 status , the grade of the tumors , whether metastatic disease was confined to bone or had also spread to other sites , and the therapy patients had received as first line metastatic treatment . for patients that had initially been diagnosed with local disease ( n = 98 ) , there was no difference between the normal platelet and thrombocytosis group in the percentage of patients who received no adjuvant treatment or only adjuvant hormonotherapy and those that had chemotherapy included in their adjuvant treatment ( p = 0.87 ) .
more patients in the thrombocytosis group had metastatic disease at diagnosis ( 70% versus 34.1% in the normal platelets group , p = 0.0003 ) .
the median overall survival of patients that had died in the whole group ( 110 of 165 patients ) was 24 months , and the mean was 28.2 months ( 95% ci 23.732.7 ) .
the median overall survival ( os ) of patients that had died in the group with normal platelet counts was 26 months ( range 1115 months ) and the mean was 30.08 months ( 95% ci 24.9735.19 ) while the median os of patients with thrombocytosis was 12.5 months ( range 254 months ) and the mean was 18.6 months ( 95% ci 10.9526.27 ) .
os was 50% ( 95% ci 41.658.3% ) and 26% ( 95% ci 1444.6% ) at 3 years in the normal and thrombocytosis group , respectively .
there were no long term survivors at 5 year in the group with thrombocytosis while in the normal platelets group 5-years survival was 22% ( 95% ci 15.3529.2% ) .
there was a statistically significant difference of the overall survival between the two groups ( logrank test p = 0.038 ) ( figure 1 ) . in the multivariate cox regression analysis , increasing age , higher grade , er / pr negativity , and thrombocytosis were statistically significantly associated with reduced overall survival while her-2 status , the presence of metastases at diagnoses , the location of metastatic disease , and the administration and type of first line metastatic treatment were not ( table 2 ) .
there was also a statistically significant difference of the progression free survival between the two groups ( logrank test p = 0.008 ) ( figure 2 ) .
in this instance the multivariate cox regression analysis showed that higher grade , er / pr negativity , the presence of metastases at diagnoses , and thrombocytosis were statistically and significantly associated with reduced progression free survival while age , her-2 status , the location of metastatic disease , and the administration and type of first line metastatic treatment were not ( table 3 ) .
an elevated platelet count may have various causes and is either primary due to essential thrombocytosis or other myeloproliferative disorders or secondary to infection , trauma or surgery , iron deficiency , or malignancy .
when other conditions are excluded , about 40% of patients with thrombocytosis of more than 400 10/l have been found to harbor an occult cancer . in this retrospective study
we have shown for the first time that thrombocytosis is associated with decreased os and pfs in patients with metastatic breast cancer . in our cohort of 165 patients
there were no significant differences between the two groups with normal platelets and thrombocytosis in the age of patients , biologic characteristics of their cancer such as er / pr and her-2 expression and grade , and no significant difference on whether they had bone only or other sites of metastases .
patients with thrombocytosis were more likely to have metastases at diagnosis with breast cancer while the normal platelet group was more likely to have developed metastatic disease at a later time in the course of their disease ( p = 0.0003 ) .
this may be due to a more aggressive biology of cases with thrombocytosis that favors earlier metastases development . in the multivariate analysis , increasing age , hormonal receptors negativity , and higher grade were associated together with thrombocytosis with decreased overall survival .
her-2 was not an independent predictor of overall or progression free survival possibly due to the fact that , for a significant minority of patients ( 23 out of 165 ) , her-2 status was unknown or ambiguous ( meaning that it was 2 + by ihc , and no fish was performed ) or due to treatment with anti - her-2 targeted therapies which improve the outcome of these patients [ 8 , 9 ] .
bone only disease also was not a significant prognostic factor in the multivariate analysis possibly due to its association with other parameters and notably hormone receptor status .
other investigators have previously detected an association of thrombocytosis with worse outcome in patients with early breast cancer .
this retrospective study is the most extended series addressing the subject in any type of cancer and included 4,300 patients that had been treated in five randomized trials of the austrian breast and colorectal cancer study group . in this report thrombocytosis
was defined as platelets more than 400 10/l and was present in 3.7% of patients .
the estimated overall survival of patients with thrombocytosis was 71 months versus 99.5 months in patients without thrombocytosis .
thrombocytosis was an independent prognostic factor for overall survival and breast cancer related survival but not for disease - free survival .
its association with worse oncologic outcomes has been also reported in esophageal squamous cell carcinoma , gastric adenocarcinoma , renal cell carcinoma , ovarian carcinoma , and other cancers [ 14 , 15 ] . in patients with esophageal squamous cell carcinoma , thrombocytosis , defined as platelets more than 293 10/l which was the mean plus 1 standard deviation of a healthy control group , was present in 21% of patients and was a significant independent prognostic factor .
this association was statistically significant for patients with stages iii and iv but not for stages i and ii disease . in gastric adenocarcinoma patients that had undergone gastrectomy with negative margins ,
thrombocytosis ( defined as platelets more than 400 10/l in this study ) was associated with worse survival and was a strong predictor specifically of hematogenous metastasis but not of locoregional recurrence or peritoneal seeding . in another series of gastric cancer patients ,
thrombocytosis was associated with worse 1-year and 3-year survival and was positively correlated with depth of tumor invasion .
metastatic renal carcinoma patients with thrombocytosis ( defined as platelets more than 400 10/l at least in one occasion during their disease ) had a mean survival of 92 months compared with 151 months in patients with normal platelets .
thrombocytosis ( defined as platelets more than 450 10/l ) was associated with more advanced stage and higher preoperative ca-125 in ovarian carcinoma .
in addition the median overall survival in patients with thrombocytosis was 2.62 years while in the group with normal platelets it was 4.65 years ( p < 0.001 ) .
in another study in ovarian cancer , thrombocytosis ( defined as platelets more than 400 10/l ) was also associated with advanced stage and grade and reduced overall survival .
thrombocytosis was significantly correlated with plasma levels of il-6 in patients with ovarian carcinoma . in mouse models bearing human ovarian cancer , human il-6
thus a model was proposed according to which ovarian cancer tumor cells produce il-6 which stimulates the liver to produce thrombopoietin , finally resulting in increased thrombopoiesis through stimulation of megakaryocyte progenitors in the bone marrow . in other cancers
serum levels of il-6 were elevated in prostate cancer patients with more advanced stage disease and correlated with decreased disease - specific survival . in breast cancer serum il-6 levels were significantly higher than in a control group of healthy women .
thus il-6 produced from tumor cells may be a pathophysiologic trigger of tumor - induced thrombocytosis across different cancer types in a manner similar to that proposed for ovarian cancer .
the mechanistic basis of platelets contribution to carcinogenesis is a subject of study . circulating tumor cells
may use platelets as a shield to protect themselves from the attack of the immune system and as an intermediary helping them to attach to endothelial cells at the destination sites of metastases .
in addition heteroaggregates constituted of platelets and tumor cells may embolize in the microcirculation and aid in the process of extravasation of tumor cells in metastatic sites .
platelets have also roles in carcinogenesis directly related to their normal function in promotion of vascular integrity .
newly formed tumor vasculature is particularly prone to dysfunction , and platelets have been shown to be indispensable for preventing hemorrhage in tumor beds .
platelets are carriers in their granules of a plethora of bioactive molecules and growth factors .
these include vascular endothelial growth factor ( vegf ) , epidermal growth factor ( egf ) , platelet - derived growth factor ( pdgf ) , hepatocyte growth factor ( hgf ) , insulin - like growth factor ( igf ) , transforming growth factor ( tgf ) , interleukin 1 ( il-1 ) , il-8 , cxc motif containing ligand 12 ( cxcl12 ) , sphingosine-1-phosphate ( s1p ) , and lysophosphatidic acid among others [ 24 , 25 ] .
platelet - derived tgf , for example , promotes an emt ( epithelial to mesenchymal transition ) program in tumor cells through smad and nf-b signaling in these cells .
emt is a program that endows epithelial cells with a mesenchymal phenotype that promotes mobility and metastasis while protecting them from anoikis ( apoptosis due to lack of adhesion ) .
platelets from patients with cancer have a higher vegf level than platelets from individuals without cancer .
in contrast circulating vegf is not elevated in cancer patients except in renal cell carcinoma if care is taken to avoid artificial platelet activation during venipuncture . as a result platelet counts may better reflect vegf concentrations in the tumor and metastases sites environment where they are activated and contribute to tumor angiogenesis . in conclusion
, this retrospective analysis of a series of metastatic breast cancer patients shows that thrombocytosis ( defined in this paper as platelets more than 350 10/l ) at the time of diagnosis of metastatic disease has prognostic value regarding overall and progression free survival .
the presence of thrombocytosis is independent of biologic characteristics that help classify breast cancer to subgroups relevant for treatment such as er / pr and her-2 expression and thus has value beyond these characteristics .
further study is needed in more extensive series to confirm these results and especially to test whether thrombocytosis can serve as a predictive marker of specific treatments . in this respect and in view of the above discussion it would be of particular interest to test thrombocytosis as a predictive marker of anti - vegf therapies .
indeed a recent study in metastatic renal cell carcinoma has shown that patients with thrombocytosis had a higher risk to present a primary refractoriness to anti - vegf treatments ( odds ratio = 1.7 , p = 0.0068 ) than patients with normal platelets .
it remains to be seen if thrombocytosis could be a predictive factor for anti - vegf therapies in other cancers and in breast cancer in particular . | background .
an elevated platelet count is often associated with malignancies , and it has been confirmed as an adverse prognostic factor in various cancers including early stage breast cancer .
we sought to determine if thrombocytosis is also a prognostic factor in metastatic breast cancer .
patients and methods . the records of 165 metastatic breast cancer patients with complete follow - up that had thrombocytosis or normal platelet counts were reviewed .
kaplan - meier curves were constructed , and the survivals of the two groups were compared using the logrank test . a cox regression analysis was used to determine if thrombocytosis is an independent factor for overall and progression free survival .
results .
there was a statistically significant difference in overall and progression free survival favoring the normal platelets group ( logrank test p = 0.038 and 0.008 , resp . ) .
thrombocytosis remained a significant adverse prognostic factor in multivariate analysis .
other independent prognostic factors for overall survival included age , er / pr status , and grade . conclusion .
thrombocytosis represents an independent adverse prognostic factor in patients with metastatic breast cancer .
thus metastatic breast cancer joins a range of cancers in which this easily measurable value can be used for clinical prognostication .
further use as a predictive value for specific treatments has a rationale and deserves to be investigated . |
proske and
gandevia described proprioception as collective information from specialized peripheral
neural endings , located at the muscle spindle , golgi tendon , or joints , which are capable of
alerting the central nervous system as to joint position , muscle force , and movement via the
dorsal roots of the spinal cord1 . in
addition , the sense of force , as one of the submodalities of proprioception , has also been
described as tension during the contraction that plays an important role in the quality of
motor function2 .
disruption in proprioception would be regarded as a risk factor to the neuromuscular
system , and may result in a variety of injuries .
for instance , age - related deficits in
proprioception of the knee joint reportedly play an important role in initiating and
advancing osteoarthritis of the knee3 , 4 . based on the literature ,
there are two main approaches used to measure the sense of force .
the first attempts to reproduce a target force as a certain level of maximum voluntary
isometric contraction ( mvic ) of a muscle group in one limb or a reference side , and
is known
as the ipsilateral remembered method ; the second aims to reproduce the same target force in
the opposite limb , and is known as the contralateral method5,6,7 .
evaluation of such a reproduced force sense requires a precise device with the capability
of measuring muscle tension in both limbs either at the same time , or within a very short
period of time . for the lower limb ,
the available instruments require the subjects to change
their position during the force sense survey .
this would interfere with the accuracy of
results , as the crucial time frame allocated to each measuring test might be inadvertently
exceeded .
furthermore , as the majority of knee studies have been devoted to other areas such
as sense of position , pathokinesiology , kinesthesia , or compromised electromyography ( emg )
patterns of knee muscles8,9,10,11 , few academic studies have addressed all major methods of
force sensing measurement , including ipsilateral remembered ( ir ) , contralateral concurrent
( cc ) , and contralateral remembered ( cr ) . because the knee is one of the joints most vulnerable to sports injuries , and due to the
lack of a comprehensive measuring device for use in force sense reproducing tasks , a new
device was designed , and its reliability for measuring sense of force was thoroughly tested .
the need for a practical , low - cost device that can target both knee joints simultaneously or
separately , with ease of use and a high rate of accuracy , unlike currently available
isokinetic devices , motivated us to design such a clinically applicable instrument ; this
device does not require highly specialized operators or application of any invasive method
to achieve results .
eighty students from tabriz university of medical sciences were included in our initial
group of participants , and 54 met our inclusion / exclusion criteria .
thirty participants ( 8
female and 22 male students ) were selected randomly ( simple randomization ) from this group
of 54 eligible candidates by using randlist 1.2 software ( registration code : 1500247846 ) .
the sample size was calculated by considering =0.05 , power=80% , and r=0.70 , with a 10%
difference .
inclusion criteria were : no history of professional sports activities , age between 2030
years , and body mass index ( bmi ) between 2225 kg / m .
the exclusion criteria
were : a history of knee pain and limited range of motion within the last six months ,
deformities , fractures , arthritis , and any systemic or neurological disorder .
then , a voluntary consent form was
signed by each participant to be officially considered for this study .
the study protocol
was approved by the ethics committee of shahid beheshti university of medical sciences
( tehran , iran ) .
the device consisted of an adjustable chair , two dynamometric mechanical arms equipped with
hinged plates , two korean manufactured load cells ( cp 500 newton model from www.dacell.com ) ,
one usb port for intercommunication between analogue and digital components of the device
using the c++ software program ( microsoft matlab ) , math works ( a sony laptop ) , vaio care
software , e series laptop ( 4192 - 71011 ) , and connecting cables .
matlab software ( microsoft matlab ,
math works ) was utilized for trend data analysis , mean calculation , and data classification
purposes ( fig .
illustration of the designed device and reference position during the measurement
process before starting the actual tests , the accuracy of the load cells was checked by using sets
of paired standard weights including 1 , 2 , 3 , 5 , 10 , 15 , 20 , 25 , and 30 kg .
the results are shown in table 1table 1.results of one - sample t - test for standard weights , using two load cellsstandardweightsloadcellsload cell 1load cell 2(n)(n)1 kg=9.8 n9.8 0.0049.8 0.0042 kg=19.6 n19.6 0.00519.6 0.0043 kg=29.4 n29.3 0.00529.4 0.0035 kg=49 n49.0 0.0449 0.0210 kg=98 n98.0
0.06196 0.0625 kg=245 n245 0.007245 0.0430 kg=294 n294 0.007294 0.04data are shown as mean sd , n : newton .
data are shown as mean sd , n : newton to minimize tactile feedback12 caused by
direct contact between the lower leg and the plates , the distal portion of the lower leg was
protected by a thick foam pad and a piece of thin leather used to cover the surface of each
hinged leg plate .
the device chair and dynamometric mechanical arms were adjusted to support 110 trunk
backward extension and 60 knee flexion , respectively .
each hinged leg plate was positioned just above the middle
malleolus of the corresponding leg .
the plates were attached to the lower leg with velcro
straps , and excessive movement was prevented by two straps fastened around the thighs and
across the trunk ( fig .
the
dominant limb was determined by the subjects ; the reference position was demonstrated ;
verbal commands and visual feedback were explained .
the study protocol included detection of maximum voluntary isometric contraction ( mvic ) and
two main measuring approaches known as ir and contralateral methods .
the latter approach
contained two subgroups known as cc and cr methods . in order to detect mvic , three single , strong voluntary isometric contractions , with
60-second rest intervals ,
each verbally prompted contraction lasted for 5 seconds , and the highest value
was taken as the mvic .
later , subjects were asked to exert only 50% of their recorded mvic
as a target force through the producing and reproducing phases of each contraction set4 , 12 , 13 .
each set of the ir method included a visually - assisted produced target force for up to 8
seconds in the reference knee joint , and a 5-second rest period , followed by a similar
contraction quality of reproduced target force within the same side minus a visual cue .
the contralateral method was subdivided in two parts , i.e. , cr and cc methods . while each
set of the cr method consisted of a visually - assisted produced target force in the reference
limb , a 5-second rest period , and a non - feedback assisted reproduced target force on the
opposite side , each set of the concurrent method followed a similar protocol , but with no
rest period .
both methods asked subjects to sustain their produced / reproduced target force
for the entire 8 seconds4 , 12 , 13 .
subjects were to alert the assessors when reaching their assumed target
force , and had to sustain this to the final second . although subjects had to complete every
method in the study protocol , this process was not accomplished in any particular order , and
methods could be covered randomly by each participant .
participants received 5-minutes rests
between any two methods , and a 3-minute rest before initiating the next set of contractions
within the same method .
to assess the test reliability in frequent measurements of force sense reproduction tasks ,
subjects were instructed to complete all study procedures twice , with only a 72-hour gap
between test dates . in total , three strong contractions were enough to determine the mvic in
extensor and flexor muscle groups of the knee joint separately before executing 36
contractions , each equal to 50% of the mvic .
therefore , the subjects were expected to cover
3 methods including [ ir , cc , and cr ] 3 sets 2 contractions 2 different muscle groups . in order to ensure accuracy and specificity of the device , a team including a
physiotherapist , designer , and biomechanical and electronic engineers met to evaluate any
force sense matching error through the study period .
the accuracy of the usb port was also as high as possible , with 16-bit resolution , and a
sampling rate of 64 per second .
the device calibration was also measured by linear regression and a one - sample
t - test , as shown in table 1 .
the
data distribution was evaluated using the shapiro - wilk test , and deemed normal after an
acceptable minimum score of 0.112 was achieved .
the difference between each produced and
reproduced target force was recorded for any set of contractions , and considered as an
absolute value to disregard the direction of the error .
the average of these values for
every three sets of contractions in any method ( flexor / extensor ) was later calculated to
determine the absolute error of the mean for that method . in order to normalize the error values as a fair percentage of the recorded mvics for all
participants , the error of the mean for each method
was divided by the mvic and the result
was multiplied by 100 [ the error of the mean / mvic 100]6 , 13 , 14 .
the values of the intraclass correlation coefficient ( icc ) and actual units of standard
error of measurement or , shown in table 3table 3.the mean standard deviation , interclass correlation coefficient ( icc ) , standard
error of measurement ( sem ) , and smallest detectable difference ( sdd ) for three force
sense measuring methods of knee flexor and extensor muscle groupsvariablemeasuring methodsfirst sessionsecond sessioniccsemsddmean sdextensor musclesir2.06 0.890.81 1.920.760.381.05cc2.10 0.902.20 0.990.80.481.32cr2.97 1.413.13 1.520.80.711.96flexor musclesir2.50 1.212.76 1.240.730.451.24cc3.01 1.382.83 1.340.810.371.02cr3.15 1.463.20 1.560.730.742.04ir : ipsilateral remembered , cc : contralateral concurrent , cr : contralateral
remembered , indicate the relative absolute reliability of this study . to determine the
actual changes in force reproducing error ,
ir : ipsilateral remembered , cc : contralateral concurrent , cr : contralateral
remembered a paired samples t - test was conducted to compare the mean variables obtained from different
dates of measurement , and spearman s correlation coefficient test was used to investigate
the existing correlation .
demographic characteristics of participants and mvic mean value of flexor and extensor
muscle groups are shown in table 2table 2.demographic characteristics and mean maximum voluntary isometric
contraction variablevariable levelsmean sd age ( years)23.4 2.5bmi ( kg / m ) 2.1 9.22mvic ( knee flexors)day 1173.3 33.7 nday 2167.8 25.4 nmvic ( knee extensors)day 1336.2 73 nday 2348.7 65 nmvic : maximum voluntary isometric contraction ;
mvic : maximum voluntary isometric contraction ; n : newton in evaluating the calibration with a linear regression test , r square=1 and r square=0.999
were used for load cells one and two , respectively .
the highest confidence rates were attributed to methods
cc=0.81 and cr , and cc=0.80 for knee flexor and extensor muscle groups , respectively ( table 3 ) .
the pooled data did not show a statistically significant difference between the first and
the second date of the study ( paired sample t - test and spearman s correlation coefficient
test ) .
however , the average value of mean sd and sem for the cr method was higher for both
muscle groups in this experiment .
the least value for sdd was attributed to the cc method in
the flexor muscle group , at 1.02 , while the cr method in the flexor muscle groups had the
highest sdd rate , at 2.04 .
absence of meaningful differences across values of mean sd for the
reproduced target force ( p<0.05 ) were convincing enough ; that no systemic bias could
interfere with the results . the device reliability for assessing force sense reproduction
across the different study test methods , 0.73 to 0.81 , was consistent with what munro
previously described as a high class of reliability16 .
in this classification , icc ranges from 0 to 0.25 and 0.26 to 0.49
are known as poor and weak , respectively , while a moderate icc must show a range of 0.50 to
0.69 . within this classification ,
the high and very high ranges of reliability are
identified as 0.70 to 0.89 and 0.90 to 0.100 , respectively16 . in order to evaluate the device reliability in this study , and for measurement of the force
sense reproduction tasks ,
the mean sd was calculated in a manner similar to that used by
docherty , dover , and their associates throughout their force sense experiments7 , 12 , 17 .
when errors associated with frequent measurements are considered , sem will assist in
determining the range within which the subject s true score is expected to lie18 .
this provides the value of measurement
error in the same units as the measurement itself .
sem is also more applicable for daily
clinical decision - making than icc itself , which can sometimes be difficult to interpret15 , 19 .
in fact , sem is inversely correlated with absolute reliability , and
thus the accuracy of the obtained measurements20 ,
21 . in this study ,
the sem recorded
through a sequence of frequent measuring methods in reproducing the target force was
relatively small , at 0.37 to 0.74 .
sdd , indicating the smallest statistically significant
change in measurement results , would enable us to estimate the actual error in force
reproducing tasks .
in fact , the smaller range of sdd would allow those true changes in
different tests to be detected more precisely , or those larger than the defined sdd to be
considered as true errors in force reproducing methods15 . in general , regulating muscle tension and the sense of force should vary among individuals .
the variations in those who perform precise work , such as musicians , are much smaller ,
whereas the reverse is true for those who perform powerful , coarse tasks22 .
the term , just - noticeable difference ( jnd ) , quantifies how different two stimuli must be
before subjects reliably report them as distinct23 .
a 510% jnd was previously reported by weber for producing and
controlling muscle force when participants were asked to hold a 975-g weight in their
hands24 , 25 .
thus , some noticeable differences in measurement of force sense
reproducing tasks through two study dates could be attributed to variations in this sense
rather than accuracy of the device . however , as no specific research has measured the
accuracy of force sense reproducing tasks of the knee joint based on this study , no direct
comparison can be determined between our findings and previous studies .
based on the results of this study , all test methods demonstrated acceptably high
reliability with a small standard error in frequent measuring tasks .
therefore , for
measurement of force sense reproduction in knee joint flexor / extensor muscle groups , this
device can be applied for diagnostic and therapeutic procedures with a great degree of
reliability .
the cost - effectiveness of this device in comparison with commercial isokinetic devices , and
its high precision to a level of one - hundredth of a newton , along with its ease of use and
comfort for operator and subjects , are major advantages . added advantages would include
simultaneous bilateral force sense evaluations on the lower limbs , which are equally
applicable to upper limb measurements with a small adjustment in the dynamometric arms of
the device .
the device was only able to measure the sense of force and reproduced force through
isometric contractions , and the sample was exclusively composed of normal subjects . | [ purpose ] the aim of this study was to determine the reliability of a newly designed
dynamometric device for use in frequent force producing / reproducing tasks on the knee
joint . [ subjects and methods ] in this cross - sectional study ( development &
reliability ) , 30 young healthy males and females ( age 23.4 2.48 years ) were selected
among students of tabriz university of medical sciences by simple randomized selection .
the study instrument was designed to measure any isometric contraction force exerted by
the knee joint flexor / extensor muscles , known as the ipsilateral and contralateral
methods .
participant knees were fixed in 60 flexion , and each participant completed the
entire set of measurements twice , 72 hours apart . [ results ]
the findings showed a good
intraclass correlation coefficient of 0.73 to 0.81 for all muscle groups .
the standard
error of measurement and smallest detectable difference for flexor muscle groups were 0.37
and 1.02 , respectively , while the values increased to standard error of measurement=0.38
and smallest detectable difference=1.05 for extensor muscle groups .
[ conclusion ] the
device designed could quantify the forces producing / reproducing tasks on the knee joint
with a high rate of reliability , and can probably be applied for outcome measurements in
proprioceptive assessment of the knee joint . |
crohn 's disease ( cd ) is one of the clinical forms of inflammatory bowel disease ( ibd ) resulting from a defective regulation of mucosal immune responses to commensal microbiota in genetically susceptible individuals .
the last years have contemplated substantial progress in the identification of the genes involved in cd predisposition , boosted by the hapmap project and genome - wide association studies . a better understanding of the biological pathways underlying cd pathogenesis will lead to the development of new therapeutic approaches that specifically target those pathways and will eventually allow personalized treatments .
therefore , an increasing need exists to predict the therapy most fitted to each patient .
since 1998 , when the us food and drug administration approved infliximab ( ifx ) for treatment of moderate or severe cd that does not respond to a conservative treatment , monoclonal antibodies to tumor necrosis factor alpha ( tnf- ) have become the hallmark treatment for refractory cd .
infliximab has proven to be effective for the treatment of both luminal and fistulizing cd . however , a lack of response or a partial response to ifx has been consistently observed and a growing need exists to identify biomarkers of response in order to achieve a more efficacious use of this expensive and potentially toxic therapy .
moreover , data from clinical trials of ifx suggest that high - risk patients and patients with active inflammation may benefit from earlier use of this drug .
clinical parameters such as concurrent therapies , smoking habits , or previous surgery seem to account for only a small amount of the variance in response to anti - tnf therapies . in a recent study by microarray analysis , pretreatment mucosal gene expression profiles predicted response to first ifx treatment in cd patients .
the study identified a 100% accurate predictive gene expression signature for response to ifx in crohn 's colitis ; class prediction analysis allowed complete separation between responders and nonresponders through a panel of 5 top significant genes : s100a8 and s100a9 , g0s2 , tnfaip6 , and il11 .
this biological therapy has been also used for the treatment of other chronic inflammatory diseases such as rheumatoid arthritis ( ra ) .
ra and cd probably share a pathogenetic background , as they have been associated with overlapping susceptibility genes .
therefore , it is reasonable to expect that common genes would anticipate the response to this therapy in both conditions .
up to date , several studies used genome - wide expression analysis to identify expression signatures predicting response to anti - tnf treatment in ra patients , but results showed little overlap .
the identified expression profiles were often not consistent with each other , and different gene sets were reported to distinguish between responders and nonresponders .
therefore , despite the original promising results in cd , the expression signature could be considered a first step towards a predictive test .
provided corroboration from a genetic standpoint is achieved , the cluster of five genes with different expression in cd responders compared to nonresponders to ifx therapy would be a useful tool to classify patients . with this hypothesis , we aimed at investigating the reported expression profile by exploring the association of tagging variants in those top - five genes with the response to ifx in an independent spanish cohort of cd patients .
once an association between polymorphisms within those genes and the response to ifx is found , a causal mechanism of ifx response would be envisaged and a simpler way to classify patients could be established , as it is much easier to perform genotyping of the involved genes than to check their levels of expression .
overall , 350 unrelated white spanish patients with active cd were consecutively recruited from 8 centers .
eligible patients were at least 18 years old , had an established diagnosis of cd , and had received at least the 3 induction doses of ifx ( 5 mg per kilogram ) at weeks 0 , 2 , and 6 .
diagnosis of cd was based on standard clinical , radiologic , endoscopic , and histological criteria .
ifx was administered to treat either moderate to severe active luminal cd or active fistulizing perianal cd .
disease phenotype was determined following the montreal classification : age at diagnosis ( a1 : 16 years ; a2 : 1740 years ; a3 : > 40 years ) , anatomic location ( l1 : terminal ileum ; l2 : colon ; l3 ; ileocolon ; l4 : upper gastrointestinal tract ; and + l4 : upper gastrointestinal modifier ) , and disease behavior ( b1 : inflammatory ; b2 : stricturing ; b3 : penetrating ; and p : perianal modifier ) .
patients were classified as responders ( remission or partial response ) or nonresponders to ifx . the response to ifx
was determined by a chronological review of the medical records and data were centrally monitored .
the response to ifx in patients with luminal disease was evaluated by the harvey - bradshaw index ( hbi ) at the beginning and 10 weeks after the first ifx dose .
partial response was defined as a decrease in the hbi of more than 3 points and absence of concomitant corticosteroids .
remission was defined as a final hbi 4 and absence of concomitant corticosteroids . in patients with perianal disease
remission was defined as the complete closure of all fistulas and partial response as a reduction ( 50% ) in the number of draining fistulas .
patients either receiving ifx for both luminal and fistulizing disease or achieving remission of any type that justified the maintenance of ifx treatment were considered responders .
all patients who did not achieve partial response or remission after the three ifx induction doses were considered nonresponders . in order to cover the highest variability within each gene showing altered expression , we chose single nucleotide polymorphisms ( snps ) by aggressive tagging from the hapmap b36 ceu population , which captured markers with r > 0.8 ( mean r = 0.93 ) and a minor - allele frequency ( maf ) > 0.1 .
genotyping of the spanish samples was carried out with predesigned taqman assays from applied biosystems ( applied biosystems inc . ,
foster city , ca , usa ) , in a 7900ht fast real - time pcr system , under conditions recommended by the manufacturer .
genotyping call - rate success was over 95% for the snps in all groups of patients .
demographic and baseline characteristics were compared between responders and nonresponders by using the mann - whitney u test or the chi - square test , whether continuous or categorical variables were considered . the statistical analysis to compare allelic and genotypic distributions
was performed using chi - square test or fisher 's exact test ( when expected values were below 5 ) .
odds ratios ( ors ) were calculated and their 95% confidence intervals were estimated using the cornfield method .
haplotypic frequencies were inferred with the expectation - maximization algorithm implemented in the haploview 4.1 software .
linkage disequilibrium was measured by calculating two parameters : r and d ( figure 1 ) .
demographic characteristics were analyzed as potential confounding factors of the ifx response using logistic regression .
baseline characteristics of the spanish patients , classified as responders or nonresponders to ifx therapy , are summarized in table 1 .
a total of 285 ( 82% ) patients were classified as responders and 62 ( 18% ) as nonresponders .
a statistically significant difference was observed between the years of evolution of disease in both groups .
tagging polymorphisms in the five previously identified genes were genotyped in the two groups of cd patients , responders , and nonresponders to ifx therapy , and frequencies are shown in table 2 .
although independently only rs11677200 in the tnfaip6 gene showed a statistically significant result , the aggressive tagging approach allowed a more thorough scrutiny of these genomic regions by analyzing the haplotypes conformed by those polymorphisms ( haplotypic frequencies in responders and nonresponders are summarized in table 3 ) . in the region where the s100a9 , s100a12 , and s100a8 genes map , the most frequent haplotype evidenced a significantly higher frequency in cd nonresponders than in responders to ifx ( p = 0.051 ; or ( 95% ci ) = 1.54 ( 0.972.43 ) ) .
additionally , one haplotype in the il11 region showed a marginal significant association with the response to the anti - tnf treatment ( p = 0.068 ; or ( 95% ci ) = 1.72 ( 0.913.21 ) ) , and trends for association could be observed for haplotypes within the other two chromosomal regions explored ( g0s2 : p = 0.15 ; or ( 95% ci ) = 2.92 ( 0.4515.23 ) and tnfaip6 : p = 0.10 ; or ( 95% ci ) = 0.71 ( 0.461.09 ) ) .
the identification of an expression profile to predict response to ifx by arijs and collaborators was focused in colonic cd patients and therefore we evaluated the specific subgroups of crohn 's patients .
tables 4 and 5 summarize the results in ileal patients and colonic and ileocolonic patients , respectively . as shown , the haplotypes associated in the overall patients were now found significantly associated in the colonic subgroup in the s100a9-s100a8 ( table 5(a ) , gcca haplotype : p = 0.025 ; or ( 95% ci ) = 1.91 ( 1.053.48 ) ) , in the g0s2 ( table 5(b ) , at haplotype : p = 0.025 ; or ( 95% ci ) = 6.31 ( 0.8247.89 ) ) and in the il11 ( table 5(d ) , ct haplotype : p = 0.025 ; or ( 95% ci ) = 2.76 ( 1.395.44 ) ) regions .
in contrast , in the tnfaip6 gene , the most frequent caga haplotype only showed a trend for association in ileal patients ( table 4(c ) , caga haplotype : p = 0.1 ; or ( 95% ci ) = 0.57 ( 0.271.17 ) ) , while no difference was detected in colonic patients ( table 5(c ) ) .
pharmacogenetics has emerged as a promising discipline which opens the possibility of a personalized medicine .
however , research has been hampered mainly due to limitations that this kind of studies still shows .
recruitment of a high number of patients with a similar treatment is not an easy task , but difficulty increases because response criteria should be centrally monitored as they were in the present study .
moreover , the high success rate of some pharmacological therapies as ifx originates a low number of nonresponders , with the consequent decrease in the statistical power to detect differences . in our patients , induction therapy with ifx achieved a very good outcome with higher rates of response than those reported in controlled trials [ 3 , 4 ] , in agreement with a high response rate to the three induction doses of ifx previously reported in a multicenter study . moreover , a better response to ifx was observed in patients with an earlier treatment ( table 1 ) , as already reported . the hierarchical cluster analysis performed by arijs and collaborators identified a profile with five differentially expressed genes which was claimed to predict response to ifx in colonic cd patients with an overall accuracy of 100% . in that work ,
the authors validated a previously published gene expression signature regarding response to ifx in ulcerative colitis patients .
il11 was the only overlapping gene between the two predictive top - five gene sets in both clinical forms of ibd .
we aimed to investigate the causal implication of the described genes in the mechanism of ifx response through the association study of those genes in a cohort of spanish cd patients .
moreover , this procedure might provide genetic markers of ifx response that would be easier to test . in our independent cohort of cd patients ,
the tagging approach allowed us to explore a higher genetic variability within the chromosomal regions where the five genes map .
two of these five top genes are s100a8 and s100a9 ; both encode members which belong to the s100 family of calcium - binding proteins and are located in a cluster on chromosome 1q21 .
calprotectin , the heterodimeric complex of s100a8 and s100a9 , shows increased expression at an early step in the neoplastic transformation during colorectal carcinogenesis and it is associated with disease activity in patients with ibd and other inflammatory conditions as rheumatoid arthritis or systemic lupus erythematosus .
moreover , fecal calprotectin concentration is considered a useful surrogate marker for mucosal healing during tnf- blocking therapy for ibd [ 21 , 22 ] . in this genetic region , as mentioned , the most frequent haplotype was found significantly associated with response to ifx , mainly in colonic patients ( 22.7% responders versus 35.9% nonresponders , p = 0.025 ) .
this interleukin is a member of the gp130 family of cytokines that stimulates t - cell dependent development of immunoglobulin - producing b cells , and that was tested as a therapy in cd . in this case , association with borderline significance has been found involving an il11 haplotype with decreased frequency in cd responders to ifx .
moreover , a significant difference was evidenced when colonic cd patients were examined ( 11.5% responders versus 27.2% nonresponders , p = 0.0012 ) .
one gene included in the predictive expression panel for ifx responsiveness is tnfaip6 ( tumor necrosis factor , alpha - induced protein 6 ) , which encodes a multifunctional protein with important roles in inflammation and tissue remodeling .
it is upregulated in many inflammatory conditions as rheumatoid arthritis , a disease that also benefits from ifx therapy , and in colorectal cancer .
another gene previously showing a differential expression profile predictive of response to ifx is g0s2 ( g0/g1 switch 2 ) , involved in lymphocyte cell cycle regulation and found upregulated in rheumatoid arthritis and psoriasis [ 26 , 27 ] .
the haplotypes studied in these two genes evidenced trends for association with the response to ifx in the overall spanish cd patients .
furthermore , only the trend for association observed in the g0s2 gene could be significantly replicated in the colonic subgroup of patients ( 0.8% responders versus 5.2% nonresponders , p = 0.036 ) .
the strategy followed in our study lends support to the reported gene expression profile predictive of anti - tnf therapy in an independent cohort of spanish cd patients .
genetic studies stand out as approaches to define pathogenic pathways and ultimately the integration of genetic together with functional data promotes a clearer understanding of the mechanisms underlying therapeutic pathways . | substantial proportion of crohn 's disease ( cd ) patients shows no response or a limited response to treatment with infliximab ( ifx ) and to identify biomarkers of response would be of great clinical and economic benefit .
the expression profile of five genes ( s100a8-s100a9 , g0s2 , tnfaip6 , and il11 ) reportedly predicted response to ifx and we aimed at investigating their etiologic role through genetic association analysis .
patients with active cd ( 350 ) who received at least three induction doses of ifx were included and classified according to ifx response . a tagging strategy was used to select genetic polymorphisms that cover the variability present in the chromosomal regions encoding the identified genes with altered expression .
following genotyping , differences between responders and nonresponders to ifx were observed in haplotypes of the studied regions : s100a8-s100a9 ( rs11205276*g / rs3014866*c / rs724781*c / rs3006488*a ; p = 0.05 ) ; g0s2 ( rs4844486*a / rs1473683*t ; p = 0.15 ) ; tnfaip6 ( rs11677200*c / rs2342910*a / rs3755480*g / rs10432475*a ; p = 0.10 ) ; and il11 ( rs1126760*c / rs1042506*g ; p = 0.07 ) .
these differences were amplified in patients with colonic and ileocolonic location for all but the tnfaip6 haplotype , which evidenced significant difference in ileal cd patients .
our results support the role of the reported expression signature as predictive of anti - tnf outcome in cd patients and suggest an etiological role of those top - five genes in the ifx response pathway . |
we
employ glass microtube structures fabricated by rolled - up nanotechnology
to infer the influence of scaffold dimensionality and cell confinement
on neural stem cell ( nsc ) migration .
thereby , we observe a pronounced
morphology change that marks a reversible mesenchymal to amoeboid
migration mode transition .
space restrictions preset by the diameter
of nanomembrane topography modify the cell shape toward characteristics
found in living tissue .
we demonstrate the importance of substrate
dimensionality for the migration mode of nscs and thereby define rolled - up
nanomembranes as the ultimate tool for single - cell migration studies . | |
the discovery that the majority of body tissues and cells harbor vitamin d receptors and that several possess the enzymatic equipment to convert the primary form in circulation into the active form , has provided new insights into the function of this steroid hormone .
there is great interest in the role that vitamin d can play in many chronic diseases , including common cancers , autoimmune , infectious , and cardiovascular.13 the sources of vitamin d are primarily the cutaneous production and food intake . in brazil ,
the dietary source is scant , since foodstuffs such as fatty fish are not available in tropical and subtropical climates .
there are no official recommendations in brazil on the fortification of foods with vitamin d.4 elderly populations present certain particularities . with aging , solar exposure
is generally limited in view of the changes in lifestyle , such as clothing and activities outside the home .
. however , more important is the lower cutaneous production of vitamin d with aging , due to the atrophic skin alterations with greater availability of the precursor 7-dehydrocholesterol . among other factors , the production of 1,25-dihydroxyvitamin d3 diminishes as a result of the decrease in kidney function with aging.57 several cutoffs related to the blood levels of 25-hydroxyvitamin d ( 25(oh)d ) have been proposed to define deficiency / insufficiency in asymptomatic individuals .
this is partially due to the variability of the concentration of vitamin d according to geographic location and the different methodologies employed in the various studies.8,9 in northeast brazil , bandeira et al found significant differences in the bone mineral density and in the serum levels of parathyroid hormone , with 25(oh)d values below 25 ng / ml in postmenopausal women.10 ensrud et al , in a large american cohort , demonstrated that 25(oh)d levels below 20 ng / ml were associated with significant hip bone loss in older men , when compared with higher levels , but this loss was not found in younger.11 other studies have demonstrated that levels above 32 ng / ml are necessary for an adequate intestinal absorption of calcium.12,13 although the literature reveals a high prevalence of vitamin d deficiency in the elderly , as far as we are aware , no consistent population studies in the tropics have evaluated its prevalence in elderly men and its relation to sun exposure and skin phototypes .
this was an analytical cross - sectional study , which involved 234 men aged over 60 years of age , randomly recruited from a basic care unit in the fifth health district ( 5th hd ) of the city of recife in the state of pernambuco , during the brazilian summer months , from october 2010 to january 2011 .
the patients eligible for the study were free - living individuals ( people who are not living in an aged care institution ) , able to walk unaided and who had not used drugs such as corticosteroids for prolonged periods ( doses 2.5 mg of prednisone or equivalent for over 3 months ) , anticonvulsants , or lithium for 1 year .
subjects with metabolic bone diseases and active neoplasms , except neoplasms of the skin such as basal cell and spino - cellular cancer , were excluded .
the study protocol was approved by the ethics in research committee of the agamenon magalhes hospital .
demographic and economic data , medications , calcium intake , and vitamin supplements were evaluated by means of a questionnaire .
exposure to the sun was calculated using the sun index ( si ; fraction of the area of the body surface exposed multiplied by the number of hours of sun exposure per week).14 skin phototypes were classified into six types , according to fitzpatrick s classification ( type i being the lightest and type vi the darkest).15 daily calcium intake was defined by asking patients to advise their calcium intake from food and supplements and classified as follows : low = intake below two portions ( 250 ml ) per day of milk and/or dairy products and without calcium supplements ( < 600 mg / day ) ; intermediate = intake of two or more portions without calcium supplementation ( 6001,000 mg / day ) ; and high = daily calcium supplementation and the consumption of milk and/or dairy products ( > 1,000 mg / day).16 when the individual did not have a calcium intake from diary but used supplements , calcium intake was classified according to the calculation of the amount of calcium contained in the supplement . in brazil , the recommended intake of calcium is 1,000 mg daily .
the questionnaire also asked whether the patient was using vitamin supplements containing vitamin d. serum 25(oh)d levels were measured by electrochemiluminescent competitive immunoassay using a diasorin liaison analyzer ( turin , italy ) , with the inter- and intra - assay coefficients of variations of 8%15% and 8%13% , respectively and a minimum limit of detection of 2 ng / ml.17 measurement of 25(oh)d by immunoassay is considered the method of choice for reasons of convenience , speed , turnaround , and cost.17 for the description of the prevalence rates , the percentages and respective 95% confidence intervals were recorded using the binomial method .
pearson s chi - square test and student s t - test with equal or unequal variances were used for percentage and mean comparisons .
the model was performed with step - wise selection of variables , maintaining in the model the variables with a significance of up to 15.0% ( p < 0.15 ) .
the following independent variables were studied : age , annual income , grouped skin phototypes ( group 1 : skin types i iii ; group 2 : skin types
iv vi ) , calcium intake , and body mass index ( bmi ) .
spss software ( v 15.0 ; ibm corp , armonk , ny , usa ) was used for the statistical calculations .
this was an analytical cross - sectional study , which involved 234 men aged over 60 years of age , randomly recruited from a basic care unit in the fifth health district ( 5th hd ) of the city of recife in the state of pernambuco , during the brazilian summer months , from october 2010 to january 2011 .
the patients eligible for the study were free - living individuals ( people who are not living in an aged care institution ) , able to walk unaided and who had not used drugs such as corticosteroids for prolonged periods ( doses 2.5 mg of prednisone or equivalent for over 3 months ) , anticonvulsants , or lithium for 1 year .
subjects with metabolic bone diseases and active neoplasms , except neoplasms of the skin such as basal cell and spino - cellular cancer , were excluded .
the study protocol was approved by the ethics in research committee of the agamenon magalhes hospital .
demographic and economic data , medications , calcium intake , and vitamin supplements were evaluated by means of a questionnaire .
exposure to the sun was calculated using the sun index ( si ; fraction of the area of the body surface exposed multiplied by the number of hours of sun exposure per week).14 skin phototypes were classified into six types , according to fitzpatrick s classification ( type i being the lightest and type vi the darkest).15 daily calcium intake was defined by asking patients to advise their calcium intake from food and supplements and classified as follows : low = intake below two portions ( 250 ml ) per day of milk and/or dairy products and without calcium supplements ( < 600 mg / day ) ; intermediate = intake of two or more portions without calcium supplementation ( 6001,000 mg / day ) ; and high = daily calcium supplementation and the consumption of milk and/or dairy products ( > 1,000 mg / day).16 when the individual did not have a calcium intake from diary but used supplements , calcium intake was classified according to the calculation of the amount of calcium contained in the supplement . in brazil , the recommended intake of calcium is 1,000 mg daily .
the questionnaire also asked whether the patient was using vitamin supplements containing vitamin d. serum 25(oh)d levels were measured by electrochemiluminescent competitive immunoassay using a diasorin liaison analyzer ( turin , italy ) , with the inter- and intra - assay coefficients of variations of 8%15% and 8%13% , respectively and a minimum limit of detection of 2 ng / ml.17 measurement of 25(oh)d by immunoassay is considered the method of choice for reasons of convenience , speed , turnaround , and cost.17
for the description of the prevalence rates , the percentages and respective 95% confidence intervals were recorded using the binomial method .
pearson s chi - square test and student s t - test with equal or unequal variances were used for percentage and mean comparisons .
the model was performed with step - wise selection of variables , maintaining in the model the variables with a significance of up to 15.0% ( p < 0.15 ) .
the following independent variables were studied : age , annual income , grouped skin phototypes ( group 1 : skin types i iii ; group 2 : skin types
iv vi ) , calcium intake , and body mass index ( bmi ) .
spss software ( v 15.0 ; ibm corp , armonk , ny , usa ) was used for the statistical calculations .
the clinico - epidemiological characteristics of the elderly patients studied are shown in table 1 .
the mean age was 69.4 6.5 years ( mean standard deviation [ sd ] ) and the majority ( 66.7% ) had dark skin ( type iv , v , or vi of fitzpatrick s classification ) .
when comparing prevalence according to skin type ( dark vs light ) for each serum 25(oh)d cutoff ( < 20 , < 25 , and < 30 ng / ml ) , we found no significant difference ( p = 0.46 ) .
using cutoffs for serum 25(oh)d levels below 20 and 30 ng / ml , the prevalence of vitamin d deficiency was 31.5% and 66.7% , respectively ( figure 1 ) .
mean serum 25(oh)d was 27.86 13.52 ng / ml ( mean sd ) .
after stratification according to age group ( 6069 , 7079 , and 80 years ) , no difference in the prevalence of vitamin d deficiency was found between them .
there was a significant difference ( p = 0.006 ) between si means in the groups with serum 25(oh)d levels below and above 20 ng / ml ( 4.46 3.69 vs 6.01 5.54 ) .
however , for the cutoffs of 25 ng / ml ( 5.07 4.31 vs 5.95 5.69 ) and 30 ng / ml ( 5.22 4.43 vs 6.12 6.14 ) , no significant difference was found .
considering si values divided into quartiles ( qs ) , the lowest quartile ( q1 ) was 1.96 and the highest ( q4 ) was 7.86 .
nonetheless , there was no statistical difference ( p = 0.113 ) in serum 25(oh)d levels between q1 ( 23.98 14.66 ) and q4 ( 29.88 13.78 ) , nor in the prevalence of hypovitaminosis d for the 20 , 25 , and 30 ng / ml cutoffs ( figure 2 ) . a low calcium intake was observed in 72% of the subjects and an annual family income of us$4,200 in 64.5% . there were no statistical differences between the groups with a low calcium intake and those with an intermediate and high intake at the three cutoffs for 25(oh)d , nor were there between the groups with an annual income of < us$4,200 and of > us$4,200 . a low level of education , defined as illiteracy or incomplete primary school education ,
was observed in 82.8% and only seven subjects ( 2.5% ) were using vitamin supplements .
table 2 presents the results of the multivariate linear regression analysis to determinate the independent factors associated with vitamin d deficiency .
the numerical variables included in the initial model were age , si , and bmi .
the dichotomous variables were income bracket , grouped skin phototype , vitamin d supplement use , calcium supplement use , and calcium intake .
the variables remaining in the model were solar index , calcium intake , and bmi .
in this study of elderly men living in the tropics , vitamin d deficiency was found to be very common .
approximately one - third of participants presented with levels < 20 ng / ml , and the majority that is , two - thirds presented with levels < 30 ng / ml .
similar results were found in the osteoporotic fractures in men study ( mros ) . in that study , 26% and 72% of the elderly men presented with levels < 20 and < 30 ng / ml , respectively . despite the similarity in results
, we had expected that the prevalence of deficiency would be much lower in our region , given that the individuals studied live in an area with abundant sunlight and the blood collection was performed in the sunniest months of the summer . since the mros study did not use si as a clinical parameter , we could not compare this variable in the two studies .
a fact that might account for the high prevalence in this region despite the high si is the increased prevalence of individuals considered to have dark skin according to fitzpatrick s classification15 as well as a high bmi .
the mros study , after bivariate analysis , demonstrated that black people are independently associated with a high prevalence of vitamin d deficiency , with a 2.4-fold greater risk of deficiency than white people .
melhus et al , in a cohort study carried out in sweden with elderly men , demonstrated that the prevalence of vitamin d deficiency , despite limited sun exposure in the winter and high latitude ( 60n ) , was rare for the 16 ng / ml cutoff.18 the mean serum 25(oh)d level was 27.5 ng / ml , which is similar to that of our patients ( 27.9 ng / ml ) . these data show that sun exposure in itself is not the preponderant factor in the prevention of 25(oh)d deficiency . in relation to sun exposure , it may be observed that in the population with greatest si ( q4 ) , when compared with the least exposed group ( q1 ) , no significant differences in mean serum 25(oh)d levels for the three cutoffs studied were observed , which suggests that the sun has little effect in preventing vitamin d deficiency in this population . in addition , the mean serum 25(oh)d in q4 was 29.8 ng / ml , demonstrating that , despite maximum sun exposure , mean 25(oh)d levels remained below 30 ng / ml .
binkley et al19 demonstrated a prevalence of 25(oh)d deficiency of 51% , using the 30 ng / ml cutoff , in a population of young surfers ( mean age 24 years ) in hawaii ( 20n ) with a mean si of 11.1 0.7 .
similarly , low serum 25(oh)d levels were observed in workers with a mean si 11.514 and in two of 18 puerto rican farmers with a weekly sun exposure of 3270 hours.20 in our study , the prevalence of dark - skinned patients ( types iv , v , and vi ) was 66.9% .
when comparing prevalence according to skin type ( dark vs light ) for each serum 25(oh ) d cutoff , we found no significant difference ( p = 0.46 ) .
harris et al found a significant difference between black and white people with regard to the prevalence of low 25(oh)d concentration.16 while 28% of elderly white men presented levels below the 20 ng / ml cutoff , the corresponding figure for elderly black men was 73.1% ( p
< 0.001).15 these data are in agreement with loomis observation that black people produce less vitamin d than white people at normal levels of sun exposure.21 the majority of the individuals in our study ( 45.7% ) had a skin color classified as intermediate ( type iii and iv ) . the number of individuals classified in the extremes for skin color was very low ( four whites and 33 blacks ) , making comparative analysis misleading . in our study
, 72% had a low calcium intake and only 2.5% ( n = 7 ) used vitamin supplements .
it should be emphasized that the number of food items enriched with vitamin d is low in brazil .
these numbers differ considerably from those of north america , where 24% of black and 44% of white elderly men and women studied were on vitamin d supplements.22 in this regard , forrest and stuhldreher demonstrated a high prevalence of vitamin d deficiency among a group of americans , particularly in those who were black , obese , and illiterate and in those with a low milk intake ( p < 0.05).23 as this study was not a population - based study , the results can not be translated to the general population .
we found a high prevalence of vitamin d deficiency in elderly men living in the tropics , despite their high exposure to the sun in the summer months . | objectivethe aim of the study reported here was to determine the prevalence of vitamin d deficiency among elderly men and its association with sun exposure and skin phototypes.subjects and methodsthis was an analytical cross - sectional study , which involved 284 men aged 60 years or over , randomly recruited from a basic care unit in the city of recife ( brazil).measurementsserum levels of 25 hydroxyvitamin d ( 25(oh)d ) , sun index , and skin phototypes were evaluated.resultsthe prevalence of vitamin d deficiency was 31.5% and 66.7% when cut points of less than 20 and 30 ng / ml , respectively , were used . mean serum 25(oh)d was 27.86 13.52 standard deviation ( sd ) ng / ml . there was no difference ( p = 0.113 ) in 25(oh ) d ( 23.98 14.66 sd vs 29.88 13.78 sd ) between individuals in the lowest quartile ( q ) of the sun index ( q1 : 1.96 ) compared with those in the highest ( q4 : 7.86 ) . when considering a cutoff of 20 ng / ml ,
the sun index was different in the two groups ( p = 0.006 ) , but there was no difference when cutoffs of 25 and 30 ng / ml were used . after adjustment , sun index and body mass index were associated positively and negatively , respectively with serum 25(oh)d independently .
most subjects ( 66.7% ) had fitzpatrick s skin phototypes iv , v , and vi .
low calcium intake was observed in 72% .
there was no difference in serum 25(oh)d levels between patients with low intake and those with intermediate and high intakes .
only 2.5% were taking a vitamin supplement.conclusionwe found a high prevalence of vitamin d deficiency among elderly men despite their high sun exposure during the summer months . |
the research was conducted at 4 islands on lake victoria ( kibuogi , ngodhe , takawiri , mfangano ) and 1 shoreline community of mbita district ( ungoye ) in western kenya ( figure ) . in this area , the pfpr210 ( community p. falciparum parasite rate standardized to the 2- to 10-year age group )
although in some area of kenya malaria has decreased , its prevalence remains high in the lake victoria basin because of the lake environment ( 810 ) . in 2009 , a total of 50%70% of households owned insecticide - treated bed nets ( 11 ) , which substantially reduce the risk for transmission of malaria parasites by providing barriers against mosquitoes .
june and october november ) ( 9 ) , in the study sites , it is highly endemic throughout the year . study sites for investigation of k13 propeller gene in plasmodium falciparum , mbita district , kenya , 20122013 .
filter paper blood spots were collected from participants ( technical appendix table 1 ) during population - wide cross - sectional malaria surveys conducted in february and august 2012 and august 2013 at the 5 study sites .
parasitic dna was extracted from the filter paper ( 12 ) , and p. falciparum dna was detected by a mitochondrial dna - based pcr ( 13 ) .
sequencing of the k13 propeller gene was attempted on the diagnostic pcr - positive specimens ( online technical appendix ) .
the prevalence of p. falciparum , as determined by pcr , in the rainy and dry seasons was 7.2%26.2% and 6.5%15.5% on the 3 small islands ( kibuogi , ngodhe , and takawiri ) , 47.3% and 31.4% on mfangano island , and 38.4% and 41.9%64.0% in ungoye , respectively ( table 1 ) . *
nine new types of point mutations were identified among these samples ( table 2 ) .
participants infected with parasites harboring a mutation on the k13 propeller gene are listed in technical appendix table 2 .
the sequences reported in this study have been deposited in the ddbj / embl / genbank databases ( accession nos.ab936059ab936067 ) .
we detected 4 novel nonsynonymous and 5 novel synonymous mutations in the highly conserved k13 propeller gene of p. falciparum parasites from western kenya .
noted that the frequency of mutant alleles strongly correlated with the prevalence of day 3 positivity after act treatment in humans in cambodia and that those mutations reflected positive selection ( 6 ) .
that study found 17 mutant alleles in the k13 propeller gene . among them , c580y , r539 t , and y493h were prevalent and strongly related to in vivo delayed parasite clearance . in our study , all the mutations found differed from those reported in cambodia , and mutant alleles were not always observed in the proceeding seasons , so some mutations could be occasionally introduced .
most of those mutations are not suitable for the life cycle of parasites , and only a few suitable for survival under the conditions of artemisinin selection pressure could be selected .
furthermore , only 1 type of mutation , a578s from mfangano island , was detected during 2 seasons , whereas other mutations were not observed in the next season , half a year later .
any family relations were not identified among the 4 participants harboring a578s mutation in february 2012 at mfangano island .
point mutations can occasionally occur on the p. falciparum k13 propeller gene as a standing variation , but most of the isolates that recently acquired the mutation may disappear because of some fitness disadvantage or the effect of a random genetic drift ( 14 ) .
we detected the mutant allele a578s in the k13 propeller gene in 2 consecutive seasons on mfangano island .
this mutation , which modifies amino acids from being hydrophobic to hydrophilic , is close to the prevalent single nucleotide polymorphism c580y from cambodia that is thought to be necessary in protein protein interactions , which could affect artemisinin susceptibility .
the genotype analyses of the parasites from this island are critical to understanding the role of this mutation and act efficiency in this geographic area .
our k13 propeller sequence analysis of p. falciparum parasites from a malaria - endemic area in kenya did not detect the predicted artemisinin - resistant genotypes , but we observed some temporal substitutions .
a limitation of our study was that the sample size was insufficient to specifically provide an understanding of this result .
the accumulation of data from this region and from other malaria - endemic areas will increase our understanding of the relationship between the k13 propeller gene and artemisinin resistance .
monitoring these molecular markers and the efficacy of antimalarial drugs is critical for increasing understanding of artemisinin resistance and predicting its spread .
demographic information about the study participants ; sequencing of the plasmodium falciparum k13 propeller gene ; and data on the participants who had parasites harboring a mutation on the k13 propeller gene . | we looked for mutations in the plasmodium falciparum k13 propeller gene of an artemisinin - resistant parasite on islands in lake victoria , kenya , where transmission in 20122013 was high .
the 4 new types of nonsynonymous , and 5 of synonymous , mutations we detected among 539 samples analyzed provide clues to understanding artemisinin - resistant parasites . |
chronic environmental conditions are a growing concern and a significant burden to the health care system due to the magnitude of the problem ( geertrudis and van den bos 1995 ; weiss and sullivan 2001 ; kipen and fiedler 2002 ; litt et al 2004 ; druss et al 2001 ; bowles 2006 ) and lack of efficient and effective management strategies ( thea and vliet 2004 ) .
the nova scotia environmental health centre ( nsehc ) is a medical facility for treating chronic conditions with probable links to the environment , such as multiple chemical sensitivity ( mcs ) , chronic fatigue syndrome ( cfs ) , fibromyalgia ( fm ) and other chronic conditions .
the management of such conditions is a controversial subject with limited documentation or evidence of support for any particular approach ( litt et al 2004 ) . for most of these conditions
there are no specific guidelines to treatment that have been shown to reverse the condition ( hessl 1987 ; ziem and mctamny 1997 ; sparks 2000 ; hoffres et al 2001 ) .
the complexity of these problems , with overlapping diagnoses , symptoms in many body systems and suspected environmental triggers , often leave the medical practitioner baffled . from our experience of managing these patients we find various contributors to impaired health , including physical , psychological , social and environmental factors affect the symptoms reported by patients .
the relative contributions of these factors may vary between patients or in individual patients over time .
the complex interactions of these factors determine the severity of the symptoms and the overall clinical presentation .
this understanding is crucial to the management of these patients where severe symptoms may exist in the absence of any objective physical or physiologic findings ( katon and walker 1998 ) .
the management scheme thus requires the involvement of a multidisciplinary team of clinicians in order to ensure that all aspects of the patient s health are addressed . adopting a symptom - based , patient - centred and multidisciplinary care approach that encompasses all aspects of the patient s health is the care model used in the management of the patients at the centre .
the crux of the treatment is in assisting the patient toward self - management by providing the necessary care , support and education .
our care model is in line with health canada s 12 determinants of health ( who ) and the chronic model of care ( wagner et al 2001 ) .
the care provided at the centre is individualistic and the care team comprises of a multidisciplinary team of clinicians ( pollard 2004 ) which includes a physician , nurse , physiotherapist , psychologist / psychotherapist , dietician and rehabilitation coordinator .
the patient is actively engaged in the multidisciplinary team decisions and in the development of the individualized care plan through regular discussions with their care coordinator at the centre , their physician .
the care management scheme at the nsehc is divided into specific phases of intervention to effectively assist the patient towards self management of their condition .
the management begins with in - depth evaluation and patient education , a phase of passive and baseline intervention by the care providers , a phase of intervention with patient being an active participant , a phase of collaborative self management phase with the involvement of the care providers and concluding with self management by patients .
care at the centre begins with a comprehensive evaluation of the patient s condition by the multidisciplinary care team .
a variety of diagnostic tests including balance assessment using a sway tester , exposure testing in a controlled environment chamber , and psychodiagnostic by a psychologist are conducted during this phase .
this information gathering phase also includes administering several validated questionnaires such as symptoms checklist inventory ( scl-90r ) , fibromyalgia impact questionnaire along with holding detailed interviews that cover a broad range of physical , psychological , social and environmental aspects of illness .
based on the outcome of the evaluation , some baseline education and information tools are offered to patients to enhance the understanding of their illness .
as patients go through this phase , further tests and inquires regarding the health condition may be done by the health care team to obtain clarity about the patient s illness .
preliminary intervention schemes that are generic in nature such as dietary intervention , stress management , rehabilitation support , building physical and emotional resiliency may be offered while the patient specific care plan is being developed by the care team . as detailed aspects of a patient s condition become evident
, the care team generates an individualized care plan for the patient which involves a focused treatment approach that directly relates to the outstanding aspect of the patient s illness which may be pain and fatigue management using a graded exercise therapy or addressing psychological symptoms with an emotion - focussed treatment approach . during this phase ,
tools and community integration schemes for self management are identified for every patient during this phase and the care team collaboratively facilitates this change for a period of time with exit point of care being self management of the condition by the patient .
treatment approaches offered to provide symptomatic relief include desensitization for classical allergies , physiotherapy for deconditioning , systemic and topical pharmaceutical agents for pain relief , intravenous nutrients for fatigue or relief of musculo - skeletal pain , heat depuration ( sauna ) and exercise . in the absence of any therapy that is known to reverse the underlying condition ,
other individualized approaches are used to help patients manage their problems and achieve a higher level of health .
stress , environmental or emotional , is addressed with counselling , craniosacral therapy or guided imagery , and on a group basis with mindfulness based stress reduction , wellness and freezeframe programs .
the treatment modalities are all designed to coach , educate and train the patients to use the techniques in the self management of their condition .
a study conducted at the centre looked at the prevalence of major symptoms in 351 patients at the centre .
general symptoms such as difficulty concentrating , fatigue , forgetfulness , and irritability were found to be present from the start of illness .
those related to irritation such as congested sinuses , itchy or burning eyes , and hoarseness or loss of voice were more common after exposure to environmental irritants ( joffres et al 2001 ) .
since the symptoms displayed by the patients affect multiple body systems , it was deemed important to develop a tool that measured and captured the changes in all of the body systems . a detailed questionnaire based on the toronto health survey questionnaire ( mckeown - eyssen et al 1994 )
was developed and validated for use at the centre on individuals with chronic environmental conditions .
this questionnaire contains modifications from the original version based on feedback to the toronto investigators , from focus groups involving patients , practitioners and pilot testing .
changes to original questionnaire included reducing the length of the questionnaire by adding more open - ended questions to capture perceived provocation of symptoms related to exposures .
the next section focussed on limitations in their daily activities due to their illness and a section on health problems documented major conditions diagnosed by a physician .
the section on symptoms addressed symptoms that the patients may have experienced since the start of their illness , frequency and intensity , and whether or not these symptoms occurred or got worse after identified exposure .
open - ended questions provided space for patients to list the type of exposures that might have been associated with occurrence of their symptoms .
this was followed by their family history , demographic characteristics , employment history , socio - economic status and information related to the completion of the questionnaire .
a validation study was conducted to determine the effectiveness and the sensitivity of the nsehc symptoms questionnaire .
it was the first attempt to describe the type , frequency , and severity of the most common symptoms experienced by an environmentally sensitive population .
a total of 385 ( 47% ) questionnaires were returned , and data were analyzed on 351 individuals .
participants tended to be women ( 80% ) , middle - aged individuals ( 37% age 4049 years ) , and those in higher educational groups ( 28% completed university ) , but there was wide variation in demographic variables .
the results from this study helped generate the top symptoms that were prevalent in this patient population .
general symptoms such as difficulty concentrating , fatigue , forgetfulness , and irritability dominated the overall prevalence of symptoms since the start of their illness . those related to irritation such as sneezing , itchy or burning eyes , and hoarseness or loss of voice were more common after exposure to environmental irritants .
overall scores were higher in women , in participants who were separated or divorced , and in low - income groups .
the top 15 symptoms reported in this study following an exposure are as shown in table 1 . as a follow up to this study
, another study was conducted to evaluate the impact of a multidisciplinary treatment approach on the top reported symptoms in the patient population .
the results from this pilot study are reported in this paper . the abbreviated version of the nsehc symptoms questionnaire , nsehc - bref was created to capture the changes in the top reported symptoms over various time periods of
follow up since the commencement of treatment at the centre . in the nsehc - bref ,
an introductory overall health related question and an open - ended question at the end used to capture any other incidents or changes that may have impacted the health status were also used .
the sections on family history , demographic characteristics , employment history , socio - economic status and information related to the completion of the questionnaire were excluded since these changes are captured in the multidisciplinary interviews and in the main symptoms questionnaire .
the nsehc - bref is seen as more of a follow up tool to measure the impact of treatment on the top reported symptoms .
the idea around capturing changes in these specific symptoms related to exposure is to study the degree of symptoms management achieved with treatment in individuals with mcs .
thus the overall objective of this study was to examine the type and extent of changes observed in the top reported symptoms as an outcome of the multidisciplinary treatment over various time periods of follow up .
the scores from nsehc - bref were compared to the scores measured using the main symptoms questionnaire .
approximately 500 patients with a diagnosis of mcs ( bartha et al 1999 ) in combination with cfs or fm were approached for participation .
patients were selected under the following categories : 6 month to 1 year of treatment at the centre , 1 to 2 years of treatment at the centre , 2 + years to a maximum of 5 years of treatment at the centre and discharged patients .
a total of 183 active patients participated in the study under the following categories : 6 months to 1 year of treatment at the centre , 1 to 2 years of treatment at the centre , 2 + to a maximum of 5 years of treatment at the centre and 109 discharged patients .
patients who agreed to participate were mailed out the abbreviated questionnaire along with the consent form .
the nova scotia environmental health centre abbreviated symptoms questionnaire , nsehc - bref was used to monitor changes in health over various time periods after enrolment to the centre .
the symptom scores were computed as the frequency of occurrence of symptoms 7 days prior to their filling out the nsehc - bref ( rarely , from time to time , most of the time , all the time , rated as 14 ) multiplied by the severity ( low , moderate , high , rated 13 ) .
the global score is a mean score computed as the sum of all scores divided by the number of symptoms .
comparison of symptom scores from the nsehc - bref was made against the detailed questionnaire scores that have already been computed for each patient at the centre .
there are 30 questions in total in the nsehc - bref with 22 questions on symptoms and 8 questions on the overall health .
approximately 500 patients with a diagnosis of mcs ( bartha et al 1999 ) in combination with cfs or fm were approached for participation .
patients were selected under the following categories : 6 month to 1 year of treatment at the centre , 1 to 2 years of treatment at the centre , 2 + years to a maximum of 5 years of treatment at the centre and discharged patients .
a total of 183 active patients participated in the study under the following categories : 6 months to 1 year of treatment at the centre , 1 to 2 years of treatment at the centre , 2 + to a maximum of 5 years of treatment at the centre and 109 discharged patients .
patients who agreed to participate were mailed out the abbreviated questionnaire along with the consent form .
the nova scotia environmental health centre abbreviated symptoms questionnaire , nsehc - bref was used to monitor changes in health over various time periods after enrolment to the centre .
the symptom scores were computed as the frequency of occurrence of symptoms 7 days prior to their filling out the nsehc - bref ( rarely , from time to time , most of the time , all the time , rated as 14 ) multiplied by the severity ( low , moderate , high , rated 13 ) .
the global score is a mean score computed as the sum of all scores divided by the number of symptoms .
comparison of symptom scores from the nsehc - bref was made against the detailed questionnaire scores that have already been computed for each patient at the centre .
there are 30 questions in total in the nsehc - bref with 22 questions on symptoms and 8 questions on the overall health .
table 3 shows the overall improvement in health in the different groups of patients tested .
patients showed statistically significant improvement in their overall health under categories such as health since ill 0.05 ( 6 months1 year ) , < 0.0001 ( 12 years , 2 + years , discharged ) ; too ill to do housework 0.05 ( 6 months1year ) , 0.008 ( 12 years ) , < 0.0001 ( 2 + and discharged ) ; limit contact with people to avoid exposures 0.9 ( 6 months1 year ) , 0.09 ( 12 years ) , 0.02 ( 2 + years ) and < 0.0001 ( discharged ) . tables 4 and 5 show changes in symptoms grouped under different categories .
stronger sense of smell and tight chest showed consistent and early changes of statistical significance with 0.02 ( 6 months1 year ) , 0.008 ( 12 years ) , < 0.001 ( 2 + and discharged ) and 0.03 ( 6 months1 year ) , 0.0007 ( 12 years ) , < 0.0001 ( 2 + ) and 0.02 ( discharged ) respectively .
nose symptoms such as usually acceptable odors were sickening and stuffy or full sinuses were observed in the 1 year and above groups .
hoarse of loss of voice and itchy eyes showed sudden improvements in 2 + and discharged groups .
table 5 shows changes in health measured in blood and gland , nervous system and muscle and joints . symptoms showing consistently significant improvement in various time periods of follow up include symptoms such as difficulty concentrating 0.01 ( 6 months1 year ) , 0.003 ( 12 years ) and < 0.0001 ( 2 + and discharged ) ; difficulty making decisions 0.007 ( 6 months1 year ) , 0.002 ( 12 years ) and < 0.0001 ( 2 + and discharged ) ; tiredness not relieved by sleep 0.002 ( 6 months1 year ) , 0.01 ( 12 years ) and < 0.0001 ( 2 + and discharged ) ; and muscle spasms and cramps 0.02 ( 6 months1 year ) , 0.002 ( 12 years ) , and 0.002 ( 2 + years and discharged ) .
symptoms such as irritability , forgetfulness and trouble finding the right words showed significant changes in the groups past 1 year of treatment . fatigue and tiredness without energy
inconsistent changes were observed in categories such as bruise easily , muscle pain and sensitive to temperature changes .
the prevalence of unexplained symptoms and reactivity is on the increase in the recent years ( caress and steinemann 2004 ) .
many patients of the centre have spent several years trying to find the right clinicians who can understand their illness and help them manage it ( geertrudis and van den bos 1995 ; druss et al 2001 ) .
when they are first seen at the centre , patients often present a very negative perspective of the health system and are very dysfunctional on account of their existing symptoms . measuring changes in chronic health conditions such as mcs is often challenging with patients having multiple diagnoses and multiple care providers .
the goal of multidisciplinary care approach at the centre is to provide an individualized care approach for its patients with timely evaluation of the presenting symptoms and integrated care delivery by multidisciplinary care providers .
black et al ( 2000 ) have described the possible effects of time over treatment in a 9-year follow up study of individuals with mcs .
however , it is important to identify the type of symptoms that change and the extent of these changes if we are to determine whether it is the treatment or simply the passage of time that contributes to improvements in health .
if there are positive changes in health , then a qualitative study can clarify the potential reasons for these changes .
it is also challenging to identify any one treatment that may have had an impact on the improvement in health symptoms ( rundall et al 2002 ; richardson and engel 2004 ) .
it may be a combination of treatments in addition to education and lifestyle changes that is impacting the health of an individual .
the objective of this study was to quantify the effects of an integrated care approach in the mcs population regardless of the type of individualized care prescribed to the patient .
the pilot study has helped us identify a useful tool that will allow us to measure health changes in patients with mcs .
the abbreviated questionnaire was used to focus the attention on the nature and extent of changes in the top reported symptoms in the mcs group .
the results are promising evidence of the effectiveness of the nsehc - bref in measuring changes at various time periods of follow up .
the pilot study showed some interesting changes in the top reported symptoms in various categories and at various time periods of follow up .
observed changes in nose and nervous systems such as improvement in stuffy sinuses and concentration match our clinical observations .
some of the inconsistencies in bruise easily and sensitivity to temperature changes raise questions on being captured as top symptoms related to exposures in this sensitive population in our previous research .
this study has provided some preliminary results on the nature and extent of symptom changes in mcs using an abbreviated questionnaire .
this study has also shown that there may be benefits in considering a more comprehensive approach to treating these individuals displaying symptoms in multiple body systems rather than a rigid treatment protocol that either treats the physical or the psychological symptoms .
the study does have its limitation which needs to be addressed in order to draw more meaningful conclusions .
there were limitations to the study design in which the primary objective was to evaluate the effectiveness an abbreviated symptoms questionnaire in measuring changes in health related to a multidisciplinary treatment approach .
so , while it is interesting to observe the type of symptoms that changed and reached statistical significance , it is impossible to comment beyond that since the same individuals were not followed at the different time periods
. it is also crucial to have controls in the study in order to determine the impact of passage of time in the observed improvements in health .
despite the study limitations , the pilot was a useful first step to observe the changes in health in mcs patients undergoing a multidisciplinary care approach with the abbreviated symptoms questionnaire .
the study helped identify an outcome tool that measures the overall effect of treatment over a period of time rather than trying to identify or isolate the effects of a particular treatment .
the outcome of this study clearly warrants further research into the efficacy of the multidisciplinary approach in mcs in a longitudinal and randomized study design . | the nova scotia environmental health centre is a treatment facility for individuals with chronic environmental conditions such as multiple chemical sensitivity , chronic fatigue syndrome , fibromyalgia , chronic respiratory conditions and in some cases chronic pain .
the premise of care is to provide a patient - centred multidisciplinary care approach leading to self - management strategies . in order to measure the outcome of the treatment in these complex problems , with overlapping diagnoses , symptoms in many body systems and suspected environmental triggers ,
a detailed symptoms questionnaire was developed specifically for this patient population and validated .
results from a pilot study in which an abbreviated symptoms questionnaire based on the top reported symptoms captured in previous research was used to measure the efficacy of a multidisciplinary care approach in individuals with multiple chemical sensitivity are presented in this paper .
the purpose of this study was to examine the extent , type and patterns of changes over time in the top reported symptoms with treatment measured using the abbreviated symptoms questionnaire .
a total of 183 active and 109 discharged patients participated in the study where the health status was measured at different time periods of follow up since the commencement of treatment at the centre .
the findings from this study were successful in generating an initial picture of the nature and type of changes in these symptoms .
for instance , symptoms such as difficulty concentrating , sinus conditions and tiredness showed early improvement , within the first 6 months of being in treatment , while others , such as fatigue , hoarseness or loss of voice , took longer while others showed inconsistent changes warranting further enquiry .
a controlled longitudinal study is planned to confirm the findings of the pilot study . |
idiopathic giant cell myocarditis is a rapid progressive disease with an enormously high mortality because of pump failure or arrhythmias.1 in many cases , clinical course is acute to fulminant , and time span between clinical presentation and start of treatment should be as short as possible .
the diagnostic tool of choice is endomyocardial biopsy ( emb ) showing typical granulomas of giant cells in histology .
however , repeat embs are frequently necessary delaying initiation of treatment.2 treatment consists of immunosuppression including cortisone and cyclosporine . here , we present a case of a young woman with fulminant myocarditis in whom gene expression profiling of giant cells was helpful in diagnosing the disease . a previously healthy woman ( 26 years )
was admitted via helicopter because of sudden onset of massive dyspnoea , nausea , and vomiting following a respiratory infection 2 weeks ago .
because of the acute beginning of dyspnoea , pulmonary embolism was excluded by computed tomography .
she was intubated and mechanically ventilated with initially high positive end expiratory pressure that led to respiratory stabilization .
echocardiography revealed a massive depression of left ventricular function with an ejection fraction ( lvef ) of 25% , while right ventricular function was normal .
inotropic therapy including levosimendan ( 0.05 g / kg / min ) and norepinephrine ( 0.2 g / kg / min ) was started .
laboratory values out of range at admission ck , creatinkinase ; hs troponin , highsensitivity troponin ; fio2 , fraction of inspiratory oxygen ; pao2 , arterial pressure of oxygen ; paco2 , arterial pressure of carbon dioxide .
invasive cardiologic workup including coronary angiography and emb was performed the following day under the suspicion of fulminant myocarditis to differentiate between lymphocytic , eosinophilic , or giant cell myocarditis .
after emb , echo showed an even more depressed lvef ( 15% ) , and the dose of norepinephrine necessary to stabilize circulation increased further ( up to 0.5 g / kg / min ) . overall , cardiogenic shock progressively deteriorated ( intermacs1 ) .
this prompted the implantation of an extracorporeal membrane oxygenation ( ecmo ) in a venoarterial manner .
after 5 days on ecmo and inotropes , the patient was stable , but lvef still was poor ( 15% ) .
preliminary results of emb showed no viral genome and no giant cells in histology ( three embs , although lymphocytes and macrophages were present ; figure
1(a ) ) .
( a ) histology with atrophic myocytes ( mean diameter : 14 m ) in regular arrangement .
( b)(e ) immunohistochemical examination including digital imaging analysis in other biopsies displays increase of tlymphocytes ( cd3 32 cells / mm , b ) , lfa1 positive cells ( lfa1 187 cells / mm , c ) , and macrophages ( mac1 468 cells / mm , d ) , but no multinuclear giant cells and no cytotoxic perforinpositive cells ( e ) .
quantification of immunohistochemical stainings by digital image analysis in two additional embs yielded the following values : cd3 + : 32 per mm ( normal values ( nv ) < 7 per mm ) , cd11a / lfa1 + : 187 per mm ( nv < 14.0 per mm ) , cd11b / mac1 + : 487 per mm ( nv < 35 per mm ) , area fraction of human leucocyte antigen class i : 14.8% ( nv < 5.5% ) , and cd54/icam1af : 7.56% ( nv < 1.2% ) , demonstrating a massive elevation of lymphocytes and macrophages ( figure
1(b)(d ) ) and expression of adhesion molecules ( no perforinpositive cytotoxic cells have been present ) .
immunohistochemical composition of cellular infiltrates corresponded to fulminant lymphocytic myocarditis , although active myocarditis ( i.e. myocytolysis ) was excluded .
however , as histological proof of multinuclear giant cells or eosinophilic cells was not successful , a myocardial gene expression profiling using a set of 16 altered genes was performed .
the results suggested the presence of multinucleated giant cells in the myocardium as key feature for giant cell myocarditis.3 this diagnostic approach was suitable to differentiate this disease from active lymphocytic myocarditis ( figure
2 ) .
expression profiles of highly upregulated ( a ) or downregulated ( b ) myocardial genes of our patient ( grey bar ) compared with inflammationfree controls ( black bar ) and to idiopathic giant cell myocarditis ( light bar ) suggested the presence of multinuclear giant cells in myocardium .
all expression values of controls free of inflammation are given as 100% and the others in relation to it .
with this information , the recommended therapy for giant cell myocarditis was started with a high cortisonedose ( 10 mg / kg body weight for 3 days followed by 4 weeks at 1 mg / kg body weight ) in combination with cyclosporine .
after 3 days of highdose cortisone and cyclosporine , lvef increased from 15% to 55% , and ecmo was explanted .
the patient was extubated and transferred to the normal ward after another 4 days .
overall , the patient left the hospital after 4 weeks to start cardiac rehabilitation .
treatment at discharge consisted of methylprednisolone ( 1 mg / kg for 4 weeks , followed by a decline of 10 mg every 2 weeks until 10 mg will be reached ) , cyclosporine ( trough level 100150 ) , pantoprazole 40 mg , calcium plus vitamin d , ramipril , eplerenone , and bisoprolol .
this treatment was continued for 12 months with repeated visits at our outpatient clinic .
all echocardiographic parameters stayed within normal ranges , throughout this time , and the patient returned to work after 10 months .
a previously healthy woman ( 26 years ) was admitted via helicopter because of sudden onset of massive dyspnoea , nausea , and vomiting following a respiratory infection 2 weeks ago .
because of the acute beginning of dyspnoea , pulmonary embolism was excluded by computed tomography .
she was intubated and mechanically ventilated with initially high positive end expiratory pressure that led to respiratory stabilization .
echocardiography revealed a massive depression of left ventricular function with an ejection fraction ( lvef ) of 25% , while right ventricular function was normal .
inotropic therapy including levosimendan ( 0.05 g / kg / min ) and norepinephrine ( 0.2 g / kg / min ) was started .
laboratory values out of range at admission ck , creatinkinase ; hs troponin , highsensitivity troponin ; fio2 , fraction of inspiratory oxygen ; pao2 , arterial pressure of oxygen ; paco2 , arterial pressure of carbon dioxide .
invasive cardiologic workup including coronary angiography and emb was performed the following day under the suspicion of fulminant myocarditis to differentiate between lymphocytic , eosinophilic , or giant cell myocarditis .
after emb , echo showed an even more depressed lvef ( 15% ) , and the dose of norepinephrine necessary to stabilize circulation increased further ( up to 0.5 g / kg / min ) .
this prompted the implantation of an extracorporeal membrane oxygenation ( ecmo ) in a venoarterial manner .
after 5 days on ecmo and inotropes , the patient was stable , but lvef still was poor ( 15% ) .
preliminary results of emb showed no viral genome and no giant cells in histology ( three embs , although lymphocytes and macrophages were present ; figure
1(a ) ) .
( a ) histology with atrophic myocytes ( mean diameter : 14 m ) in regular arrangement .
( b)(e ) immunohistochemical examination including digital imaging analysis in other biopsies displays increase of tlymphocytes ( cd3 32 cells / mm , b ) , lfa1 positive cells ( lfa1 187 cells / mm , c ) , and macrophages ( mac1 468 cells / mm , d ) , but no multinuclear giant cells and no cytotoxic perforinpositive cells ( e ) .
quantification of immunohistochemical stainings by digital image analysis in two additional embs yielded the following values : cd3 + : 32 per mm ( normal values ( nv ) < 7 per mm ) , cd11a / lfa1 + : 187 per mm ( nv < 14.0 per mm ) , cd11b / mac1 + : 487 per mm ( nv < 35 per mm ) , area fraction of human leucocyte antigen class i : 14.8% ( nv < 5.5% ) , and cd54/icam1af : 7.56% ( nv < 1.2% ) , demonstrating a massive elevation of lymphocytes and macrophages ( figure
1(b)(d ) ) and expression of adhesion molecules ( no perforinpositive cytotoxic cells have been present ) .
immunohistochemical composition of cellular infiltrates corresponded to fulminant lymphocytic myocarditis , although active myocarditis ( i.e. myocytolysis ) was excluded .
however , as histological proof of multinuclear giant cells or eosinophilic cells was not successful , a myocardial gene expression profiling using a set of 16 altered genes was performed .
the results suggested the presence of multinucleated giant cells in the myocardium as key feature for giant cell myocarditis.3 this diagnostic approach was suitable to differentiate this disease from active lymphocytic myocarditis ( figure
2 ) .
expression profiles of highly upregulated ( a ) or downregulated ( b ) myocardial genes of our patient ( grey bar ) compared with inflammationfree controls ( black bar ) and to idiopathic giant cell myocarditis ( light bar ) suggested the presence of multinuclear giant cells in myocardium .
all expression values of controls free of inflammation are given as 100% and the others in relation to it .
with this information , the recommended therapy for giant cell myocarditis was started with a high cortisonedose ( 10 mg / kg body weight for 3 days followed by 4 weeks at 1 mg / kg body weight ) in combination with cyclosporine .
after 3 days of highdose cortisone and cyclosporine , lvef increased from 15% to 55% , and ecmo was explanted .
the patient was extubated and transferred to the normal ward after another 4 days .
overall , the patient left the hospital after 4 weeks to start cardiac rehabilitation .
treatment at discharge consisted of methylprednisolone ( 1 mg / kg for 4 weeks , followed by a decline of 10 mg every 2 weeks until 10 mg will be reached ) , cyclosporine ( trough level 100150 ) , pantoprazole 40 mg , calcium plus vitamin d , ramipril , eplerenone , and bisoprolol .
this treatment was continued for 12 months with repeated visits at our outpatient clinic .
all echocardiographic parameters stayed within normal ranges , throughout this time , and the patient returned to work after 10 months .
histological proof of multinucleated giant cells is the gold standard , but a recent publication showed that only 68% of first embs are diagnostic and repeat embs are necessary to confirm diagnosis.2 in our case , repeat emb has been discussed but gene expression profiling , a method previously published by lassner and coworkers , proved beneficial and a repeat biopsy could be avoided.3 , 4 however , the complete lack of myocytolysis ( i.e. histological confirmation of myocarditis ) in our patient was surprising . nevertheless , lymphocytic myocarditis could be diagnosed with immunohistochemical tools . we can not completely rule out that lvef may have recovered by prolongation of ecmo and inotropic therapy .
however , the finding of gene profiles of giant cells in biopsy specimen prompted the initiation of treatment for giant cell myocarditis and lvef recovered within 48 h. we think that a full recovery without immunosuppression is highly unlikely .
as shown here , the new technique of gene expression profiling may be helpful in patients with fulminant myocarditis to confirm the presence of giant cells in specimen without histological evidence of the disease , laying path to early initiation of immunosuppression and avoidance of repeat embs as shown in our case report .
therefore , we conclude that gene expression profiling of giant cell myocarditis in patients with fulminant myocarditis and negative histological findings may be helpful laying path to tailored immunosuppressive treatment for this disease .
drs lassner and schultheiss report a patent pending named verwendung von micrornas oder genen als marker zur identifizierung , diagnose und therapie einzelner nichtischmischer kardiomyopathien oder speichererkrankungen in germany . | abstracta healthy woman with acute onset of pulmonary oedema and severely depressed left ventricular function underwent endomyocardial biopsy under the clinical suspicion of fulminant myocarditis . while awaiting the results of biopsy , the situation deteriorated to interagency registry for mechanically assisted circulatory support ( intermacs ) and extracorporeal membrane oxygenation was implanted .
finally , immunohistochemistry in biopsy specimen corresponded to fulminant lymphocytic myocarditis , although active myocarditis was excluded .
furthermore , gene expression profiling identified giant cell myocarditis although multinuclear cells were absent .
this prompted the start of immunosuppression with cortisone and cyclosporine .
the patient fully recovered . |
it is characterized by excessive lateral translation of the patella relative to the femur and induction of pain in full knee extension1,2 ) . in most cases , patellar tracking
is assessed in non - weight - bearing conditions where the patient is placed in the sitting position for clinical examination and in the supine or sitting position with the knee in flexion for radiographic assessment , failing to replicate the patellofemoral joint motion during weight - bearing activities3 - 5 ) . as a result
, the risk of misdiagnosis increases , and improper treatment or unnecessary surgery may be carried out based upon non - weight - bearing assessments .
there is limited understanding of the overall kinematics of the patellofemoral joint under weight - bearing conditions .
patellofemoral joint alignment and motion were assessed in the supine position under non - weight - bearing conditions in most studies in the literature , except for a few reports6 - 9 ) .
in addition , patellofemoral joint motion is also affected by quadriceps femoris contraction10 ) and joint loading11,12 ) .
thus , we believe that thorough investigation on the influence of weight - bearing as opposed to non - weight - bearing is essential for comprehensive understanding of the patellofemoral joint . in a recent research , baldini et al.5 ) identified radiographic changes from the supine merchant ( supine - m ) view and standing weight - bearing merchant ( standing - m ) view obtained after total knee arthroplasty : lateral tilt and subluxation of the patella were neutralized and reduced under weight - bearing , indicating the efficacy of weight - bearing radiography in patellofemoral joint assessment5 ) . in this study
, we hypothesized that weight - bearing conditions would result in changes in the patellofemoral alignment , such as patellar tilt angle ( tilt ) and lateral patellar displacement ( lat - pd ) , in patients without patellofemoral joint pain and radiologic abnormalities other than the complaint of knee pain .
of the patients who visited our clinic with a major complaint of knee pain , 44 patients without patellofemoral joint pain and radiographic abnormalities were included in this study .
there were 22 males and 22 females with a mean age of 27.8 years ( range , 18 to 39 years ) . in all patients ,
supine - m and standing - m views were taken on the same occasion from the 44 left knees and 44 right knees .
the supine - m view was taken with the patient placed in the supine position maintaining 45 of femorotibial angle and the x - ray beam directed at 30 angle from the horizon .
the standing - m view was taken with the patient maintaining 45 of flexion while standing and the x - ray beam directed vertical to the floor .
the angle between the femur and tibia was maintained during the procedure by using an orthopaedic goniometer .
care was taken to ensure the reflected x - ray beam to have the same angle with respect to the patellofemoral joint on both the supine - m and standing - m views ( fig .
the supine - m and standing - m views were obtained using a general method on the same occasion .
the tilt ( + angle : lateral tilt ) , patellofemoral angle ( + angle : lateral divergence ) , congruence angle ( cong , + angle : increased incongruence ) , and lat - pd ( + angle : lateral displacement ) were measured on each view and evaluated for statistical analysis . to analyze the influence of various factors on discrepancy in the supine - m and standing - m view measurements , possible influencing factors ( gender , age ,
femoral neck offset [ f - off ] , femoral length [ fl ] , tibial length [ tl ] , mechanical axis of the lower limb , thigh width [ tw ] , calf width [ cw ] , and radiographic q - angle [ rad - q ] ) were investigated .
radiographic measurements for each parameter were performed twice by one investigator with an interval of one week using picture archiving communication system ( infinitt , seoul , korea ) and the mean values were used for analysis ( fig .
the tilt was defined as the angle between a line connecting the two edges of the patella ( corner - to - corner line ) and the horizontal line .
increased tilt was associated more lateral patellar tilt , and conversely decreased tilt was related to more medial patellar tilt .
the cong was measured as the angle between a line that bisects the sulcus angle and a line that is drawn from the lowest point in the intercondylar groove through the articular ridge of the patella .
patella lateral subluxation distance ( sub - d ) was defined as the distance from the lowest point of the femoral trochlea to the median ridge of the patella : if the median ridge was located lateral to the lowest point of the trochlea , the distance was assigned a positive value , whereas if located medial , a negative value was given .
lateral patellofemoral angle ( lat - pf ) was defined as the angle created by the intercondylar line of the femur and a line drawn parallel to the lateral articular surface of the patella .
lat - pd was measured as the distance from the medial end of the patella to the most anterior point of the medial condyle of the femur .
a positive value was given if the medial end of the patella was located laterally , whereas a negative value was assigned for medial location13,14 ) .
additional measurements were performed to investigate factors that could influence the values of the above - described five parameters measured from the supine - m and the standing - m views .
the f - off was measured as the distance from the center of the rotation of the femoral head to the long axis of the femur .
the fl was measured from the center of the femoral intercondylar fossa through the long axis of the femur to the tip of the bone on the weight - bearing full leg standing view .
similarly , the tl was measured from the tibial intercondylar eminence through the long axis to the distal end of the tibia on the weight - bearing full leg standing view .
the tw and cw were measured inward from the most protruded portion of the thigh and calf , respectively , on the weight - bearing full leg standing view .
the rad - q was defined as the angle between a line extending from the tibial tubercle to the mid - patella and a line from the anterior superior iliac spine to the mid - patella on the weight - bearing full leg standing view .
the intraobserver agreement percentage and kappa coefficient were 93% and 0.86 , respectively , and the lowest values of all the parameters were higher than those suggested by the icc .
statistically significant differences between the values obtained from the supine - m and standing - m views were investigated using paired t - test .
possible influencing factors including gender , age , f - off , fl , tl , mechanical axis of the lower limb , tw , cw , and rad - q were assessed using univariate and multivariate regression analysis .
of the patients who visited our clinic with a major complaint of knee pain , 44 patients without patellofemoral joint pain and radiographic abnormalities were included in this study .
there were 22 males and 22 females with a mean age of 27.8 years ( range , 18 to 39 years ) . in all patients ,
supine - m and standing - m views were taken on the same occasion from the 44 left knees and 44 right knees .
the supine - m view was taken with the patient placed in the supine position maintaining 45 of femorotibial angle and the x - ray beam directed at 30 angle from the horizon .
the standing - m view was taken with the patient maintaining 45 of flexion while standing and the x - ray beam directed vertical to the floor .
the angle between the femur and tibia was maintained during the procedure by using an orthopaedic goniometer .
care was taken to ensure the reflected x - ray beam to have the same angle with respect to the patellofemoral joint on both the supine - m and standing - m views ( fig .
the supine - m and standing - m views were obtained using a general method on the same occasion . the tilt ( + angle : lateral tilt ) ,
patellofemoral angle ( + angle : lateral divergence ) , congruence angle ( cong , + angle : increased incongruence ) , and lat - pd ( + angle : lateral displacement ) were measured on each view and evaluated for statistical analysis . to analyze the influence of various factors on discrepancy in the supine - m and standing - m view measurements , possible influencing factors ( gender , age , femoral neck offset [ f - off ] , femoral length [ fl ] , tibial length [ tl ] , mechanical axis of the lower limb , thigh width [ tw ] , calf width [ cw ] , and radiographic q - angle [ rad - q ] ) were investigated .
radiographic measurements for each parameter were performed twice by one investigator with an interval of one week using picture archiving communication system ( infinitt , seoul , korea ) and the mean values were used for analysis ( fig .
the tilt was defined as the angle between a line connecting the two edges of the patella ( corner - to - corner line ) and the horizontal line .
increased tilt was associated more lateral patellar tilt , and conversely decreased tilt was related to more medial patellar tilt .
the cong was measured as the angle between a line that bisects the sulcus angle and a line that is drawn from the lowest point in the intercondylar groove through the articular ridge of the patella .
patella lateral subluxation distance ( sub - d ) was defined as the distance from the lowest point of the femoral trochlea to the median ridge of the patella : if the median ridge was located lateral to the lowest point of the trochlea , the distance was assigned a positive value , whereas if located medial , a negative value was given .
lateral patellofemoral angle ( lat - pf ) was defined as the angle created by the intercondylar line of the femur and a line drawn parallel to the lateral articular surface of the patella .
lat - pd was measured as the distance from the medial end of the patella to the most anterior point of the medial condyle of the femur .
a positive value was given if the medial end of the patella was located laterally , whereas a negative value was assigned for medial location13,14 ) .
additional measurements were performed to investigate factors that could influence the values of the above - described five parameters measured from the supine - m and the standing - m views .
the f - off was measured as the distance from the center of the rotation of the femoral head to the long axis of the femur .
the fl was measured from the center of the femoral intercondylar fossa through the long axis of the femur to the tip of the bone on the weight - bearing full leg standing view .
similarly , the tl was measured from the tibial intercondylar eminence through the long axis to the distal end of the tibia on the weight - bearing full leg standing view .
the tw and cw were measured inward from the most protruded portion of the thigh and calf , respectively , on the weight - bearing full leg standing view .
the rad - q was defined as the angle between a line extending from the tibial tubercle to the mid - patella and a line from the anterior superior iliac spine to the mid - patella on the weight - bearing full leg standing view .
pasw ver . 17.0 ( spss inc . , chicago , il , usa ) was used for statistical analysis .
the intraobserver agreement percentage and kappa coefficient were 93% and 0.86 , respectively , and the lowest values of all the parameters were higher than those suggested by the icc .
statistically significant differences between the values obtained from the supine - m and standing - m views were investigated using paired t - test .
possible influencing factors including gender , age , f - off , fl , tl , mechanical axis of the lower limb , tw , cw , and rad - q were assessed using univariate and multivariate regression analysis .
the mean tilt was significantly decreased from 5.32.5 ( range , 1.5 to 9.8 ) on the supine - m view to 3.72.0 ( range , 0.1 to 8.5 ) on the standing - m view ( p=0.002 ) .
the mean cong was reduced from -3.54.5 ( range , -11.0 to 11.2 ) on the supine - m view to -8.72.4 ( range , -12.0 to -2.8 ) on the standing - m view ( p<0.001 ) , indicating increased congruence of the knee joint .
the mean sub - d was significantly shorter on the standing - m view ( -2.01.8 mm ; range , -5.8 to 4.6 mm ) than on the supine - m view ( -1.02.8 mm ; range , -6.6 to 4.6 mm ) ( p=0.039 ) .
the mean lat - pf was notably increased from 6.83.8 ( range , 1.1 to 15.8 ) on the supine - m view to 11.34.7 ( range , 1.0 to 20.9 ) on the standing - m view ( p<0.001 ) .
the mean lat - pd was significantly shorter on the standing - m view ( -3.53.7 mm ; range , -9.6 to 8.2 mm ) than on the supine - m view ( -1.93.6 mm ; range , -8.4 to 8.5 mm ) ( p<0.001 ) ( table 1 , fig .
the mean values of the possible influencing factors were : 51.56.3 mm ( range , 36.9 to 69.6 mm ) for the f - off ; 447.433.2 mm ( range , 397.0 to 498.2 mm ) for the fl ; 368.226.8 mm ( range , 321.8 to 410.6 mm ) for the tl ; 189.111.7 mm ( range , 160.2 to 215.6 mm ) for the tw ; 122.38.7 mm ( range , 105.3 to 137.3 mm ) for the cw ; 4.31.3 ( range , 1.8 to 7.1 ) for the rad - q ; and 1.51.6 ( range , -2.1 to 4.1 ) for the mechanical axis .
however , radiological q - angle showed a statistically significant correlation with the difference between the patellofemoral indices on weight - bearing and non - weight bearing positions , with regards to the lateral patellar subluxation ( p=0.043 , =0.094 ) , lat - pd ( p=0.026 , =0.112 ) , and congruency angle ( p<0.001 , =0.488 ) , obtained using univariate and multivariate regressing analysis ( table 2 ) .
on the comparison between the supine - m and standing - m views , the tilt was decreased , the lat - pf was increased , and the sub - d and lat - pd were decreased on the standing - m view .
these results were consistent with our hypothesis that there would be a notable difference between the two views , suggesting the clinical significance of radiographic discrepancy in the assessment of the patellofemoral joint . in a recent study , draper et al.4 ) reported that lat - pd was significantly reduced under weight - bearing conditions in patients with patellofemoral joint pain , although tilt did not exhibit significant changes4 ) .
thus , it is our understanding that a diagnosis based on supine - m view alone could be inaccurate , eventually resulting in inappropriate treatment or unnecessary surgery .
still , there is no agreement on the factors that may account for the difference under weight - bearing and non - weight - bearing conditions .
it has been considered that complex interactions of various factors could be attributable to the discrepancy .
above all , the two most frequently encountered causes are the influence of the quadriceps femoris on the patella and the bone geometry of the patella and femur and patellofemoral joint motion during closed - chain contraction ( weight - bearing condition ) .
regarding the influence of the quadriceps femoris on the patella , the movement of the patella is mostly dependent on the quadriceps femoris . considering that hip alignment and quadriceps femoris activation and strength vary during different movement conditions
, weight - bearing could result in such radiographic changes in the patellofemoral joint4 ) .
in addition , the onset of electromyographic ( emg ) activity of the four components of the quadriceps femoris is more concurrent in closed - chain contraction than in open - chain isometric contraction .
the onset of vastus medialis obliquus is significantly delayed in open - chain contraction and exhibits a lower amplitude of emg than the other components15 ) .
this unbalanced onset of quadriceps activation could result in a lateral orientation of the patella in the spine position compared to the closed - chain contraction15 ) .
some previous studies showed that active quadriceps femoris contraction10 ) and weight - bearing condition16 ) could result in more lateral displacement and tilt of the patella than passive knee motion under non - weight - bearing condition .
although the exact weight - bearing condition was not reproduced in these studies , their results were similar to those measured on the weight - bearing radiographs in our study .
regarding the bone geometry and joint motion , patellar kinematics is influenced by the axial and rotational alignment of the lower limb17,18 ) .
the normal knee function is related to pivoting in the medial and lateral sides of the knee and thus patellofemoral joint motion is affected by either the medial or lateral side pivoting19 ) .
it is well documented in the literature that knee joint motion under weight - bearing condition , or in closed - chain contraction , is different from that under non - weight - bearing condition .
when the knee flexes with the foot on the floor , the femur is slightly externally rotated .
then , when the knee is completely extended , the femur is internally rotated and the knee joint is in its most stable position to support the body weight .
conversely , when the femur is fixated on a table with the foot off the ground , knee flexion results in internal rotation of the tibia whereas knee extension elicits external rotation of the tibia20 - 23 ) .
koo et al.19 ) reported in a biomechanical study that the knee joint center of rotation is predominantly located on the lateral side and the femur is externally rotated and anteriorly translated relative to the tibia during normal walking ( closed - chain contraction ) .
in addition , knee joint motions are dependent on weight - bearing and non - weight - bearing activities : the center of rotation of the knee is in the medial compartment during a non - weight - bearing activity , whereas it is shifted laterally during a weight - bearing activity24 ) .
recent studies demonstrated that internal rotation of the tibia is correlated with sliding of the joint on the medial compartment and lateral pivoting occurs during a weight - bearing activity19,25 ) .
limitations of our study include that the patellofemoral joint was not assessed during normal ambulation and the patients had no patellofemoral pain .
we believe that the influence of weight - bearing on radiographic changes of the patellofemoral joint should be confirmed in further studies involving patients with patellofemoral joint pain to establish the clinical significance of our study .
the standing - m view showed increased medial tilt of the patella , decreased lateral displacement of the patella and increased congruence compared to those on the supine - m view .
this study showed that weight - bearing conditions have an influence on the patellofemoral joint indices .
therefore , we believe that radiographic assessment of the patellofemoral joint should be based on both the supine - m and standing - m views . | purposethis research was conducted to compare supine and standing merchant views , to observe radiographic changes of the patellofemoral joint according to weight - bearing conditions , and to evaluate correlation factors affecting patellofemoral joint indices.materials and methodsforty - four patients without skeletal problems were selected for this study .
patellar tilt angle , lateral patellofemoral angle , congruence angle , lateral subluxation distance and lateral patellar displacement were measured on each radiograph and evaluated for statistical significance .
possible correlation factors that can affect the radiographic discrepancy were analyzed using the univariate and multivariate regression analysis.resultsin the standing merchant view , the patellar tilt angle , congruence angle and lateral patellar displacement were significantly decreased ( p<0.001 ) , whereas the lateral patellofemoral angle was significantly increased ( p<0.001 ) compared to that in the supine merchant view . thigh width ( p<0.001 , r2=0.22 ) and radiographic q - angle ( p<0.001 , r2=0.34 ) were found correlated with the radiographic discrepancy of congruence angle .
discrepancy of the lateral patella displacement increased as radiographic q - angle increased ( p=0.027 , r2=0.112).conclusionscompared with the supine merchant view , standing ( weight - bearing ) merchant view showed decreased patella tilt angle , congruence angle , and lateral patella displacement .
the results indicate that both supine and standing merchant views should be considered in the radiographic evaluation of the patellofemoral joint . |
laser photocoagulation is widely used for treating various retinal diseases , including diabetic retinopathy , branch retinal vein occlusion , and central serous retinopathy .
unfortunately , conventional laser treatments are destructive procedures used to create whole - layer damage to retinal tissue .
such invasive treatments are not suitable for macular diseases , including macular edema , because laser scars generally grow larger and create permanent scotomas .
laser scar enlargement , subretinal fibrosis , and subretinal neovascular membranes have been reported as serious complications of laser treatment [ 48 ] .
micropulse lasers were invented to reduce laser - induced burns that can occur with conventional laser treatment .
lasers in the micropulse mode exert pulses of short laser applications , thereby diminishing the heat generated at the target site . in fact , retinal coagulation scars are not visible upon fundoscopic examination , autofluorescent imaging , or optical coherence tomography [ 11 , 12 ] when the eyes are treated with a subthreshold micropulse laser .
many studies have shown the clinical efficacy of this treatment [ 10 , 1317 ] ; however , few studies have investigated the molecular mechanisms responsible for fluid absorption from retinal tissue .
although thermal stimulation of the retinal pigment epithelium ( rpe ) is thought to be the key trigger for fluid absorption from an edematous retina , the molecular mechanisms of this process are not yet understood .
notably , lavinsky et al . found slight morphological changes following laser irradiation with a low - energy output in dutch - belted rabbits .
these changes were reversible ( i.e. , they did not cause permanent damage ) when photocoagulation spots ( visible burns ) were barely visible on the retinal surface .
these data suggested that biological cascades within the rpe may contribute to the therapeutic effects of sublethal photocoagulation during the treatment of macular edema .
heat shock protein ( hsp ) family members , which act as chaperone proteins , aid in refolding denatured proteins and are protective against apoptosis and inflammation [ 2024 ] .
in particular , elevated expression of hsp is known to occur with increases in temperature .
upregulation of hsp70 expression induced by laser irradiation is thought to play an important role in the improvement of macular edema .
intriguingly , nondamaging laser irradiation induces hsp70 expression within the rpe of hemizygous c57bl/6 mice , whereas low - energy laser irradiation induces rpe cell death in dutch - belted rabbits .
unfortunately , whether sublethal laser energy induces hsp70 expression in human retinal tissue is still unknown . in this study , as a first step towards understanding the molecular mechanisms through which micropulse laser application affects retinal tissue , we constructed a laser irradiation system to directly expose cultured human retinal pigment epithelial arpe-19 cell layers to a sublethal dose of laser energy .
the expression of hsp70 at both the mrna and protein levels was examined over time .
the arpe-19 immortal human rpe cell line at passage 19 ( atcc crl-2303 , lot number : 60279299 ) was purchased from american type culture collection ( manassas , va , usa ) , and all experiments were performed using the cells at passages 22 through 26 .
cells were maintained in dulbecco 's modified eagle 's medium : nutrient mixture f-12 ( dmem / f-12 , gibco , life technologies , carlsbad , ca , usa ) supplemented with 10% fetal bovine serum ( one shot fbs ; gibco , life technologies ) and 1% penicillin - streptomycin ( pen strep ; gibco , life technologies ) at 37c in a 5% co2 incubator .
cell culture media were replaced 2 - 3 times a week . for laser applications ,
3 10 cells were seeded on the glass bottoms ( 12 mm in diameter ) of 35 cm glass - bottomed culture dishes ( asahi techno glass , tokyo , japan ) and left to stand for 30 min .
two milliliters of dmem / f-12 was then added , and media were replaced on the fourth day of culture .
diode lasers are primarily used for targeting of rpe cells in photocoagulation treatment in the clinical setting .
therefore , we constructed an experimental system that made it possible to irradiate a fully confluent layer of cultured cells . in order to directly irradiate cultured cell layers with a laser , as is performed in the clinical setting , a slit - lamp adapter for an 810 nm diode laser ( oculight sl ; iris medical , mountain view , ca , usa ) was placed on top of a slit - lamp system created by cutting a slit - lamp microscope barrel ( 900 bm ; haag - streit diagnostics , bern , switzerland ) and vertically fixing it onto a culture dish . after laser irradiation , cells were inspected with a confocal microscope ( tcs sp8 ; leica microsystems , mannheim , germany ) equipped with a life cell imaging chamber ( stage top incubator ; tokai hit , shizuoka , japan ) and/or a phase - contrast microscope ( dmi3000b ; leica microsystems ) .
measurements of photocoagulated areas were performed using an image analyzer ( las 4.2 ; leica microsystems ) and image j 1.47 public domain software ( national institutes of health , bethesda , md , usa ) . a live / dead cell imaging kit ( molecular probe , life technologies ) was used to visualize live and dead cells after laser irradiation .
the live / dead reagent , composed of calcein - am and ethidium homodimer-1 , was directly added to conditioned media on cultured cells after laser irradiation and incubated for 15 min at 25c . to discriminate between live and dead cells ,
the rpe cell layer was inspected with a confocal microscope with the appropriate filters in place .
live cells were able to hydrolyze calcein - am and were therefore stained green , while dead cells were permeable to ethidium homodimer-1 and were therefore stained red .
all rna isolation experiments were performed using a purelink rna mini kit with dnase ( ambion , life technologies ) according to the manufacturer 's instructions .
first - strand cdna was synthesized from 200 ng of total rna using a high capacity rna - to - cdna kit ( applied biosystems , life technologies ) . for quantification of hsp70 mrna
, one - twentieth of the above - mentioned cdna was analyzed using taqman fast advanced master mix ( applied biosystems , life technologies ) and taqman gene expression assay probe - primer sets for hspa1a ( hs00359163_s1 ; applied biosystems , life technologies ) .
the level of hsp70 mrna was determined by the 2 method using gapdh ( hs02758991_g1 , applied biosystems , life technologies ) as an internal control .
cells were fixed with 4% paraformaldehyde ( thermo fischer scientific , waltham , ma , usa ) and permeabilized using 0.1% triton x-100 .
cells were treated with image - it fx signal enhancer ( molecular probe , life technologies ) , blocked in 10% normal goat serum ( molecular probe , life technologies ) , and incubated with an anti - hsp70 antibody ( 3a3 ; abcam , cambridge , uk ) or anti - gapdh antibody ( loading control ; abcam ) .
these primary antibodies were visualized with alexa fluor 488 goat anti - mouse igg antibody ( molecular probe , life technologies ) , which served as a secondary antibody .
samples were counterstained with slowfade gold antifade mountant with 4,6-diamidino-2-phenylindole ( dapi ; molecular probe , life technologies ) .
student 's t - tests and mann - whitney u tests were used to test mean differences for statistical significance .
each experiment was conducted more than three times . differences with p values of less than 0.05
cells were irradiated on glass - bottomed dishes via a dichroic mirror and a perpendicular laser ( figures 1(a ) and 1(b ) ) . in order to uniformly expose the cell layer to laser energy ,
several methods of spreading black paper under culture dishes when performing laser irradiation in arpe-19 cells have been previously reported [ 29 , 30 ] .
additionally , culture media without phenol red were used to avoid blocking of the emitted diode laser light .
the culture dish was placed on a mechanical stage so that the dish could be moved into the proper laser irradiation position in a flat x - y plane .
the stage was moved in 0.1 mm steps , and the cell layer was accurately irradiated with the laser in equal intervals .
the position of the laser irradiation site was indicated by a red light pointing in the direction of the x- or y - axis .
the position along the z - axis was then determined using the focal point of the laser .
the focal point was defined as the position in which the laser aiming beam was most clearly projected onto the surface of the black paper .
when a cell layer was irradiated with a conventional continuous - wave mode laser , the laser irradiation site and its periphery gradually changed into a coagulation - like form .
the formation of sparse annular intercellular spaces around the laser irradiation site was also observed in bright - field images obtained with a confocal microscope 2 h after laser irradiation ( figure 1(c ) and supplementary material 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/729792 ) .
these cell groups in the bright - field images remained viable , as indicated by green fluorescence staining with calcein - am , an indicator of cell survival .
in contrast , the laser irradiation site and nearby cells were dead , as indicated by red fluorescence staining with ethidium homodimer-1 , an indicator of cell death ( figure 1(d ) ) .
a laser provides intermittent irradiation in the micropulse mode , with the percentage of irradiation time set at a fixed number .
the duty cycle is 15% , with the laser irradiating for 0.3 ms during each 2.0 ms period .
when the total micropulse laser exposure duration was 1000 ms , the duty cycle was repeated 500 times ( figure 2(a ) ) .
figure 2(b ) shows phase - contrast images of irradiation sites 2 h after micropulse laser irradiation was performed with the following laser settings : duty cycle of 15% , laser exposure duration between 500 and 1000 ms , and laser power between 600 and 800 mw .
laser irradiation was performed at 1 mm intervals along the x- and y - axes on each cell layer .
laser exposure duration was shortened with each 1 mm position change along the x - axis , but laser power remained constant .
laser power was decreased with each 1 mm position change along the y - axis , but laser exposure duration remained constant . when the laser power was fixed at 800 mw , the coagulation area gradually decreased as the laser exposure time was shortened ( 1000 to 500 ms : 184 8.8 to 81 13.5 m ) . however , coagulation - like lesions were no longer visible with phase - contrast microscopy when the laser power was 600 mw and the laser exposure duration was 600 ms .
additionally , coagulation area proportionally increased with laser power within the range examined ( spearman 's correlation coefficient : r > 0.8 , p < 0.05 , figure 2(c ) ) .
based on the observed changes in micropulse laser irradiation lesions in a cultured cell layer ( figure 2(b ) ) , the optimal , reproducible sublethal laser settings were determined to be a laser exposure duration of 1000 ms , a duty cycle of 15% , and a laser power of 600 mw .
bright - field images , captured with a confocal microscope , revealed that cell morphology changed in sublethal photothermal stimulation sites where intercellular spaces were sparse ( figure 2(d ) ) .
however , almost all cells within sublethal photothermal stimulation sites were alive , even 24 h after micropulse laser irradiation ( figure 2(d ) ) .
additionally , the coagulation - like area was significantly smaller for lesions created in the micropulse mode than for those created in the continuous - wave mode under the same energy conditions ( mann - whitney u test : p < 0.001 , figures 2(e ) and 2(f ) ) . to investigate whether sublethal photocoagulation induced hsp expression , which is important in reducing macular edema , we used qrt - pcr to examine the expression levels of hspa1a mrna , which encodes hsp70 protein , in a cultured cell layer irradiated with a sublethal irradiation dose using a micropulse laser ( figure 2(d ) ) .
cells were examined until 24 h after irradiation from each of the 81 irradiation positions in the x - y plane . in the pretreated irradiation group ,
the expression of hsp70 mrna was only slightly increased over baseline , even 24 h after irradiation ( figure 3(a ) ) . because the clinical efficacy of macular edema treatment has been shown to be better with high - density micropulse laser irradiation than with standard laser treatment
, we also examined the effects of varying the number of laser irradiation lesions on hspa1a mrna levels by changing the spacing of laser irradiation lesions on the cultured cell layer , which resulted in an increase in the number of lesions from 81 to 477 . as expected , a significant increase in hspa1a mrna expression was observed 3 h after irradiation in cell layers with a higher number of lesions ( figure 3(b ) ) .
actual hsp70 protein levels in cultured cell layers were also examined after sublethal laser irradiation using immunocytochemistry with an anti - hsp70 antibody ( figure 3(c ) ) .
immunofluorescent signals were present in concentric areas around laser irradiation sites . however , gapdh , used as an endogenous control for qrt - pcr analyses of hspa1a mrna , was uniformly present throughout the cultured cell layer . the presence of cells within laser irradiation sites was confirmed with dapi counterstaining .
even 24 h after photothermal stimulation , hsp70 immunostaining persisted at laser irradiation sites .
in this study , we established a system in which one layer of densely cultured cells could be irradiated with a diode laser .
moreover , we confirmed that sublethal micropulse photothermal stimulation induced hsp70 expression in a cultured layer of arpe-19 cells .
previous reports in the literature have shown that the size of coagulation lesions created using a continuous - wave laser is dependent on laser duration and power in dutch - belted rabbits [ 31 , 32 ] .
our in vitro study results are consistent with this and revealed that this is also true for coagulation - like lesions made with a micropulse laser ( figures 2(b ) and 2(c ) and supplementary material 2 ) . using the same amount of energy , more tissue damage occurred in lesions created with a continuous - wave laser than in those created with a micropulse laser ( figures 2(e ) and 2(f ) ) .
it is thought that micropulse lasers cause less damage than continuous - wave lasers because the pulses of very short , intermittent energy may allow for heat dissipation within tissue .
although numerous clinical studies have shown the efficacy of subthreshold micropulse laser treatments , the laser settings ( i.e. , laser power and pulse duration ) used in these studies have not been consistent [ 10 , 1317 ]
. moreover , the optimal laser energy for subthreshold laser treatments has not yet been established , and surgeons often use continuous - wave laser modes for titrating laser settings [ 16 , 17 , 33 ] .
our study revealed that laser thermal damage caused by continuous - wave and micropulse laser modes differed ( figures 2(e ) and 2(f ) ) .
our results also suggested that laser power and exposure time titrations for the treatment of retinal disease should be performed using subthreshold micropulse photocoagulation and not continuous - wave laser photocoagulation .
sublethal micropulse photothermal stimulation sites had cellular groups with sparse intercellular spaces that did not have any dead cells , even 24 h after laser irradiation , as determined using cell viability assays ( figure 2(d ) ) .
we presumed that these intercellular spaces represented the loss of cell - to - cell adhesion in the rpe layer , which may contribute to a dysfunctional blood retinal barrier in vivo , directly increasing fluid and molecule movement between the retina and choroidal tissues .
restoring the blood retinal barrier with photothermal stimulation may lead to improvements in macular edema .
future research on proteins related to rpe cell adhesion and rpe tight junctions ( e.g. , occludin , claudin , and zonula occludens-1 ) is needed in rpe cell layers .
several studies have shown that heated tissue remaining viable after conventional continuous - wave laser treatment undergoes a stress response , which includes production of beneficial intracellular biological factors ( e.g. , pedf , tsp-1 , sdf1 , and hsp ) with antiangiogenic and restorative functions [ 25 , 30 , 34 ] . because hsp may block the activity of apoptotic and inflammatory pathways that cause cellular damage [ 2024 ] , upregulation of hsp70 expression induced by laser irradiation
is thought to be important for reducing macular edema following clinical laser therapy [ 25 , 26 ] . in the current study ,
sublethal micropulse photothermal stimulation was shown to induce upregulation of hsp70 , the major hsp in mammalian cells . moreover ,
expression of hsp70 mrna , quantified by real - time qrt - pcr , was temporarily upregulated in preconditioned cultured cell layers for 12 h following laser irradiation .
immunostaining indicated that expression of hsp70 proteins in cultured cell layers occurred concentrically around laser - irradiated sites .
this study is the first to report hsp70 expression and upregulation in cultured cells following sublethal micropulse laser energy application .
the beneficial effects of subthreshold micropulse photocoagulation for diabetic macular edema are not observed immediately in the clinical setting but begin to become evident 3 months following therapy [ 10 , 1317 ] .
therefore , sublethal micropulse photothermal stimulation that induces hsp70 upregulation may facilitate rpe remodeling and contribute to improvements in macular edema .
first , we examined a monolayer of cultured cells , which may not have the same vertical heat conduction properties as an intact eye . secondly , arpe-19 cells exhibit some characteristics and features that are different from those of human rpe cells .
therefore , the laser irradiation system developed and used in this study should be further examined in arpe-19 cells and in human rpe cells derived from retinal tissue and induced pluripotent stem cells .
in conclusion , while the therapeutic mechanism underlying the efficacy of micropulse laser therapy has not yet been determined , our data showed that hsp70 may be a trigger for improving rpe health and function .
moreover , hsp70 may be used as a biological marker to determine therapeutic laser levels for subthreshold micropulse photocoagulation .
in addition , testing laser irradiation systems in a cultured layer of human rpe cells is advantageous because changes in cell morphology , molecular composition , cellular transcriptomes , and cellular proteomes after sublethal photothermal stimulation can be precisely assessed using live imaging microscopy , dna microarrays , and mass spectrometry techniques . | purpose / aim of the study .
subthreshold micropulse diode laser photocoagulation is an effective treatment for macular edema .
the molecular mechanisms underlying treatment success are poorly understood .
therefore , we investigated the effects of sublethal laser energy doses on a single layer of densely cultured arpe-19 cells as a model of the human retinal pigment epithelium ( rpe ) .
materials and methods .
a single layer of densely cultured human arpe-19 cells was perpendicularly irradiated with a micropulse diode laser .
nonirradiated cells served as controls .
sublethal laser energy was applied to form a photocoagulation - like area in the cultured cell layers .
hsp70 expression was evaluated using quantitative polymerase chain reaction and immunocytochemistry .
results .
photocoagulation - like areas were successfully created in cultured arpe-19 cell layers using sublethal laser energy with our laser irradiation system .
hsp70
mrna expression in cell layers was induced within 30 min of laser irradiation , peaking at 3 h after irradiation .
this increase was dependent on the number of laser pulses .
hsp70 upregulation was not observed in untreated cell layers .
immunostaining indicated that hsp70 expression occurred concentrically around laser irradiation sites and persisted for 24 h following irradiation .
conclusion .
sublethal photothermal stimulation with a micropulse laser may facilitate hsp70 expression in the rpe without inducing cellular damage . |
obesity , defined as a body mass index ( bmi ) 30 kg / m , is a chronic disease with major health and economic implications and is recognized as one of the greatest contributors of excessive morbidity and mortality in the 21st century .
approximately 30% of the american population is obese , making it the leading nutritional disorder in our society .
this trend has escalated to epidemic proportions with a disproportionate increase in persons with superobesity , defined as those with a bmi 50 kg / m . in 1991 , the national institutes of health issued a consensus statement concluding that in the morbidly obese , bariatric surgery is the most successful intervention for long - term weight loss .
significant weight loss following bariatric surgery reduces the inherent obesity - specific comorbidities , lowers cardiovascular risk , and provides a survival benefit in this escalating patient population [ 3 , 4 ] .
weight loss surgery has become increasingly utilized , with greater than 225,000 procedures performed in the united states in 2008 according to the american society of metabolic and bariatric surgery . due to the malabsorption induced by the procedure ,
in conjunction with a reduced gastric volume and alterations in eating behaviors , there is an increased risk of developing certain mineral and vitamin deficiencies .
retinol deficiency is more commonly associated with malabsorptive weight loss surgical interventions , and fewer studies have reported this outcome with roux - en - y gastric bypass surgery [ 6 , 7 ] .
routine postoperative laboratory surveillance at our institution identified a direct recurring coupling of deficiencies of serum retinol and prealbumin concentration ; this association has not been previously described in the gastric bypass literature .
we performed a retrospective chart review of 122 obese patients ( 96 women , 26 men ) who underwent roux - en - y gastric bypass surgery at william beaumont hospital in royal oak , michigan , usa .
fifty - four charts had incomplete data and were excluded from analysis ; the remaining 69 subjects served as our study population .
the patient population was preapproved for surgery from a multidisciplinary perspective at the william beaumont hospital weight control center .
the surgeries were performed between october 2005 and july 2007 by two affiliated bariatric surgeons .
data were obtained from william beaumont hospital 's electronic chart system ( one chart , epic systems corporation ) and operative reports .
serum markers of nutrition were assessed including : prealbumin , albumin , total protein , and retinol .
roux limb measurements were analyzed as a potential mediator of malabsorption , and liver function studies were obtained .
patients were interviewed at baseline and follow - up intervals by our team of bariatric dietitians for nutritional compliance with dietary protein intake .
confidentiality was protected by assigning patients ' anonymous numbers , and the study was approved by the hospital 's human investigation committee .
our protocol at the william beaumont hospital weight loss center involves regular follow - up outpatient visits at 6 weeks , 3 , 6 , 9 , and 12 months with a multidisciplinary team that includes a dietitian , exercise physiologist , psychologist , and bariatrician . the dietitian works to optimize dietary intake and assess for food intolerances .
routines recommendations include consuming a higher daily intake of lean protein ( approximately 1.2 g / kg of ideal body weight ) , which generally corresponds to 5580 and 70110 grams for women and men , respectively . other important dietary principles include avoidance of excessive sugars ( > 5 grams per serving ) , which can promote a dumping syndrome , and inclusion of < 30% of daily intake from fat per day to avoid steatorrhea .
patients are also counseled to minimize the consumption of partially saturated and hydrogenated fats .
we routinely recommend a chewable multivitamin twice daily , calcium citrate 500 mg three times daily with 400 iu of vitamin d , ferrous sulfate 30 mg daily separated by 2 hours from the calcium compound , and 1000 mcg of cyanocobalamin daily to prevent vitamin and mineral deficiencies .
laboratory surveillance of commonly reported mineral and vitamin deficiencies as well as protein levels prompted a tiered response of additional treatments as needed .
demographics and baseline characteristics are reported as means standard deviation ( sd ) or counts with percent frequencies as appropriate .
spearman correlations were completed between weight change and vitamin a levels with all the continuous variables at both 6 weeks and 1 year .
univariate comparisons between patients meeting their protein goal and those that did not were made using either a test for normally distributed data or wilcoxon rank tests for the outliers .
categorical variables were examined using pearson 's chi - square as appropriate ( expected frequency > 5 ; otherwise fisher 's exact tests were used ) .
weight loss at 6 weeks and 1 year following surgery was 20.1 9.1 kg and 44.1 17.1 kg , respectively ( figure 1(a ) ) . at 6 weeks and 1 year , 35% and 18% of patients were vitamin a deficient ( < 325 mcg / l ) .
similarly , 34% and 19% had low prealbumin levels ( < 18 mg / dl ) at these time intervals ( figure 1(b ) ) .
vitamin a directly correlated with prealbumin levels at 6 weeks ( r = 0.67 , p < .001 ) , and 1-year ( r = 0.67 , p < .0001 ) .
there was no significant correlation between roux length measurement and serum vitamin a concentrations , at 6 weeks and 1 year ( r = 0.008 and 0.008 , resp .
; p = .96 for both ) . similarly , the correlations between the roux length and prealbumin concentrations at 6 weeks or 1 year were insignificant ( r = 0.08 ; p = .55 and r = 0.001 ; p = .99 ) . achieving dietary protein intake goal , defined as average daily dietary protein intake within 5 grams of intake goal or greater on average , was also not associated with serum levels of vitamin a at 6 weeks or 1 year ( p = .41 ; p = .24 , resp . ) .
no significant correlations between vitamin a levels and markers of liver function ( aspartate aminotransferase and alanine aminotransferase ) were observed at 6 weeks ( p = .29 , .98 , resp . ) or at 1 year ( p = .34 , .99 , resp . ) .
vitamin k assessments were not included in the study methodology and can not be commented on .
we examined the associations between zinc , protein , and vitamin a in various forms and did not identify any significant relationships .
vitamin a is an essential fat - soluble vitamin absorbed through the small intestine as either retinol ( animal derived ) or carotene ( plant and vegetable derived ) .
subsequently , it is converted to retinyl palmitate and hydrolyzed to bound retinyl binding protein that transports vitamin a to tissues .
several potential mechanisms may exacerbate vitamin a deficiency in a postoperative gastric bypass patient .
first , the deficiency may arise from surgically bypassing the duodenum and first portion of the jejunum , promoting an iatrogenically induced malabsorption .
second , drastic decreases in the dietary intake of many micronutrients like carotenoids and retinol , especially in early recovery , are likely to occur . in addition , traditional dietary recommendations after gastric bypass include a low - fat diet which potentially limit the absorption of fat - soluble vitamins .
this patient subset may also be at risk due to confounding nonalcoholic steatohepatitis , higher rates of cirrhosis , or both , which may interfere with maintaining vitamin a storage and production .
finally , higher levels of oxidative stress may also occur after gastric bypass surgery , which can interfere with vitamin a absorption and processing .
vitamin a deficiency is rarely described in western society ; however , worldwide , it remains the most common etiology of visual disturbances , including blindness .
vitamin a deficiency has been ascribed to a wide variety of ophthalmologic complications including conjunctival and corneal xerosis , keratomalacia , retinopathy , visual loss , and nyctalopia .
moreover , retinol supports photosensitive pigmented cells of the retinal rods and cones that are necessary for optimal visual acuity .
serum retinol levels and protein - calorie malnutrition have been correlated among children and infants in developing nations .
vitamin a deficient children treated with an augmented dietary protein intake demonstrated an increase in serum protein markers as well as serum retinol levels . in this extreme clinical scenario ,
retinol deficiency was successfully managed through this dietary intervention alone , without the addition of vitamin a rich foods or vitamin a supplementation .
the present findings support the intimate interaction that serum retinol levels have to carrier proteins which determine the bioavailability of serum retinol concentration and reinforce the interdependent relationship of these nutritional markers . as such , total body stores of vitamin a may not be truly deficient ; in reality , limited access to nutritional protein binding and transport capacity may underlie these serum retinol findings .
these data suggest that addressing the nutritional protein levels is necessary in conjunction with deficient serum retinol concentrations .
there are several case series describing vitamin a deficiency with visual disturbances involving patients who have had gastric or intestinal surgery [ 6 , 7 , 10 ] .
none of our patients complained of visual disturbance ; however , ocular complaints may have been underreported in the scope of this paper because many clinical features especially early in the course of retinol deficiency can be vague or nonspecific and may not have been recognized as clinically relevant .
it is important to acknowledge this potential complication after bariatric surgery in patients who undergo longstanding iatrogenic malabsorption with limited nutritional protein stores and consider appropriate diagnostic testing and referral for ophthalmologic assessment , when appropriate .
accordingly , our population was limited to those patients who were compliant with their baseline evaluation , 1-year follow - up exam and serial laboratory testing .
subjects with missing preoperative or postoperative lab values were excluded from the analysis , which may have biased our study in representing a more compliant subset of patients . because we captured these data in the scope of clinical management ,
multivitamin brands and additional retinol supplementation may have varied according to patient preference and our treatment methodology .
we also did not account for other potential confounding variables , including physical activity , nutritional compliance , or the duration of supplementation .
nevertheless , vitamin a deficiency is of escalating interest in clinical centers and will likely be the focus of future research .
we noted a striking , direct relationship between postoperative nutritional protein levels and vitamin a concentrations in our gastric bypass populations .
vitamin a deficiency is common after gastric bypass and is directly associated with a low serum prealbumin concentration , a measure of protein - calorie malnutrition .
this fat - soluble vitamin deficiency should be considered in postoperative patients and deficiency should be strongly suspected in those with evidence of protein calorie malnutrition .
improving awareness and understanding of total body vitamin a utilization is of paramount importance in the ongoing medical management of this at - risk population . | introduction .
few data are available on vitamin a deficiency in the gastric bypass population
.
methods .
we performed a retrospective chart review of gastric bypass patients ( n = 69 , 74% female ) .
the relationship between serum vitamin a concentration and markers of protein metabolism at 6-weeks and 1-year post - operative were assessed .
results .
the average weight loss at 6-weeks and 1-year following surgery was 20.1 9.1 kg and 44.1 17.1 kg , respectively . at 6 weeks and 1 year after surgery , 35% and 18% of patients were vitamin a deficient , ( < 325
mcg / l ) .
similarly , 34% and 19% had low pre - albumin levels ( < 18 mg / dl ) , at these time intervals .
vitamin a directly correlated with pre - albumin levels at 6 weeks ( r = 0.67 , p < 0.001 ) and 1-year ( r = 0.67 ,
p < 0.0001 ) .
there was no correlation between the roux limb length measurement and pre - albumin or vitamin a serum concentrations at these post - operative follow - ups .
vitamin a levels and markers of liver function testing were also unrelated .
conclusion .
vitamin a deficiency is common after bariatric surgery and is associated with a low serum concentration of pre - albumin .
this fat - soluble vitamin should be measured in patients who have undergone gastric bypass surgery and deficiency should be suspected in those with evidence of protein - calorie malnutrition . |
methylene blue dye ( mbd ) has been successfully used worldwide with few complications in patients undergoing breast surgery .
we herein describe 2 different complications in 2 patients : 1 ) skin and parenchymal necrosis when mbd was injected subdermally and 2 ) mycoplasma infection caused by contaminated mbd in breast reduction surgery .
several publications advocate the use of subdermal mbd injections for sln mapping without complications 1 - 3 ; however , several publications report complications ranging from blue staining of the skin and fat necrosis 4 - 6 . the american society of clinical oncology ( asco ) guidelines advocate the use of blue dye in conjunction with radioisotope for sln mapping as this combined strategy yields the highest rates of successful sln mapping 7 .
some surgeons prefer mbd over lymphazurin ( isosulphan blue dye ) because lymphazurin is more expensive , sometimes unavailable due to national shortages , and may rarely cause anaphylaxis 3 , 8 .
our objective is to bring to light the fact that although mbd is relatively safe , it is not without potential for serious complications .
the 2 patients described in these case reports were seen at the university of arizona cancer center during 2008 and referred to a breast surgical oncologist for further care .
we evaluated and managed complications of mbd injected by 2 referring surgeons for different indications . a 67-year - old woman with a t1b n0 m0 infiltrating ductal carcinoma of the left breast
was treated by a surgeon at the referring institution : she underwent a left segmental mastectomy and sentinel lymph node ( sln ) biopsy for a 10 mm primary tumor located at the 9 o'clock position . the initial operating surgeon injected mbd in a subareolar subdermal fashion , complicated by mbd skin necrosis .
the patient initially presented describing pain and tenderness of her left breast that persisted for two months following her first surgery .
we noted a sinus in the 8 o'clock position , draining purulent fluid ; erythema surrounding the areola , extending toward the axilla ; and fluctuance in the central breast . on breast ultrasound examination , an area of loculated fluid
culture results of this fluid showed corynebacteria and anaerobic gram negative rods , which was clinically believed to be due to infection of the chronic open wound secondary to mbd necrosis .
we subsequently admitted the patient to the hospital to perform an incision and drainage ( i&d ) of her left breast unresolving abscess cavity .
we made an incision in the area of maximal fluctuance in the left breast in the periareolar region at 5 o'clock . to explore the borders of the cavity
, we used a lacrimal probe ; we noted that the cavity was connected to the sinus in the 8 o'clock position .
the cavity measured approximately 8 cm x 7 cm x 2.5 cm and contained necrotic tissue .
then , we made a separate incision over the sinus in the 8 o'clock position and completely excised the tract .
once we debrided the cavity , we placed a wound vacuum - assisted closure ( vac ) device for negative pressure treatment with suction drainage 9 , 10 through 2 separate incisions ( figures 1 - 4 ) .
a 45-year - old pre - menopausal woman underwent bilateral breast reductions performed by the referring surgeon in 2007 .
centers for disease control and prevention ( cdc ) have reported contamination of mbd with mycobacteria chelonae in facelift patients in 200311 . however , to our knowledge , infectious contamination of mbd has not been previously reported in any breast surgery patients .
the patient 's plastic surgeon that had previously performed her breast reductions cultured mycobacteria chelonae directly from a contaminated bottle of mbd ( personal correspondence , microbiology reports ) .
this plastic surgeon had a series of 24 mycoplasma chelonae infections secondary to mbd on several of his cosmetic surgery patients during a period of 3 months in 2007 ( personal correspondence , microbiology reports ) .
this was never reported to the cdc or elsewhere in the medical literature to date .
our patient 's follow - up mammogram showed distortion of the right breast in the upper quadrant , which was thought to be scarring from prior surgery , along with a separate mass , located in the 8 o'clock position , reported with the dimensions of 1.2 cm .
we performed an ultrasound - guided core biopsy of that mass and identified nottingham grade 2 infiltrating lobular carcinoma with associated lobular carcinoma in situ .
a bilateral breast magnetic resonance imaging ( mri ) showed a broad region ( 6.2 x 2.7 cm ) of abnormal enhancement in the right breast , which was suspicious for lobular carcinoma ( figures 6 - 7 ) . to evaluate the extent of disease
, we performed 3 mri - guided core biopsies : 2 were negative for malignancy , and 1 revealed lobular carcinoma in situ .
thus , mri was proven to overestimate her extent of disease secondary to her breast infections due to breast reduction .
the patient elected to undergo bilateral total skin - sparing mastectomies and right axillary sln dissection .
final pathology of the right breast revealed a 1.8-cm , multifocal , grade 2 , invasive lobular carcinoma with associated lobular carcinoma in situ .
margins were 2 cm from the tumor , with none of the 4 slns involved .
she enrolled in the trial assigning individualized options for treatment ( tailor rx ) and was randomized to undergo chemotherapy .
her chronic lingering infection was an important factor in the decision to use systemic therapy with cyclophosphamide , methotrexate , and 5-fluorouracil ( cmf ) , to avoid neutropenia .
her history of bilateral breast infections requiring numerous debridements also factored into her decision making to select bilateral mastectomies .
she has completed chemotherapy , underwent breast reconstruction and is maintained on tamoxifen hormonal therapy .
a 67-year - old woman with a t1b n0 m0 infiltrating ductal carcinoma of the left breast was treated by a surgeon at the referring institution : she underwent a left segmental mastectomy and sentinel lymph node ( sln ) biopsy for a 10 mm primary tumor located at the 9 o'clock position . the initial operating surgeon injected mbd in a subareolar subdermal fashion , complicated by mbd skin necrosis .
the patient initially presented describing pain and tenderness of her left breast that persisted for two months following her first surgery .
we noted a sinus in the 8 o'clock position , draining purulent fluid ; erythema surrounding the areola , extending toward the axilla ; and fluctuance in the central breast . on breast ultrasound examination , an area of loculated fluid
culture results of this fluid showed corynebacteria and anaerobic gram negative rods , which was clinically believed to be due to infection of the chronic open wound secondary to mbd necrosis .
we subsequently admitted the patient to the hospital to perform an incision and drainage ( i&d ) of her left breast unresolving abscess cavity .
we made an incision in the area of maximal fluctuance in the left breast in the periareolar region at 5 o'clock . to explore the borders of the cavity
, we used a lacrimal probe ; we noted that the cavity was connected to the sinus in the 8 o'clock position .
the cavity measured approximately 8 cm x 7 cm x 2.5 cm and contained necrotic tissue .
then , we made a separate incision over the sinus in the 8 o'clock position and completely excised the tract .
once we debrided the cavity , we placed a wound vacuum - assisted closure ( vac ) device for negative pressure treatment with suction drainage 9 , 10 through 2 separate incisions ( figures 1 - 4 ) .
a 45-year - old pre - menopausal woman underwent bilateral breast reductions performed by the referring surgeon in 2007 .
she required multiple debridements , which resulted in extensive scar tissue and granuloma formation . the u.s .
centers for disease control and prevention ( cdc ) have reported contamination of mbd with mycobacteria chelonae in facelift patients in 200311 . however , to our knowledge , infectious contamination of mbd has not been previously reported in any breast surgery patients .
the patient 's plastic surgeon that had previously performed her breast reductions cultured mycobacteria chelonae directly from a contaminated bottle of mbd ( personal correspondence , microbiology reports ) .
this plastic surgeon had a series of 24 mycoplasma chelonae infections secondary to mbd on several of his cosmetic surgery patients during a period of 3 months in 2007 ( personal correspondence , microbiology reports ) .
this was never reported to the cdc or elsewhere in the medical literature to date .
our patient 's follow - up mammogram showed distortion of the right breast in the upper quadrant , which was thought to be scarring from prior surgery , along with a separate mass , located in the 8 o'clock position , reported with the dimensions of 1.2 cm .
we performed an ultrasound - guided core biopsy of that mass and identified nottingham grade 2 infiltrating lobular carcinoma with associated lobular carcinoma in situ .
a bilateral breast magnetic resonance imaging ( mri ) showed a broad region ( 6.2 x 2.7 cm ) of abnormal enhancement in the right breast , which was suspicious for lobular carcinoma ( figures 6 - 7 ) . to evaluate the extent of disease
, we performed 3 mri - guided core biopsies : 2 were negative for malignancy , and 1 revealed lobular carcinoma in situ .
thus , mri was proven to overestimate her extent of disease secondary to her breast infections due to breast reduction .
the patient elected to undergo bilateral total skin - sparing mastectomies and right axillary sln dissection .
final pathology of the right breast revealed a 1.8-cm , multifocal , grade 2 , invasive lobular carcinoma with associated lobular carcinoma in situ .
margins were 2 cm from the tumor , with none of the 4 slns involved .
she enrolled in the trial assigning individualized options for treatment ( tailor rx ) and was randomized to undergo chemotherapy .
her chronic lingering infection was an important factor in the decision to use systemic therapy with cyclophosphamide , methotrexate , and 5-fluorouracil ( cmf ) , to avoid neutropenia .
her history of bilateral breast infections requiring numerous debridements also factored into her decision making to select bilateral mastectomies .
she has completed chemotherapy , underwent breast reconstruction and is maintained on tamoxifen hormonal therapy .
we are aware of only a few published complications of mbd in breast surgery patients ( table 1 ) .
varghese et al 1 , described safely injecting mbd in the subdermal plane in the subareolar region with 97.6% identification rate .
their only reported complication was temporary tattooing from mbd , which was also seen on goavert et al series 12 .
mathelin et al 2 and soni et al 3 both used mbd safely without adverse reactions in sln mapping .
stradling et al 5 was the first to describe local skin reactions to mbd injection for sln mapping , including skin necrosis in 5 of 24 patients .
zakaria et al 4 and salhab et al 6 both reported skin and fat necrosis in a small percentage of patients who received mbd injections for sln mapping .
komenaka et al 13 described a palpable mass at the site of injection of mbd .
nearly all of the masses resolved by one year , however , one resolved after 18 months .
they reported capsular contraction associated with mbd , after immediate reconstruction using breast prosthesis . in their case
they attributed local inflammatory reaction from the mbd as the main cause of the capsular contraction .
one of our patients ( case 1 ) presented with a well - developed region of fat necrosis with cavity formation .
fat necrosis is an important diagnostic consideration as it may persist and can clinically mimic carcinoma . early in its development ,
fat necrosis is composed of disrupted fat cells and hemorrhage with an influx of histiocytes , some becoming multinucleate as they ingest debris . after several weeks the affected area develops peripheral fibrosis , often with calcification and forming a tumor - like lesion which may clinically mimic carcinoma 15 .
electrocautery has evident thermal effect around the edges and involved areas and does not cause such extensive destruction beyond the local tissue perimeter , with several centimeters of fat necrosis , as in the case of our patient .
we are aware of 1 report of mbd contamination with mycobacteria chelonae that caused infections in patients who had undergone facelifts 11 .
to our knowledge , our case represents the initial report of contaminated mbd in breast surgery patients .
this is of particular concern due to the impact that contaminated mbd had on the surgical and adjuvant management of breast cancer for our patients .
our 2 case reports confirm the findings of previous publications that complications of mbd are capable of causing tissue necrosis . of note ,
both of our patients required extensive surgical debridements , revealing that mbd does indeed have side effects not considered in the previously published literature . in our case report # 2 , the discovery of a complicating mycobacterial infection led to delayed adjuvant treatment and had a significant impact on our choice for systemic treatment .
historically , giuliano et al 16 , described using blue dye as peritumoral injection with good results ; peritumoral injection of blue dye is the most common approach to lymphatic mapping with vital blue dyes .
there are a limited number of studies indicating high success rates of identifying slns using subareolar injection of blue dye 20 - 22 .
rodier et al 23 , using both blue dye and radiolabelled isotope , found that using periareolar injection was equivalent to using peritumoral injection in identifying sln .
there are several studies supporting the different sites of injection for blue dye , but subareolar and dermal injections have been proven to cause more local side effects , like discoloration of the breast , that can last several months 24 . in our case
it caused more than just discoloration but led to substantial tissue loss due to necrosis .
lymphazurin had long been held as the standard for sentinel lymph node mapping in breast cancer25 .
however , mbd has demonstrated equivalent efficacy in lymphatic mapping compared to lymphazurin 3 , 26 - 28 , is less expensive , more readily available and is not associated with the potentially deleterious side effects of severe allergic reaction , including anaphylaxis 3 , 8 , 29 as has been reported with lymphazurin .
raut et al reported an incidence of severe anaphylactoid reaction to lymphazurin of 1.1 % ; preoperative prophylaxis reduced the severity , but not the overall incidence ( 0.5% ) of adverse reactions to lymphazurin blue dye 30 .
blue urticaria and facial edema were the reactions observed after preoperative prophylaxis in that study .
as described in the above cases and table , mbd is not without its own potential for complications .
the risk for complications of mbd should be taken into consideration when selecting a blue dye for lymphatic mapping .
either may be used in combination with radioactive colloid and a gamma probe for sentinel lymph node mapping . according to the asco guidelines , the greatest proportion of successful mappings and the lowest false negative rates
are associated with the use of blue dye and radiolabelled colloid used in conjunction 7 .
however , golshan and colleagues 31 have recently published data to show that sentinel nodes can be successfully identified with mbd only .
additionally , they recommend the use of mbd over lymphazurin , due to the latter 's known potentially deleterious side effects of severe allergic reaction , anaphylaxis or even death .
the dosing and concentration of mbd , in successful identification of sln , has been described as 1 - 5 milliliters ( ml ) of 1% methylene blue 3 , 5 , 31 or 1.25mg / ml 2 , 4 .
mbd may indeed become the most prevalent vital blue dye for these reasons and due to intermittent national shortages of lymphazurin blue dye 3 .
the importance of understanding its proper usage and potential complications , therefore , can not be understated .
we are aware of only a few published complications of mbd in breast surgery patients ( table 1 ) .
varghese et al 1 , described safely injecting mbd in the subdermal plane in the subareolar region with 97.6% identification rate .
their only reported complication was temporary tattooing from mbd , which was also seen on goavert et al series 12 .
mathelin et al 2 and soni et al 3 both used mbd safely without adverse reactions in sln mapping .
stradling et al 5 was the first to describe local skin reactions to mbd injection for sln mapping , including skin necrosis in 5 of 24 patients .
zakaria et al 4 and salhab et al 6 both reported skin and fat necrosis in a small percentage of patients who received mbd injections for sln mapping .
komenaka et al 13 described a palpable mass at the site of injection of mbd .
nearly all of the masses resolved by one year , however , one resolved after 18 months .
they reported capsular contraction associated with mbd , after immediate reconstruction using breast prosthesis . in their case
they attributed local inflammatory reaction from the mbd as the main cause of the capsular contraction .
one of our patients ( case 1 ) presented with a well - developed region of fat necrosis with cavity formation .
fat necrosis is an important diagnostic consideration as it may persist and can clinically mimic carcinoma . early in its development ,
fat necrosis is composed of disrupted fat cells and hemorrhage with an influx of histiocytes , some becoming multinucleate as they ingest debris . after several weeks the affected area develops peripheral fibrosis , often with calcification and forming a tumor - like lesion which may clinically mimic carcinoma 15 .
electrocautery has evident thermal effect around the edges and involved areas and does not cause such extensive destruction beyond the local tissue perimeter , with several centimeters of fat necrosis , as in the case of our patient .
we are aware of 1 report of mbd contamination with mycobacteria chelonae that caused infections in patients who had undergone facelifts 11 . to our knowledge
this is of particular concern due to the impact that contaminated mbd had on the surgical and adjuvant management of breast cancer for our patients .
our 2 case reports confirm the findings of previous publications that complications of mbd are capable of causing tissue necrosis .
of note , both of our patients required extensive surgical debridements , revealing that mbd does indeed have side effects not considered in the previously published literature . in our case report # 2 , the discovery of a complicating mycobacterial infection led to delayed adjuvant treatment and had a significant impact on our choice for systemic treatment .
the site of injection of blue dyes is still controversial . historically , giuliano et al 16 , described using blue dye as peritumoral injection with good results ; peritumoral injection of blue dye is the most common approach to lymphatic mapping with vital blue dyes .
there are a limited number of studies indicating high success rates of identifying slns using subareolar injection of blue dye 20 - 22 .
rodier et al 23 , using both blue dye and radiolabelled isotope , found that using periareolar injection was equivalent to using peritumoral injection in identifying sln .
there are several studies supporting the different sites of injection for blue dye , but subareolar and dermal injections have been proven to cause more local side effects , like discoloration of the breast , that can last several months 24 . in our case
it caused more than just discoloration but led to substantial tissue loss due to necrosis .
lymphazurin had long been held as the standard for sentinel lymph node mapping in breast cancer25 .
however , mbd has demonstrated equivalent efficacy in lymphatic mapping compared to lymphazurin 3 , 26 - 28 , is less expensive , more readily available and is not associated with the potentially deleterious side effects of severe allergic reaction , including anaphylaxis 3 , 8 , 29 as has been reported with lymphazurin .
raut et al reported an incidence of severe anaphylactoid reaction to lymphazurin of 1.1 % ; preoperative prophylaxis reduced the severity , but not the overall incidence ( 0.5% ) of adverse reactions to lymphazurin blue dye 30 .
blue urticaria and facial edema were the reactions observed after preoperative prophylaxis in that study .
as described in the above cases and table , mbd is not without its own potential for complications .
the risk for complications of mbd should be taken into consideration when selecting a blue dye for lymphatic mapping
. both mbd and lymphazurin are vital blue dyes . either may be used in combination with radioactive colloid and a gamma probe for sentinel lymph node mapping . according to the asco guidelines , the greatest proportion of successful mappings and the lowest false negative rates
are associated with the use of blue dye and radiolabelled colloid used in conjunction 7 . however
, golshan and colleagues 31 have recently published data to show that sentinel nodes can be successfully identified with mbd only .
additionally , they recommend the use of mbd over lymphazurin , due to the latter 's known potentially deleterious side effects of severe allergic reaction , anaphylaxis or even death .
the dosing and concentration of mbd , in successful identification of sln , has been described as 1 - 5 milliliters ( ml ) of 1% methylene blue 3 , 5 , 31 or 1.25mg / ml 2 , 4 .
mbd may indeed become the most prevalent vital blue dye for these reasons and due to intermittent national shortages of lymphazurin blue dye 3 .
the importance of understanding its proper usage and potential complications , therefore , can not be understated .
the use of mbd in breast surgery patients is not without risk . in both of these patients ,
mbd was integral to complications requiring surgical debridements for local wound problems . in each patient 's case ,
awareness should be raised regarding mbd 's potential to elicit tissue necrosis resulting in significant morbidity , cosmetically unsatisfactory results and even delayed cancer treatment . | introduction : methylene blue dye has been used worldwide successfully with few complications in breast surgery .
we present two different complications involving methylene blue : 1 ) skin and parenchymal necrosis when dye was injected in a subdermal fashion and 2 ) mycoplasma infection caused by contaminated methylene blue in breast reduction surgery.methods : we present two cases seen at the university of arizona during 2008 and referred to a breast surgeon for management .
we evaluated and managed complications of methylene blue dye injected by 2 referring surgeons for different indications . a review of the literature was performed.results : the first case is a 67 year old female diagnosed with infiltrating ductal carcinoma of the left breast for which she was treated by her initial surgeon with left segmental mastectomy and sentinel node biopsy .
the operating surgeon injected methylene blue in a subareolar subdermal fashion ( distant from the primary tumor ) ; unfortunately the patient suffered skin and breast necrosis requiring multiple surgical debridements and finally achieving delayed primary closure .
the second case is a 45 year old female with infiltrating lobular carcinoma with a history of mycoplasma infection secondary to methylene blue injected for breast reduction surgery .
she required multiple debridements and had granulomas masquerading as cancer on mri that confounded her extent of disease.conclusions : the use of methylene blue dye in breast surgery is not without risk . in both cases methylene blue was responsible for complications requiring surgical debridement for local wound problems . in each case
severe necrosis and infection were present .
methylene blue may cause not only significant morbidity , but may also produce cosmetically unsatisfactory results . |
is a lecturer at the institute of chemical technology and engineering at the pozna university of technology in poland .
her areas of scientific interest include the technique of separation , adsorption , surface , and interfacial tension and surfactants .
is a lecturer in the department of technology and instrumental analysis at the faculty of commodity science pozna university of economics .
her areas of scientific interest include essential oils , adjutants , household and personal care products ingredients , surface , and antimicrobial properties of surfactants .
is a lecturer in the department of technology and instrumental analysis at the faculty of commodity science pozna university of economics .
her areas of scientific interest include cosmetic ingredients , surface and interfacial tension , and characterization of surfactants .
surfactants are one of the most important groups of organic chemicals , and are used in vast amounts in domestic and industrial applications [ 1 , 2 ] . they are designed to remove dirt , sweat , sebum , and oils from the skin and other surfaces .
these surfactants exhibit excellent surface properties , i.e. , low surface tension and a critical micelle concentration .
zwitterionic surfactants are good wetting agents and can be high , moderate or low foaming surfactants .
probably the most important class of amphoteric surfactants is amidopropyl betaines , in particular cocamidopropyl betaine ( capb ) .
cocamidopropyl betaine is predominately used as a cosmetic ingredient and as a detergent ( 50 % of the produced volume in europe29,500 tons / year ) .
for example it is used in cosmetics and personal hygiene products , mainly in rinse - off products such as shampoos , but also in roll - on deodorants , contact lens solutions , toothpaste , makeup removers , bath gels , skincare products , cleansers , liquid soaps , anti - dandruff products , and exfoliating and peel - off products , in the preparations for the treatment of acne and conditioners , as well as eye make - up products and antiseptics [ 713 ] .
moreover it is use as a detergent includes hand washing agents , and hand dish washing agents . due to the mild nature and low degree of skin irritation
cocobetaine is also present with other surfactants ( anionic , nonionic ) in commercially available products .
an example of such a combination is a mixture of cocobetaine and sodium lauryl sulfate ( sls ) as the primary surfactant , which reduces the irritation of the skin and mucous membranes , strengthens the feeling on the skin , and increases the organoleptic sensations [ 5 , 10 , 12 , 14 , 15 ] .
it is easily soluble in water and its 10 % solution has an acidic reaction with a ph of about 56 .
1general formula of cocobetaine general formula of cocobetaine a literature survey shows that chaotropic ions influence some properties like solubilization , micellization and rheology .
the addition of salts to aqueous surfactant solutions may result in a modification of both intramicellar and intermicellar interactions .
accordingly , a fundamental understanding of how salts affect the behavior of aqueous surfactant solutions may lead to a more effective utilization of this material in various practical applications .
studies on the effect of sodium chloride on the micellar properties are mainly focused on the cationic and anionic surfactants [ 1822 ] .
sodium chloride is used as a thickener in shampoos and conditioners containing sodium lauryl sulfate .
it is a contributing factor to the eye irritation experienced with most shampoos , and it may also cause dry and itchy scalp .
the addition of salts to the aqueous solution of the surfactants changes the solution properties , such as the critical micellar concentration , as well as the phase behavior of the surfactant solution , as well as the critical micelle concentration . because salts are added to the personal care products , it is important to assess the impact of salt on the behavior of such solutions .
the aim of this study was to check the effect of sodium chloride on the surface and wetting properties of aqueous solutions of capb . in the presence of sodium chloride , cocamidopropyl betaine
sodium chloride is used as a thickener in shampoos and conditioners containing sodium lauryl sulfate .
it is a contributing factor to the eye irritation experienced with most shampoos , and it may also cause dry and itchy scalp .
the addition of salts to the aqueous solution of the surfactants changes the solution properties , such as the critical micellar concentration , as well as the phase behavior of the surfactant solution , as well as the critical micelle concentration . because salts are added to the personal care products , it is important to assess the impact of salt on the behavior of such solutions .
the aim of this study was to check the effect of sodium chloride on the surface and wetting properties of aqueous solutions of capb . in the presence of sodium chloride , cocamidopropyl betaine
the sample of cocobetaine was purchased from one of the cosmetics companies making it available on the polish market .
it was stored in a refrigerator and not used after the expiration date specified by the producer . according to the manufacturer 's information , the commercial capb consists of 30 % cocamidopropyl betaine and 70 % water , without sodium chloride .
the samples of cocobetaine used to measure surface tension were prepared as aqueous and salt solutions ( nacl , from poch s.a , poland ) of surfactant .
the surface tension measurement was made for stock aqueous and salt solutions and for the following 18 solutions obtained by a serial dilutions method .
the measurement was carried out for aqueous and sodium chloride solutions with four concentrations : 0.01 , 0.05 , 0.2 and 2 m using a pendent drop tensiometer tracker ( i.t .
the tensiometer captures the image of an air drop immersed in the aqueous solution of surfactant and records the drop shape as a function of time .
the drop shape is determined by the surface tension of the liquid , gravity , and the density difference between the air and surrounding medium .
the drop image is analysed with a profile fitting method in order to determine the contact angle and the surface tension .
measurements of surface tension were performed at constant temperature for aqueous solutions at 297 k. measurement duration depends on concentration of surfactants in the sample and was conducted to reach equilibrium .
additional physicochemical analyses on the basis of the surface tension data were carried out using the szyszkowski and frumkin equation .
contact angle measurements were performed with the same equipment for water or aqueous salt solutions at a concentration of cocobetaine 1 , 0.01 , 0.0001 g / dm .
surfaces used for measurements of contact angle were : aluminium , paraffin , pvc ( poly(1-chloroethylene ) ) , ptfe ( poly(1,1,2,2-tetrafluoroethylene ) ) , glass , pe ( polyethene ) , pmma ( poly(methyl 2-methylpropenoate ) ) .
the foaming properties of the water and aqueous salt solutions of cocobetaine were investigated in a glass apparatus using the ross - miles method in accordance with the standard astm00a51e47 . after a creating column of foam ,
foam volumes recorded after 10 min were the basis for determining the stability of the produced foam . the measurements of foaming properties were made for water and aqueous salt solutions for six different concentrations of cocobetaine .
during the measurement of the surface tension by the drop shape method , the dependence of surface tension versus time was observed . because the adsorption process of surfactants is dynamic , exact equilibrium values of the surface tension are necessary to determine the adsorption isotherms . for surfactants with low surface activity the adsorption reaches its equilibrium in time on the order of seconds to hours .
it was found that the time to reach the adsorption equilibrium decreases with the increase in the concentration of cocobetaine in the aqueous solutions and changed from about 600 to 2,400 s at the lowest measured concentration . in fig . 2 , the plots of the surface tension versus the log of concentration are presented .
the minimum values of the surface tension were similar ( about 25 mn / m ) regardless of the sodium chloride concentrations . from these curves critical micellar concentration ( cmc ) values
they were calculated for a molar mass of 356 which corresponds to the derivative of cocobetaine with 12 carbon atoms in the alkyl chain . as seen in fig . 2 and table 1 , values of the cmc increase with increasing concentrations of salt in the aqueous solutions .
the presence of the electrolyte generally causes a decrease in the cmc of most surfactants .
addition of electrolytes decreases the repulsion between similarly charged ionic head groups within a micelle and therefore , the detergent monomers can pack tightly and the cmc is reduced .
the change in the cmc of these types of surfactants on the addition of electrolyte has been attributed mainly to the salting out or salting in of the hydrophobic groups in the aqueous solvent by the electrolyte , rather than to the effect of the latter on the hydrophilic groups of the surfactant .fig .
2surface tension isotherms for aqueous and salt solutions of cocobetainetable 1adsorption parameters for cocobetaine in water / air systemsadsorption parameterunit2 m nacl0.2 m nacl0.05 m nacl0.01 m naclwatercmcmol / dm
7.02 10
5.60 10
2.23 10
0.89 10
0.28 10
a
sz
mol / dm
1.16 10
1.24 10
3.16 10
2.79 10
4.65 10
b
sz 10
3.703.964.515.565.67k or sz0.9770.9910.9880.9930.990
a
f
mol / dm
3.93 10
3.85 10
1.11 10
7.95 10
1.96 10
b
f 10
2.135.836.856.437.06
a2.23 10
2.73 10
01.58 10
7.59 10
k or f,0.9870.9960.9990.9950.993 10
mol / m
1.101.171.331.641.66
a
min
nm
1.501.421.251.010.998g
ads
kj / mol55.950.247.942.541.3 surface tension isotherms for aqueous and salt solutions of cocobetaine adsorption parameters for cocobetaine in water / air systems as was mentioned above , in the presence of sodium chloride in an aqueous solution of cocamidopropyl betaine could be described as the inner salt or as the respective sodium salt , and that could be a reason for the different behavior of this compound in systems with nacl compared to systems with water . in the commercial products , salts
are often added , and this should be taken into account to explain the different surface properties of cocamidopropyl betaine .
the measured change of the surface tension with concentration was fitted with the szyszkowski isotherm:1\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$\gamma^{\text{sz } } = \gamma_{0 } \left [ { 1 - b_{\text{sz } } \ln \left ( { \frac{c}{{a_{\text{sz } } } } + 1 } \right ) } \right]$$\end{document}sz=01-bszlncasz+1where 0 is the interfacial tension for concentration c = 0 and asz and bsz are the adsorption coefficients . using the szyszkowski adsorption coefficients asz , bsz ,
the surface excess at the saturated interface ( ) , the minimum molecular area in the adsorption layer at the saturated interface ( amin ) and the gibbs free energy of adsorption ( gads ) could be estimated .
on the other hand , the szyszkowski isotherm does not take into consideration the mutual interactions between the adsorbed molecules , therefore , the frumkin isotherm might be more suitable for describing the experimental data as it does introduce such interaction .
( 2 ) in which a characterizes the mutual interactions between the adsorbed molecules : 2\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$\gamma^{\text{f } } = \gamma_{0 } \left [ { 1 - b_{\text{f } } \ln \left ( { \frac{c}{{a_{\text{f } } } } + 1 } \right ) - a'\left ( { \frac{c}{{c + a_{\text{f } } } } } \right)^{2 } } \right]$$\end{document}f=01-bflncaf+1-acc+af2 the surface tension measurements showed that the cocobetaine is surface active in all aqueous solutions and depends on the salts concentration . the results obtained , presented in table 1 indicate that in the case of using water as the solvent , the surface concentration at the saturated interface is higher in comparison to the solution with various concentration of nacl . in consequence ,
the statistical area occupied by the adsorbed molecules of cocobetaine at the saturated air / water interface is smaller in comparison to the system with salt .
salt addition ( 0.1 m nacl ) has resulted in a slight increase in the limiting area per molecule .
the results obtained suggest that the structures of the adsorbed monolayers formed at the interfaces considered are quite different .
the molecules of the cocobetaine are much closer together at the air / water interface than in the case of the air / salt solution one .
the different populations at the interface with the salt and the non - salt aqueous solution suggests a different orientation of the adsorbed molecules at the interfacial monolayer .
as indicated by the data presented in table 1 , the magnitude of gads is also affected by the type of aqueous solution .
the values of gads calculated in systems with nacl are more negative in comparison to the system without salt , and decrease with increasing salt concentration . thus a less pronounced tendency for adsorption
the results obtained suggest more spontaneous adsorption processes of molecules of cocobetaine in systems with nacl in aqueous solutions .
the analysis of the mutual interactions between the adsorbed molecules on the ground of the magnitude of a values indicated that the interactions are insignificant and can be neglected in all the systems considered , irrespective of the concentration of nacl in aqueous solution .
the efficiency of adsorption of cocobetaine in the various aqueous solutions is clearly different , too .
this can be concluded from studies on surface excess isotherms shown in fig . 3 which demonstrated higher efficiency of adsorption of cocobetaine molecules in water as diluents .
in all cases , the surface excess isotherms increases monotonically to a constant value , characterized by the saturation of the phase boundary .
presentation of the adsorption isotherm in a semi - logarithmic system allows showing distinct differences between the various bulk concentrations of cocobetaine in various aqueous solutions , at which the saturation of the air / aqueous solution is observed . as can be seen the cocobetaine molecules in salts solution ( 0.05 , 0.2 and 2 m ) saturate phase boundaries at a much higher concentration compared to the water or 0.01 m nacl solution.fig .
3surface excess isotherms for aqueous and salt solutions of cocobetaine surface excess isotherms for aqueous and salt solutions of cocobetaine another important attribute of surfactants is the ability to wet out solid surfaces .
the wetting properties are the ability to test product to dissolve in water , to lower the surface tension between liquid and solid , and to remove air from liquids and solids by aqueous solutions [ 33 , 34 ] .
one way to characterize the wetting of solid surfaces by a liquid is to measure the contact angle .
solid surfaces with high surface energy are completely wetted by the liquids with high surface tension , respectively . as it can be seen in fig .
the presence of salt in the solutions effects only on wetting properties of teflon , polyethylene and paraffin surfaces .
other examined surfaces were wetted by the aqueous and salt solutions almost to the same extent.fig .
4contact angles of 0.1 % cocobetaine s solutions for different surfaces contact angles of 0.1 % cocobetaine s solutions for different surfaces bargeman and van voorst vader found that there is a linear relationship between the adhesional tension ( cos( ) ) and surface tension ( ) of aqueous solutions of surfactants:\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$\gamma \times { \cos } ( \theta ) = a \times \gamma + b,$$\end{document}cos()=a+b , where a and b are constants .
they have also proposed that for nonpolar solids , such as a teflon and surfactant system , the value of constant a is 1 .
the results of adhesional tension vs surface tension of aqueous solutions of cocobetaine for teflon plate are presented in fig .
5 confirms the conclusion of bargeman and van voorst vader that there is a linear relationship between these parameters . when all the lines have been drawn the figure was unreadable ,
regardless of the solvent used values of slope ranged are between 0.74 and 1.2 . however ,
in contrast the a constant was positive in the case of contact angle measurements for the series of zwitterionic surfactants , alcohol hexadecyl polyoxyethylene glycidyl ether glycine betaines on the surface of the quartz plates ( polar solids ) .fig .
5dependence between the adhesional and surface tension of cocobetaine in different solutions dependence between the adhesional and surface tension of cocobetaine in different solutions foaming ability is a typical property of surfactant solutions .
the ability to produce foam is of great importance in cosmetics and household products . in the process of cleaning and washing the important role played by the quantity and durability of the produced foam .
the comparison of volume of foam aqueous and salt solutions of cocobetaine shows ( fig .
6 ) that the volume of foam for aqueous solutions is much higher than for appropriate salt ones . thus , as well as
surface properties , the ability of the foaming surfactant solution is better for water in comparison to the solution with addition of sodium chloride .
the graph also shows that height of foam column is much lower for salt solutions then for aqueous solutions regardless of the time of measurement . comparing the foaming capacity of aqueous solutions cocobetaine with aqueous solutions of soap ,
this is much lower amount of foam than for the same solutions of cocobetaine .fig .
6volume of foam for cocobetaine solutions , closed
square water , closed
circle 0.01 m nacl , closed
triangle 0.05 m nacl , closed
diamond 0.2 m nacl , closed
star 2 m nacl volume of foam for cocobetaine solutions , closed
square water , closed
circle 0.01 m nacl , closed
triangle 0.05 m nacl , closed
diamond 0.2 m nacl , closed
star 2 m nacl stability of foam was assessed by calculating the ratio of the initial foam volume to its value after 5 and 10 min .
main indicators applied to estimate the stability of the foam are : h5/h1index , h10/h1 index and constant speed of falling foam ( k ) .
table 2 presents indexes estimated for several concentrations of cocobetaine aqueous and salt solutions .
it can be also seen that the reduction of the foam in time is rapid for 2 m salt solutions of cocobetaine .
the foam created for this solution disappears much faster than for aqueous solutions.table 2stability of foam indicators for cocobetaine solutionsaqueous solution0.01 m salt solution0.05 m salt solution0.2 m salt solution2 m salt solutionconcentration of cocobetaineh5/h1 indexh10/h1 indexh5/h1 indexh10/h1 indexh5/h1 indexh10/h1 indexh5/h1 indexh10/h1 indexh5/h1 indexh10/h1 index0.1250.970.950.970.930.930.910.910.890.890.780.250.970.940.950.920.930.880.930.900.930.790.50.970.940.940.910.940.870.920.890.900.8010.950.920.930.910.940.870.940.880.930.83 stability of foam indicators for cocobetaine solutions
based on the research and analysis carried out , it can be concluded that properties of cocamidopropyl betaine depend on the concentration of salt in the surfactant solutions .
experimental data showed that cocobetaine exhibits good surface properties and can be used as a substance which reduce surface tension even at low initial concentrations .
the presence of salt in the solutions lessens their ability to reduce surface tension , decreases the cmc and increases the adsorption parameters and structure of the adsorbed monolayer .
the experimental data showed that salt solutions of cocobetaine create a much smaller column of foam than aqueous solutions . | surfactants are important ingredients of personal care products and household products .
the main characteristic of these compounds is to decrease the surface tension of solvent and resulting many properties such as contact angle , foam properties etc .
the coexistence of other ingredients in the product may affect the properties of surfactants .
one of the main components contained in almost every personal care and household product is sodium chloride .
the main aim of this work was to determine the effect of this salt on some surface and usage properties of cocamidopropyl betaine ( capb ) . from our experiments
it was shown that the effect of added sodium chloride in the aqueous solutions of capb on the properties is the opposite to the one described in the literature for cationic and anionic surfactants , i.e. , cmc increases with increasing ionic strength , foam height decreases with increasing salt concentration .
our investigation showed that sodium chloride makes worse the properties of the capb solutions examined in this work . |
as the common benign tumor in male , benign prostatic hyperplasia ( bph ) develops as a age - related phenomenon in almost all men , starting at approximately 40 years of age .
clinically , bph leads to a progressive enlargement of the prostate gland , causing both obstructive and irritative lower urinary tract symptoms ( luts ) .
the lifetime risk for a 50-year - old man to undergo a prostatectomy for bph has been estimated to be as high as 40% .
bph is a specific histopathologic entity characterized by stromal and epithelial cell hyperplasia . for over a century , there have been two known etiologic factors for the pathogenesis of bph : aging and androgens .
moreover , family history , ethnicity , smoking , type ii diabetes mellitus ( dm ) , high - density lipoprotein cholesterol ( hdl - c ) , high insulin content , high blood pressure , and obesity have been reported to be risk factors .
many reports have discussed the correlations of metabolic syndrome ( ms ) and prostate volume . however , despite such existing epidemiological and pathophysiologic evidence , there are many disputes in considering ms as a risk factor for bph .
another reason is that most studies that analyzed the relationship between ms and bph were conducted in older men more than 50 years of age .
therefore , unlike the previous studies that reviewed bph and ms in old age groups , in the present study , we analyzed its associations with ms and components of ms by investigating the prostate volume of relatively healthy , young male subjects ( in their fourth to fifth decades ) without underlying disease .
among 75,368 men older than 30 and younger than 50 years of age who came to the health promotion center at the kangbuk samsung hospital for a routine health check - up between march 2009 and june 2010 , data were retrospectively obtained from 986 men who underwent transrectal ultrasonography ( trus ) of the prostate .
trus was performed in subjects who came to the health promotion center as part of a basic examination included in a health screening program or as a test selected by the subjects themselves .
the health screening program available at our health promotion center includes anthropometric measurements ( height , weight , and waist circumference [ wc ] ) , blood test ( a complete blood cell count , basic chemistry , serologic test , blood coagulation test , thyroid function test , and assay for tumor markers ) , stool / urine analysis , abdominal ultrasonography , gastrofiberscopy , chest radiography , pulmonary function test , electrocardiography , and detailed clinical examination .
all subjects were also asked to complete a questionnaire designed to assess sociodemographic factors , comorbidities , and current or past medications .
the institutional review board of the kangbuk samsung hospital approved all the procedures involved in sampling and data collection , and written informed consent was obtained from all participants . among the 986 men ,
138 men were excluded from the study for reasons including taking medications for bph ( n=47 ) , having a history of type i insulin - dependent dm ( n=22 ) , having abnormal serum prostate - specific antigen ( psa ) ( 4 ng / ml ; n=11 ) , or having a suspicion of prostate cancer by trus or digital rectal examination ( dre ; n=1 ) .
metabolic syndrome was defined by using the criteria established by the national cholesterol education program - adult treatment panel iii - american heart association / national heart , lung , and blood institute ( ncep - atpiii - aha / nhlbi ) statement , published in 2005 .
central obesity was defined as wc 90 cm in men or 80 cm in women by the modified atp iii guideline that the who - western pacific region ( wpr ) and the international association for the study of obesity ( iaso ) presented for asian populations in 2000 .
ms was diagnosed when at least three of the following criteria were present : wc of 90 cm , triglyceride ( tg ) levels of 150 mg / dl or undergoing treatment for hypertriglyceridemia , hdl - c levels of < 40 mg / dl or undergoing treatment for low hdl - c , blood pressure ( bp ) of 130/85 mm hg or undergoing treatment for hypertension , and fasting plasma glucose ( fpg ) of 100 mg / dl or undergoing treatment for hyperglycemia .
all parameters were measured on fresh serum obtained after the subjects had fasted for 12 hours overnight .
serum total cholesterol , tg , hdl - c , and fpg were measured by enzymatic methods .
the prostate volume was calculated according to the prostate ellipsoid formula , multiplying the largest anteroposterior ( height , h ) , transverse ( width , w ) , and cephalocaudal ( length , l ) prostate diameters by 0.524 ( hxwxlx /6 ) by using trus ( aloka , prosound-5sv ) .
all analyses were performed by using the pasw ver . 17.0 ( spss inc . ,
associations were assessed between each of the metabolic components , as well as ms , and prostate volume .
prostate volumes were compared in men with and without ms , and with and without each metabolic component , by using the mann - whitney u - test .
continuous variables were expressed as the meanstandard deviation ( sd ) , and because most variables in this report were not normally distributed , we preferred to use nonparametric statistics , namely , the median value .
a multiple logistic regression model was performed to examine the association between ms and prostate volume .
we used the risk ratio with 95% confidence intervals ( cis ) to evaluate the association between significant variables and prostate volume .
among 75,368 men older than 30 and younger than 50 years of age who came to the health promotion center at the kangbuk samsung hospital for a routine health check - up between march 2009 and june 2010 , data were retrospectively obtained from 986 men who underwent transrectal ultrasonography ( trus ) of the prostate .
trus was performed in subjects who came to the health promotion center as part of a basic examination included in a health screening program or as a test selected by the subjects themselves .
the health screening program available at our health promotion center includes anthropometric measurements ( height , weight , and waist circumference [ wc ] ) , blood test ( a complete blood cell count , basic chemistry , serologic test , blood coagulation test , thyroid function test , and assay for tumor markers ) , stool / urine analysis , abdominal ultrasonography , gastrofiberscopy , chest radiography , pulmonary function test , electrocardiography , and detailed clinical examination .
all subjects were also asked to complete a questionnaire designed to assess sociodemographic factors , comorbidities , and current or past medications .
the institutional review board of the kangbuk samsung hospital approved all the procedures involved in sampling and data collection , and written informed consent was obtained from all participants . among the 986 men ,
138 men were excluded from the study for reasons including taking medications for bph ( n=47 ) , having a history of type i insulin - dependent dm ( n=22 ) , having abnormal serum prostate - specific antigen ( psa ) ( 4 ng / ml ; n=11 ) , or having a suspicion of prostate cancer by trus or digital rectal examination ( dre ; n=1 ) .
metabolic syndrome was defined by using the criteria established by the national cholesterol education program - adult treatment panel iii - american heart association / national heart , lung , and blood institute ( ncep - atpiii - aha / nhlbi ) statement , published in 2005 .
central obesity was defined as wc 90 cm in men or 80 cm in women by the modified atp iii guideline that the who - western pacific region ( wpr ) and the international association for the study of obesity ( iaso ) presented for asian populations in 2000 .
ms was diagnosed when at least three of the following criteria were present : wc of 90 cm , triglyceride ( tg ) levels of 150 mg / dl or undergoing treatment for hypertriglyceridemia , hdl - c levels of < 40 mg / dl or undergoing treatment for low hdl - c , blood pressure ( bp ) of 130/85 mm hg or undergoing treatment for hypertension , and fasting plasma glucose ( fpg ) of 100 mg / dl or undergoing treatment for hyperglycemia .
all parameters were measured on fresh serum obtained after the subjects had fasted for 12 hours overnight .
serum total cholesterol , tg , hdl - c , and fpg were measured by enzymatic methods .
the prostate volume was calculated according to the prostate ellipsoid formula , multiplying the largest anteroposterior ( height , h ) , transverse ( width , w ) , and cephalocaudal ( length , l ) prostate diameters by 0.524 ( hxwxlx /6 ) by using trus ( aloka , prosound-5sv ) .
all analyses were performed by using the pasw ver . 17.0 ( spss inc . , chicago , il , usa ) .
associations were assessed between each of the metabolic components , as well as ms , and prostate volume .
prostate volumes were compared in men with and without ms , and with and without each metabolic component , by using the mann - whitney u - test .
continuous variables were expressed as the meanstandard deviation ( sd ) , and because most variables in this report were not normally distributed , we preferred to use nonparametric statistics , namely , the median value .
a multiple logistic regression model was performed to examine the association between ms and prostate volume .
we used the risk ratio with 95% confidence intervals ( cis ) to evaluate the association between significant variables and prostate volume .
the mean value of each component of ms and psa are shown in table 1 .
the median prostate volumes in the ms and non - ms groups were 18.4 cc ( range , 14.3 - 23.1 cc ) and 17.8 cc ( range , 13.6 - 21.6 cc ) , respectively .
in addition , there were no statistically significant differences in the prostate volume according to the presence of ms in the two age subgroups ( 30 - 39 and 40 - 49 years ; table 2 ) .
men with abdominal obesity ( wc90 cm ) and men with abnormal fpg levels ( 100 mg / dl ) had a larger prostate volume than did the normal control group .
the median prostate volumes in the abdominal obesity group and the normal wc group were 19.5 cc ( range , 15.6 - 23.7 cc ) and 17.5 cc ( range , 13.3 - 21.2 cc ) , respectively ( p<0.05 ) .
the prostate volumes according to normal and abnormal fpg level were 16.9 cc ( range , 12.7 - 20.4 cc ) and 18.9 cc ( range , 14.3 - 22.7 cc ) , respectively ( p<0.05 ) .
when the prostate volume was further analyzed according to age subgroups , the prostate volumes in men with abdominal obesity or abnormal fpg levels were also significantly larger than in the normal control groups ( p<0.05 ) ( table 2 ) .
the results of the multivariate analysis after adjustment for age and each ms factor showed that abdominal obesity ( or=2.305 ; 95% ci : 1.470 - 3.614 , p=0.001 ) and an abnormal fpg level ( or=1.441 ; 95% ci : 1.303 - 1.643 , p=0.001 ) were strongly associated with enlarged prostate volume ( 20 cc ) ( table 3 ) .
in the united states , the prevalence rate of ms has been reported variously ranging from 2.4% to 43.5% . according to data reported in united states in 2005 , the prevalence rate of ms according to the ncep - atp iii criteria reached 34.6% , and a prevalence rate of 28%
ms is a constellation of multiple metabolic and cardiovascular disease risk factors , at the center of which is insulin resistance ( a state in which muscle , liver , and fat tissues have reduced sensitivity to insulin ) and compensatory hyperinsulinemia .
investigators have reported a strong influence of age on the presence of ms , which affects 43.5% of those aged 60 to 69 years .
have histological evidence of bph and , after age 70 , the proportion increases to 80% . historically , the development of bph focused on the interplay between genetic and hormonal factors .
more recently , however , a growing body of evidence has demonstrated a strong and independent link between bph and obesity and ms .
furthermore , this association implies that modifiable risk factors may play a role in the pathogenesis of these entities . despite the high prevalence of bph in aged men ,
although the etiology of bph is not well understood , several theories have been proposed to explain the pathogenesis of bph .
testosterone - dihydrotestosterone ( dht ) signaling and mesenchymal - epithelial interactions are required for normal prostaticgrowth and are known to play an important role in the progression of disease .
many risk factors for bph , such as insulin , insulin - like growth factors ( igfs ) , and dyslipidemia , might act through androgen - independent mechanisms .
the underlying cause of ms also continues to challenge experts , but both insulin resistance and central obesity are considered to be significant factors .
recent studies suggest that hyperinsulinemia secondary to insulin resistance and the components of ms are risk factors for the development of bph [ 14 - 16 ] . furthermore , ms may play a role in bph pathogenesis .
several experimental and clinical reports indicate the critical role of obesity and insulin - resistance - associated complications in the pathogenesis of bph .
the increased incidence of bph in insulin - resistant and diabetic populations strengthens the relationship between these two pathological conditions and makes this an increasingly relevant problem .
insulin resistance is a condition in which a normal level of insulin elicits a subnormal response .
it is a condition that is associated with a group of disorders such as obesity , dyslipidemia , elevated fasting glucose level , hyperinsulinemia , and hypertension .
in addition to type 2 dm and cardiovascular disease , patients with insulin resistance syndrome are at higher risk for bph .
the insulin - resistance - associated disorders such as obesity , dyslipidemia , hypertension , sympathetic overactivity , and hyperinsulinemia have been implicated in the pathogenesis of bph .
obesity has been implicated in the etiology of bph because of its influence on metabolic and endocrine changes .
recent findings indicate that obesity substantially increases the risk for bph . in the study conducted by dahle et al ,
an increased serum insulin level was related to increased bph risk , and parson et al reported the correlations of prostate volume with body mass index , elevated fasting blood sugar level , and dm .
the health professionals follow - up study , a prostate cancer prevention trial , and a case - control study of italian men showed a positive association between obesity and bph .
the fasting serum level of insulin in the patients with higher waist - to - hip ratio was significantly high , indicating the involvement of insulin in the progression of disease .
several independent studies have further supported the association between obesity and hyperinsulinemia . in a population - based study conducted in korea , jang
et al reported that there was a significant correlation between each ms factor and prostate volume .
koo et al also reported that ms is associated with prostate volume - related factors , but not to voiding dysfunction in korean men 60 years of age or older . among the subcategories of ms , they reported that obesity is the factor most strongly related to prostate volume .
few reports argue that obesity is associated with increased estrogen - to - androgen ratio and sympathetic activity , both of which are individually hypothesized to promote the development of prostatic hyperplasia .
obesity can augment prostatic growth either by promoting the development of insulin resistance and secondary hyperinsulinemia or by increasing the estrogen - to - androgen ratio .
one more pathway that explains the increased risk for bph in hyperinsulinemia is the igf axis .
igf-1 is a strong mitogen and increases cell proliferation and induces apoptosis in many tissues , including prostatic stroma and epithelium . in this study
, we found that obesity and dm were risk factors for bph in young men , but the presence of ms was not . according to the study of gupta
et al , ms had no contributing effect on bph in a prospective study of the causal relationship of ms and bph through long - term follow - up observation ( mean of 15.6 years ) .
our report is the only study to assess whether relatively young men with ms had an increased risk of bph , and we found no relationship between the two .
apart from abnormal fpg and obesity , none of the other markers of the ms were associated with an increased risk of bph .
the results from other prospective studies have not shown a consistent relationship between any of the ms markers and bph .
thus , considering the evidence from the longitudinal studies , it is unlikely that a causative association exists between the ms and bph .
we did not find any relationship between prostate volume and hdl - c or tg . in the study conducted by rohrmann
et al using data from nhanes iii , they also did not find any relationship between luts and hdl - c , low density lipoprotein , tg , or total cholesterol .
overall , dyslipidemia is likely to have a limited role , if any , in the etiology of bph .
in contrast , gann et al , in a smaller prospective study involving 640 men , found an increased odds of bph with a higher diastolic bp and no effect with systolic bp .
the mechanism of the association between bp and bph / luts remains unknown , but a role of sympathetic tone has been hypothesized . unlike the previous studies , the present study analyzed the substantial correlations of bph with ms in relative younger men aged 30 to 49 years .
the present data show the possibility of young adults with obesity or dm having a larger prostate volume , which is similar to the clinical observation that men suffering from dm and obesity have a larger prostate gland volume than do men without these conditions at old ages . according to the study of berry et al ,
the prostate volume doubling time was 4.5 years among bph patients aged 30 to 50 years , but the reported the doubling times of the patients aged 50 - 71 years was 10 years and that of patients aged more than 70 years was 100 years .
also , they also reported that a fast growth speed of prostate volume in younger men with bph .
this study suggests that it is possible that the elements of ms may have different effects on the younger and the older .
also , if young men have obesity and dm , they are predisposed to early and severe development of bph owing to the effect of obesity and dm on the naturally rapidly growing prostate compared with old men .
therefore , more caution is needed when young men have obesity and dm for early diagnosis and treatment of bph .
also , modification of lifestyles and high - fat diets in young men with obesity and dm are needed to prevent the development of bph . in our study , the analyses were cross - sectional , and thus causal and longitudinal relations were not addressed .
there was the potential for selection bias because the subjects were the populations who visited the health promotion center for routine medical examination and not for the treatment or diagnosis of luts / bph .
also , we did not consider the subject 's lifestyle patterns of diet habits , exercise , and hormonal status including testosterone . despite the aforementioned limitations , however
, this study is valuable in that it proposed the possibility of early or severe bph development in young men with obesity or dm .
in young men aged 30 to 49 years old , the presence of ms was not correlated with prostate volume .
however , men with abnormal fpg and wc had larger prostates than did normal groups . the risk of prostate enlargement increased with increasing fpg and wc .
obesity and dm could be more important factors than ms in prostate volume enlargement in relatively young adults . | purposethe metabolic syndrome ( ms ) has been accepted as an important cause of benign prostatic hyperplasia ( bph ) in old age . however , there are no studies of the influence of ms on prostate volume in relatively young adults .
we evaluated the relationship between parameters of ms and prostate volume in men under 50 years of age who visited our health promotion center.materials and methodsa total of 968 men aged 30 to 49 years were enrolled from march 2009 to june 2010 .
prostate volume by transrectal ultrasonography of the prostate , serum prostate - specific antigen , digital rectal examination , urinalysis , and ms - related parameters were investigated .
we evaluated the correlations of prostate volume with ms and ms - related parameters.resultsprostate volume was not significantly larger in the ms group ( 18.4 cc ; range : 14.3 - 23.1 cc ) than in the non - ms group ( 17.8 cc ; range , 13.6 - 21.6 cc ) .
the prostate volumes in subjects with abnormal fasting plasma glucose ( fpg ) ( 18.9 cc ; range , 14.3 - 22.7 cc ) and abnormal waist circumference ( wc ) ( 19.5 cc ; range , 15.6 - 23.7 cc ) were significantly larger than those of subjects with normal parameters ( 16.9 [ range , 12.7 - 20.4 ] cc and 17.5 [ range , 13.3 - 21.2 ] cc , respectively ; p=0.001 ) .
the logistic regression analysis showed the fpg level and wc to have a significantly positive correlation with the prostate volume ( odds ratios : 1.441 [ 95% ci : 1.303 - 1.643 ] and 2.305 [ 95% ci : 1.470 - 3.614 ] , respectively).conclusionsgroups with abnormal fpg and wc had larger prostate volumes than did normal groups .
the abnormal fpg and wc could be more important factors than ms in prostate volume enlargement in relatively young adults . |
physical dependency is described as an adaptive state that manifests itself as intense physical disturbance when drug use is suspended .
most people believe that addiction or drug dependency is a male phenomenon and females are less involved in these issues . in the islamic republic of iran ,
although there is no valid census about drug dependency in iranian women , but the ministry of health reports that there is one female drug dependent per 8 males in iran .
the administrators of prisons declare that 50% of female prisoners are in jails regarding drugs and addiction .
a study in the united states of america has shown that 7.7% of males in comparison with 5% of females were drug abusers ( the sex difference was lower than 3% ) . in the united states ,
almost 4.5 million women drink alcohol , 3.5 million abuse prescription drugs , and more than 3 million abused illicit drugs .
addiction in women can expose them to malnutrition , high blood pressure , cancer , and some other dangerous diseases like hepatitis or aids .
comparing to males , women may be more prone to aids or other sexual transmitted diseases . besides , they are more susceptible to other gynecological diseases and their complications [ 4 , 5 ] .
this study was aimed at evaluating extramarital sexual relations in vulnerable women who have substance dependency ( substance dependence in prison ) , invulnerable women of substance dependence ( substance dependence with no history of being in prison ) , and control groups ( women with no history of addiction or being in prison ) in mashhad , a metropolitan in the north east of iran .
this cross - sectional study was done on 236 girls and young women aged 1625 years in 2012 , in three groups : vulnerable women who have substance dependency ( crimes that had made women incarcerated were considered as vulnerability in this study ) , invulnerable women who have substance dependency ( substance dependent women without history of incarceration ) , and control group ( women with no history of dependency or being in prison , individuals who had come to health centers for primary health care ) .
the sample size was calculated based on the mean difference in two groups based on a previous study , by considering = 0.05 and = 0.2 , and 80 persons were calculated for each group . in a first step the calculated sample size for each group was divided into 2 subgroups : 1620 and 2125 years old ( to insure including women younger than 20 ) . in the second step , by using a random sampling method the participants were selected from a list of prisoners ( group 1 ) . at the last step substance dependent women who were referred to welfare centers ( group 2 ) and women who had come to health centers for primary health care ( group 3 ) were selected based on age matching to the first group . all interviewers had attended a training class to eliminate the interinterviewer bias .
all participants filled the informed consent and then demographic and familial characteristics , alcohol consumption , and main social and sexual behaviors were obtained .
all analyses were done using statistical package for social sciences ( spss ) version 11.5 .
demographic and social characteristics of participants are demonstrated in table 1 . as shown in this table , education , occupation status , personal income ,
family income , lodging , marital status , and alcohol consumption are statistically different in three groups .
regarding extramarital sexual relations there was a statistically significant difference between three groups ( 43.8% in vulnerable group , 55.8% in invulnerable group , and 1.4% in control group ; chi = 52.21 , p < 0.001 ) .
there was no statistically significant difference between vulnerable and invulnerable groups ( chi = 2.29 , p = 0.15 ) .
the extramarital sexual relation based on marital status in all the substance dependent subjects is shown in figure 1 .
the kind of sexual relation , safe ( considered as using condoms ) and unsafe ( considered as not using condoms ) , is demonstrated in figure 2 . among 36 extramarital sexual relations in vulnerable women who have substance dependency ,
41.7% were unsafe , and among 43 extramarital sexual relations in invulnerable women who have substance dependency 18.6% were unsafe . in the control group , only one person had an extramarital sexual relation and it was safe .
there was a statistically significant difference between vulnerable and invulnerable groups ( p = 0.03 ) ( figure 2 ) .
as shown in figure 3 , the type of substances which was abused was statistically different in substance dependent participants according to having or not having an extramarital sexual relation ( p = 0.01 ) .
alcohol consumption was statistically different in two groups based on safe or unsafe sexual relations ( chi = 9.95 , p = 0.003 ) .
43.8% of vulnerable women who have substance dependency had extramarital sexual relations ; this percentage was 55.8% in invulnerable women who have substance dependency and 1.4% in the control group .
teets had described the frequency of rape in female drug abusers and found that , from 60 interviewed women , 73% had a history of rape and 45% had been raped more than once . in 35% of participants raping
had been happening when the victims are affected by the substances which were abused . in 2010 , perez del rio et al . studied the relation of drug abuse and showed that in female addicts there is an implicit relation between drug abuse and sexual abuse in childhood or adulthood ( 37.8% ) and in some cases this sexual relation was for earning money ( 51.4% ) .
, it was shown that crystal and methamphetamine abuse was higher in addicts who had extramarital sexual relations and alcohol consumption was higher in unsafe sexual intercourse group .
although we should notice that crystal and methamphetamine were the most common substances which were abused in all the drug dependent subjects , the type of abused substances was statistically different in two substance dependent women ( p < 0.01 ) .
results of this study showed that condom use in sexual relation was not statistically different when the man had abused drugs in comparison with time they had not used illicit drugs ( 48.3% versus 49% ) .
the differences between the results of that study and our study may be due to gender difference . in one study in tanzania , fisher et al .
studied safe and unsafe sexual relations in subjects who had drunk alcohol 2 hours before the intercourse comparing to the ones who had not .
results showed that condom usage failure ( in males , females , or both ) was more than 5 times higher in individuals who had drunk alcohol before the sexual relation especially when the woman had drunk alcohol .
alcohol consumption was higher in some situations : the first sexual relation in women , sexual relations in unknown or with less control era , and people whose parents had abused drugs or alcohol before sexual relations . surprisingly , condom usage was not related to alcohol consumption but the probability of failure was much higher . in this study we did not assess the condom use failure , so if we did it , the result could be the same , but condom usage was related to alcohol consumption and this difference may be because of different cultural contexts and sexual behaviors in societies . in this study the extramarital relation was higher in drug dependent women .
results showed that out of 27 addict women , 14 had a history of extramarital sexual relation .
dependent women had a lower educational level but relatively had high personal income and being married was a protective factor for extramarital relation . in another study in texas , it was demonstrated that addict women had lower education and income than addict men .
the higher rate of extramarital relation in single females was the same as the result of this study .
the results of another study showed that nearly 25.5% of subjects had high risk sexual behaviors in which 47% had not used condoms in the last sexual intercourse .
high risk sexual behaviors were higher in heroin addicts , lower ages , and higher income groups . in this
study the extramarital relation was higher in higher personal income but this study is a cross - sectional study and we can not conclude causality from this type of study .
one reason may be that this group has an extramarital sexual relation to earn money .
motazakker et al . studied the high risk behaviors in 384 addicts who were receiving methadone as a treatment .
intravenous method of drug abuse was used by 58 subjects ( 15.1% ) , snuff in 235 subjects ( 61.4% ) , and oral method in 91 subjects ( 23.6% ) .
102 subjects ( 26.6% ) had a history of alcohol consumption for more than 5 years , 88 subjects ( 22.9% ) had tattoos , 6 subjects ( 1.6% ) had needle sharing , 19 subjects ( 5% ) had razor blade sharing , and 62 subjects ( 16.1% ) had a history of extramarital sexual relation .
the pattern of drug abuse was as follows : heroin in 127 ( 33.1% ) , opium in 194 ( 50.5% ) , crystal in 2 ( 0.5% ) , crack in 26 ( 6.8% ) , and cannabis in 2 ( 0.5% ) individuals .
the result of this study emphasizes the role of drug dependency in sexual behaviors . in this study
there was a statistically significant difference in extramarital sexual relation based on marital status and being married seems to be a protective factor from extramarital sexual relation .
based on our study results and some other similar studies [ 1518 ] we can conclude that higher rates of extramarital sexual relations and unsafe sexual intercourse can be the consequence of drug dependency in women .
poverty , the need for money to buy drugs , high rates of divorce in addict women , the need for higher doses to increase sexual arousal , and finally psychological problems with sexual self - esteem form a vicious cycle that can harm this group even more .
addiction , poverty , and family breakdown are among the most important social determinants of health that need more attention , especially in this specific group substance dependent women . | introduction . addiction in women can expose them to malnutrition , high blood pressure , cancer , and some other dangerous diseases like hepatitis , aids , or other sexual transmitted diseases .
the aim of this study was to assess illegal sexual relations in three groups of women .
methods .
this is a cross - sectional study that was done on 236 girls and young women aged 1625 years in 2012 in three groups : vulnerable women who have substance dependency ( crimes that had made women incarcerated were considered as vulnerability in this study ) , invulnerable women who have substance dependency ( substance dependent women without a history of incarceration ) , and a control group ( women with no history of substance dependency or being in prison ) .
results .
43.8% of vulnerable women who have substance dependency had extramarital sexual relations ; this percentage was 55.8% in invulnerable women who have substance dependency and 1.4% in the control group .
crystal and methamphetamine abuse was higher in addicts who had extramarital sexual relations and alcohol abuse was correlated with unsafe sexual intercourse ( r = 0.36 , p = 0.001 ) .
there was a statistically significant difference in extramarital sexual relation based on marital status ( p < 0.001 ) .
conclusions .
poverty , drug dependency , divorce , and alcohol consumption make women prone to other high risk behaviors that need more attention . |
it is a common misconception that microorganisms isolated in pure culture from an environment represent the numerically dominant and/or functionally significant species in that environment . in fact , microorganisms isolated using standard cultivation methods are rarely numerically dominant in the communities from which they were obtained : instead , they are isolated by virtue of their ability to grow rapidly into colonies on high - nutrient artificial growth media , typically under aerobic conditions , at moderate temperatures .
easily isolated organisms are the ' weeds ' of the microbial world and are estimated to constitute less than 1% of all microbial species ( this figure was estimated by comparing plate counts with direct microscopic counts of microorganisms in environmental samples ; it has been called the " great plate - count anomaly " ) .
given that the study of a microorganism is simpler if you have it in pure culture on an agar plate , it is not surprising that most of what we know about microbiology comes from the study of microbial weeds .
for example , approximately 65% of published microbiological research from 1991 to 1997 was dedicated to only eight bacterial genera , escherichia ( 18% ) , helicobacter ( 8% ) , pseudomonas ( 7% ) , bacillus ( 7% ) , streptococcus ( 6% ) , mycobacterium ( 6% ) , staphylococcus ( 6% ) and salmonella ( 5% ) , all of which are relatively simple to grow on agar plates .
intuitively , it seems unlikely that this handful of organisms can be representative of the approximately 5,000 validly described prokaryotic species , but exactly how unrepresentative are they ? and if more than 99% of microorganisms in the environment are unculturable using standard techniques , how representative are cultivated microorganisms of prokaryotic diversity as a whole ? to answer these questions , we need a framework for placing prokaryotic species and genera in a broader evolutionary context .
the pioneering work of carl woese and colleagues on comparative analysis of small - subunit ribosomal rnas ( 16s and 18s rrnas ) provided an objective framework for determining evolutionary relationships between organisms and thereby ' quantifying ' diversity as sequence divergence on a phylogenetic tree .
woese found that cellular life can be divided into three primary lineages ( domains ) , one eukaryotic ( eucarya , also called eukaryota ) and two prokaryotic ( bacteria and archaea ) , and he also defined 11 major lineages ( phyla or divisions ) within the bacterial domain on the basis of 16s rrna sequences obtained from cultivated organisms .
this analysis revealed distant relationships not suspected from phenotypic characterization , such as the association between the genera bacteroides and flavobacterium .
the leading reference source in prokaryotic taxonomy , bergey 's manual of systematic bacteriology , has adopted a 16s rrna framework to classify prokaryotes , replacing the previous ad hoc scheme that was based on traditional phenotypic characterization .
the manual proposes a standardized prokaryote nomenclature that has mostly been fitted to a classical taxonomic hierarchy ( species , genus , family , order , class , phylum ) ; i will adhere to this system as far as possible in this article ( see the taxonomic outline available at ) .
the phylum is the highest - level grouping in the bacterial and archaeal domains and , therefore , is a useful rank for overviewing prokaryotic diversity .
the eight most intensively studied prokaryotic genera listed in the introduction are members of only three bacterial phyla : proteobacteria ( escherichia , helicobacter , pseudomonas , salmonella ) , firmicutes ( bacillus , streptococcus , staphylococcus ) and actinobacteria ( mycobacterium ) . moreover , the top 25 most - studied genera are all members of these three phyla , with the exceptions of chlamydia and borrelia ( clinically important genera of the bacterial phyla chlamydiae and spirochaetes , respectively ) . in a recent study , 177 environmental , veterinary and clinical isolates that were not identifiable by traditional phenotypic characterization were evaluated by comparative 16s rrna analysis .
the isolates included a large number of different genera and species , but at the phylum level all except one of the 177 were members of only four bacterial phyla : proteobacteria ( 82 isolates ) , firmicutes ( 61 ) , actinobacteria ( 29 ) and bacteroidetes ( 4 ) .
this cultivation bias towards four bacterial phyla ( the ' big four ' ) is also reflected in microbial culture collections ; for example , 97% of prokaryotes deposited in the australian collection of microorganisms are members of the big four ( figure 1a ) .
in fact , it is a challenge to obtain isolates that do not belong to the big four , and these four phyla therefore dominate our present understanding of microbiology .
a logical question to ask is how many prokaryotic phyla there are altogether , in order to estimate how biased a sampling of four may be .
in the mid 1980s , norman pace and colleagues outlined a molecular approach that bypassed the need to cultivate a microorganism in order to determine the sequence of its 16s rrna gene ( 16s rdna ) .
essentially , bulk nucleic acids are extracted directly from environmental samples , 16s rdna sequences are isolated from the bulk dna , typically via pcr ( using primers broadly targeting 16s rdnas ) and cloning , and these sequences are compared with known sequences ( figure 2 ) .
gene sequences obtained in this manner ( ' environmental clone sequences ' ) can then be assigned a location in a phylogenetic tree and can thus act as a marker for the organism from which they were obtained .
the approach can be brought full circle by applying 16s rrna - targeted nucleic - acid probes specific for the organisms of interest to visualize and quantify the target group in the environmental sample using techniques such as whole - cell fluorescence in situ hybridization ( fish ) and membrane hybridization ( figure 2 ) .
many researchers have applied the rrna approach to a wide variety of environmental samples over the past decade and , perhaps not surprisingly given the great plate - count anomaly , the number of recognized bacterial phyla has exploded from the original estimate of 11 in 1987 to 36 in 1998 . this increase is due not only to environmental sequences that have filled out the tree , but also to a steady trickle of sequences from ' exotic ' cultured organisms , particularly thermophiles , that highlight new lineages .
figure 3a presents a recent conservative estimate of bacterial diversity at the phylum level ; it is conservative because it includes only phyla for which at least four near - full - length 16s rdna sequences ( over 1,300 nucleotides ) are known .
the total number of phylum - level lineages in this tree is 35 , 22 ( 63% ) of which have one or more cultivated representatives and 13 ( 37% ) of which are known only from environmental sequences .
there are at least another ten phylum - level lineages , however , that are present in the bacterial domain but are not shown in figure 3a because they are represented by too few and/or only partial sequences .
these lineages include cultivated bacteria such as chrysiogenes and dictyoglomus , which are recognized as representing independent phyla in the taxonomic outline of bergey 's manual of systematic bacteriology .
as more 16s rdna sequences accumulate from both cultured and uncultured prokaryotes , the boundaries of existing phyla are being challenged and need to be re - evaluated .
for example , the bacterial phylum firmicutes , as currently defined , may not be monophyletic and may comprise at least four distinct phylum - level lineages that include the haloanaerobiales , thermoanerobacteriales , and sulfobacillus groups .
higher - level associations between bacterial phyla have not been resolved in 16s rdna trees , with the exceptions of the sister - group affiliations of the bacteroidetes and chlorobi , and of the chlamydiae and verrucomicrobia .
this is presumably because such relationships are beyond the resolution that can be obtained from the 16s rrna molecule and/or the current inference methods .
recently , trees based on concatenated ribosomal proteins obtained from complete genome sequences have suggested higher - order associations between chlamydiae and spirochaetes , between thermotogae and aquificae , and between actinobacteria , deinococcus - thermus and cyanobacteria .
the phylum verrucomicrobia is also likely to be a member of the same group as chlamydiae and spirochaetes , given that it is a sister group to chlamydiae ; this prediction can be tested when a completed genome sequence becomes available for the verrucomicrobia .
several ' candidate ' phyla , comprising only environmental clone sequences , have developed into large groups with sequence divergences similar to or greater than those within the big four phyla ( examples include op11 and ws6 ) , and yet we know nothing about these lineages beyond a crude outline of their environmental distribution . most have not even been ( knowingly ) observed under the microscope . in a preliminary investigation of one candidate phylum , tm7
, we determined that representatives of the group had typical gram - positive cell envelopes and that they may have archaea - like streptomycin resistance . detailed study of lineages like this one may yield insights into the evolutionary history of gram - positive bacteria ( including , perhaps , a radical proposal that gram - positive bacteria are related to archaea ) , which so far appear to have a restricted phylum - level distribution within the bacterial domain ( actinobacteria and firmicutes ) .
tm7 bacteria have also been implicated in human subgingival ( gum ) disease , which might promote their study .
the archaea are formally divided into two phyla , crenarchaeota and euryarchaeota , from 16s rrna phylogeny , but these groupings may be artifacts because analysis of concatenated ribosomal protein sequences suggests that euryarchaeota , at least , is not a monophyletic group .
figure 3b presents a current estimate of the major lineages in the archaeal 16s rdna tree below the level of the crenarchaeota and euryarchaeota ( indicated to the right of the tree ) , using the same criteria and annotation used for the bacterial tree ( figure 3a ) .
the total number of phylum - level lineages in the archaeal tree is 18 , of which 8 ( 44% ) have cultivated representatives and 10 ( 56% ) have none .
a higher tally of 23 phyla is arrived at if lineages not meeting the selection criteria are included in the estimate .
these include methanopyri , currently represented by a single sequence , and environmental group c3 , which has no full - length representatives .
most archaeal research has concentrated on the cultivated methanogenic ( such as methanococci ) and thermophilic ( such as thermoprotei and thermococci ) lineages ( figure 3b ) .
as is the case with the bacteria , most candidate archeal phyla are completely uncharacterized at this point .
a notable exception is candidate phylum c1 ( figure 3b ) , which contains cenarchaeum symbiosum , an uncultured archaeon that has been amenable to detailed study , including partial genome sequencing , because it exists as a near monoculture in a marine sponge .
the advent of large - scale dna sequencing has provided unprecedented access to molecular data for inferring the tree of life .
currently , complete genome sequences of prokaryotes have been obtained only from pure cultures and hence , at the phylum level , microbial genomics reflects the bias towards the big four phyla ( figures 1b,3 ) . this bias ( 71% from the big four ) is not as extreme as in culture collections ( 97% ; figure 1 ) because phyla containing human pathogens , such as chlamydiae and spirochaetes , are better represented by genome sequences ( figure 3a ) , as are archaea ( figure 3b ) .
increasing efforts are being made to select phylogenetically diverse prokaryotes ( archaea for example ) for genome sequencing , using the 16s rrna phylogeny as a guide .
but is selection solely on the basis of an exotic location in a 16s rrna tree justified ?
the implicit assumption is that the evolutionary history of 16s rrna represents the evolutionary history of the whole organism ( the whole genome ) , but the concept of a unified organismal phylogeny has been significantly compromised by the finding of widespread lateral gene transfer ( lgt ) between organisms .
lgt appears to affect the informational genes ( those involved in transcription and translation ) to a lesser extent than metabolic and other operational genes , leading to the hypothesis that a core set of vertically transmitted informational genes define organismal phylogeny .
recent evidence suggests that this may not be the case for the euryarchaeota , however ; here , informational genes are apparently no less subject to lgt than operational genes .
reliable detection of lgt by comparison of gene trees is complicated by gene duplication and loss , and different methods for detecting lgt are not particularly consistent .
the extent to which lgt blurs organismal phylogenies is therefore unclear at this point . at one extreme ,
if genomes are largely chimeric assemblages of genes with different histories , then any random sampling of organisms should provide a representative ' window ' into genome space . on the other hand ,
if a core of vertically transmitted genes ( which includes 16s rdna ) defines the organism , then striving to obtain genome sequences from all major lineages in the 16s rrna tree seems justified .
either way , a more complete sampling of phyla defined using 16s rrna should help to resolve the issue .
the number of prokaryote genome - sequencing projects completed or in progress as of 20 august 2001 is shown for each phylum - level lineage in the bacterial ( figure 3a ) and archaeal ( figure 3b ) domains .
phylum - level lineages comprising only environmental clone sequences ( figure 3 ) also need to be sampled for genome sequences ; this could best be achieved by obtaining one or more representatives of each phylum in pure culture .
the classical approach to cultivating microorganisms is to prepare a solid or liquid growth medium containing an appropriate carbon source , energy source and electron acceptor depending on the physiological type of organism being isolated .
the medium is then inoculated with a suitable source of microorganisms and left to incubate at a desired growth temperature until organisms multiply to the point at which we become aware of their presence by colony formation or increased turbidity .
this approach is not phylogenetically directed , however , and , as discussed above , typically ends up collecting fast - multiplying microbial weeds . to isolate representatives of novel environmental lineages , a directed form of cultivation is required . in one such approach , the first step is to select a target group and design group - specific oligonucleotide probes to detect or visualize the target organisms in environmental samples ( figure 2 ) .
the probes can be used to screen a range of samples and , hopefully , to identify a habitat that is a rich source of the target group .
the target organisms then need to be either selectively enriched on the basis of their phenotype or physically isolated from other non - target organisms present in the sample . as it is likely that we know nothing about the physiology of the target environmental group , physical isolation is the preferred route .
several methods have been used successfully to physically isolate microorganisms , including sample dilution , filtration , micromanipulators and optical tweezers , density - gradient centrifugation , and cell sorting using flow cytometry ( for an excellent review , see )
. sample dilution may work when the target organism is numerically dominant in a microbial community .
the sample is simply diluted until only the target organism remains , albeit at a much lower cell density than in the starting material .
sample filtration separates cells according to size , so if the target group is particularly large or small , this might be useful for initial sorting away from the primary inoculum .
micromanipulators and optical tweezers are instruments for physically moving single cells or tight clusters of cells from a mixture of cells to fresh growth medium , where the cell(s ) can grow in isolation .
these methods are most suitable for isolation of large , morphologically conspicuous microorganisms , such as filaments .
density - gradient centrifugation separates cells according to buoyant density and may be useful for initial sorting of communities to enrich for the target organisms . cell sorting by flow cytometry is a high - throughput method for quickly isolating target cells from a mixed culture ; it is most suitable for singly - occurring cells because cell aggregates can interfere with the hydrodynamic focusing in the apparatus .
when individual cells are being isolated ( by micromanipulators or optical tweezers ) , the isolation procedure can not be directly monitored by fish because cells are killed ( by fixation with paraformaldehyde ) and isolated cells must be viable for the next step in culturing ; procedures in which subsamples can be sacrificed ( such as filtration or density - gradient centrifugation ) can be monitored by fish . once individual target cells have been physically isolated , a range of growth conditions can be tested to try to promote growth without the complication of overgrowth by non - target cells .
strategies include using habitat - simulating growth media , diffusion - gradient enrichments and longer incubation times ( reviewed in ) .
common growth media , such as tryptic soy agar , poorly simulate most natural habitats because they are overly substrate - rich ; media that more closely resemble the inoculum habitat will therefore have a greater chance of supporting target - organism growth .
the use of cell - free filtrates of the inoculum habitat as the basis for the growth medium is one way of achieving this .
diffusion - gradient enrichments facilitate rapid determination of the optimal growth conditions for two parameters at a time , such as ph and nutrient concentrations , usually applied as gradients over a solid or semisolid medium at right angles to each other .
finally , simply allowing inoculated growth media to incubate for longer periods than the standard overnight to two - week period may increase the chance of successful isolation of target organisms ( see below ) . throughout the process
a phylogenetically directed isolation approach has been successfully demonstrated for an archaeal clone sequence , psl91 , obtained from a hot spring .
sequence - specific fish probes were designed and applied to an enrichment from the hot spring , and grape - like cell clusters were highlighted by fish .
clusters demonstrating this morphotype were then physically isolated using optical tweezers , grown in pure culture in a liquid medium and confirmed as the target archaeon by fish .
the psl91 sequence represents a member of the thermoprotei , however ( figure 3b ) , and this phylum contains other cultivated representatives , including one genus , desulfurococcus , relatively closely related to psl91 ( 96% 16s rdna sequence identity ) .
this may have provided physiological clues as to how to grow the target organism , given that close phylogenetic relatives often ( but not always ) have similar phenotypes . in some instances ,
for example , we discovered that micromanipulated filaments belonging to candidate phylum tm7 ( figure 3a ) could form colonies visible to the naked eye on low - nutrient solid media ( r2a ) under aerobic conditions ; the only catch was that they took 50 days to do so ( p.h .
this may be the case for a wide range of uncultivated organisms , with simple removal of the target organism from the weeds in the inoculum , and a little patience , being all that is required for success .
there are likely to be many prokaryotes that will never be brought into pure culture , however , such as organisms that live in obligately interdependent relationships , because the conditions for their growth are too exacting ( and thus can not be reproduced in the laboratory ) . for such organisms ,
genomes of uncultured prokaryotes can be accessed by a relatively straightforward adaptation of the rrna approach ( figure 2 ) .
high - molecular - weight dna extracted from environmental samples can be cloned directly into large - insert cloning vectors , such as cosmids or bacterial artificial chromosomes ( bacs ) . with careful handling of the environmental dna , this results in access to large contiguous portions of microbial genomes - 35 - 40 kilobases ( kb ) for cosmids and up to 200 kb for bacs - without the need for cultivation .
bacs have the additional advantage that heterologous expression of some of the insert genes may be possible in the escherichia coli host harboring the vector .
clones can be sequenced using shotgun or chromosome - walking methods and comparatively analyzed ( figure 2 ) .
if a 16s rrna gene or another conserved gene is identified in a clone then the phylogenetic identity of the genome segment can be determined .
perhaps the most impressive application of this approach to date is the discovery of proteorhodopsin in an uncultured lineage of marine bacterioplankton belonging to the gammaproteobacteria .
an open reading frame encoding proteorhodopsin was found on a 130 kb genomic fragment together with a 16s rdna sequence identifying its owner as a member of the ' sar86 ' group in the gammaproteobacteria .
members of the sar86 group had been detected on numerous occasions in culture - independent surveys of marine habitats , but no function could be inferred for them because there are no close cultivated representatives for the group .
the discovery of proteorhodopsin , which is phylogenetically related to the light - driven proton pump bacteriorhodopsins , suggests that the sar86 lineage lives phototrophically in the marine environment .
ideally , we would like to reconstruct entire genomes from uncultured prokaryotes using large - insert cloning - vector approaches .
this is a daunting task given the species complexity of most microbial communities and the genomic microheterogeneity within prokaryotic populations .
it will probably be an impossible task for habitats such as soil , containing thousands of individual genomes .
it remains to be seen , however , whether it is possible to reconstruct complete genomes from a low - diversity microbial community . in conclusion ,
several major lineages of bacteria ( but not archaea ) containing isolated representatives lack even a single sequenced genome . over a third of phylum - level
prokaryotic lineages are represented exclusively by sequences of uncultured prokaryotes that have been repeatedly detected in culture - independent habitat surveys over the past decade .
the mere existence of such large phylogenetically conspicuous groups , about which we know virtually nothing , should be reason enough to study them .
yet there remains a reluctance amongst many microbiologists to accept these ' virtual bacteria ' as bona fide members of the microbial world . by analogy , imagine that we were unaware of the metazoa until a few years ago , when we began detecting them in environmental surveys using phylogenetic markers .
imagine that metazoa - specific probes were designed to allow us to see this new group under the ' macroscope ' .
what do these creatures do for a living ? what other organisms remain to be discovered in this group ?
this is approximately the stage we are at in the description of candidate prokaryotic phyla . at the very least ,
uncharacterized prokaryotic phyla will probably contain members with impressive physiological repertoires and interesting evolutionary histories , worthy of study and of genome sequencing .
i thank mark ragan , norman pace and george garrity for providing comments on the manuscript .
pie charts showing the phylum - level distribution of prokaryotic isolates ( a ) in the australian collection of microorganisms and ( b ) in the prokaryote genome sequences completed or in progress as of 20 august 2001 . ' full - cycle ' rrna approach to characterizing microorganisms in their natural settings without the need for cultivation .
access to whole genomes of uncultivated organisms is also possible using the same basic approach but with large - insert cloning vectors , such as bacs , which remove the need for pcr .
evolutionary distance dendrograms of ( a ) bacterial and ( b ) archaeal diversity derived from comparative analysis of 16s rrna gene sequences .
the trees were constructed using the arbsoftware package and a sequence database modified from the march 1997 arb database release using 50% consensus sequence filters for each domain and the olsen correction and neighbor - joining options .
this modified database will be available from the ribosomal database project user - submitted alignments download site .
major lineages ( phyla ) are shown as wedges with horizontal dimensions reflecting the known degree of divergence within that lineage .
phyla with cultivated representatives are in gray and , where possible , named according to the taxonomic outline of bergey 's manual .
phyla known only from environmental sequences are in white ; because they are not formally recognized as taxonomic groups , they are usually named after the first clones found from within the group .
note that environmental groups e2 and e3 defined in are part of the thermoplasmata phylum in the archaeal tree in ( b ) . the number of genome sequences completed or in progress for each phylum is given in brackets after the phylum name , with the exception of methanopyrus kandleri , which is not included in the tree because it is represented by a single sequence .
bacterial phyla with cultured representatives but without representative sequenced genomes where possible , the nomenclature of the taxonomic outline for bergey 's manual of systematic bacteriology has been used . | our understanding of prokaryote biology from study of pure cultures and genome sequencing has been limited by a pronounced sampling bias towards four bacterial phyla - proteobacteria , firmicutes , actinobacteria and bacteroidetes - out of 35 bacterial and 18 archaeal phylum - level lineages .
this bias is beginning to be rectified by the use of phylogenetically directed isolation strategies and by directly accessing microbial genomes from environmental samples . |
breast cancer is a heterogeneous disease comprising numerous distinct entities with different biological features and clinical behaviors ( reis - filho and pusztai , 2011 , weigelt and reis - filho , 2009 ) .
invasive ductal carcinomas of no special type ( idc - nst ) , the commonest histologic type of breast cancer , have been shown to be heterogeneous at the transcriptomic and genomic levels , and can be classified into several molecular subtypes ( cancer genome atlasn , 2012 , reis - filho and pusztai , 2011 ) .
intrinsic gene classification is the most widely used molecular taxonomy for idc - nsts , and classifies breast cancers into luminal a , luminal b , her2-enriched and basal - like subtypes ( parker et al . , 2009 ,
these subtypes have been shown not only to be associated with distinct clinical outcomes , but also with different risk factors , clinicopathologic features and repertoires of genetic aberrations ( cancer genome atlasn , 2012 , parker et al . , 2009 , reis - filho and pusztai , 2011 ) .
the complexity and diversity of breast cancers have also been documented at the histologic level ( weigelt et al .
, 2010c , weigelt and reis - filho , 2009 ) , however this information has not been fully explored in molecular subtyping studies , which have primarily focused on idc - nsts . based on the cytological and architectural features of invasive breast cancers , the world health organization ( who ) recognizes the existence of 21 special histologic subtypes in addition to idc - nsts ( lakhani et al . , 2012 ) .
genomic and transcriptomic analyses of special histologic types of breast cancer conducted by our group and others ( bertucci et al .
, 2008 , duprez et al . , 2012 , geyer et al . , 2010 , gruel et al . , 2010 , horlings et al . , 2013 ,
lacroix - triki et al . , 2010 , lopez - garcia et al .
, 2010b , marchio et al . , 2009 , marchio et al . , 2008 , vincent - salomon et al . , 2007 ,
, 2010b , weigelt et al . , 2008 , weigelt et al .
, 2009b , wetterskog et al . , 2012 ) have demonstrated that tumors from each of the special histologic types of breast cancer are more homogeneous amongst themselves than idc - nsts .
in addition , some of the histologic special types have been shown to be driven by recurrent fusion genes resultant of chromosomal translocations .
for example , secretory carcinomas of the breast are characterized by the t(12 ; 15 ) translocation , which results in the formation of the etv6-ntrk3 fusion gene ( tognon et al . , 2002 ) , and > 90% of adenoid cystic carcinomas of the breast display the t(6 ; 9 ) translocation , which leads to the fusion of myb with nfib ( persson et al . , 2009 ,
papillary carcinoma ( pc ) is a rare ( < 1% ) special histologic type of breast cancer that often affects postmenopausal women and has an overall favorable outcome ( grabowski et al .
pcs comprise a morphologically heterogeneous group of lesions , all of which share a growth pattern characterized by the presence of arborescent fibrovascular stalks lined by a layer of neoplastic epithelial cells devoid of an intervening myoepithelial cell layer , a feature that distinguishes them from benign intraductal papillomas and papillary carcinomas in situ ( collins and schnitt , 2008 , hill and yeh , 2005 , pal et al . , 2010 , weigelt et al . , 2010a ) .
papillary neoplasms of the breast include three histologic subtypes , namely encapsulated papillary carcinoma ( epc ) , solid papillary carcinoma ( spc ) and invasive papillary carcinoma ( ipc ) .
epc is a well - circumscribed lesion where the involved duct is surrounded by a thick fibrous capsule ; in epcs , the neoplastic cells are arranged in papillary fronds in the majority of cases , however areas with cribriform and/or solid patterns are not uncommonly found ( lakhani et al . , 2012 ,
spc is also a well - circumscribed lesion that is densely cellular and composed of expansile nodules of epithelial cells , and ipc comprises papillary lesion with neoplastic cells arranged in finger - like projections with clear invasion into adjacent stroma .
although the classification of epcs and spcs as invasive or in situ disease still remains a matter of controversy , these tumors have the potential to disseminate to axillary lymph nodes and , albeit rarely , distant metastatic deposits of pcs have been documented ( rakha et al . , 2011 ,
, it has been proposed that epcs and spcs should be considered forms of invasive breast cancer with excellent outcome ( lakhani et al . , 2012
our group has previously demonstrated that pcs are preferentially estrogen receptor ( er ) and progesterone receptor ( pr ) positive , lack her2 gene amplification , and display relatively simple genomes in terms of their repertoires of gene copy number aberrations ( duprez et al . , 2012 ) .
in addition , the similarities in the gene copy number profiles of pcs and grade- and er - matched idc - nsts have led us to suggest that pcs may be best positioned as part of the spectrum of er - positive idc - nsts , rather than a distinct entity ( duprez et al . , 2012 ) .
on the other hand , the transcriptomic characteristics of pcs and whether these tumors would differ from idc - nsts at the gene expression level remain to be determined .
therefore , the primary aims of this study were i ) to investigate whether pcs would constitute a molecular entity distinct from grade- and er - matched idc - nsts at the transcriptomic level , and ii ) to define whether pcs would be driven by recurrent fusion genes or pathognomonic mutations .
in addition , we carried out an exploratory , hypothesis - generating analysis to investigate whether epcs , spcs and ipcs would display distinct transcriptomic and genomic profiles .
nineteen pcs of the breast were retrieved from institut curie , paris , france ( from 1995 to 2009 ) . in this study , we included pcs from patients diagnosed and managed in the above institution , whose tumors were < 5 cm and who had no clinical and/or radiological evidence of distant metastases .
exclusion criteria were ( a ) patients with multiple tumors , either ipsi- or contra - lateral , ( b ) patients who received neoadjuvant chemotherapy , ( c ) patients for whom all histologic slides and blocks were not available for review , ( d ) tumors not consistent with the final diagnosis of epc , spc or ipc , and ( e ) tumors whose frozen samples contained < 50% of tumor cell content . for sixteen cases , both dna and rna of sufficient quality and quantity were available for microarray - based gene expression and copy number profiling ; for three cases only rna could be extracted due to limited frozen tissue availability ( supplementary table s1 ) .
samples were anonymized prior to analysis and the study was approved by local research ethics committees of the authors ' institutions .
all cases were independently reviewed by two pathologists ( av - s and jsr - f ) , who subtyped the tumors into epc , spc and ipc following the who criteria ( lakhani et al . , 2012 ) , and graded the tumors according to the nottingham grading system ( elston and ellis , 1991 ) .
histologic grade- and er - matched idc - nsts , whose frozen samples contained > 50% of tumor cells , were retrieved from the files of institut curie , paris , france .
sixteen idc - nsts were matched 1:1 according to histologic grade and er status with the pcs included in this study ( table 1 ) . for gene expression profiling analysis , power calculations employing the approach described by dobbin et al .
( dobbin et al . , 2008 ) revealed that 16 samples of pcs and idc - nsts would result in the identification of genes significantly differentially expressed between the groups with a tolerance = 0.10 .
eight cases of pcs ( 3 ipcs , 3 epcs , 2 spcs ) , from which sufficient rna of acceptable quality was available , were subjected to paired - end massively parallel rna - sequencing ( supplementary table s1 ) , with the aim of identifying highly recurrent fusion genes and pathognomonic mutations .
power calculations , assuming that if pcs were driven by a recurrent fusion gene in a way akin to secretory carcinomas ( which harbor the etv6-ntrk3 fusion gene in > 95% of cases ) ( tognon et al . , 2002 ) or adenoid cystic carcinomas of the breast ( which harbor the myb - nfib fusion gene in > 90% of cases ) ( persson et al . , 2009 ,
2012 ) , and that a pathognomonic driver event ( i.e. pathognomonic fusion gene or mutation ) would be present in at least 70% of cases ( an estimate that is conservative ) ) , revealed that based on a binomial distribution , by sequencing 8 samples we would have been able to identify a recurrent driver event ( i.e. in two or more cases ) with > 95% statistical power .
pathognomonic driver event present in 50% of cases would be detectable with > 80% power if 8 samples were subjected to rna - sequencing .
immunohistochemical profiles of the included cases of pcs and grade- and er - matched idc - nsts were assessed on 3 m thick sections , using antibodies against er , pr and her2 as previously described ( duprez et al . , 2012 ) .
the results of immunohistochemical analyses were interpreted by two pathologists ( av - s and jsr - f ) .
antibody clones , dilutions , antigen retrieval methods , scoring systems and cut - offs used are described in supplementary table s2 .
dna and rna were extracted using the dneasy blood and tissue kit ( qiagen , hamburg , germany ) and trizol ( invitrogen , life technologies , paisley , uk ) respectively , according to the manufacturers ' guidelines .
dna and rna quantity and quality were analyzed with the qubit fluorometer ( invitrogen ) and the agilent bioanalyzer ( agilent , santa clara , ca , usa ) . only samples with rna integrity ( rin )
> 6 ( bioanalyzer , agilent , santa clara , ca , usa ) were used for downstream analyses .
gene copy number profiling was performed using the snp array 6.0 platform ( affymetrix , santa clara , ca , usa ) as per manufacturer 's instructions .
raw data were analyzed using the crmav2 algorithm in the aroma.affymetrix package ( bengtsson et al . , 2009 ) and absolute ( carter et al . , 2012 ) . in all cases ,
absolute defined estimates of tumor cellularity were > 50% ( median 71% , range 57%93% ) .
log2 ratios obtained from crmav2 were then smoothed using the circular binary segmentation ( cbs ) algorithm in the dnacopy package ( olshen et al . , 2004 ) in r. cbs - smoothed log2 ratios were then used in all subsequent analyses to define gains , losses and amplifications .
homozygous deletions were defined as a cbs - smoothed log2 ratio < 1 and using absolute ; only homozygous deletions identified by both methods were considered valid .
low - level gain was defined as a cbs - smoothed log2 ratio of > 0.15 and 0.5 , and high - level gains / amplification were defined as a log2 ratio > 0.5 .
to identify statistically significant differences between the genomic profiles of the different subtypes of pcs , we compared the copy number profiles of epc , spc and ipc using categorical data and a multi - fisher 's exact test adjusted by the false discovery rate as previously described ( duprez et al . , 2012 ) .
hierarchical clustering analysis was performed with categorical states ( i.e. gains , losses , high - level gains / amplifications ) using euclidean distance and the ward 's algorithm .
gene expression profiling was performed with the humanht-12 v4 expression beadchip kit ( illumina inc , san diego , ca , usa ) as per manufacturer 's instructions .
raw data were robust - spline normalized using the lumi package in r ( du et al . , 2008 ) .
illumina transcript probes with detection p - value<0.01 in fewer than 20 samples of both pcs and idc - nsts or without unambiguous mapping information were excluded , resulting in a set of 17,296 probes for further analysis . for intrinsic subtyping using the pam50 classification ( parker et al . , 2009 , weigelt et al . , 2010d ) , the gene expression profiles of pcs and idc - nsts were normalized together with 997 breast cancers of the metabric discovery cohort , as described by curtis et al .
samples were then assigned to one of the five subtypes using spearman 's rank correlation and the pam50 centroids curated for the illumina platform strictly as described by curtis et al .
( curtis et al . , 2012 ) and following the university of north carolina ( unc ) guidelines ( https://genome.unc.edu/pubsup/breastgeo/guide%20to%20intrinsic%20subtyping%209-6-10.pdf ) . for unsupervised clustering analysis
, we included only probes with a median absolute deviation ( mad ) > 0.6 and excluded replicate probes mapping to the same genes by keeping only the probe with the greatest mad , resulting in a set of 2310 probes for clustering .
gene expression values for the series of pcs and idc - nsts were median centered prior to clustering .
hierarchical clustering was performed using the ward 's or the complete linkage algorithm with pearson correlation as the distance metric .
the stability of the clusters was estimated by performing 1000 iterations of bootstrapping using the r package pvclust ( suzuki and shimodaira , 2006 ) . to determine the genes significantly differentially expressed between pcs and idc - nsts , and between different subtypes of pcs
, we used the significance analysis of microarrays ( sam ) software implemented in the samr r package .
we included probes with a mad > 0.3 and excluded replicate probes as described above , resulting in a set of 11,108 probes for sam .
probes were median - centered prior to sam analysis and 1000 permutations were performed to estimate the false discovery rate ( fdr ) .
the raw and processed data are available on geo ( gse55640 ) , and the r script and code are available in the supplementary materials . to identify genes whose expression levels correlate with copy number changes , cbs - smoothed log2 ratios from snp 6.0 data were used to assign the snp 6.0 states for each of the 17,296 probes in the gene expression dataset using the median values for all probes that overlap with the genomic position of each gene .
this resulted in a 1:1 matrix of expression ratios and snp 6.0 cbs values , which were subsequently employed for downstream statistical analysis . to define genes that were up - regulated when gained or amplified , and down - regulated when lost , we performed a mann whitney u test using categorical gene copy number states ( i.e. gain vs. no gain , loss vs. no loss or amplification vs. no amplification ) as the grouping variable and the expression of genes as the dependent variable .
p - values were corrected using benjamini and hochberg adjustment ( benjamini and hochberg , 1995 ) to minimize type i or alpha errors , and adjusted p - values<0.05 were considered significant . significantly regulated pathways and networks in the gene expression data
were determined using the ingenuity pathway analysis ( ipa ) program ( http://www.ingenuity.com ) and the database for annotation , visualization and integrated discovery ( david , http://david.abcc.ncifcrf.gov ) . for ipa analysis ,
the fold difference of transcripts differentially expressed identified by sam was mapped to networks available in the ingenuity database and ranked by score .
the score indicates the likelihood of the genes in a network being found together due to chance . using a 99% confidence level ,
scores of 3 are considered significant ( lopez - garcia et al . , 2010b ) .
paired - end massively parallel rna - sequencing ( 2x 54bp cycles ) was performed according to the standard illumina mrna paired - end library protocol on a genome analyzer ii ( illumina ) as previously described ( natrajan et al . , 2014 ) .
data were aligned to the genome using bowtie 2 ( langmead and salzberg , 2012 ) .
chimerascan and defuse were used to identify mate - pairs supporting novel chimeric transcripts as previously described ( iyer et al . , 2011 , mcpherson et al . , 2011 ,
high - confidence chimeric transcripts detected using both algorithms were considered as potential candidates . nominated in - frame fusion gene candidates identified by rna - sequencing were prioritized for validation in the index cases by rt - pcr .
the split sequences ( sequences encompassing the breakage point / fusion junctions ) were used as reference to design primer sets for each fusion gene pair ( supplementary table s3 ) . for this ,
100 ng of total rna was reverse transcribed using superscript iii ( invitrogen ) and pcr was performed using the gotaq green master mix kit ( promega , usa , madison , wi , usa ) . for each sample ,
triplicate reactions containing 100 ng rna were subjected to reverse transcription as described previously ( arriola et al . ,
qrt - pcr was performed for ret , igfbp5 , tmprss6 , erp27 , nfkbiz and gbp3 ( assay on demand ids : hs01120030_m1 , hs00181213_m1 , hs00542184_m1 , hs00328370_m1 , hs00230071_m1 , hs00544385_m1 , respectively ; applied biosystems , foster city , usa ) using taqman chemistry on the steponeplus real - time pcr system ( applied biosystems ) , based on the standard curve method ( weigelt et al . , 2010b ) .
target gene expression levels were normalized to the geometric mean of two reference genes ( tfrc and mrpl19 ; hs00174609_m1-tfrc , hs00608522_g1-mrpl19 ) ( natrajan et al . , 2012 ) .
all statistical comparisons were performed using a mann whitney u test ( graphpad prism 6 ) . for the identification of mutations from rna - sequencing data ,
pcr duplicates were removed using samtools ( li et al . , 2009 ) .
local re - alignment and base quality re - calibration were performed using the genome analysis tool kit ( gatk ) ( mckenna et al . , 2010 ) .
mutations were called using the gatk haplotype caller ( mckenna et al . , 2010 ) , where a minimum of 5 variant reads , 10 total read depth , and a genotype quality ( gq ) of 40 were required to call a mutation . to remove germline polymorphisms and sequencer - specific artifacts , we filtered out mutation calls from 13 unrelated , normal female rna samples , whose rna - sequencing results were generated on the same sequencer using the same protocols . only single nucleotide variants ( snvs ) or insertions and deletions ( indels ) supported by at least 2 reads supporting the reference alleles were included , as those devoid of a reference allele were highly enriched for germline variations .
further , we filtered out mutations catalogued in dbsnp as having > 1% global population frequency and all germline variants identified in the 1000 genomes project .
the functional effects of missense mutations were predicted using mutation assessor ( reva et al . , 2011 ) ,
chasm ( carter et al . , 2009 ) , fathmm ( shihab et al . , 2013 ) and polyphen-2 ( adzhubei et al . , 2010)/mutation taster ( schwarz et al . , 2010 ) .
a mutation was considered potentially pathogenic if at least two predictors classified it as pathogenic .
mutationtaster and polyphen-2 were used in combination , given that these two algorithms are underpinned by the same bioinformatic principles and their results , albeit not identical , are remarkably similar .
genes recurrently mutated in pcs and likely to constitute somatic mutations were validated at the genomic level using sanger sequencing .
briefly , 50 ng tumor dna was amplified with specific primers ( supplementary table s4 ) and sequencing performed using the bigdye terminator v 1.1 kit ( applied biosystems ) , as previously described ( hernandez et al . , 2012 ) .
histopathologic review of the 16 pcs subjected to snp6 and gene expression profiling revealed that 7 ( 44% ) , 6 ( 37% ) and 3 ( 19% ) cases were of histologic grades 1 , 2 and 3 , respectively , and 4 ( 25% ) , 7 ( 44% ) and 5 ( 31% ) cases were of spc , epc , and ipc subtype , respectively ( table 1 ) .
immunohistochemical analysis revealed that all pcs were er - positive and lacked her2 expression in accord with our previous results ( duprez et al . , 2012 ) ( table 1 and supplementary table s1 ) ; 81% of pcs were er - positive / pr - positive / her2-negative and 19% of cases were er - positive / pr - negative / her2-negative ( supplementary table s1 ) .
pam50 subtyping classified 10 ( 63% ) , 5 ( 31% ) and 1 ( 6% ) pcs as of luminal a , luminal b and basal - like subtypes , respectively . all but one epc and all but one ipc were classified as of luminal a subtype , whereas all spcs were classified as of luminal b ( table 1 ) .
unsupervised hierarchical clustering of pcs and grade- and er - matched idc - nsts revealed that pcs preferentially clustered together ( two - tailed fisher 's exact test , p - value = 0.002 ; figure 1 ) , irrespective of the clustering algorithm employed .
these data suggest that although pcs and grade- and er - matched idc - nsts are similar at the gene copy number level ( duprez et al . ,
2012 ) , their global patterns of gene expression are distinct . sam analysis further supported this observation , given that 8.3% of the significantly regulated probes ( 795 unique genes ) were differentially expressed between pcs and histologic grade- and er - matched idc - nsts ( fdr < 10% ) , of which 780 unique genes were expressed at significantly lower levels in pcs than in grade- and er - matched idc - nsts ( supplementary table s5 ) .
ipa and david gene ontology and pathway analysis employing these 780 genes revealed a significant enrichment for genes involved in cellular growth and proliferation ( e.g. kit , aldh1a3 , elf4 , ccnd2 and map4k4 , right - tailed fisher exact test p - value<0.0001 , figure 2a , supplementary table s5 ) .
this is consistent with the observation that pcs are a form of er - positive breast cancer with an excellent outcome ( rakha et al . , 2011 ) , given that the expression levels of proliferation - related genes have been shown to constitute one of the strongest predictors of outcome of er - positive breast cancers ( reis - filho and pusztai , 2011 ) .
other genes expressed at significantly lower levels in pcs than in grade- and er - matched idc - nsts included those involved in cell assembly and organization ( e.g. lamb1 , actn1 and collagen genes such as col1a2 , col6a1 , col8a1 , col12a1 , right - tailed fisher exact test p - value<0.0001 , figure 2b ) and cellular movement and migration ( e.g. mmp3 , mmp7 and thbs4 , right - tailed fisher exact test p - value<0.0001 , figure 2c ) .
consistent with these observations , david kegg pathway analysis revealed that the cell adhesion molecules ( cams ) pathway was the most significantly enriched for genes expressed at lower levels in pcs than in idc - nsts ( data not shown ) .
by contrast , 15 genes involved in cellular homeostasis ( e.g. qsox1 ) and angiogenesis ( e.g. vegfa , esm1 , slc4a11 and vcam1 ) were expressed at significantly higher levels in pcs than in grade- and er - matched idc - nsts ( supplementary table s5 ) .
we have previously demonstrated that the patterns of gene copy number alterations found in pcs are relatively simple and similar to those found in idc - nsts of the same histologic grade and er - status ( duprez et al . , 2012 ) . here , using a platform with a higher resolution , we confirmed our previous observations that pcs displayed genomic features consistent with those of low - grade , er - positive idc - nsts ( i.e. 16q losses , 16p gains and 1q gains ; figure 3 and supplementary figure s1 ) . after excluding regions mapping to known copy number polymorphisms ( http://dgv.tcag.ca/dgv/app/home ) , we observed recurrent amplifications in 11q13.3 ( n = 2 ) and 8p11.23-p11.22 ( n = 2 ) loci ( supplementary figure s1 and supplementary table s6 ) .
consistent with our previous observations ( duprez et al . , 2012 ) , her2 and esr1 gene amplification was not observed in the pcs studied . to define genes that are up - regulated and gained , or down - regulated and lost in pcs
we identified 214 genes that were significantly over - expressed when gained ( mann whitney u test , adjusted p - value<0.05 ) , and these were significantly enriched for cellular functions including cellular growth and proliferation
( e.g. ago2 and capn2 , right - tailed fisher 's exact test p - value<0.01 , ipa ) and cell death and survival
( e.g. lmna , stk3 and ptk2 ; right - tailed fisher 's exact test p - value<0.01 , ipa , and p - value<0.01 , david ; supplementary table s7 ; supplementary figure s2a ) . on the other hand ,
275 genes were found to be significantly down - regulated when lost ( mann whitney u test , adjusted p - value<0.05 ) .
functional annotation of these genes revealed a significant enrichment for genes involved in cell cycle progression
( e.g. asah1 and birc2 , right - tailed fisher 's exact test p - value<0.01 , ipa ) and cellular death and survival ( e.g. cdt1 and max , right - tailed fisher exact test p - value<0.01 , ipa ; supplementary table s7 ; supplementary figure s2b ) .
we further sought to identify potential amplicon drivers in pcs on the basis that amplicons drivers are frequently overexpressed when amplified .
we identified 182 genes expressed at significantly higher levels when amplified in pcs ( mann whitney u test , p - value<0.05 ; supplementary table s8 ) .
this list included genes mapping to the 8p11.2-p12 amplicon ( i.e. prosc ) and the 11q13 amplicon ( i.e. cttn and fadd ) , which have been previously shown to be overexpressed when amplified in idc - nsts ( adelaide et al . , 2007 , chin et al . , 2006 , chin et al .
amplification correlation in other histologic special types of breast cancer ( horlings et al . , 2013 ) .
when p - values were corrected for multiple testing , however , only 16 genes were shown to be significantly overexpressed when amplified , including rsf1 , which plays a role in the rsf chromatin - remodeling complex . taken together , our analyses demonstrate that the transcriptomic profiles of pcs differ from those of grade - matched er - positive idc - nsts , with down - regulation of proliferation - related , cell assembly and organization and cellular movement and migration genes , and overexpression of genes related to homeostasis and angiogenesis . to determine whether pcs of the breast are driven by a highly recurrent fusion gene
, 2011 , mcpherson et al . , 2011 ) revealed a total of 1156 fusion transcripts ( involving 999 unique gene combinations ) and 396 fusion transcripts ( involving 339 unique gene combinations ) , respectively , of which 33 unique gene combinations were identified by both defuse and chimerascan ( supplementary table s9 ) .
we next sought to identify potential gain - of - function fusion genes that may underpin pcs . in a way akin to the oncogenic fusion genes identified in special histologic types of breast cancer , leukemias , lymphomas and sarcomas , we prioritized fusion transcripts that were identified by both algorithms , involved open reading frames with known associated functions , and that harbored intact functional domains . using the above criteria
, we identified 14 in - frame fusion transcripts , 12 of which were successfully validated by rt - pcr ( figure 4 , supplementary figure s4 ; supplementary table s10 ) .
these validated fusion transcripts did not map to regions harboring amplifications and were only detected and validated in the index cases .
only one of these confirmed fusion transcripts , mapkapk3-hemk1 , harbored intact functional domains encoded by the first 4 exons of the protein kinase mapkapk3 and the last 7 exons of the methyltransferase hemk1 ( figure 4d ) .
in addition to the kinase domain , this chimeric transcript has a conserved dna methylase domain , which has been reported to methylate the mitochondrial translation release factor mtrf1l ( ishizawa et al . , 2008 ) .
taken together , our results demonstrate that pcs are not underpinned by a highly recurrent fusion gene , at variance with some other special histological types of breast cancer .
to assess the constellations of expressed mutations in pcs , mutational analysis of the rna - sequencing data was performed .
given the challenges in the de novo identification of mutations based on the analysis of rna - sequencing data , we have focused on the 35 significantly mutated genes in common types of breast cancers as reported by tcga ( cancer genome atlas , 2012 ) .
this analysis revealed that five of these genes were mutated in pcs ( pik3ca , tp53 , map3k1 , akt1 , and ctcf ; table 2 ) .
the pik3ca ( h1047r ) mutation ( case papi13 ) and tp53 ( r273h ) mutation ( case papi17 ) are known hotspot mutations ( muller and vousden , 2013 , samuels et al . , 2004 ) . to identify further potentially pathogenic mutations
, we defined genes that were recurrently mutated in the 8 pcs analyzed and predicted to be pathogenic using five mutation function predictor algorithms ( i.e. mutation assessor ( reva et al . , 2011 ) , chasm ( carter et al . , 2009 ) , fathmm ( shihab et al . , 2013 ) and polyphen-2 ( adzhubei et al . , 2010)/mutation taster ( schwarz et al . , 2010 ) ) .
after filtering out single nucleotide variants and insertions and deletions present in dbsnp at a > 1% global population frequency , and all germline variants identified in the 1000 genomes project , we identified 156 potential somatic mutations ( supplementary table s11 ) . out of these , lama5 and macf1 were found to be recurrently mutated ( i.e. in two samples each ) in the pcs analyzed and considered to be potentially pathogenic mutations by at least two of the mutation function prediction algorithms employed in this study . these mutations were validated in the index cases by sanger sequencing ( supplementary figure s5 ) .
mutations affecting lama5 and macf1 were not restricted to papillary carcinomas , however , as also 1.1% and 2.4% of the common types of breast cancers in the tcga cohort harbored a mutation in these genes , respectively ( gao et al . , 2013 )
this analysis suggests that the papillary phenotype is not underpinned by a highly recurrent expressed mutation ( i.e. present in > 50% of samples ) affecting a gene expressed in pcs .
we next carried out a hypothesis - generating , comparative exploratory analysis of the transcriptomic profiles of pc subtypes to ascertain whether histologic subtypes of pcs ( i.e. epc , spc and ipc ) would have distinctive transcriptomic profiles .
unsupervised hierarchical clustering demonstrated that epcs preferentially clustered together ( fisher 's exact test , p = 0.0406 , figure 5a ) , irrespective of the clustering algorithm employed ( figure 5b ) .
it should be noted , however , that all epcs were of histologic grade 1 , and a significant enrichment for tumors of histologic grade 1 ( fisher 's exact test , p = 0.04 ) was also observed in the cluster enriched for epcs .
sam analysis using 11,108 probes revealed that less than 0.2% of genes were differentially expressed between epcs and spcs / ipcs ( fdr < 10% ; supplementary table s12 ) .
functional annotation of these genes with both david and ipa revealed that several genes involved in cell migration , such as plat and ctsf , were down - regulated in epcs in comparison with spcs and ipcs .
furthermore a comparison between spcs and epcs suggested that , consistent with the reported neuroendocrine phenotype of spcs , genes related to neuroendocrine differentiation in human cancers , including ret , ascl1 and dok7 , were expressed at significantly higher levels in spcs than in epcs ( mann whitney u test p - value<0.01 , supplementary table s13 ) .
consistent with these findings , immunohistochemical analysis revealed that three out of four spcs displayed expression of synaptophysin , whereas only one out of seven epcs focally displayed synaptophysin expression ( p = 0.0878 , fisher 's exact test ; supplementary figure s6 ) .
probably due to the small sample size of the groups analyzed , no differentially expressed genes were identified in the other comparisons performed between the different histologic subtypes of pc ( data not shown ) .
to validate the findings obtained by microarray - based gene expression profiling , the expression of genes identified to be significantly differentially expressed between pc histologic subtypes ( i.e. ret , igfbp5 , tmprss6 , erp27 , nfkbiz and gbp3 ) was assessed using qrt - pcr in the 16 pcs included in this study .
this analysis confirmed that erp27 , nfkbiz , gbp3 and igfbp5 were expressed at significantly higher levels in epcs than in spcs and ipcs ( supplementary figure s7 ; mann whitney u test p - values , 0.0002 , 0.0008 , 0.0035 and 0.0003 , respectively ) , and that ret and tmprss6 were expressed at significantly higher levels in spcs than in epcs and ipcs ( supplementary figure s7 ; mann whitney u test p - values : 0.0059 and 0.02 , respectively ) .
genomic analysis of epcs , spcs and ipcs revealed that these histologic subtypes of pcs have similar patterns of copy number losses , gains and amplifications ( figure 3 , figure 6 ) , consistent with our previous observations ( duprez et al . , 2012 ) .
taken together , although the distinct histologic subtypes of pcs display rather similar patterns of gene copy number alterations , epcs differ from other pc subtypes in regards to the expression of genes related to cell migration , whereas spcs display transcriptomic features consistent with neuroendocrine differentiation .
here we demonstrate that pcs of the breast are er - positive , her2-negative breast cancers , that largely display a luminal phenotype and gene copy number aberrations consistent with those of low - grade er - positive breast cancers ( i.e. , 16q losses , 16p gains , and 1q gains ) ( lopez - garcia et al . , 2010a ) . despite the genomic similarities between pcs and idc - nsts ,
pcs were shown to have gene expression profiles distinct from those of idc - nsts of the same histologic grade and er - status and to display lower levels of expression of proliferation - related , cell assembly and organization and cellular movement and migration genes .
we have also demonstrated that pcs , at variance with some other special histologic types of breast cancer , are not underpinned by a highly recurrent expressed fusion gene or highly recurrent expressed mutation ( i.e. present in > 50% of samples ) . in addition , a hypothesis generating , exploratory analysis of the transcriptomic profiles of subtypes of pcs revealed that epcs express a subset of genes involved in cell migration at significantly lower levels than spcs and ipcs , and that spcs display transcriptomic and immunohistochemical features consistent with those associated with neuroendocrine differentiation in human cancers .
previous genomic analyses of other special histologic types of breast cancer have revealed that micropapillary ( marchio et al . , 2009 ,
, 2008 , natrajan et al . , 2014 ) , mucinous ( horlings et al . , 2013 , lacroix - triki et al . ,
2010 ) , and adenoid cystic ( wetterskog et al . , 2012 ) carcinomas display different patterns of genomic alterations when compared to grade- and er - matched idc - nsts . here
we have confirmed , employing a platform with a higher resolution , our previous observations ( duprez et al . , 2012 ) that although pcs harbor fewer gene copy number alterations than idc - nsts of the same histologic grade and er - status , the patterns of gene copy number aberrations found in pcs are similar to those observed in idc - nsts . despite the genomic similarities between pcs and idc - nsts , pcs expressed proliferation - related genes , including ccnd2 , kit and map4k4 , at significantly lower levels than grade- and er - matched idc - nsts .
given that the number and complexity of gene copy number aberrations ( ciriello et al .
, 2013 , reyal et al . , 2012 , vincent - salomon et al . , 2013 ) and
the levels of expression of proliferation - related genes are prognostic in er - positive luminal cancers ( reis - filho and pusztai , 2011 ) , our findings may provide a biological rationale for the more indolent clinical behavior of pcs .
functional annotation of the genes expressed at significantly lower levels in pcs also revealed an enrichment for pathways and networks related to cell adhesion and cellular movement and migration , including several laminins , integrins , collagens and matrix metalloproteinases .
the genes found to be differentially expressed in this study may also partially explain the differences in clinical behavior ( i.e. indolent behavior , favorable prognosis and limited invasiveness ) between pcs and idc - nsts of similar histologic grade .
in particular , pcs displayed lower levels of expression of genes related to cell adhesion , migration and movement , including mmp2 and mmp7 and genes that are found in the basement membrane ( e.g. laminins , integrins , collagens ) .
( rakha et al . , 2012 ) in that pcs display mmp2 and mmp7 expression levels lower than those found in idc - nsts ( rakha et al . , 2012 ) , and that pcs , in particular epcs , exhibit an expression pattern of invasion - associated markers intermediate between dcis and idc - nsts ( rakha et al . , 2012 ) .
the transcriptomic analysis performed in this study has also revealed that pcs display higher levels of expression of genes related to angiogenesis ( e.g. vegfa , esm1 and slc4a11 ) than grade- and er - matched idc - nsts .
this observation is consistent with the notion that pcs are well - vascularized lesions ( wagner et al . , 2004 ) and that histologically these tumors are characterized by the presence of neoplastic cells arranged in papillary fronds containing fibrovascular cores .
rna - sequencing analysis of 8 pcs ( 3 epcs , 2 spcs , and 3 ipcs ) revealed that pcs are neither underpinned by a highly recurrent fusion gene or a highly recurrent mutation .
of the 35 significantly mutated genes present in the tcga analysis of breast cancers ( cancer genome atlas , 2012 ) , five ( pik3ca , tp53 , map3k1 , akt1 , ctcf ) were also found to be mutated in the pcs subjected to rna - sequencing .
although potentially pathogenic mutations in lama5 and macf1 were found to be recurrent in pcs , they were present in only two out of 8 pcs tested , and could also be found in a subset of the common - type breast cancers analyzed by the tcga , suggesting that pcs are unlikely to be driven by a highly recurrent mutation in a way akin to lobular carcinomas of the breast , which harbor cdh1 gene mutations in > 60% of cases
first , the small number of cases subjected to gene expression profiling could have resulted in a type ii or error in the class comparison analysis of the subtypes of pcs ; therefore , this comparative analysis should be perceived as exploratory and hypothesis - generating .
although only 8 pcs were subjected to rna - sequencing for the discovery of a highly recurrent expressed fusion gene or mutation , a type ii or error is unlikely , given that with 8 samples we should have been able to identify a recurrent fusion gene at a prevalence similar to that of etv6-ntrk3 in secretory carcinomas ( tognon et al .
, 2002 ) or myb - nfib in adenoid cystic carcinomas ( wetterskog et al . , 2012 ) , or a recurrent mutation found at a prevalence similar to foxl2 mutations in granulosa cell tumors of the ovary ( shah et al .
it should be noted that if different subtypes of papillary carcinomas would be driven by distinct fusion genes or mutations , we would not be sufficiently powered to detect these events with the eight cases ( i.e. three ipcs , three epcs and two spcs ) subjected to massively parallel sequencing analysis in this study . given that rna - sequencing was only performed in the neoplastic tissues , only expressed mutations could be identified and the de novo discovery of somatic mutations was not trivial ; however , our analysis revealed mutations in pcs that have also been found in er - positive luminal idc - nsts ( cancer genome atlas , 2012 ) . finally , given the retrospective nature of this cohort of pcs , a detailed correlation between the genomic or transcriptomic features of pcs identified in this study with the outcome of pc patients could not be performed .
in fact , given that pcs have an indolent clinical course and rarely metastasize , future analyses investigating the impact of the transcriptomic differences between pcs and idc - nsts of the same histologic grade and er - status identified here on the outcome of breast cancer patients are warranted . despite these limitations , our study confirms that pcs of the breast preferentially display a luminal phenotype , have relatively simple genomic profiles , and are unlikely to be underpinned by a highly recurrent fusion gene or a pathognomonic expressed mutation . in addition , our findings offer insights into the molecular basis of the relatively indolent behavior and distinct morphological features of the three subtypes of pcs .
when compared to that of idc - nsts of the same grade and histologic type , pcs express proliferation - related genes at lower levels , features consistent with those of er - positive cancers with an indolent clinical behavior , and display lower levels of expression of genes related to cell adhesion , migration and movement , suggestive of a less invasive phenotype .
sp , ckyn and cae interpreted results and drafted the manuscript ; sp , ckyn , lgm , cfc , rn , f - cb and pmw carried out experiments and analyzed data ; om and av - s provided samples and interpreted data . | papillary carcinoma ( pc ) is a rare type of breast cancer , which comprises three histologic subtypes : encapsulated pc ( epc ) , solid pc ( spc ) and invasive pc ( ipc ) .
microarray - based gene expression and affymetrix snp 6.0 gene copy number profiling , and rna - sequencing revealed that pcs are luminal breast cancers that display transcriptomic profiles distinct from those of grade- and estrogen receptor ( er)-matched invasive ductal carcinomas of no special type ( idc - nsts ) , and that the papillary histologic pattern is unlikely to be underpinned by a highly recurrent expressed fusion gene or a highly recurrent expressed mutation . despite displaying similar patterns of gene copy number alterations ,
significant differences in the transcriptomic profiles of epcs , spcs and ipcs were found , and may account for their different histologic features . |
residual provisional cements and debris on prepared abutment teeth may negatively influence the performance of the definitive luting agent.1 apart from the choice of restorative materials , clinical outcome may be influenced by factors such as tooth preparation , preparation coarseness , type of luting agent , fit of restoration , type of provisional cement and also cleansing techniques used to remove the remnants of provisional cements .
indirect restorations usually require temporization for protection of the pulp and to restore the patients esthetic and functional needs .
terata2 showed that both residual zinc oxide eugenol and non - eugenol containing temporary cements reduced the tensile bond strength of resin luting agents .
for this reason , it is imperative to remove any remnants of the provisional luting agent .
grasso et al.3 showed that pumice cleansing was known to be more effective than other cleansing techniques such as explorer / air - water technique or with 0.12% chlorhexidine gluconate .
hlsmann et al.4 showed that 17% edta solution has a good cleaning effect on the root canal walls and removes the smear layer by dissolving the inorganic components .
for this reason , the resin must wet the dentinal surface to produce sound adhesion .
the manner in which liquid spreads on a surface expresses the wettability of the surface .
high wettability provides intimate contact and enhanced adhesion.5 contamination of provisional cement on dentin surface may interfere with the spreading and penetration of resin through the dentinal tubules.6 to increase the bond strength of resin , acids have been used to demineralize the dentin surface and to remove the subsequent debris and remnants of provisional cements present in it . this in vitro study
was aimed to evaluate 1 ) the shear bond strength of resin luting agent to dentin surfaces cleansed with different agents like pumice , ultrasonic scaler with chlorhexidine gluconate , edta .
in addition mode of failure was observed under scanning electron microscope , 2 ) the influence of these cleansing methods on wetting properties of the dentin by axisymmetric drop shape analysis - contact diameter technique ( adsa - cd ) , and 3 ) cleanliness of resulting dentin surfaces under a field emission scanning electron microscope .
freshly extracted 40 unrestored , caries free third molars were cleaned and the coronal portion was separated mesiodistally at the central fossa with a water cooled diamond coated disc .
the resultant 80 specimens were then mounted with the buccal or lingual surfaces facing upward with autopolymerizing resin ( denture base polymer resin , dpi - rr cold cure , new delhi , india ) ( fig .
the buccal and lingual surfaces were prepared with a standard - grit diamond rotary cutting instrument until the dentin surface was reached and preparation was finished with a fine - grit diamond instrument . to simulate the provisional restoration , discs ( 3 mm 1 mm )
were made with autopolymerizing resin ( dpi , self cure tooth moulding powder , new delhi , india ) ( fig .
2 ) and luted with one of the provisional cements :
group a - zinc oxide eugenol ( dental products of india ltd , new delhi , india)group b - non eugenol cement ( lot no : 409274 , rely x temp ne , 3 m espe , germany ) .
group a - zinc oxide eugenol ( dental products of india ltd , new delhi , india ) group b - non eugenol cement ( lot no : 409274 , rely x temp ne , 3 m espe , germany ) .
after that , provisional cements were mechanically removed with explorer until the dentin surface appeared macroscopically clean and then specimens were divided into 4 subgroups depending upon the cleansing methods used to clean the dentin surface
subgroup i control group air water spraysubgroup ii cleaned with pumice prophylaxissubgroup iii cleaned with ultrasonic scaler using 0.2% chlorhexidine gluconate as irrigantsubgroup iv scrubbed with cotton pellet soaked in 17% edta ( lot no:110224 glyde , dentsply , usa ) for 15 seconds
subgroup i control group air water spray subgroup ii cleaned with pumice prophylaxis subgroup iii cleaned with ultrasonic scaler using 0.2% chlorhexidine gluconate as irrigant subgroup iv scrubbed with cotton pellet soaked in 17% edta ( lot no:110224 glyde , dentsply , usa ) for 15 seconds after this , the cleansed specimens were equally divided into three groups and subjected to shear bond test , contact angle test and sem respectively . to test the bond strength of composites bonded on to the cleansed dentin surface the following procedures were carried on : a composite material ( esthet x micromatrix restorative dentsply , l.d .
caulk division , milford , de , usa ) was placed into a plastic transparent mould of 3 mm diameter and 1 mm height and cured for 20 seconds .
dentin surfaces were etched , dentin adhesive and dentin bonding agent were applied according to manufacturer 's instructions .
resin luting agent ( lot no : p2 1188 , variolink ii , dual curing luting composite system , ivoclar vivadent , liechtenstein ) was placed in between composite and dentin surface .
each specimen was polymerized for 40 seconds at a distance of 1 mm using visible light polymerizing unit ( blue phase c5 , ivoclar vivadent , liechtenstein ) and specimens were stored in distilled water at room temperature for 24 hours .
the type of failure was observed by scanning electron microscope at a magnification of 1000. the nature of failure was noted as adhesive , cohesive or mixed ( figs .
the dentin surfaces that were contaminated with provisional cement and cleansed with different techniques were wetted with water and dentin wetting was analyzed by measuring the contact angle of water .
this technique permits measurement of the contact angle of sessile drops when they have a flat profile .
one 10 l drop of " deionized " water was placed on the cleansed dentin surface of the prepared specimen ( fig . 8) and
the drop image was captured with a micro video system when the drop was in equilibrium .
the video signal was transmitted to a computer that provided the contact angle values ( fig . 9 ) .
the mean , standard deviation and test of significance of mean values between the groups were studied .
multiple range tukey hsd procedure was employed to identify the significant groups at 5% level and pair t - test was used to compare the shear bond strength and contact angle values among the groups . to evaluate the effect of the cleansing technique on the dentin surface , specimens were prepared as already mentioned .
all specimens were allowed to dry overnight and were then gold - sputtered and examined under scanning electron microscope at 15 kv .
freshly extracted 40 unrestored , caries free third molars were cleaned and the coronal portion was separated mesiodistally at the central fossa with a water cooled diamond coated disc .
the resultant 80 specimens were then mounted with the buccal or lingual surfaces facing upward with autopolymerizing resin ( denture base polymer resin , dpi - rr cold cure , new delhi , india ) ( fig .
the buccal and lingual surfaces were prepared with a standard - grit diamond rotary cutting instrument until the dentin surface was reached and preparation was finished with a fine - grit diamond instrument . to simulate the provisional restoration , discs ( 3 mm 1 mm )
were made with autopolymerizing resin ( dpi , self cure tooth moulding powder , new delhi , india ) ( fig .
2 ) and luted with one of the provisional cements :
group a - zinc oxide eugenol ( dental products of india ltd , new delhi , india)group b - non eugenol cement ( lot no : 409274 , rely x temp ne , 3 m espe , germany ) .
group a - zinc oxide eugenol ( dental products of india ltd , new delhi , india ) group b - non eugenol cement ( lot no : 409274 , rely x temp ne , 3 m espe , germany ) .
after that , provisional cements were mechanically removed with explorer until the dentin surface appeared macroscopically clean and then specimens were divided into 4 subgroups depending upon the cleansing methods used to clean the dentin surface
subgroup i control group air water spraysubgroup ii cleaned with pumice prophylaxissubgroup iii cleaned with ultrasonic scaler using 0.2% chlorhexidine gluconate as irrigantsubgroup iv scrubbed with cotton pellet soaked in 17% edta ( lot no:110224 glyde , dentsply , usa ) for 15 seconds
subgroup i control group air water spray subgroup ii cleaned with pumice prophylaxis subgroup iii cleaned with ultrasonic scaler using 0.2% chlorhexidine gluconate as irrigant subgroup iv scrubbed with cotton pellet soaked in 17% edta ( lot no:110224 glyde , dentsply , usa ) for 15 seconds after this , the cleansed specimens were equally divided into three groups and subjected to shear bond test , contact angle test and sem respectively .
to test the bond strength of composites bonded on to the cleansed dentin surface the following procedures were carried on : a composite material ( esthet x micromatrix restorative dentsply , l.d .
caulk division , milford , de , usa ) was placed into a plastic transparent mould of 3 mm diameter and 1 mm height and cured for 20 seconds .
dentin surfaces were etched , dentin adhesive and dentin bonding agent were applied according to manufacturer 's instructions . resin luting agent ( lot no : p2 1188 , variolink ii , dual curing luting composite system , ivoclar vivadent , liechtenstein ) was placed in between composite and dentin surface .
each specimen was polymerized for 40 seconds at a distance of 1 mm using visible light polymerizing unit ( blue phase c5 , ivoclar vivadent , liechtenstein ) and specimens were stored in distilled water at room temperature for 24 hours .
the type of failure was observed by scanning electron microscope at a magnification of 1000. the nature of failure was noted as adhesive , cohesive or mixed ( figs .
for contact angle measurements , the dentin surfaces that were contaminated with provisional cement and cleansed with different techniques were wetted with water and dentin wetting was analyzed by measuring the contact angle of water . for contact angle measurement ,
this technique permits measurement of the contact angle of sessile drops when they have a flat profile .
one 10 l drop of " deionized " water was placed on the cleansed dentin surface of the prepared specimen ( fig . 8) and
the drop image was captured with a micro video system when the drop was in equilibrium .
the video signal was transmitted to a computer that provided the contact angle values ( fig .
the mean , standard deviation and test of significance of mean values between the groups were studied .
multiple range tukey hsd procedure was employed to identify the significant groups at 5% level and pair t - test was used to compare the shear bond strength and contact angle values among the groups .
to evaluate the effect of the cleansing technique on the dentin surface , specimens were prepared as already mentioned .
all specimens were allowed to dry overnight and were then gold - sputtered and examined under scanning electron microscope at 15 kv .
in group a , the mean shear bond strength value of group iv was significantly higher than the mean shear bond strength value of all other groups ( table 1 ) . in group b , the mean shear bond strength value of group iv was significantly higher than the mean shear bond strength value of group ii followed by group iii and group i ( table 2 ) .
the failure types for group a is presented in table 4 and for group b in table 5 . in group a , the mean contact angle of group iv was significantly lower than the group i and group iii ( table 6 ) . in group b ,
the mean contact angle value of group iv was significantly lower than the mean contact angle of group ii followed by group iii and group i ( table 7 ) .
the sem observation of the specimen showed that dentinal tubules were completely covered by the smear layer and the remnants of the provisional cements on the dentin surface .
widespread remnants of the provisional cements were seen on the micrograph of the untreated dentin surface .
the use of pumice prophylaxis for both zinc oxide eugenol and freegenol groups produced a smoother and cleaner surface than for other groups , despite formation of large remnant particles .
the remnants smeared to the surface were more evident on the dentin cleansed with the ultrasonic scaler with 0.2% chlorhexidine gluconate irrigant ( figs .
the adverse effects of residual eugenol from the provisional cements on the bonding characteristics of subsequent restorations have been well documented.7 - 10 several investigators2,3 have hypothesized that different cleaning techniques for the removal of the provisional cement remnants from the dentin , like air - water spray , pumice prophylaxis , and use of cleansing agents affect the bond strength of resin luting agent to the dentin and the water contact angle of the dentin surface .
after tooth preparation , the dentin is covered with a smear layer that is composed primarily of cut , mineralized collagen fibrils . when eugenol containing , provisional cement is applied on the smear layer , eugenol leaches into and through the smear layer to the dentin tubules , contaminating the dentin surface.11 various techniques of removing the smear layer
have been used in literature.12 - 14 in comparison , edta and pumice prophylaxis have been found to be more effective in cleaning the remaining dentinal surface of residual debris and the smear layer.3,12,15,16 cameron17 claimed that cleanliness is achieved following ultrasonically agitated irrigation with distilled water .
dentin bonding is achieved by resin penetration into dentinal tubules , and into interfibrillar spaces .
higher shear bond strengths have been achieved with dual cure cements rather than the chemical cure cements18 and lafuente et al.19 showed that dual cements had higher bond strength to dentin than to enamel .
the rationale of using this material is to have a material with extended working time and capable of reaching a high degree of conversion to have a better bond .
hlsmann et al.4 have reported that 17% edta has a good cleansing effect on dentin and removes the smear layer by dissolving the inorganic compounds .
the shear bond strength achieved with pumice prophylaxis was found to be higher than ultrasonic cleansing and air - water spray ( control ) , which is in accordance with the studies by grasso et al.3 ultrasonic scaling with 0.2% chlorhexidine gluconate has showed lower shear bond strength compared to other cleansing regimes which is in accordance with the study by abott et al.15 the shear bond strength of the freegenol group ( group b ) in our study also shows that 17% edta has better shear bond strength values compared to pumice and ultrasonic scaler with 0.2% chlorhexidine gluconate . here in this group edta
has good efficiency in removing the remnants of provisional cement and the smear layer covering the dentinal tubules compared to eugenol group ( table 3 ) .
specimens treated with freegenol cement have beneficial effects on the shear bond strength values compared to those treated with zinc oxide eugenol cement which is in accordance with the studies conducted by various investigators.7 - 9,20 acid etching is a procedure that changes substrate conditions because it eliminates the dentin mineral content and exposes the collagen layer.7 on the etched substrate , adhesive resins penetrate and forms the hybrid layer.21 acid etching dissolves the residual eugenol , along with the microscopic remnants.7 also conditioning of dentin surface with 37% phosphoric acid removes the smear layer and smear plugs and opens the dentinal tubules , in addition to demineralizing the intertubular and peritubular dentin . in this present study , although all specimens were etched with 37% phosphoric acid , and adhesive resins were used , shear bond strengths showed significant differences .
it was concluded that the cleansing techniques had considerable effect on the bond strength of the resin luting agent .
the modes of failures for specimens were of 3 types : adhesive , cohesive and mixed .
mixed and cohesive fractures were the most common types of failure for all groups ( tables 4 , 5 ) . however , the control specimens of both the groups showed adhesive failures .
edta and pumice shows cohesive failure for both zinc oxide eugenol and freegenol cements ( figs .
the lowest contact angle were obtained with 17% edta for both zinc - oxide eugenol and freegenol groups , which indicates that edta is more effective at removal of the remnant of the provisional cement and the smear layer , so edta creates appropriate physical and chemical interactions between the resin cement and dentin .
sem observation shows that 17% edta was the most effective solution to remove the smear layer .
kielbassa et al.11 showed that diffusion of eugenol may be more than 200 m in depth , so that acid etching may not eliminate the effects of eugenol .
that negative effects of eugenol on dentin adhesion are not based on the wetting process but on adhesion test results.5 the result of this present study suggests that the shear bond strength and wettability of dentin were affected not only by the acid etching and or dentin primers but also dentin cleansing techniques . in this study
, edta has been used as an effective dentinal cleansing agent even in the presence of eugenol in an in vitro situation .
the contact angle measurement , in an in vivo situation , would consider various factors like thermal and mechanical changes , oral fluid contaminations , organic and inorganic residues , etc .
further , a three dimensional model of the bonding surfaces would better reflect the failures which occur as a combination of various compressive , tensile and shearing forces .
future advancements in research methodologies including the finite element method for a 3d model and a digitized adsacd technique could be incorporated to achieve higher levels of accuracy and relevance .
within the limitation of this study , it was concluded that
significantly different shear bond strength were found with the different cleansing techniques evaluated.specimens treated with edta showed the highest shear bond strength for both zinc oxide eugenol and freegenol groups .
higher shear bond strength values were also obtained for pumice prophylaxis when compared to control group and ultrasonic scaler group.edta showed the lowest contact angle for both the groups.sem showed that edta was the most effective solution to remove the smear layer , and pumice prophylaxis leaves large remnant particles . also , mode of failure seen was predominantly cohesive for both edta and pumice prophylaxis .
specimens treated with edta showed the highest shear bond strength for both zinc oxide eugenol and freegenol groups .
higher shear bond strength values were also obtained for pumice prophylaxis when compared to control group and ultrasonic scaler group .
sem showed that edta was the most effective solution to remove the smear layer , and pumice prophylaxis leaves large remnant particles .
also , mode of failure seen was predominantly cohesive for both edta and pumice prophylaxis . | purposeto evaluate the shear bond strength of resin luting agent to dentin surfaces cleansed with different agents like pumice , ultrasonic scaler with chlorhexidine gluconate , edta and the influence of these cleansing methods on wetting properties of the dentin by axisymmetric drop shape analysis - contact diameter technique ( adsa - cd).materials and methodsforty coronal portions of human third molar were prepared until dentin was exposed .
specimens were divided into two groups : group a and group b. provisional restorations made with autopolymerizing resin were luted to dentin surface with zinc oxide eugenol in group a and with freegenol cement in group b. all specimens were stored in distilled water at room temperature for 24 hrs and provisional cements were mechanically removed with explorer and rinsed with water and cleansed using various methods ( control - air - water spray , pumice prophylaxis , ultrasonic scaler with 0.2% chlorhexidine gluconate , 17% edta ) .
contact angle measurements were performed to assess wettability of various cleansing agents using the adsa - cd technique .
bond strength of a resin luting agent bonded to the cleansed surface was assessed using instron testing machine and the mode of failure noted .
sem was done to assess the surface cleanliness .
data were statistically analyzed by one - way analysis of variance with tukey hsd tests ( =.05).resultsspecimens treated with edta showed the highest shear bond strength and the lowest contact angle for both groups .
sem showed that edta was the most effective solution to remove the smear layer .
also , mode of failure seen was predominantly cohesive for both edta and pumice prophylaxis.conclusionedta was the most effective dentin cleansing agent among the compared groups . |
a 42-year - old male patient visited the oral medicine and radiology unit of nhdc , mumbai in 2009 .
he was referred by his family dentist , complaining pain in the left mandibular angle region .
he had difficulty in chewing on the left side and the mouth opening was normal . on the examination ,
the left mandibular third molar appeared completely erupted and well aligned , however a tooth - like structure with black discoloration appeared to be embedded distal to the third molar ( fig .
the provisional diagnosis was pericoronitis and a lateral oblique radiograph was advised in order to evaluate the region accurately .
the patient did not show any structure beyond the third molars in three other quadrants .
the lateral oblique radiograph for the left mandible revealed the presence of a partially impacted supernumerary tooth distal to the third molar .
the shape of the supernumerary tooth was similar with a molar but smaller in size , single rooted and distally inclined , thus it could be defined as a supplementary distomolar .
we decided to extract the fourth molar since the patient had discomfort due to pericoronitis and irreversible pulpitis .
a 36-year - old male patient visited our department with a complaint of missing tooth in the lower left premolar area .
on the clinical examination , it was noted that besides the missing tooth , the patient also had a supernumerary tooth distal to the right maxillary third molar which was placed palatal and distal to the third molar .
the roots of the third molar and the supernumerary tooth were overlapped and difficult to visualize them independently , however the morphology of the crown resembled a mini - sized molar , hence it was diagnosed as a supplementary distomolar ( fig .
as there was no complaint and the fourth molar was not clinically impeding the patient 's day to day activity , it was decided not to give any further treatment .
a 36-year - old male patient visited our department with a complaint of replacement of missing teeth .
in addition , a supernumerary premolar - like tooth , distal to the right maxillary second premolar was observed , as well as a second premolar in the left maxilla was missing .
the upper third molars in both sides were erupted and well aligned in the arch , while the mandibular third molars in both sides were not found clinically . in the lower right quadrant , a total of four , well - aligned , premolar - like teeth were observed .
the tooth distal to the canine appeared as the first premolar , the tooth distal to that appeared as a mini - sized second premolar , and the following two teeth had the same anatomy and size with the second premolar .
thus , it was difficult to distinguish them as rather permanent or supplementary . in the lower left quadrant , the first premolar had caries and the second premolar was retained root state , followed by two more teeth ( fig .
3a ) . due to the destruction of the crown , it was difficult to distinguish which was the permanent or the supplementary tooth .
panoramic radiograph confirmed these clinical findings and additionally revealed an impacted supernumerary tooth mesial to the first molar in the left mandible ( fig .
3b ) . there was no impacted lower third molar in the mandible . it was decided not to extract the erupted supernumerary teeth since the patient had no complaint and the occlusion was not impeded .
however , before giving prosthesis in the upper right first molar region , alignment of the supernumerary had to be considered .
the patient was referred for endodontic treatment of the caries lesion of the lower right premolar and to an oral surgeon for extraction of the retained root .
also , the opinion on the impacted supernumerary tooth was requested if it could affect the lower left first molar or the impacted supernumeraries would develop into cystic lesions .
a 35-year - old male patient visited our department with a chief complaint of mild pain and food impaction in the upper right posterior region . on the examination , the upper right first molar had caries . on further intraoral examination
, it was found that the patient had three supernumerary teeth lingual and distal to the second premolar in the lower right quadrant ( fig .
since the patient had pain on the upper right first molar , the endodontist suggested that the patient needed to extract the supplementary teeth in the lower right mandible to prevent food impaction on that area .
the patient was referred to an oral surgeon to extract them and panoramic radiograph was taken .
the radiograph showed an impacted supernumerary tooth on the left lower mandibular area , which was placed apical to the lower left second premolar ( fig .
an intraoral radiograph of the same region also showed the presence of a dilacerated premolar - like tooth ; its root was placed apical to the mesial root of the mandibular first molar , and the crown was in the apical and buccal aspect of the left mandibular second premolar .
a 20-year - old male patient visited our department for routine dental check up , prophylaxis , and replacement of the missing left lower molars which had been extracted . on intraoral examination
, the left mandibular first and second molars were missing and a rotated supplementary premolar - like tooth , distal to the second premolar , was observed . on the right side of the mandible ,
the first molar was missing and there were two supplementary premolar - like teeth which were rotated and placed lingual to the premolars ( fig .
panoramic radiograph confirmed these clinical findings , and additionally revealed an impacted supernumerary tooth below the left mandibular premolars , with the crown facing towards the midline .
the apex of the impacted supernumerary tooth was below and distal to the supplementary tooth ( fig .
the patient was referred to a prosthodontist to get an opinion if the presence supernumeraries in the lower right region might impede the placement of prosthesis .
the patient was also informed not to extract the impacted supernumerary tooth as it was asymptomatic .
a 42-year - old male patient visited the oral medicine and radiology unit of nhdc , mumbai in 2009 .
he was referred by his family dentist , complaining pain in the left mandibular angle region .
he had difficulty in chewing on the left side and the mouth opening was normal . on the examination ,
the left mandibular third molar appeared completely erupted and well aligned , however a tooth - like structure with black discoloration appeared to be embedded distal to the third molar ( fig .
the provisional diagnosis was pericoronitis and a lateral oblique radiograph was advised in order to evaluate the region accurately .
the patient did not show any structure beyond the third molars in three other quadrants .
the lateral oblique radiograph for the left mandible revealed the presence of a partially impacted supernumerary tooth distal to the third molar .
the shape of the supernumerary tooth was similar with a molar but smaller in size , single rooted and distally inclined , thus it could be defined as a supplementary distomolar .
we decided to extract the fourth molar since the patient had discomfort due to pericoronitis and irreversible pulpitis .
a 36-year - old male patient visited our department with a complaint of missing tooth in the lower left premolar area .
on the clinical examination , it was noted that besides the missing tooth , the patient also had a supernumerary tooth distal to the right maxillary third molar which was placed palatal and distal to the third molar .
the roots of the third molar and the supernumerary tooth were overlapped and difficult to visualize them independently , however the morphology of the crown resembled a mini - sized molar , hence it was diagnosed as a supplementary distomolar ( fig .
as there was no complaint and the fourth molar was not clinically impeding the patient 's day to day activity , it was decided not to give any further treatment .
a 36-year - old male patient visited our department with a complaint of replacement of missing teeth .
in addition , a supernumerary premolar - like tooth , distal to the right maxillary second premolar was observed , as well as a second premolar in the left maxilla was missing .
the upper third molars in both sides were erupted and well aligned in the arch , while the mandibular third molars in both sides were not found clinically . in the lower right quadrant , a total of four , well - aligned , premolar - like teeth were observed .
the tooth distal to the canine appeared as the first premolar , the tooth distal to that appeared as a mini - sized second premolar , and the following two teeth had the same anatomy and size with the second premolar .
thus , it was difficult to distinguish them as rather permanent or supplementary . in the lower left quadrant , the first premolar had caries and the second premolar was retained root state , followed by two more teeth ( fig .
3a ) . due to the destruction of the crown , it was difficult to distinguish which was the permanent or the supplementary tooth
panoramic radiograph confirmed these clinical findings and additionally revealed an impacted supernumerary tooth mesial to the first molar in the left mandible ( fig .
it was decided not to extract the erupted supernumerary teeth since the patient had no complaint and the occlusion was not impeded .
however , before giving prosthesis in the upper right first molar region , alignment of the supernumerary had to be considered .
the patient was referred for endodontic treatment of the caries lesion of the lower right premolar and to an oral surgeon for extraction of the retained root .
also , the opinion on the impacted supernumerary tooth was requested if it could affect the lower left first molar or the impacted supernumeraries would develop into cystic lesions .
a 35-year - old male patient visited our department with a chief complaint of mild pain and food impaction in the upper right posterior region . on the examination , the upper right first molar had caries . on further intraoral examination
, it was found that the patient had three supernumerary teeth lingual and distal to the second premolar in the lower right quadrant ( fig .
4a ) . since the patient had pain on the upper right first molar , the endodontist suggested that the patient needed to extract the supplementary teeth in the lower right mandible to prevent food impaction on that area .
the patient was referred to an oral surgeon to extract them and panoramic radiograph was taken .
the radiograph showed an impacted supernumerary tooth on the left lower mandibular area , which was placed apical to the lower left second premolar ( fig .
an intraoral radiograph of the same region also showed the presence of a dilacerated premolar - like tooth ; its root was placed apical to the mesial root of the mandibular first molar , and the crown was in the apical and buccal aspect of the left mandibular second premolar .
a 20-year - old male patient visited our department for routine dental check up , prophylaxis , and replacement of the missing left lower molars which had been extracted . on intraoral examination , the left mandibular first and second molars were missing and a rotated supplementary premolar - like tooth , distal to the second premolar , was observed . on the right side of the mandible ,
the first molar was missing and there were two supplementary premolar - like teeth which were rotated and placed lingual to the premolars ( fig .
panoramic radiograph confirmed these clinical findings , and additionally revealed an impacted supernumerary tooth below the left mandibular premolars , with the crown facing towards the midline .
the apex of the impacted supernumerary tooth was below and distal to the supplementary tooth ( fig .
the patient was referred to a prosthodontist to get an opinion if the presence supernumeraries in the lower right region might impede the placement of prosthesis .
the patient was also informed not to extract the impacted supernumerary tooth as it was asymptomatic .
yusof in 19907 reported that the premolar region in the lower arch was the most common area having supernumerary teeth . in our cases ,
three cases showed the presence of the multiple supernumerary teeth in the premolar area of all the quadrants and two cases showed an extra molar distal to the third molar .
the occurrence of non - syndromic supernumerary teeth is more often in maxilla than in mandible,8 in male than in female,9 in permanent dentition than in primary dentition , and unilaterally than bilaterally.10 patients with non - syndromic supernumerary teeth might be related to heredity factor , therefore familial history should be carefully examined .
hegde and munshi11 and mason et al12 reported the displacement , rotation , ectopic eruption , and malocclusion of adjacent teeth due to supernumerary teeth in their studies , and similar alterations were noted in our cases , related to the position and orientation of the supernumerary teeth .
the clinical and radiographic examinations are important for a substantial treatment plan which varies from simple extractions to orthodontic / prosthetic treatment for obtaining correct occlusion .
however , it is difficult to establish an ideal treatment for those of multiple supernumeraries cases .
patient with non - syndromic supernumerary teeth need to have periodical radiographic observation if he / she is asymptomatic .
then , it is advisable to remove these supernumerary teeth immediately if it starts to show any pathologic change . in all the five reported cases except for case 1 , the patients had no discomfort associated with the supernumerary teeth .
however , we still emphasize the importance of a precise clinical history and radiographic examination when patients with multiple supernumerary teeth visit for consultation since most of them are associated with other dental anomalies and syndromes .
then the related clinical problems should be considered primarily than treating the presence of the supernumerary teeth . | hyperdontia or supernumerary teeth without associated syndrome is a rare phenomenon , as supernumerary teeth are usually associated with cleft lip and palate or other syndromes such as gardner 's syndrome , cleidocranial dysplasia , and so on .
five patients with supernumerary teeth visited our department .
they had no familial history or other pathology , certain treatment protocols was modified due to the presence of supernumerary teeth .
non - syndromic supernumerary teeth , if asymptomatic , need to have periodical radiographic observation .
if they showed no variation as they impacted in the jaw , careful examination is necessary because they may develop into pathological status such as dentigerous cysts .
the importance of a precise clinical history and radiographic examination for patients with multiple supernumerary teeth should be emphasized . |
infantile hemangioma ( ih ) is the most common benign tumor of infancy,1 affecting nearly 10% of children under 1 year old and 30% of premature babies .
prematurity , older maternal age , preeclampsia , and multiple gestations have been suggested to be risk factors of the development of ih.2 approximately 60% of hemangiomas are located at the head and neck regions,3 and they present with a 2.4:1 ratio of females to males.4 their large size , facial location , and/or segmental morphology are the major risk factors associated with the development of complications including ulceration , functional impairment , and permanent disfigurement.5 as most ihs can involute spontaneously , there is still great controversy over the management of ih .
however , consensus was achieved in that when the tumor gives rise to infection , ulceration , necrosis and bleeding resulting in serious disfigurement , dysfunctions and even threatening life , treatment is urgently required .
the lesions that potentially impair vital function or cause life - threatening complications are described as alarming hemangiomas.6 there is no gold standard for alarming hemangioma .
recombinant interferon alpha ( ifn- ) , an inhibitor of angiogenesis , has been used successfully in treating complicated ih , particularly in hemangiomas that have failed to respond to oral propranolol.7,8 however , concerns of irreversible neurotoxicity , especially spastic diplegia , handicap the application of ifn-.911 here , we present a study where we treated eleven chinese cases of alarming hemangioma with ifn-2a .
the study was approved by the ethics committee at the ninth people s hospital , shanghai jiao tong university ( shanghai , people s republic of china ) .
eleven consecutive infants ( nine girls and two boys ) with alarming hemangiomas in the head and neck region who failed to respond to a 2-week course of propranolol or corticosteroid therapy , and who received a subcutaneous injection of ifn- in our institution between january 2009 and december 2010 were enrolled in this study .
all patients had large ( > 5 cm in diameter ) or multiple hemangiomas ( 2 sites ) in the head and neck region ; in three patients , the hemangioma caused airway obstruction .
the demographic data regarding each patient s age , sex , site of lesion , and duration of treatment are shown in table 1 .
the diagnosis of hemangiomas was established mainly by obtaining the patient s history , noting clinical manifestations , and performing a physical examination .
all patients underwent ultrasonography , and four patients had a magnetic resonance imaging ( mri ) examination to obtain more details of the lesion ( when the diagnosis was uncertain , mri was performed ) .
after informed consent had been obtained from the parents according to institutional guidelines , the patients were treated with ifn-2a for a period of 24.5 months .
ifn-2a was administrated subcutaneously once daily at a dose of 310 units / m per day .
periodical baseline electrocardiogram , neurologic examination , and laboratory evaluations of hepatic transaminases were performed .
the size and extent of the deep - seated lesions were monitored by serial color doppler ultrasonography ; the percent change of the superficial lesions compared the pre- and posttreatment tumor size using photographs .
the patients were observed at an interval of 3 weeks during the therapy course ; follow - up visits were continued for up to 2 years .
the final results were assessed by two other physicians from the department of oral oncology , depending on the clinical examinations , photographs , and doppler ultrasonography taken before and after treatment ; the response to ifn-2a therapy was evaluated according to the following scale ( that was modified following achauer et al12 ) : scale 1 , poor response ( shrinkage 0%25% ) ; scale 2 , fair response ( shrinkage 26%50% ) ; scale 3 , good response ( shrinkage 51%75% ) ; scale 4 , excellent response ( shrinkage 76%100% ) .
eleven consecutive infants ( nine girls and two boys ) with alarming hemangiomas in the head and neck region who failed to respond to a 2-week course of propranolol or corticosteroid therapy , and who received a subcutaneous injection of ifn- in our institution between january 2009 and december 2010 were enrolled in this study .
all patients had large ( > 5 cm in diameter ) or multiple hemangiomas ( 2 sites ) in the head and neck region ; in three patients , the hemangioma caused airway obstruction .
the demographic data regarding each patient s age , sex , site of lesion , and duration of treatment are shown in table 1 .
the diagnosis of hemangiomas was established mainly by obtaining the patient s history , noting clinical manifestations , and performing a physical examination .
all patients underwent ultrasonography , and four patients had a magnetic resonance imaging ( mri ) examination to obtain more details of the lesion ( when the diagnosis was uncertain , mri was performed ) .
after informed consent had been obtained from the parents according to institutional guidelines , the patients were treated with ifn-2a for a period of 24.5 months .
ifn-2a was administrated subcutaneously once daily at a dose of 310 units / m per day .
periodical baseline electrocardiogram , neurologic examination , and laboratory evaluations of hepatic transaminases were performed .
the size and extent of the deep - seated lesions were monitored by serial color doppler ultrasonography ; the percent change of the superficial lesions compared the pre- and posttreatment tumor size using photographs .
the patients were observed at an interval of 3 weeks during the therapy course ; follow - up visits were continued for up to 2 years .
the final results were assessed by two other physicians from the department of oral oncology , depending on the clinical examinations , photographs , and doppler ultrasonography taken before and after treatment ; the response to ifn-2a therapy was evaluated according to the following scale ( that was modified following achauer et al12 ) : scale 1 , poor response ( shrinkage 0%25% ) ; scale 2 , fair response ( shrinkage 26%50% ) ; scale 3 , good response ( shrinkage 51%75% ) ; scale 4 , excellent response ( shrinkage 76%100% ) .
all eleven patients received the treatment for a duration of 24.5 months ( median : 3 months ) .
eight patients had received medication for 3 months , one for 4.5 months , and two for 2 months .
the age at initiation of ifn-2a therapy ranged from 3 days to 8 months ( median : 4 months ) .
it was observed that the most apparent regression of tumors happened at 1 month after medication administration ; the tumor shrank further with the red color fading . nine patients ( 82% ) achieved scale 4 ( figures 1a , b , 2a , and b ) and two patients ( 18% ) achieved scale 3 . the adverse effects included low fever ( six cases ; 54.5% ) , diarrhea ( two cases ; 18.2% ) , and anorexia ( one case ; 9.1% ) , as well as neutropenia and high levels of aminotransferases to varying degrees , which resolved after stopping the medication .
neurotoxicity such as spastic diplegia and other motor developmental disturbances were not found in this series .
no patients experienced the rebound phenomenon , and no patients required the restarting of ifn or another therapy in the following 2 years .
in this series of eleven infants with alarming hemangiomas treated with ifn-2a , rapid and good response to treatment was obtained with few reversible adverse effects .
in addition to its antiviral effect , ifn- was also used as an inhibitor of angiogenesis given its antiangiogenic properties.13 since its efficacy in pulmonary hemangiomatosis was first reported by white et al14 in 1989 , ifn- was chosen as a therapeutic modality for hemangiomas , especially for life - threatening cases or steroids - insensitive patients . according to other reports,1517
the rate of response to ifn in hemangiomas varies from 80%100% , which is very similar to our overall response rate of 100% ; our result was as good as those found in these studies ( p>0.05 ) , and the duration time was shorter ( p<0.05 ) .
the common adverse effects of ifn include influenza - like symptoms of fever , somnolence , anorexia , diarrhea or constipation , and a high level of aminotransferases ; these symptoms are always slight and they can be reversed following the discontinuation of ifn .
although exceptional , neurotoxicity is still the main concern in the treatment of ih with ifn .
epilepsy , spastic diplegia , and lower limb disability had been reported after ifn injection .
chang et al16 first reported in 1997 that one patient who received treatment with ifn-2b developed mild gross motor delay , which improved 2 months after the cessation of therapy .
the pathogenesis of ifn - related neurotoxic effects is unclear , but given its potential seriousness , many experts have limited the use of ifn- for alarming hemangiomas . according to a meta - analysis of a large sample , which was carried out by michaud et al9 in 2004 , eleven of 441 children ( 2.5% ) receiving ifn therapy for the treatment of vascular lesions developed spastic diplegia ;
perhaps there were not enough patients in our study to see this outcome , or it is also possible that the short duration of therapy decreased the risk for these children .
considering that the durations we used were only 24.5 months ( median : 3 months ) , we suggest that the short - term use of ifn- should be safe .
the current treatments of ihs include drug therapy , cryosurgery , laser therapy , and surgical excision .
oral corticosteroids ( for example , prednisolone , prednisone ) used to be the mainstay of therapy for hemangiomas , but their clinical application was limited by the adverse effects such as cushing s syndrome and growth retardation . since the impressive effect of propranolol in treating hemangioma was reported by laut - labrze et al18 in 2008 ,
many studies ( including our own experiences ) have confirmed its effectiveness and have reported few side effects in hemangiomas.1922 nowadays , propranolol has been our first - line medicine for treating ih , but there are still some patients with alarming hemangiomas who may be concomitant with a contraindication for propranolol such as asthma , cardiac failure , or failure to respond to this medicine . for these cases ,
the rapid and impressive responses , as well as the few side effects observed in this series without relapse after treatment with ifn-2a prompts us to suggest that the short - term ( < 4.5 months ) use of ifn-2a can be chosen as an alternative option for alarming hemangiomas in the head and neck region , which are resistant to corticosteroid or propranolol treatment . | objectiveto evaluate the efficacy and adverse effects of interferon-2a in the treatment of alarming infantile hemangiomas in the head and neck region.patients and methodsfrom january 2009december 2010 , a subcutaneous injection of interferon-2a was applied to eleven infants with giant multifocal or segmental hemangiomas at a dose of 3 million units / m2 per day .
all patients did not respond to propranolol or corticosteroids .
the age at initiation of interferon-2a therapy ranged from 3 days to 8 months ( median : 4 months ) .
the duration of therapy ranged from 24.5 months ( median : 3 months ) .
eight patients received medication for 3 months , one patient for 4.5 months , and two patients for 2 months.resultsnine patients had a reduction in tumor mass of 95% ; two patients tumors decreased in size by 75% .
the overall response rate was 100% .
the main adverse effects included fever , diarrhea , and anorexia , which resolved after stopping the medication .
no serious adverse effect was observed.conclusionshort-term treatment with interferon-2a can be used as a safe and effective treatment for alarming infantile hemangiomas that are resistant to propranolol or corticosteroids , and that endanger the proper functioning of the affected organ or the patient s life . |
age - related changes in human salivary glands have been described by some studies in humans
and animals
.
the literature regarding aging alterations in human sublingual glands is limited by the number of reports , and some phenomena are still unclear . despite the different methodologies used in the investigations of age - related microscopic changes in the salivary glands , the consensus is that there is a decrease in the volume of parenchyma that is accompanied by an increase in fat and connective tissue
.
this aging process also includes acinar atrophy and an increase in the number of duct - like structures ( dls )
( figure 1 ) . the term duct - like structures
is commonly used because there is no evidence that the structures are true ducts . for many researchers ,
the appearance of dls with age represents the final process of acinar atrophy , and the making the distinction between a duct and a dls can be very difficult
.
furthermore , the dls do not represent a specific phenomenon of senescence , and they also appear in atrophic glands
and glandular neoplasms
.
figure 1a : normal gland 26 years old ( group i ) . preserved acini and absence of duct - like structures ( dls ) are observed ; b : altered gland presence of dls , absence of acini and substitution of the glandular parenchyma by connective tissue 68 years old ( group ii ) ( hematoxylin and eosin 100x )
some authors also believe that , during the aging process , atrophic acini can resemble ducts , and the distinguishing between them can be very difficult or even impossible
, demonstrating that the morphology of aged salivary glands is not completely established .
regarding immunohistochemical studies related to the salivary glands , most of them only aim to characterize their epithelial and/or myoepithelial components
or to study pathological processes such as neoplasias
.
it is evident that the literature regarding age - related changes in salivary glands is not abundant . in humans , studies are scarce and the sublingual gland is rarely contemplated .
the increase in the number of dls allows speculation that a mitotic process is occurring within the cells or even a transition of an acinus to a duct
because the acini disappear while the dls increase in number .
the aim of this work is to evaluate the phenotype and the cell proliferation index of dls in human sublingual glands during the aging process .
this study was approved by the human research ethics committee of the bauru school of dentistry at the university of so paulo ( usp ) ( process n 59/2010 ) and also by the so paulo death verification service ( usp ) , where the glands were obtained .
the methods and the exclusion criteria were the same as those used by azevedo , et al .
individuals with the following systemic disorders or base diseases were excluded from the study : lymphoma , leukemia , mucoviscidosis , rheumatic diseases , sjgren s syndrome , obesity , cachexia , diabetes mellitus , alcoholic cirrhosis , collagen diseases , history of head or neck cancer - related surgery , history of cytotoxic drug administration , previous radiotherapy of the head or neck during the last 3 months and macroscopic autolysis .
data regarding systemic involvement and cause of death were obtained from the family and the autopsy reports .
the main causes of death included pulmonary edema , bronchopneumonia , acute myocardial infarction , cerebral vascular accident , ischemic heart disease , cerebral edema and congestive heart failure .
the interval between the time of death and the autopsy ranged from 6:05 to 92:55 h , with a mean time of 16:46 h. the cadaver glands were completely removed along with a portion of the overlying mucosa of the mouth floor .
the glands from the cadavers were divided into two groups : group i ( control ; 0 - 30 years old ) and group ii ( 60 - 90 years old ) .
the paraffin - embedded material was cut into 3 - 5 m - thick sequential slices . for the study of the cellular phenotype of the dls ,
antibodies against cytokeratin 19 ( ck 19 ; clone rck108 ; isotype igg1/mouse ; monoclonal ; dako , dako inc , carpinteria , ca , usa , cod#m0888 ; dilution 1:100 ) and the s-100 protein ( s-100 ; isotype igg / rabbit ; polyclonal ; dako , dako inc , carpinteria , ca , usa , cod#z0311 ; dilution 1:2000 ) were used .
the anti - ck 19 antibody was used to stain the cells of the ductal system
and anti s-100 to stain the serous acinar cells
.
the histochemical markers periodic acid - schiff ( pas ) and alcian blue ( ph 2.5 ) were used to stain the mucous acinar cells . for the analysis of the cell proliferation capacity of the dls , the ki-67 ( ki-67 ; clone mib-1 ; isotype igg1/mouse ; monoclonal ; dako , dako inc , carpinteria , ca , usa , cod#m720 ; dilution 1:400 ) antibody was used
.
the immunohistochemical study was performed using the polymeric horseradish peroxidase ( hrp)-linker antibody conjugate technique .
the material was incubated in a humidity chamber at 4c for 18 hours . considering the large s-100 protein family , the polyclonal rabbit anti - s-100
was used , which reacts strongly with all family members , including human s100a and b sub - units .
the specimens were counterstained with harris hematoxylin , and the slides were mounted using adhesive resin ( entellan , merck , darmstadt , hessen , germany ) and coverslips .
for all immunohistochemical markers , both a positive and a negative control were prepared according to the manufacturers recommendations .
a digital image - capture computer system ( axiocam mrc , zeiss , jena , thuringia , germany ) and a binocular microscope ( axioskop 2 plus , zeiss , jena , thuringia , germany ) with a 40x objective were used .
the images were analyzed using the software supplied with the microscope ( axiovision 4.5 , zeiss , jena , thuringia , germany ) .
each microscopic field was previously standardized in this program to avoid differences in the expression intensity of the antibodies . in each
captured microscopic field , the dls were counted to establish their percentage in the staining profile .
the most representative areas of the event ( spots ) , identified using a lower magnification , were selected .
when the dls were diffusely distributed and spread out , 20 spots were randomly selected .
in group i , all microscopic fields containing dls were captured and in group ii , 20 fields containing dls were randomly captured ( figure 2 ) .
true ducts or acini that were easily recognized were excluded from the count .
figure 2schematic representation of the selection of spots .
a : due to the small quantity of duct - like structures ( dls ) in group i , all microscopic fields containing dls were captured ; b : a spot containing three dls ; c : 20 fields containing dls were randomly captured in group ii ; d : diffuse distribution of dls in group ii
for the localization and the counting of the dls , slides stained with hematoxylin - eosin ( h.e . ) were used .
we considered dls to be those with shrunken seromucous cells , granule depletion and widened lumens that were adjacent to ductal epithelial cells and resembled intralobular non - striated ducts , which is consistent with the characterization used in our previous study
.
we excluded true excretory ducts , which exhibited a dilated lumen lined by cuboidal cells with central nuclei and an eosinophilic cytoplasm
.
a section of each gland was analyzed with each studied molecule , totaling 60 slides for each staining ( 30 per group ) .
therefore , a total of 360 slides were analyzed [ 60 for each marker : h.e . ,
pas , alcian blue ( ph 2.5 ) , ck 19 , s-100 and ki-67 ] .
a semi - quantitative scoring system was applied to the immunohistochemical and histochemical analysis [ adapted from soini , et al .
both analyses resulted in a final score that represented no expression , weak expression or strong expression .
in each microscopic field , the percentage of positive ( with partial or total staining ) dls was calculated for each marker .
scores ranging from 0 to 4 were attributed to those percentage values : 0 ) absence of positive dls ; 1 ) < 25% of positive dls ; 2 ) 26 - 50% of positive dls ; 3 ) 51 - 75% of positive dls ; 4 ) > 75% of positive dls .
then , for the dls positive samples , the partial or total covering and the intensity of the immunostaining in the positive cells were calculated following the same method applied previously . finally , the sum of the values attributed to the quantity , covering and intensity of the immunostaining resulted in a final score where 3 - 7 represented weak to moderate expression and 8 - 12 was a strong expression of the antibody .
basically , the pas and alcian blue staining in the dls followed the same scoring system , except for the intensity values , which did not exist for these stains .
the cellular proliferative activity in the dls , revealed by ki-67 staining , was calculated as the percentage of positive cells to establish a cellular proliferation index in the same previously captured fields .
the comparison between groups and between markers was accomplished by the student s t - test or the mann - whitney test . the correlation among the staining groups
the exclusion criteria were the same as those used by azevedo , et al .
individuals with the following systemic disorders or base diseases were excluded from the study : lymphoma , leukemia , mucoviscidosis , rheumatic diseases , sjgren s syndrome , obesity , cachexia , diabetes mellitus , alcoholic cirrhosis , collagen diseases , history of head or neck cancer - related surgery , history of cytotoxic drug administration , previous radiotherapy of the head or neck during the last 3 months and macroscopic autolysis .
data regarding systemic involvement and cause of death were obtained from the family and the autopsy reports .
the main causes of death included pulmonary edema , bronchopneumonia , acute myocardial infarction , cerebral vascular accident , ischemic heart disease , cerebral edema and congestive heart failure .
the interval between the time of death and the autopsy ranged from 6:05 to 92:55 h , with a mean time of 16:46 h. the cadaver glands were completely removed along with a portion of the overlying mucosa of the mouth floor .
the glands from the cadavers were divided into two groups : group i ( control ; 0 - 30 years old ) and group ii ( 60 - 90 years old ) .
the paraffin - embedded material was cut into 3 - 5 m - thick sequential slices .
for the study of the cellular phenotype of the dls , antibodies against cytokeratin 19 ( ck 19 ; clone rck108 ; isotype igg1/mouse ; monoclonal ; dako , dako inc , carpinteria , ca , usa , cod#m0888 ; dilution 1:100 ) and the s-100 protein ( s-100 ; isotype igg / rabbit ; polyclonal ; dako , dako inc , carpinteria , ca , usa , cod#z0311 ; dilution 1:2000 ) were used .
the anti - ck 19 antibody was used to stain the cells of the ductal system
and anti s-100 to stain the serous acinar cells
.
the histochemical markers periodic acid - schiff ( pas ) and alcian blue ( ph 2.5 ) were used to stain the mucous acinar cells . for the analysis of the cell proliferation capacity of the dls , the ki-67 ( ki-67 ; clone mib-1 ; isotype igg1/mouse ; monoclonal ; dako , dako inc , carpinteria , ca , usa , cod#m720 ; dilution 1:400 ) antibody was used
.
the immunohistochemical study was performed using the polymeric horseradish peroxidase ( hrp)-linker antibody conjugate technique .
the material was incubated in a humidity chamber at 4c for 18 hours . considering the large s-100 protein family , the polyclonal
rabbit anti - s-100 was used , which reacts strongly with all family members , including human s100a and b sub - units .
the specimens were counterstained with harris hematoxylin , and the slides were mounted using adhesive resin ( entellan , merck , darmstadt , hessen , germany ) and coverslips .
for all immunohistochemical markers , both a positive and a negative control were prepared according to the manufacturers recommendations .
a digital image - capture computer system ( axiocam mrc , zeiss , jena , thuringia , germany ) and a binocular microscope ( axioskop 2 plus , zeiss , jena , thuringia , germany ) with a 40x objective were used .
the images were analyzed using the software supplied with the microscope ( axiovision 4.5 , zeiss , jena , thuringia , germany ) .
each microscopic field was previously standardized in this program to avoid differences in the expression intensity of the antibodies . in each captured microscopic field
the most representative areas of the event ( spots ) , identified using a lower magnification , were selected .
when the dls were diffusely distributed and spread out , 20 spots were randomly selected .
in group i , all microscopic fields containing dls were captured and in group ii , 20 fields containing dls were randomly captured ( figure 2 ) .
a : due to the small quantity of duct - like structures ( dls ) in group i , all microscopic fields containing dls were captured ; b : a spot containing three dls ; c : 20 fields containing dls were randomly captured in group ii ; d : diffuse distribution of dls in group ii
for the localization and the counting of the dls , slides stained with hematoxylin - eosin ( h.e . ) were used .
we considered dls to be those with shrunken seromucous cells , granule depletion and widened lumens that were adjacent to ductal epithelial cells and resembled intralobular non - striated ducts , which is consistent with the characterization used in our previous study
.
we excluded true excretory ducts , which exhibited a dilated lumen lined by cuboidal cells with central nuclei and an eosinophilic cytoplasm
.
a section of each gland was analyzed with each studied molecule , totaling 60 slides for each staining ( 30 per group ) .
therefore , a total of 360 slides were analyzed [ 60 for each marker : h.e . ,
pas , alcian blue ( ph 2.5 ) , ck 19 , s-100 and ki-67 ] .
a semi - quantitative scoring system was applied to the immunohistochemical and histochemical analysis [ adapted from soini , et al .
both analyses resulted in a final score that represented no expression , weak expression or strong expression . in each microscopic field ,
the percentage of positive ( with partial or total staining ) dls was calculated for each marker .
scores ranging from 0 to 4 were attributed to those percentage values : 0 ) absence of positive dls ; 1 ) < 25% of positive dls ; 2 ) 26 - 50% of positive dls ; 3 ) 51 - 75% of positive dls ; 4 ) > 75% of positive dls . then , for the dls positive samples , the partial or total covering and the intensity of the immunostaining in the positive cells were calculated following the same method applied previously . finally , the sum of the values attributed to the quantity , covering and intensity of the immunostaining resulted in a final score where 3 - 7 represented weak to moderate expression and 8 - 12 was a strong expression of the antibody .
basically , the pas and alcian blue staining in the dls followed the same scoring system , except for the intensity values , which did not exist for these stains .
the cellular proliferative activity in the dls , revealed by ki-67 staining , was calculated as the percentage of positive cells to establish a cellular proliferation index in the same previously captured fields .
the comparison between groups and between markers was accomplished by the student s t - test or the mann - whitney test . the correlation among the staining groups
the kappa coefficients ( k ) for the s-100 , ck 19 , pas and alcian blue variables were 87.50% , 78.13% , 96.88% , and 98.88% , respectively .
this variation could be observed among different glands or even among different lobes of the same gland .
overall , the glands of young people were intact , with serous and mucous acini well delineated and absent or few and sparse dls ( figure 1a ) . in the glands of the elderly ,
certain sections had preserved lobes while others had characteristics inherent to senility , which is indicated by the intense replacement of the glandular parenchyma by fibrous and fatty tissue and many dls ( figure 1b ) . in group
i , from the 30 slides analyzed , only 12 had dls , whereas in group ii , only 2 slides did not show these structures . when comparing both groups ,
only the immunostaining for ck 19 showed a statistically significant difference between them ( p=0.033 ) , with stronger expression in the elderly group .
although the expression for s-100 was also stronger in group ii , the difference was not significant ( p=1.000 ) ( table 1 ) .
table 1comparison between groups regarding histochemical and immunohistochemical staininggroups iiip ( n=12)(n=28 ) median1 quartile3 quartilemedian1 quartile3 quartile s-1000.0000.0006.0001.2860.0003.8751.000ck-199.0006.00011.62510.8409.08312.0000.033*pas0.0000.0000.0000.0000.0000.4580.536aa0.0000.0001.8750.0000.0000.9170.907mann - whitney test ( p<0.05 ) .
alcian blue serous acini ( identified by s-100 expression ) were present in the dls with a lower frequency and a weaker intensity than the ck 19 expression levels .
in contrast , the expression of ck 19 was strong in the dls and very similar to the immunostaining in the ducts .
the serous acini exhibited strong immunostaining for s-100 and moderate , weak or absent expression of ck 19 .
the ducts were invariably negative for s-100 and positive for ck 19 ( figure 3 ) .
figure 3a : duct - like structures ( dls ) with weak or absent s-100 expression .
a strong expression of s100 seen in the nerve cells ( short arrow ) can be regarded as the positive control .
serous cells with strong expression ( arrow ) ( group ii ) ; b : strong expression of ck 19 in some cells in the dls .
serous cells with weak expression ( group ii ) ; c : positive s-100 expression in serous acinar cells ( arrows ) and negative in ducts and dls ( short arrows ) ( group i ) ; d : strong ck 19 expression in ducts and dls ( arrows ) and weak or moderate expression in serous acinar cells ( group ii )
regarding group i , the immunostaining of the dls for ck 19 was stronger than for s-100 , with a statistically significant difference observed ( p=0.004 ) ( table 2 ) .
table 2comparison between s-100 and ck 19 immunostaining of duct - like structures in groups i and iigroup i
s-100ck 19 meansdsemmeansdsemp2.5833.3700.9737.6254.2161.2170.004 *
group ii
s-100
ck 19
median
1
quartile
3
quartile
median
1
quartile
3
quartile
1.2860.0003.87510.8409.08312.000<0.001 *
student s t - test ( p<0.05 ) .
ii , the same circumstance was observed and this difference was even more significant ( p<0.001 ) ( table 2 ) .
this condition was also seen in the descriptive microscopic analysis because in the senile glands , the dls strongly expressed ck 19 ( figure 4a ) , showing a ductal pattern . in some sections
, we also observed the presence of areas with a mixture of acinar and ductal cells ( figure 4b ) .
figure 4a : similar ck 19 expression between ducts ( * ) and duct - like structures ( group ii ) .
the ductal cells are stained with the anti- ck 19 ( group ii ) .
when the pearson correlation coefficient was applied , the correlation between ck 19 and s-100 was absent ( =-0.163 ; p=0.315 ) .
regarding the pas and alcian blue staining for mucous acini , both markers worked similarly , where the staining was evident in the mucous acini and quite rare in the dls ( figure 5a ) .
when there was staining , it was restricted to a small portion of the structure and was classified as partial ( figure 5b ) .
these markers also showed , in some cases , the presence of mucin within the ducts or dls ( figure 5c ) .
there was no statistically significant difference between pas and alcian blue ( p=0.270 ) .
figure 5a : mucous acini stain for periodic acid - schiff ( pas ) and alcian blue .
the isolated duct - like structures ( dls ) is negative for both markers ( arrows ) group i ( 21 years old ) ; b : partial staining of a dls for pas and alcian blue ( arrows ) .
group ii ( 76 years old ) ; c : negative dls for pas and alcian blue . presence of mucus in the lumen of a dls ( arrows ) .
group ii ( 76 years old )
in both groups , there was no immunostaining for the ki-67 antibody in any dls ( figure 6 ) .
the expression of various basal cells of the epithelium of the oral mucosa worked as a positive controlfor the antibody used .
figure 6absence of nuclei expression of ki-67 in the duct - like structures ( group ii )
this variation could be observed among different glands or even among different lobes of the same gland .
overall , the glands of young people were intact , with serous and mucous acini well delineated and absent or few and sparse dls ( figure 1a ) . in the glands of the elderly ,
certain sections had preserved lobes while others had characteristics inherent to senility , which is indicated by the intense replacement of the glandular parenchyma by fibrous and fatty tissue and many dls ( figure 1b ) . in group
i , from the 30 slides analyzed , only 12 had dls , whereas in group ii , only 2 slides did not show these structures .
when comparing both groups , only the immunostaining for ck 19 showed a statistically significant difference between them ( p=0.033 ) , with stronger expression in the elderly group .
although the expression for s-100 was also stronger in group ii , the difference was not significant ( p=1.000 ) ( table 1 ) .
table 1comparison between groups regarding histochemical and immunohistochemical staininggroups iiip ( n=12)(n=28 ) median1 quartile3 quartilemedian1 quartile3 quartile s-1000.0000.0006.0001.2860.0003.8751.000ck-199.0006.00011.62510.8409.08312.0000.033*pas0.0000.0000.0000.0000.0000.4580.536aa0.0000.0001.8750.0000.0000.9170.907mann - whitney test ( p<0.05 ) .
alcian blue serous acini ( identified by s-100 expression ) were present in the dls with a lower frequency and a weaker intensity than the ck 19 expression levels .
in contrast , the expression of ck 19 was strong in the dls and very similar to the immunostaining in the ducts .
the serous acini exhibited strong immunostaining for s-100 and moderate , weak or absent expression of ck 19 .
the ducts were invariably negative for s-100 and positive for ck 19 ( figure 3 ) .
figure 3a : duct - like structures ( dls ) with weak or absent s-100 expression .
a strong expression of s100 seen in the nerve cells ( short arrow ) can be regarded as the positive control .
serous cells with strong expression ( arrow ) ( group ii ) ; b : strong expression of ck 19 in some cells in the dls .
serous cells with weak expression ( group ii ) ; c : positive s-100 expression in serous acinar cells ( arrows ) and negative in ducts and dls ( short arrows ) ( group i ) ; d : strong ck 19 expression in ducts and dls ( arrows ) and weak or moderate expression in serous acinar cells ( group ii )
regarding group i , the immunostaining of the dls for ck 19 was stronger than for s-100 , with a statistically significant difference observed ( p=0.004 ) ( table 2 ) .
table 2comparison between s-100 and ck 19 immunostaining of duct - like structures in groups i and iigroup i
s-100ck 19 meansdsemmeansdsemp2.5833.3700.9737.6254.2161.2170.004 *
group ii
s-100
ck 19
median
1
quartile
3
quartile
median
1
quartile
3
quartile
1.2860.0003.87510.8409.08312.000<0.001 *
student s t - test ( p<0.05 ) .
ii , the same circumstance was observed and this difference was even more significant ( p<0.001 ) ( table 2 ) .
this condition was also seen in the descriptive microscopic analysis because in the senile glands , the dls strongly expressed ck 19 ( figure 4a ) , showing a ductal pattern . in some sections
, we also observed the presence of areas with a mixture of acinar and ductal cells ( figure 4b ) .
figure 4a : similar ck 19 expression between ducts ( * ) and duct - like structures ( group ii ) .
b : mixed structure with ductal and acinar cells . the ductal cells are stained with the anti- ck 19 ( group ii ) .
when the pearson correlation coefficient was applied , the correlation between ck 19 and s-100 was absent ( =-0.163 ; p=0.315 ) . regarding the pas and alcian blue staining for mucous acini ,
both markers worked similarly , where the staining was evident in the mucous acini and quite rare in the dls ( figure 5a ) .
when there was staining , it was restricted to a small portion of the structure and was classified as partial ( figure 5b ) .
these markers also showed , in some cases , the presence of mucin within the ducts or dls ( figure 5c ) .
there was no statistically significant difference between pas and alcian blue ( p=0.270 ) .
figure 5a : mucous acini stain for periodic acid - schiff ( pas ) and alcian blue .
the isolated duct - like structures ( dls ) is negative for both markers ( arrows ) group i ( 21 years old ) ; b : partial staining of a dls for pas and alcian blue ( arrows ) .
group ii ( 76 years old ) ; c : negative dls for pas and alcian blue . presence of mucus in the lumen of a dls ( arrows ) .
in both groups , there was no immunostaining for the ki-67 antibody in any dls ( figure 6 ) .
the expression of various basal cells of the epithelium of the oral mucosa worked as a positive controlfor the antibody used .
figure 6absence of nuclei expression of ki-67 in the duct - like structures ( group ii )
the sublingual glands used in this work are the same as those used by azevedo , et al .
( 2005).in regards to the gender of the cadavers and the side of the glands , previous studies have already demonstrated that there are no differences in these variables
.
we consider our samples to be very rich and consistent because the sublingual glands were removed from the cadavers after a short post - mortem period .
moreover , the sublingual glands are rarely addressed in previous studies , where they are typically derived from rats
. only the works of the present line of research use human sublingual glands
. because dls are not an exclusive phenomenon of senescence , group i was used in this study as a control to observe whether there were differences in the staining pattern of dls in this group in comparison to group ii . when a gland from group i exhibited dls , the number was very low and often limited to the extremity of the lobe .
we believe that the presence of dls in the glands of young individuals may be due to some injury , i.e. , small traumas to the mouth floor during feeding and microbstructions of the peripheral ductal system .
moreover , these structures have been shown in studies that induce glandular atrophy through the duct obstruction
or in pathological processes such as neoplasms
. therefore , despite the presence of dls upon cellular senescence , they are not exclusive in this age group , which justifies why we opted to include group i in the present study . in regards to normal salivary glands , several studies demonstrate that ck 19 is strongly expressed in all parts of the ductal system and shows weak response to serous and mucous acinar cells
. for geiger , et al .
( 2002 ) , ck 19 is expressed in ductal cells but completely absent in acinar cells . regarding the s-100 protein ,
studies in normal salivary glands and tumors showed strong expression in serous acinar cells and weak expression in the ductal segments
. in this study , ck 19
was strongly expressed in true ducts and most of the dls . the ck 19 expression in
the serous acini was weaker or absent and was not detected in the mucous acini .
however , the s-100 protein was expressed in serous acinar cells and absent in true ducts and mucous acinar cells .
the difference in the immunostaining of ck 19 in the dls between groups was significant ( p=0.033 ) ( table 1 ) .
the expression of this protein was higher in the elderly , indicating the possibility that , over time , the dls begin to express the molecules of the ductal system more strongly . in both groups
, there was a statistically significant difference between ck 19 and s-100 ( tables 4 and 5 ) with more intense ck 19 staining , demonstrating a ductal pattern of the dls .
it is possible that the dls increase their ductal pattern with age while they simultaneously decrease the expression of s-100 .
regarding the cell proliferation activity , we did not find any cells positive for ki-67 in the dls of both groups ( figure 6 ) .
the effectiveness of the antibody used was proven by the presence of nuclear staining in various basal cells of the oral mucosa epithelium in the same sections .
( 2002 ) who found a proliferation index from 0% in acinar and ductal cells from young and senile human labial glands .
( 2002 ) , the staining for pcna in ductal cells is high in the early stages of glandular atrophy and subsequently decreases . instead
, the dls are commonly observed in the later stages of atrophy , suggesting that both mitosis ( early stages ) and the transition from acinar to ductal cells ( later stages ) contribute to the increase in the number of ducts in the atrophic sublingual glands .
these findings may explain the absence of immunostaining for ki-67 in the dls , demonstrating that they represent a late stage of atrophy ( or aging ) where mitosis is no longer observed .
however , even in the dls of young glands , there was no evidence of cell proliferation , which is difficult to explain unless the authors used another marker ( pcna ) . based on our results
, we do not support the hypothesis that mitosis explains the increase in the number of dls .
( 2002 ) that the dls represent the final process of acinar atrophy , we can infer that , in this investigation , s-100 was expressed in the dls due to the presence of serous acinar cells that remained from the atrophy process . unlike serous cells , there was rarely any trace of mucous cells in the dls ( figure 5b ) .
therefore , we hypothesized that , if these structures are actually products of an acinar regression , this acinus is mixed , where mucous cells degenerate while the serous portion is more resistant and occasionally remains active .
this finding corroborates other studies in humans
, in which the serous cells showed greater resistance to autolysis compared to the mucous cells . frequently , the areas of mixed serous and ductal cells were visualized as illustrated in figure 4b .
( 2002 ) demonstrated that evidence of a mixed phenotype suggests the transition and differentiation of cells through metaplasia .
this transition from one acinus to a duct may be a result of epithelial metaplasia , which is defined as the transition from a reactive differentiated epithelium to another type of differentiated epithelium characterized by an impaired function or an alteration that is not physiologically normal in that place
.
salivary glands exhibit a variety of metaplastic changes
and this could explain the cellular phenotypic changes resulting from aging . for mintz and mooradian
( 1987 ) ,
the sublingual glands underwent squamous metaplasia of the ductal epithelium with age . according to ihrler , et al .
( 2002 ) , the majority of salivary gland metaplasias develop from basal cells of striated and excretory ducts that have an enormous capacity for pluridirectional morphogenetic differentiation . for us
, our results strongly suggest metaplasia to explain this age - related change in human salivary glands .
we believe in a dynamic process that arises in the acinus and culminates in a complete transformation of a duct . because the acinar cells originate from the primitive ductal cells , the embryological phenomenon maybe the way that the reverse process can occur . extrapolating this data to the clinic
, we understand that the acini fail to work as secretory units and start to conduct the saliva . at this moment
this could explain the decrease in the function of the saliva with age . according to dayan , et al .
( 2000 ) , the decrease in acini volume may explain the decrease in secretion by the salivary glands with age . according to scott
( 1977 ) ,
the junction of all age - related microscopic changes may explain the decrease in glandular function .
however , despite proof that acini decrease with age , certain studies support that saliva gland functions remain stable in healthy elderly patients
.
( 2014 ) demonstrated that symptoms of oral dryness ( xerostomia ) are not associated with age - related degenerative changes in human labial salivary glands .
the authors suggest that the autonomic innervation of labial salivary glands may play a role in the occurrence of xerostomia and that the integrity of labial salivary gland acini is related to the parenchymal autonomic innervation
.
one hypothesis is that the secretion reserve capacity of the glands can be affected by external factors such as radiation or drug use , but not by senescence
. because there is a difficulty in collecting saliva coming from the sublingual gland and lacked studies with direct tests of salivary flow
, we can not firmly conclude if there is a decrease in salivary flow of the sublingual gland with age .
although the clinical impact of the age - related changes in the salivary glands is still unclear , we think this work represents a contribution to pathologists .
because these structures are commonly found in mouth floor , pre - prosthetic surgery specimens and in neoplasias of the major and minor salivary glands , these findings may help explain the phenomenon of the appearance of multiple dls when elaborating microscopic pathology reports .
the present study demonstrated that duct - like structures present a ductal phenotypic profile and do not present cell proliferation activity .
they may represent a regressive process arising from acini or represent the result of metaplasia . | there are several age - related microscopic changes in the salivary glands , including the increase in the number of duct - like structures ( dls )
. however , the true origin and the phenotype of the dls are not known .
objective to evaluate the phenotype and the cell proliferation index of the dls of human sublingual glands.material and methods sixty sublingual glands obtained from human cadavers were divided into two groups - 0 - 30 and 61 - 90 years old .
the phenotype was estimated by immunostaining for cytokeratin 19 ( ck 19 ) and the s-100 protein as well as by the presence of mucin and glycogen .
the cell proliferation index was determined by the ki-67 antibody .
the histochemical techniques used periodic acid - schiff ( pas ) and alcian blue . in each captured microscopic field , the dls were counted to establish a percentage for the staining profile .
the statistical analysis was accomplished using student s t - test , the mann - whitney test and pearson s correlation coefficient ( p<0.05).results comparing both groups , only ck 19 showed a statistically significant difference ( p=0.033 ) , with the strongest expression in the elderly group .
there was no significant difference between pas and alcian blue ( p=0.270 ) . in both groups ,
the immunostaining for ck 19 was stronger than that for s-100 ( p=0.004;p<0.001 ) , but there was no correlation between the two immunomarkers ( =-0.163 ; p=0.315 ) .
there was no immunostaining for ki-67.conclusions dls demonstrate a ductal phenotypic profile and do not present cell proliferation activity .
dls may represent a regressive process arising from acini or represent the result of metaplasia . |
rna recombination is one of the major factors responsible for the generation of new rna viruses and retroviruses
. the biological mechanisms of recombination differ across organisms , but in broad terms recombination results in the creation of mosaic sequences where the evolutionary history at each site may be different .
recombination , defined as the exchange of genetic information between two nucleotide sequences , is an important process that influences biological evolution at many different levels .
recombination explains a considerable amount of genetic diversity in natural populations and , in general , genes located in regions of the genome with low levels of recombination have low levels of polymorphism ( posada and crandall , 2001 ) .
it has been shown that rna recombination enables the exchange of genetic material , not only between the same or similar viruses but also between distinctly different viruses ( worobey and holmes , 1999 ) .
sometimes , it also permits crossovers between viral and host rna ( greene and allison , 1994 ; aaziz and tepfer , 1999 ; baroth et al .
taking into account the structure of viral genomic molecules and the location of crossover sites , three basic types of rna recombination were distinguished : homologous , aberrant homologous and non - homologous ( lai , 1992 ; alejska et al . , 2001 ) .
the former two occur between two identical or similar rnas ( or between molecules displaying local homology ) , while the latter involves two different molecules .
most of the collected data suggest that rna recombinants are formed according to a copy choice model ( alejska et al . , 2001 ) .
a viral replication complex starts nascent rna strand synthesis on one template , called rna donor , and then switches to another template , called rna acceptor .
accordingly , two main factors are thought to affect rna recombination : the structure of recombining molecules and the ability of the viral replicase to switch templates . through generations ,
viral populations evolve under various selective forces at different regions and sites that display different functional constraints .
a stringent and robust criterion for detecting adaptive evolution in a protein - coding gene is an accelerated nonsynonymous ( dn , amino acid replacing ) rate relative to the synonymous ( ds , silent ) rate of substitutions , with the rate ratio = dn / ds > 1 . as silent mutations
do not change the amino acid whereas replacement mutations do , the difference in their fixation rates provides a measure of selective pressure on the protein . amongst positive - strand plant rna viruses ,
the family tombusviridae encompasses several viruses with an important economical impact . according to the 8 ictv ( international committee on taxonomy of viruses ) report ( fauquet et al . , 2005 )
, the family tombusviridae includes the following genera : tombusvirus , carmovirus , necrovirus , dianthovirus , machlomovirus , avenavirus , aureusvirus and panicovirus . according to the baltimore classification
, the viruses in this family are classified as type iv viruses , and are part of the luteovirus supergroup ( habili and symons , 1989 ) .
the rna is contained in an icosahedral ( t = 3 ) capsid , composed of 180 units of a single coat protein 2742 kda in size ; the virion measures 2835 nm in diameter , and is not enveloped .
all tombusviridae have a positive- sense , single - stranded linear genome , with the exception of dianthoviruses , whose genome is bipartite .
the 5 terminus is capped only in carnation mottle carmovirus , red clover necrotic mosaic dianthovirus and maize chlorotic mottle machlomovirus .
the polymerase orf encodes an amber stop codon that is the site of a readthrough event within orf 1 ( except in dianthoviruses , where readthrough occurs via a frameshift ) , producing two products necessary for replication .
the replication process of members of family tombusviridae comprises the following steps : ( i ) the virus penetrates into the host cell , ( ii ) the viral genomic rna is uncoated and released into the cytoplasm , ( iii ) the viral rna is translated to produce the two proteins necessary for rna synthesis ( replication and transcription ) , ( iv ) a negative - sense complementary ssrna is synthesized using the genome rna as a template , ( v ) a new genomic rna is synthesized using the negative - sense rna as a template , ( vi ) the rna - dependant rna polymerase ( rdrp ) recognizes internal subgenomic promoters on the negative - sense rna , to transcribe the 3 co - terminal subgenomic rnas that will generate the capsid and movement protein , ( vii ) new virus particles are formed ( white and nagy , 2004 ) . the main objective of this work was to determine and characterize virus evolution mechanisms of the tombusviridae based on the occurrence of putative recombination events and positive selection in their full - length genome .
this was achieved by the analysis of 79 accessions obtained from genbank . as a result
the sequences of the entire genome of 79 accessions cataloged in genbank were used in this study ( table 1 ) .
the nucleotide sequences were aligned using programs clustalw 2.0.9 and clustalx 2.0.9 ( larkin et al . , 2007 ) with default configuration .
their phylogenetic relationships were determined with the maximum - likelihood ( ml ) algorithm incorporated in the mega version 5 program ( tamura et al . , 2011 ) under assumption of the substitution models proposed by jukes and cantor ( 1969 ) ( jc ) , hasegawa et al .
( 1985 ) ( hky85 ) , and tamura and nei ( 1993 ) ( tn93 ) . bootstrap analyses with 500 replicates were performed to assess the robustness of the branches . using the mega4.1 program ( kumar et al . , 2008 ) , positive selection was inferred by the counting method described by nei and gojobori ( 1986 ) and , later on , by suzuki and gojobori ( 1999 ) . according to this method , the phylogenetic tree of sequences analyzed was used . for the parsimony method ,
the total numbers of synonymous ( cs ) and nonsynonymous ( cn ) substitutions as well as the average numbers of synonymous ( ss ) and nonsynonymous ( sn ) sites per codon over the phylogenetic tree for each codon site were computed according to the maximum parsimony principle ( fitch , 1971 ; hartigan , 1973 ) .
the null hypothesis of selective neutrality ( rs = rn or = 1 ) was tested for each site by computing the probability ( p ) of obtaining the observed or more biased values for cs and cn , which were assumed to follow a binomial distribution with the probabilities of occurrence of synonymous and nonsynonymous substitutions given by ss/(ss + sn ) and sn/(ss + sn ) , respectively .
positive selection is inferred when p < 0.05 and cn / sn > cs / ss ( suzuki , 2006 ) .
potential recombination events between diverged nucleotide sequences were explored using two programs : rdp v3.31 ( martin et al . , 2005b ) and recco ( maydt and lengauer , 2006 ) .
rdp incorporates several published recombination detection methods into a single suite of tools : rdp ( martin and rybicki , 2000 ) , geneconv ( padidam et al . , 1999 ) , bootscan ( martin et al . , 2005a ) , maxchi ( smith , 1992 ) , chimaera ( posada and crandall , 2001 ) , siscan ( gibbs et al . , 2000 ) , and 3seq ( boni et al . , 2007 ) . in all cases ,
the algorithm developed and described by maydt and lengauer ( 2006 ) as being a fast , simple and sensitive method for detecting recombination in a set of sequences and locating putative recombination breakpoints is based on cost minimization .
the only parameter considered is , representing the cost of mutation relative to recombination .
when changes from 0 to 1 , the cost of mutation weighted by increases , and the cost for recombination weighted by 1 - decreases .
examination of the recco program output regarding the occurrence of recombination events in the complete genome of the tombusviridae family , revealed that three out of five aureusviruses were putative recombinants ( polv.pigeonpea , jcsmv.iran , mawlmv.usa ) .
in contrast , clsv ( unknown isolate ) and clsv.canada did not show any recombinant signal ( table 2 ) . within the genus aureusvirus ,
the most frequently recombining virus was polv.pigeonpea ( 33 putative recombination sites ) , whereas only 28 possible recombination signals were detected in the genome of viruses jcsmv.iran and mawlmv.usa .
similarly , the only representative of the genus avenavirus ( ocsv ) was a potential recombinant with 175 putative sites .
the rdp package confirmed these results for both genera . among the carmoviruses , 14 out of 30 members were possible recombinants . according to recco ,
the most frequently recombining virus was jinrsv with 134 putative events , while mensv.nagasaki and mensv.nk had the lowest number of recombination sites ( two putative sites ) .
the rdp v3.31 algorithm confirmed the occurrence of possible recombination events only for accessions mensv.nk and mensv.nh .
recombination investigations of genus dianthovirus based on recco analysis showed that only 80% of them were possible recombinants ( carrsv - rna 1 and 2 , rcnmv - rna 1 and 2 , rcnmv.can .
however , the rdp package did not predict recombination in the dianthoviruses ( table 3 ) . although the most frequently recombining necrovirus was rvx ( 166 putative sites ) , rcnmv.rna 2 had only two putative sites .
based on recco analysis , 50% of the necroviruses ( bbsv.val25.iran , lwsv , tnv-a.fmb , tnv - a.c , tnv-d.hungarian , and ommv-gp.portugal , ) were possible recombinants .
conversely , bbsv , bbsv.co.usa , bbsv.xinjiang , olv-1.citrus , olv-1.gm6.portugal , and tnv-d.rhotamsted were not possible recombinants .
these results were congruent with those obtained with the rdp package . while the most frequently recombining virus was lwsv ( 39 sites ) , tnv.a.c recombined into two sites .
regarding the sole representative of genus panicovirus ( pmv ) , the results obtained by the two methods ( recco and rdp v3.31 ) were incongruent .
indeed , with recco , 108 possible sites were detected , whereas no recombination signals were found with the rdp package .
a similar situation was observed with regard to the newly proposed carmoviruses ( nlvcv.alaska , pcrpv.gr 57 , plpv.pv-0193 ) . according to recco analysis , although nlvcv.alaska was the most frequently recombining virus ( 65 sites ) , plpv.pv-0193 recombined only into 38 sites ( table 4 ) .
regarding the members of genus tombusvirus , there was an agreement between the two methods indicating that 80% of the analyzed accessions were putative recombinants .
while cblv had the highest number of putative recombination signals ( 67 sites ) , tbsv.cherry had only two recombination sites .
furthermore , it is noteworthy that the two representatives of genus machlomovirus ( mcmov , and mcmov.nebraska ) were not recombinants as assessed by the two methods of analysis used in this study .
seeking for the recombination frequency in the genome of the tombusviridae , two - thirds of the aureusviruses ( jcsmv.iran , and mawlmv.usa ) showed that in most cases , their breakpoint length was a single residue .
in contrast , the breakpoint length of most putative recombination sites of polv.pigeonpea was between three and 37 nucleotides ( table 2 ) . also , the breakpoint length of the major recombination sites of the single representative of genus avenavirus ( ocsv ) consisted of a single residue . in about 50% of the members of the genus carmovirus ,
as opposed to that , the breakpoint interval of the remaining members exceeded three residues reaching a size as long as 82 residues ( mensv.nh ) . in 62% of the investigated dianthoviruses , the breakpoint length exceeded three nucleotides reaching 100 residues ( carrsv.rna 1 ) ( table 3 ) . in the necroviruses ,
the breakpoint interval distribution was similar i.e. , 50% of the breakpoints consisted of a single residue , while the remaining breakpoints were between three and 77 nucleotides . for the sole member of the genus panicovirus ( pmv )
, most of the recombination sites had a breakpoint length of a single residue ( 45 ) ( table 3 ) .
as for the tombusviruses , 75% showed a breakpoint length exceeding three residues up to 161 nucleotides ( amocv.bari ) ( table 4 ) .
maximum composite likelihood estimate of the nucleotide substitution pattern were made using the mega4.1 program .
the results for tombusviridae showed that the rates of different transitional substitutions varied from 3.18 to 14.61 , and those of transversional substitutions varied from 6.6 to 8.57 .
the nucleotide frequencies were : 0.269 ( a ) , 0.258 ( t / u ) , 0.207 ( c ) , and 0.266 ( g ) .
the transition / transversion rate ratios were k1 = 1.705 ( purines ) and k2 = 0.482 ( pyrimidines ) .
the overall transition / transversion bias was r = 0.547 , where r = [ agk1 + tck2]/[(a+g)(t+c ) ] .
in all these analyses , the codon positions included were first + second + third + noncoding .
all positions containing gaps and missing data were excluded from the dataset ( complete deletion option ) .
the purpose of this test is to indentify sequences which do not fit the neutral theory model at equilibrium between mutation and genetic drift .
tajima s test compares a standardized measure of the total number of segregating sites ( the polymorphic dna sites ) in the sampled dna and the average number of mutations between pairs in the sample .
the high genetic stability of viruses can be attributed to negative or purifying selection to maintain the functional integrity of the viral genome .
the degree of negative selection in genes , or the degree of functional constraint for the maintenance of the encoded protein sequence , can be estimated , as mentioned above , by the ratio between the nucleotide diversities in nonsynonymous and synonymous positions ( dn / ds ) . for most coding genes
the dn / ds ratio is < 1 which is consistent with negative selection against protein change .
in contrast , a dn / ds ratio > 1 may be an indication that adaptive or positive selection is driving gene divergence . in this study
, pairwise comparisons of all screened accessions showed that , none of the members of the genera machlomovirus and panicovirus , and unclassified tombusviridae was under positive selection . on the contrary , the genera aureusvirus ( jcmsv.iran ) , avenavirus ( ocsv ) , carmovirus ( carmov.china , carmov.indian ) , dianthovirus ( carrsv - rna 2 , rcnmv - rna 2 , scnmv - rna 2.59 , scnmv - rna 2.38 ) , necrovirus ( bbsv , bbsv.val25.iran ) , tombuvirus ( galv.nipplefruit , pelv , tbsv.statice , pnsv ) along with the unassigned tombusviridae ( plpv.pv.0193 ) were under positive selection ( table 5 ) .
it is worth pointing out that , in the viruses with a segmented genome , positive selection was detected only in rna 2 , suggesting that probably reassortment events occurred .
all these results were obtained by testing neutrality in sequence pairs with fisher s exact test .
the probability of rejecting the null hypothesis of strict - neutrality ( dn = ds ) in favor of positive selection for each sequence pair was determined .
the variance of the difference ( dn - ds ) was computed using the bootstrap method ( 500 replicates ) .
all analyses were made using the nei - gojobori method incorporated in the mega program .
all positions containing gaps and missing data were excluded from the dataset ( complete deletion option ) .
the phylogenetic relationships among members of the family tombusviridae , based on the sequences of their complete genome , were inferred using a maximum likelihood algorithm under the assumption of three models of substitution ( jc , hky85 , tn93 ) .
the inferred phylogeny showed that each taxonomical genus in the family tombusviridae constituted a homogenous group clearly distinct from the others .
however , the results obtained in this study evidenced a few differences in terms of virus species composition within each taxonomical genus compared to the current classification adopted by the ictv .
in fact , three viruses considered by the ictv as unassigned ( plpv.pv-0193 ) and unclassified tombusviridae ( nlvcv.alaska , pcrpv.gr 57 ) showed a close phylogenetic relationship to known members of the genus carmovirus .
the first subgroup comprised viruses : tcv , ccfv , jinrv , hcrsv , plpv , pcrpv , nlvcv , scv , afbv , pfbv , cpmov , symomv and carmov , and the second subgroup encompassed viruses : mensv , and psnv .
furthermore , it was proposed that genus necrovirus should be constituted by two distinct subgroups named tentative subgroup i ( bbsv , tnv.d , lwsv ) and tentative subgroup ii ( ommv , tnv.a , olv-1 ) ( figure 1 ) .
it should be noted that here ommv is an integral part of subgroup i rather than an unclassified necrovirus .
in contrast , genus aureusvirus encompassed members that evolved in a homogenous manner : clsv , polv , mawlmv , and jcsmv . similarly , the following members of genus tombusvirus also formed a coherent ensemble : mansv , cblv , lnv.l , lnv.zantedeschia , pelv , cnv , cymrsv , amocv , tbsv.statice , tbsv.nipplefruit , tbsv.pepper , tbsv.cherry , galv , pnsv , and carirsv .
in fact , according to this classification , mansv was considered as an unassigned tombusviridae , whereas lnv and pnsv were included in the unclassified tombusvirus group . concerning genus dianthovirus which clearly was not monophyletic , the clustering pattern showed two distinct clades representing their rnas 1 and 2 , as illustrated in figure 1 .
examination of the recco program output regarding the occurrence of recombination events in the complete genome of the tombusviridae family , revealed that three out of five aureusviruses were putative recombinants ( polv.pigeonpea , jcsmv.iran , mawlmv.usa ) .
in contrast , clsv ( unknown isolate ) and clsv.canada did not show any recombinant signal ( table 2 ) . within the genus aureusvirus ,
the most frequently recombining virus was polv.pigeonpea ( 33 putative recombination sites ) , whereas only 28 possible recombination signals were detected in the genome of viruses jcsmv.iran and mawlmv.usa .
similarly , the only representative of the genus avenavirus ( ocsv ) was a potential recombinant with 175 putative sites .
the rdp package confirmed these results for both genera . among the carmoviruses , 14 out of 30 members were possible recombinants . according to recco ,
the most frequently recombining virus was jinrsv with 134 putative events , while mensv.nagasaki and mensv.nk had the lowest number of recombination sites ( two putative sites ) .
the rdp v3.31 algorithm confirmed the occurrence of possible recombination events only for accessions mensv.nk and mensv.nh .
recombination investigations of genus dianthovirus based on recco analysis showed that only 80% of them were possible recombinants ( carrsv - rna 1 and 2 , rcnmv - rna 1 and 2 , rcnmv.can .
however , the rdp package did not predict recombination in the dianthoviruses ( table 3 ) . although the most frequently recombining necrovirus was rvx ( 166 putative sites ) , rcnmv.rna 2 had only two putative sites .
based on recco analysis , 50% of the necroviruses ( bbsv.val25.iran , lwsv , tnv-a.fmb , tnv - a.c , tnv-d.hungarian , and ommv-gp.portugal , ) were possible recombinants .
conversely , bbsv , bbsv.co.usa , bbsv.xinjiang , olv-1.citrus , olv-1.gm6.portugal , and tnv-d.rhotamsted were not possible recombinants .
these results were congruent with those obtained with the rdp package . while the most frequently recombining virus was lwsv ( 39 sites ) , tnv.a.c recombined into two sites .
regarding the sole representative of genus panicovirus ( pmv ) , the results obtained by the two methods ( recco and rdp v3.31 ) were incongruent .
indeed , with recco , 108 possible sites were detected , whereas no recombination signals were found with the rdp package .
a similar situation was observed with regard to the newly proposed carmoviruses ( nlvcv.alaska , pcrpv.gr 57 , plpv.pv-0193 ) . according to recco analysis , although nlvcv.alaska was the most frequently recombining virus ( 65 sites ) , plpv.pv-0193 recombined only into 38 sites ( table 4 ) .
regarding the members of genus tombusvirus , there was an agreement between the two methods indicating that 80% of the analyzed accessions were putative recombinants .
while cblv had the highest number of putative recombination signals ( 67 sites ) , tbsv.cherry had only two recombination sites .
furthermore , it is noteworthy that the two representatives of genus machlomovirus ( mcmov , and mcmov.nebraska ) were not recombinants as assessed by the two methods of analysis used in this study .
seeking for the recombination frequency in the genome of the tombusviridae , two - thirds of the aureusviruses ( jcsmv.iran , and mawlmv.usa ) showed that in most cases , their breakpoint length was a single residue .
in contrast , the breakpoint length of most putative recombination sites of polv.pigeonpea was between three and 37 nucleotides ( table 2 ) . also , the breakpoint length of the major recombination sites of the single representative of genus avenavirus ( ocsv ) consisted of a single residue . in about 50% of the members of the genus carmovirus ,
as opposed to that , the breakpoint interval of the remaining members exceeded three residues reaching a size as long as 82 residues ( mensv.nh ) . in 62% of the investigated dianthoviruses , the breakpoint length exceeded three nucleotides reaching 100 residues ( carrsv.rna 1 ) ( table 3 ) . in the necroviruses ,
the breakpoint interval distribution was similar i.e. , 50% of the breakpoints consisted of a single residue , while the remaining breakpoints were between three and 77 nucleotides . for the sole member of the genus panicovirus ( pmv )
, most of the recombination sites had a breakpoint length of a single residue ( 45 ) ( table 3 ) .
as for the tombusviruses , 75% showed a breakpoint length exceeding three residues up to 161 nucleotides ( amocv.bari ) ( table 4 ) .
maximum composite likelihood estimate of the nucleotide substitution pattern were made using the mega4.1 program .
the results for tombusviridae showed that the rates of different transitional substitutions varied from 3.18 to 14.61 , and those of transversional substitutions varied from 6.6 to 8.57 .
the nucleotide frequencies were : 0.269 ( a ) , 0.258 ( t / u ) , 0.207 ( c ) , and 0.266 ( g ) .
the transition / transversion rate ratios were k1 = 1.705 ( purines ) and k2 = 0.482 ( pyrimidines ) .
the overall transition / transversion bias was r = 0.547 , where r = [ agk1 + tck2]/[(a+g)(t+c ) ] .
in all these analyses , the codon positions included were first + second + third + noncoding .
all positions containing gaps and missing data were excluded from the dataset ( complete deletion option ) .
the purpose of this test is to indentify sequences which do not fit the neutral theory model at equilibrium between mutation and genetic drift .
tajima s test compares a standardized measure of the total number of segregating sites ( the polymorphic dna sites ) in the sampled dna and the average number of mutations between pairs in the sample .
the high genetic stability of viruses can be attributed to negative or purifying selection to maintain the functional integrity of the viral genome .
the degree of negative selection in genes , or the degree of functional constraint for the maintenance of the encoded protein sequence , can be estimated , as mentioned above , by the ratio between the nucleotide diversities in nonsynonymous and synonymous positions ( dn / ds ) . for most coding genes
the dn / ds ratio is < 1 which is consistent with negative selection against protein change .
in contrast , a dn / ds ratio > 1 may be an indication that adaptive or positive selection is driving gene divergence . in this study ,
pairwise comparisons of all screened accessions showed that , none of the members of the genera machlomovirus and panicovirus , and unclassified tombusviridae was under positive selection . on the contrary , the genera aureusvirus ( jcmsv.iran ) , avenavirus ( ocsv ) , carmovirus ( carmov.china , carmov.indian ) , dianthovirus ( carrsv - rna 2 , rcnmv - rna 2 , scnmv - rna 2.59 , scnmv - rna 2.38 ) , necrovirus ( bbsv , bbsv.val25.iran ) , tombuvirus ( galv.nipplefruit , pelv , tbsv.statice , pnsv ) along with the unassigned tombusviridae ( plpv.pv.0193 ) were under positive selection ( table 5 ) .
it is worth pointing out that , in the viruses with a segmented genome , positive selection was detected only in rna 2 , suggesting that probably reassortment events occurred .
all these results were obtained by testing neutrality in sequence pairs with fisher s exact test .
the probability of rejecting the null hypothesis of strict - neutrality ( dn = ds ) in favor of positive selection for each sequence pair was determined
the variance of the difference ( dn - ds ) was computed using the bootstrap method ( 500 replicates ) .
all analyses were made using the nei - gojobori method incorporated in the mega program .
all positions containing gaps and missing data were excluded from the dataset ( complete deletion option ) .
the phylogenetic relationships among members of the family tombusviridae , based on the sequences of their complete genome , were inferred using a maximum likelihood algorithm under the assumption of three models of substitution ( jc , hky85 , tn93 ) .
the inferred phylogeny showed that each taxonomical genus in the family tombusviridae constituted a homogenous group clearly distinct from the others .
however , the results obtained in this study evidenced a few differences in terms of virus species composition within each taxonomical genus compared to the current classification adopted by the ictv .
in fact , three viruses considered by the ictv as unassigned ( plpv.pv-0193 ) and unclassified tombusviridae ( nlvcv.alaska , pcrpv.gr 57 ) showed a close phylogenetic relationship to known members of the genus carmovirus . moreover , the viruses belonging to this genus were divided into two distinct subgroups .
the first subgroup comprised viruses : tcv , ccfv , jinrv , hcrsv , plpv , pcrpv , nlvcv , scv , afbv , pfbv , cpmov , symomv and carmov , and the second subgroup encompassed viruses : mensv , and psnv .
furthermore , it was proposed that genus necrovirus should be constituted by two distinct subgroups named tentative subgroup i ( bbsv , tnv.d , lwsv ) and tentative subgroup ii ( ommv , tnv.a , olv-1 ) ( figure 1 ) .
it should be noted that here ommv is an integral part of subgroup i rather than an unclassified necrovirus .
in contrast , genus aureusvirus encompassed members that evolved in a homogenous manner : clsv , polv , mawlmv , and jcsmv .
similarly , the following members of genus tombusvirus also formed a coherent ensemble : mansv , cblv , lnv.l , lnv.zantedeschia , pelv , cnv , cymrsv , amocv , tbsv.statice , tbsv.nipplefruit , tbsv.pepper , tbsv.cherry , galv , pnsv , and carirsv .
in fact , according to this classification , mansv was considered as an unassigned tombusviridae , whereas lnv and pnsv were included in the unclassified tombusvirus group .
concerning genus dianthovirus which clearly was not monophyletic , the clustering pattern showed two distinct clades representing their rnas 1 and 2 , as illustrated in figure 1 .
this study evidenced the prediction of putative recombination events in the genome of several members of the family tombusviridae and demonstrated that tombusviruses and carmoviruses are highly recombinant compared to viruses of the other genera .
for this purpose , two methods were chosen ( recco and rdp v.3.31 ) , based on the fact that they are appropriate for the mosaic structure of viruses as reported in previous works ( boulila , 2009 ; 2010 ) . in this study , using the recco algorithm , it was demonstrated that the viruses belonging to the following genera contained putative recombination signals in their genome : aureusvirus , avenavirus , carmovirus , dianthovirus , necrovirus , panicovirus , and tombusvirus .
these results were in good agreement with those obtained by the rdp package except for members of genus diantovirus . by both methods , the two representatives of genus machlomovirus ( mcmov , mcmov.nebraska )
the most frequently recombining viruses were : ocsv , rvx.rna 1 , jinrsv , and pmv with 175 , 166 , 134 , and 108 putative recombination sites , respectively .
mensv.nagasaki , mensv.nk , rcnmv.rna 2 , tnv - a.c , and tbsv.cherry ( 2 sites ) , mensv.mnsv-al , carrsv - rna 2 , and tnv-a.fm1b ( 3 sites ) , mensv.kochi , bbsv-val25.iran , and tnv-d.hungarian ( 4 sites ) , carmov , mensv.mnsv-264 , and mensv.nh ( 5 sites ) , rcnmv.rna 1.can , tbsv.nipplefruit , and tbsv.pepper ( 7 sites ) , and rcnmv.rna 1 ( 8 sites ) showed the lowest frequency of recombination breakpoints .
furthermore , this study showed that recombination may occur between viruses belonging to different genera .
for example : oat chlorotic stunt avenavirus ( ocsv ) and melon necrotic spot carmovirus ( mensv ) may give rise to pothos latent aureusvirus ( polv ) .
similarly , ocsv itself may result from a recombination between turnip crinkle carmovirus ( tcv ) and maize white line mosaic aureusvirus ( mawlmv ) ( table 2 ) .
seemingly , these viruses could contain part of their sequences particularly in the coat protein - encoding gene of each other . such an event
was largely studied for cucumber necrosis tombusvirus ( cnv ) and melon necrotic spot carmovirus ( mensv ) ( riviere and rochon , 1990 ) . on the other hand ,
investigations of selective pressure acting on protein expression of virus genes led to the identification of positive selection in 17 accession pairs involving 15 different lineages .
it is worth mentioning that numerous viruses : jcsmv.iran , ocsv , carrsv - rna 2 , rcnmv - rna 2.can , bbsv.val25.iran , galv.nipplefruit , tbsv.statice , pnsv , and plpv.pv.0193 evolved under both mechanisms : recombination and positive selection between which synergism might be occurring . such a synergism between recombination and natural selection may have played a major role in darwinian molecular evolution .
the evolutionary history of the tombusviridae has shown that the 79 accessions split into eight clearly separated clusters representing the eight genera of the tombusviridae family . from the present phylogenetic study ,
at least two taxonomic implications can be drawn : ( i ) three viruses ( nlvcv.alaska , pcrpv.gr 57 , plpv.pv-0193 ) currently considered by the ictv as : one unassigned tombusviridae ( plpv.pv-0193 ) , and two unclassified tombusviridae ( nlvcv.alaska and pcrpv.gr 57 ) .
all of them should be included in genus carmovirus ; ( ii ) in addition to the viruses belonging to genus carmovirus which have formed two separated subgroups , the members of genera necrovirus , and dianthovirus evolved separately and divided into two distinct subgroups as shown in figure 1 .
in contrast , members of genera aureusvirus , and tombusvirus formed separately a single ensemble .
the evolutionary relationships among viruses are a reliable approach for classification . as stated by stuart et al .
( 2004 ) ( who reported similar results regarding the genetic divergence of components of genus necrovirus ) , the comparison of complete genomes is a more balanced approach that should provide a more precise scheme of relatedness . on the other hand , it should be pointed out that , in genus dianthovirus , the genetic divergence between rnas 1 and 2 is correlated to the final products synthesized and their use by the virus to survive . for example : rna silencing is a small rna - guided sequence- specific gene activation mechanism in eukaryotes that is involved in different biological phenomena ( e.g. development , heterochromatin formation and defense against molecular parasites such as viruses ) .
these rna silencing suppressors have been identified in the following genera : aureusvirus , carmovirus , tombusvirus , and dianthovirus ( voinnet e al . , 1999 ;
2003 ; mrai et al . , 2005 ; takeda et al . , 2005 ) .
the dianthoviral suppressor consists of multiple components including p27 , p88 ( encoded by two orfs in rna 1 ) and viral rna ( takeda et al . , 2005 ) .
moreover , sequence variability of the coat protein - coding gene ( rna 1 ) may be linked to the interaction between this structural protein and the host and vector which themselves show a major diversity among diathoviruses . in contrast , the orf in rna 2 encodes the movement protein .
finally , to the author s best knowledge , this is the largest study in the literature so far on recombination potentially occurring in the entire genome of all currently known members of the family tombusviridae as well as positive selection operating on protein expression and their phylogenetic reconstruction . | a detailed study of putative recombination events and their evolution frequency in the whole genome of the currently known members of the family tombusviridae , comprising 79 accessions retrieved from the international databases , was carried out by using the recco and rdp version 3.31 algorithms .
the first program allowed the detection of potential recombination sites in seven out of eight virus genera ( aureusvirus , avenavirus , carmovirus , dianthovirus , necrovirus , panicovirus , and tombusvirus ) , the second program provided the same results except for genus dianthovirus . on the other hand , both methods failed to detect recombination breakpoints in the genome of members of genus machlomovirus . furthermore , based on fisher s exact test of neutrality , positive selection exerted on protein - coding genes was detected in 17 accession pairs involving 15 different lineages .
except genera machlomovirus , and panicovirus along with unclassified tombusviridae , all the other taxonomical genera and the unassigned tombusviridae encompassed representatives under positive selection .
the evolutionary history of all members of the tombusviridae family showed that they segregated into eight distinct groups corresponding to the eight genera which constitute this family .
the inferred phylogeny reshuffled the classification currently adopted by the international committee on taxonomy of viruses .
a reclassification was proposed . |
one of the fundamental building blocks necessary for the development of nanotechnology are semiconductor nanodots ( nds ) , nanocrystals ( ncs ) or nanoparticles ( nps ) viz .
these are completely quantised , zero dimensional ( 0d ) semiconductors with discrete energy levels whose electronic and optoelectronic properties are size dependent .
semiconductor qds have been traditionally obtained by strained layer growth using molecular beam epitaxy ( mbe ) or chemical vapour deposition ( cvd ) . however , during recent years nds , ncs and nps have also been obtained using complex chemical synthesis involving solid , liquid and gas reactions and a broad variety of methods including hydrothermal and colloidal synthesis .
so far , nds , ncs , nps have been realised from group iv elemental semiconductors like si and ge but also iii - v and ii - vi compound semiconductors like gaas , inp , inn gan , zns , cds and oxides like zno [ 14 - 17 ] , sno2 and in2o3[19 - 28 ] . among the iii - v s , nitride ( n ) semiconductors are especially attractive due to the fact that their energy band gap can be adjusted over a very wide range e.g. between 0.7 ev for inn up to 3.4 ev in gan by changing the composition of the ternary semiconductor inxga1xn over 0 x 1 . on the other hand , among the oxides , in2o3 is a very important wide - band gap semiconductor ( eg = 3.55 ev ) which is widely used for the fabrication of devices such as electro - optic modulators , solar cells , electro - chromic mirrors and sensors . to date
in2o3 ncs have been obtained via cvd using in2o3:in in a flow of ar : ch4 at 950 c by jia et al .
or just ar between 950 and 1300 c by cheng et al . and guha et al . .
similarly , chains of in2o3 ncs have been obtained by direct oxidation of in which was heated to 900 c .
high yields are critical in order to successfully fabricate nano - devices and low temperatures desirable for compatibility with mainstream silicon processes but also those involving polymers .
one of the shortcomings in the synthesis of in2o3 nanostructures obtained by cvd is the fact that until now high temperatures have been used .
gel method by zhou et al . , which took , however , > 100 h and involved annealing between 500 and 850 c .
in addition , a high yield - low temperature synthesis of single crystalline tin doped indium oxide octahedrons via a low temperature , catalyst - free process was also demonstrated by wei et al . , who obtained nanostructures as low as 450 c using a solution of indium acetate and tin chloride .
therefore , there is active interest in the low temperature synthesis of in2o3 ncs and an active , growing interest in inn qds which have been grown by mbe or metal organic cvd ( mocvd ) predominantly on gan , aln , al2o3 .
very few investigations have been carried out on the synthesis of colloidal inn nps while recently indium oxynitride which can be thought of as an alloy between inn and in2o3 was investigated and proposed for optoelectronic device applications . in this work ,
we have investigated the synthesis of in2o3ncs by direct oxidation of in with o2 in an atmospheric pressure ( apcvd ) reactor and the limitations that arise as a consequence of the direct oxidation process .
we find that during direct oxidation of in by o2the transfer of in into the gas stream is inhibited by the formation of an in2o3shell around the in powder which melts at 157 c .
the in2o3shell breaks up only at high growth temperatures ( tg ) i.e.tg > 900 c , thereby releasing in into the gas stream which in turn reacts with o2leading to the formation of a high yield of in2o3octahedrons with sizes of 500 nm but also chains of these nanostuctures .
we find that including nh4cl into the in under a gas flow of n2leads to the sublimation of nh4cl into nh3and hcl at around 338 c which results into the complete transfer of the in precursor into the gas stream of n2where it reacts with hcl and forms incl that adsorbs onto the si(111 ) and reacts with h2o and o2forming in2o3nanopyramids at temperatures as low as 500 c . upon carrying out the reaction under nh3as opposed to n2we obtain inn nanoparticles with an average diameter of 300 nm .
the low temperature growth of inn and in2o3ncs directly on si(111 ) using a common type of reaction could be exploited for the synthesis of inn / in2o3hetero - nanostructures for a diverse range of applications such as solar cells , sensors , etc .
the in - based nanostructures were grown using an apcvd reactor which consists of four mass flow controllers ( mfc s ) and a horizontal quartz tube furnace , capable of reaching a maximum temperature of 1100 c .
more specifically , we usednsi(111 ) andnsi(111 ) covered with a thin layer of au that had a thickness of a few nm s .
the si(111 ) samples had an area of 1 cmand initially they were immersed in hf , rinsed in de - ionised water and dried with nitrogen prior to the deposition of the au layer in order to remove the native oxide and surface contamination . the au layer was deposited via sputtering at a rate of 0.1 nm / s using an ar plasma under a pressure < 10 mbar . for the purpose of optical measurements
all of the in - based ncs were grown directly on quartz . in the case of direct oxidation at high temperatures ,
fine in powder ( aldrich , mesh-100 , 99.99% ) was weighed and loaded into a quartz boat together with a au / si(111 ) and plain si(111 ) sample . the au / si(111 ) sample
was positioned about 5 mm over the in and the si(111 ) about 10 mm downstream from the in powder .
then the boat was loaded into the quartz tube reactor and positioned directly above the thermocouple used to measure the heater temperature at the centre of tube .
after loading the boat at room temperature ( rt ) , ar ( 99.999% ) was introduced at a flow rate of 500 standard cubic centimetres per minute ( sccm ) for 10 min , in order to purge the tube . following this ,
the temperature was ramped to the desired growth temperature ( tg ) in a reduced ar flow of 100 sccm . upon reachingtgthe flow of ar
was reduced to 90 sccm and o2introduced at a flow of 10 sccm for another 60 min after which the o2flow was cut off and the quartz tube was allowed to cool down over at least 60 min in an inert gas flow of ar , 100 sccm .
the sample was removed only when the temperature was lower than 100 c . for the synthesis of in2o3 ncs at low temperatures , an equimolar mixture of in powder ( aldrich , mesh-100 , 99.99% ) and nh4cl ( 99% , vwr ) was prepared and loaded in the centre of the quartz boat . a au / si(111 )
sample was positioned over the mixture of in and nh4cl and a si(111 ) sample without au downstream from the mixture as described above .
after loading the boat at room temperature ( rt ) , nitrogen n2 ( 99.999% ) was introduced at a flow rate of 500 sccm for 15 min in order to purge the tube . following this ,
the temperature was ramped to the desired growth temperature ( tg ) in a n2 flow of 100 sccm s . upon reaching tg the flow of n2
was maintained at 100 sccm for a further 60 min after which the quartz tube was allowed to cool down over at least 60 min .
finally the inn ncs were obtained by using an equimolar mixture of in : nh4cl and exactly the same growth conditions that led to the formation of inn nanowires by direct nitridation of in at 600 c under a gas flow of 250 sccm nh3 which is described in detail elsewhere .
the morphology of the nanostructures were examined with a tescan scanning electron microscope ( sem ) and the crystal structure and phase purity of the nanostructures were investigated using a shimadzu , xrd-6000 , x - ray diffractometer and cu , ka source .
a scan of 2 in the range between 10 and 80 was performed for the nanostructures grown under different conditions . for the xrd we used al and stainless steel ( fe3c ) holders to place the samples on the goniometer . in some of the xrd patterns
the characteristic peaks of the holder material were detected and identified . finally , optical spectroscopy was carried out using a standard spectrophotometer uv / v ( perkin elmer lambda 950 ) in the reflection mode at near normal incident to minimise scattering effects from the sample .
in the case of direct oxidation of in with o2we observed the formation of in2o3ncs only fortg 900 c and a typical sem image of in2o3ncs grown attg = 1000 c is shown in fig .
1a . following the reaction we found that the in powder had expanded significantly into a porous like solid which disintegrated easily into a powder while a bright yellow dust corresponding to the in2o3ncs was found on the si(111 ) but also in plentiful supply on the sides of the quartz tube . the in2o3ncs are octahedrons and had the tendency to form large dendrites consisting of in2o3nc chains as shown in fig .
the small in2o3ncs have a diameter of 500 nm , while the sizes of the larger octahedrons are almost 2500 nm .
in2o3ncs grown on si(111 ) by direct oxidation of in with o2at 1000 c ( a ) isolated ncs with min diameter of 500 nm ( b ) dendrite structure consisting of chains of ncs ( c ) side view of nc chains the surface density of the in2o3ncs across the surface of the si(111 ) was very uniform and the density of the dendrites was dependent on the amount of in and the distance of the silicon from the in .
the in2o3ncs grown attg = 1000 c on silicon exhibited clear peaks in the x - ray diffraction spectra shown in fig .
2and have a cubic structure , while the optical bandgap is near 3.5 ev as determined from the reflection spectrum shown in fig .
3 . xrd spectrum of in2o3ncs grown on si(111 ) ( i ) by direct oxidation of in by ar:10% o2at 1000 c , i.e. cvd71 , top pattern and ( ii ) by reaction of in with nh4cl at 600 c under a flow of n2i.e .
enhanced in2o3peaks are obtained from the sample grown at the higher temperature reflection spectrum of the in2o3ncs grown at 1000 c directly on quartz as already stated above , no nanostructures were obtained for tg < 900 c in the presence of o2 and the in powder which melts at 157 c forms a well - defined sphere in the quartz boat found after each growth run .
the surface of the in had a grey , non - reflective appearance as opposed to the highly reflective surface of in which we always obtained during the growth of inn from in and nh3 .
in contrast to the above findings , the reaction of in and nh4cl occurred efficiently even at temperatures as low as 400 c , since no trace of the in : nh4cl mixture whatsoever was found in the boat after removing the latter from the quartz tube .
in addition , a dark yellow powder was always found near the cool end of the reactor which rapidly turned into metallic grey upon exposure to the ambient air . a typical sem image of in2o3ncs obtained on si(111 ) at 600 c after the reaction of in with nh4cl under n2is shown in fig .
4b . interestingly , no nano - pyramids were obtained on the si(111 ) that was covered with a few nm s of au and positioned directly over the in : nh4cl mixture .
( a ) in2o3ncs as small as 200 nm were grown on si(111 ) at 600 c via the reaction of in and nh4cl .
the large pyramids have a base almost equal to 2000 nm ( b ) coalescence of in2o3ncs into small flower like structures at 500 c ( a ) sem image of the inn ncs grown at 600 c on si(111 ) ( b ) high magnification of the hexagonally shaped inn ncs with diameters 200300 nm ( c ) xrd spectrum corresponding to single phase hexagonal inn
to date in2o3 ncs have been obtained using cvd either by direct oxidation of in or by reduction of in2o3 at high temperatures [ 19 - 21 ] . in the case of direct oxidation of in with
this shell inhibits the transfer of in into the gas stream , so it is necessary to increase the temperature above 900 c in order to break the shell and ensure an adequate transfer of in into the gas stream .
this leads to an apparent expansion of the in very similar to that which occurs during the nitridation of al when it reacts with nh3 . in the latter case ,
when al is heated up in a flow of n2:nh3 it leads to the formation of aln around the source of al which in turn inhibits the transfer of al into the gas stream and consequently the growth of aln . at high temperatures the molten al expands and breaks the surrounding aln shell thereby releasing al .
after cool down the residual al appears to have expanded and has a porous like appearance .
the formation of aln on the al source has been shown to be inhibited by incorporating nh4cl in the al .
the decomposition of nh4cl enhances the porosity of the al melt thereby promoting the transfer of al into the gas stream . on the other hand ,
the reaction of nh4cl and al leads to the formation of alcl3 which is a gas and this reacts in turn with nitrogen giving aln according to,(1)(2)(3 ) in the case of in , we find that incorporation of nh4cl leads to the complete transfer of the solids of in and nh4cl into the gas stream of n2 .
indium has a low melting point of 156 c and once molten it forms a sphere whose size depends on the initial amount of powder .
the complete elimination of the in and nh4cl is a direct consequence of the sublimation of the nh4cl into nh3 and hcl which occurs at 338 c . according to chaiken et al .
, the sublimation rate of nh4cl increases by a factor of 10 when changing the temperature from t = 100600 c and the typical sublimation weight loss of nh4cl is over 90% when heated for 60 min .
sublimation is endothermic and the temperature is expected to be reduced only by a few tens c in the case of nh4cl .
consequently , the sublimation of nh4cl enhances the porosity of the in resulting into an efficient transfer of in from the interior of the melt into the gas stream where it subsequently reacts with the hcl thereby forming incl , incl2 and incl3 .
however , gaseous incl3 at temperatures > 400 c suffers decomposition and yields incl and incl2 while the amount of incl increases with increasing temperature .
thus , during growth for t > 400 c incl3 decomposes predominantly into incl according to,(4 ) incl is yellow , changes to red at 120 c and has a relatively low melting point of 216 c .
the incl molecules adsorb on the si(111 ) surface and react with h2o possibly absorbed by the nh4cl to form in2o3ncs according to,(5 ) a similar reaction involving spraying of incl3 .
h2o in ethanol was recently employed for the deposition of a polycrystalline thin film of in2o3 on stripe - patterned si at 280 c .
the in2o3 ncs have a stable , light yellow colour , even after exposure to the ambient air
. however , most of the incl is carried downstream as evidenced by the large amounts of dark yellow powder found only near the cool end of the reactor . both incl and incl2
decompose into in and cl when reacting with h2o and so lead to the separation of metallic indium .
therefore , the rapid change of the dark yellow powder to metallic grey upon exposure to the ambient air is attributed to the humidity of the air , i.e. h2o .
it is important to point out that the in2o3 ncs were also obtained via the sublimation of nh4cl in in under the presence of o2 for t < 900 c .
in contrast , no in2o3 ncs were obtained in the case of the oxidation of in alone by o2 for t < 900 c .
the formation of in2o3 pyramidal ncs on si(111 ) was confirmed by the xrd spectrum shown in fig .
2 , only the in2o3 reflections are observed ensuring the absence of a second phase .
2 correspond to the in2o3 peaks also observed by du et al . who have grown in2o3 structures by dehydration of in(oh)3 after heat treatment at 500 c for 4 h. the in2o3 peaks have higher intensities for growth temperatures above 600 c . on the other hand , the sublimation of nh4cl in the in under a gas flow of nh3 does not yield pyramidal ncs . upon carrying out the reaction of in with nh4cl in a gas flow of 250 sccm of nh3 as
opposed to n2 at 600 c we obtained single phase , hexagonal structured inn nps with an average diameter of 300 nm as shown in fig . 5b and confirmed by xrd shown in fig .
5c where the ( 002 ) , ( 101 ) , ( 103 ) and ( 112 ) reflections of the inn , si ( 111 ) and the stainless steel holder peaks are observed . in this case
the incl reacts with the nh3 giving inn similar to the recent investigation of kumagai et al .
on hydride vapour phase epitaxy of inn epitaxial layers via the reaction of incl3 with nh3 .
these inn ncs are also very similar in size and morphology with those obtained by mocvd on gan .
an interesting aspect of the in2o3ncs obtained from the reaction of in with nh4cl is that they self - assemble into well - defined circles fort 600 c , as shown in fig .
6a and coalesce near the periphery leading to the formation of m size , flower - like structures , as shown in fig .
6b att = 500 c resembling a wreath . upon reducing the temperature to 400 c
we do not find anymore isolated in2o3ncs but only m size flower - like structures which now fill the wreaths , as shown in fig .
this type of self - assembly was not observed for the in2o3ncs grown by direct oxidation of in with o2att > 900 c which aggregate into dendrites that consist of nc - chains .
a possible explanation for this type of self - assembly is wetting of the si(111 ) surface by in .
the wetting is not uniform across the sample but instead the in forms wet circular regions on the si(111 ) surface which tend to expand as the growth proceeds thereby leading to the coalescence of the ncs and the formation of the wreath - like structures shown in fig .
d . these wreath - like structures become larger when decreasing the temperature and the individual flower - like structures may grow up to 20 m , fig .
acoalescence of in2o3ncs at the periphery of a well - defined circle and formation of flower - like structuresbcoalescence of in2o3ncs into flowers and formation of a wreath spanning over 20 m , obtained attg = 500 cclarge wreath full of flower - like structures spanning over almost 200 m obtained attg = 400 c andd20 m isolated flower - like structure
we have investigated the synthesis of in2o3ncs by direct oxidation of in with o2and also by the incorporation of nh4cl in the in under n2 .
the synthesis of in2o3ncs by direct oxidation of in with o2is limited by the formation of an oxide shell surrounding the in which can be eliminated only attg > 900 c as a consequence of the expanding melt therefore allowing an efficient transfer of in into the main gas stream .
attg > 1000 c we obtain 500 nm size in2o3ncs but also dendrite structures consisting of nc chains .
the high temperature requirement can be alleviated by the addition of nh4cl into the in which breaks down into nh3and hcl by sublimation thus resulting into the complete transfer of the in into the gas stream and the formation of incl which reacts with h2o and o2 . in this way we obtained in2o3ncs at temperatures as low as 500 c under n2but also inn by simply changing from n2to nh3 .
this method is potentially interesting for the synthesis of narrow and wide - band gap inn and in2o3ncs at low temperatures but also for nano - engineering of heterostructures and the fabrication of novel devices such as third generation solar cells . | indium oxide ( in2o3 ) nanocrystals ( ncs ) have been obtained via atmospheric pressure , chemical vapour deposition ( apcvd ) on si(111 ) via the direct oxidation of in with ar:10% o2at 1000 c but also at temperatures as low as 500 c by the sublimation of ammonium chloride ( nh4cl ) which is incorporated into the in under a gas flow of nitrogen ( n2 ) .
similarly inn ncs have also been obtained using sublimation of nh4cl in a gas flow of nh3 . during oxidation of in under a flow of o2the transfer of in into the gas stream
is inhibited by the formation of in2o3around the in powder which breaks up only at high temperatures , i.e.t > 900 c , thereby releasing in into the gas stream which can then react with o2leading to a high yield formation of isolated 500 nm in2o3octahedrons but also chains of these nanostructures .
no such ncs were obtained by direct oxidation fortg < 900 c .
the incorporation of nh4cl in the in leads to the sublimation of nh4cl into nh3and hcl at around 338 c which in turn produces an efficient dispersion and transfer of the whole in into the gas stream of n2where it reacts with hcl forming primarily incl .
the latter adsorbs onto the si(111 ) where it reacts with h2o and o2leading to the formation of in2o3nanopyramids on si(111 ) .
the rest of the incl is carried downstream , where it solidifies at lower temperatures , and rapidly breaks down into metallic in upon exposure to h2o in the air . upon carrying out
the reaction of in with nh4cl at 600 c under nh3as opposed to n2 , we obtain inn nanoparticles on si(111 ) with an average diameter of 300 nm . |
upper - limb pain and dysfunction are frequent complaints associated with manual wheelchair
propulsion , which is physically demanding and involves repetitive movements1 .
about 73% of wheelchair users suffer from
chronic upper - limb pain , which is primarily attributed to two factors : wheelchair propulsion
and transfers2 .
therefore , selecting the
appropriate wheelchair and seating system is one of the most important decisions for those
who spend long periods of time in their wheelchairs3 .
active wheelchair users report shoulder , elbow , and wrist / hand
pain , chiefly shoulder pain4 .
the chair
backrest is designed to decrease the stresses on the vertebral column by : allowing
relaxation of the erector spinae muscles , maintaining lumbar lordosis , and improving
comfort5 .
while biomechanical variables
are important criteria for wheelchair users , they have not been studied sufficiently .
prevention is the best way to reduce chronic pain , and researchers have suggested various
methods of preventing the development of problems during wheelchair propulsion .
back belts
might have positive effects on sustaining an erect trunk and lumbar lordosis by supporting
the force from the knees , but they may secondarily lead to knee discomfort .
it is also important to
investigate how the backrest thickness affects comfort , especially in a dynamic setting
during wheelchair propulsion .
therefore , this study investigated the effects of backrest
thickness on the lumbar load and comfort by assessing the surface electromyography ( semg )
activity of the shoulder muscles during wheelchair propulsion with lumbar support .
all of the subjects were informed of
the study purpose and methods before participating , and provided their informed consent
according to the principles of the declaration of helsinki .
our study consisted of three randomized backrest
conditions : no pad ; a 3-cm - thick lumbar pad ; and a 6-cm - thick lumbar pad .
the lumbar pads of
the backrests used in this study had a density of 27 kg / m .
the location of the
lumbar pad was standardized by aligning it at the mid - lumbar level ( l3 ) .
they propelled the wheelchair
30 times with their hands , the upper body in an upright position at an average rate of once
per second . for stable propulsion ,
the semg activity of the upper extremity muscles was
collected , amplified , digitized , and analyzed using an me6000-biosignal monitor ( mega
electronics , kuopio , finland ) .
the electrode locations were as follows : anterior deltoid ,
anterior aspect of the arm , approximately 4 cm below the clavicle ; upper trapezius , slightly
lateral to and halfway between the cervical spine at c-7 and the acromion ; posterior
deltoid , 2 cm below the lateral border of the spine of the scapula and angled obliquely to
the arm ; and biceps brachii , attached at the middle muscle belly over the short and long
heads .
the emg data were converted into root mean square ( rms ) values in a window comprising
300 ms of data .
we obtained the reference voluntary contraction ( rvc ) using the submaximal
normalization method .
spss 22.0 ( spss , chicago , il , usa ) was used to analyze the differences
in shoulder muscle activities .
smirnov test was performed to test for a
normal distribution before using parametric statistics . the significance of differences
among wheelchair propulsion with no lumbar pad , the 3-cm lumbar pad , and the 6-cm lumbar pad
was tested using one - way repeated - measures analysis of variance ( anova ) .
for the significant main
effect , the bonferroni correction was used to identify the specific mean differences .
the normalized emg data obtained while performing the wheelchair propulsion task with no
pad , a 3-cm - thick lumbar pad , and a 6-cm - thick lumbar pad revealed significant differences
in the activation of the anterior deltoid , upper trapezius , posterior deltoid , and biceps
brachii muscles .
muscle activation of the anterior deltoid , upper trapezius , and biceps
brachii muscles significantly decreased when the 3-cm pad was used compared to none , and
significantly increased in the anterior deltoid , upper trapezius , posterior deltoid , and
biceps brachii when the participants used the 6-cm pad compared to the 3-cm pad .
there were
no significant differences in muscle activation between the no pad and the 6-cm lumbar pad
conditions ( table 1table 1.muscle activations during wheelchair propulsion with the different lumbar pad
conditions ( n=15)muscle%rvc ( meansd)none3-cm pad6-cm padanterior deltoid70.1322.1549.8116.8266.0221.72upper trapezius100.2218.2083.8215.33110.8220.53posterior deltoid105.3123.7195.8516.77100.1818.90biceps brachii110.8916.3590.3015.19118.7013.49p<0.05 ) .
many studies have indicated that there is a relationship between wheelchair propulsion and
upper limb injuries1 , 8 .
previous study has shown that a lumbar support increased ( or
preserved ) the comfort while sitting .
especially , it indicated that a 23 change in lumbar
posture influences the compressive load at l4l5 when performing spinal loading work9 .
our present results show that there was a
significant decrease in the activation of the anterior deltoid , upper trapezius , and biceps
brachii muscles when the participants used the 3-cm pad compared to no pad , while the
activation of the anterior deltoid , upper trapezius , posterior deltoid , and biceps brachii
significantly increased when the participants used the 6-cm pad compared to the 3-cm pad .
generally ,
the push phase mainly uses the anterior deltoid , pectoralis major , and biceps brachii ,
whereas the recovery phase primarily uses the upper trapezius and middle and posterior
deltoid10 , 11 .
one possible explanation for our result is the effect of muscle
activation on the propulsion efficiency when performing wheelchair propulsion with the 3-cm
pad compared with no pad or a 6-cm pad .
it has also been reported that an appropriate lumbar
pad provides a biomechanical advantage to the shoulder during seated work .
a decrease in
muscle load might elicit the optimal curvature of the lumbar spine when propelling a
wheelchair .
this suggests that although users might not benefit from a lumbar pad to improve
their temporal muscle load , long - term users develop musculoskeletal shoulder pain .
goosey et
al . indicated that a lower propelling frequency could explain the effectiveness of pushing
economy .
a high frequency is associated with more shifts during the deceleration and
acceleration phases , and increased inertial limb moments . as a result
, a lower frequency
leads to less muscle activity , possibly reducing rotator cuff fatigue , while maintaining the
stability of the humeral head4 , 12 , 13 . in this study ,
the backrest was rated as more comfortable , and it had better dampening capability , which
allowed leverage in the contact area between the lumbar spine and backrest during wheelchair
propulsion .
the results of our present study confirm that an appropriate lumbar pad allows
more effective coordination of the shoulder muscles and increases comfort during wheelchair
propulsion . | [ purpose ] this study investigated the optimal thickness of the wheelchair backrest for
lumbar load and increased comfort .
[ subjects ] fifteen healthy people participated .
[ methods ] the study examined three randomized backrest conditions : no pad ; a 3-cm - thick
lumbar pad ; and a 6-cm - thick lumbar pad .
the location of the lumbar pad was standardized
at the mid - lumbar level ( l3 ) .
participants were instructed to propel the wheelchair using
only the handrims .
[ results ] activation of the anterior deltoid , upper trapezius , and
biceps brachii muscles was significantly reduced when the participants used the 3-cm pad
compared to no pad , while it was significantly increased in the anterior deltoid , upper
trapezius , posterior deltoid , and biceps brachii when the participants used the 6-cm pad
compared to the 3-cm pad .
muscle activation did not differ significantly between the no
pad and the 6-cm lumbar pad conditions .
[ conclusion ] a lumbar pad decreased the activation
of the upper extremity muscles .
we believe that padding of the appropriate thickness will
lead to effective muscle activation while propelling a wheelchair and decrease the risk of
musculoskeletal disease . |
the successful development of novel first - in - class therapeutic agents in the cns has been lagging with respect to other disease areas .
moreover , only 8% of cns drugs that enter phase 1 are approved , with about 65% of the failures due to lack of efficacy or sufficient differentiation in phase iii .
this high degree of failure is caused by the extreme complexity of the human brain neurobiology and the increasing realization that the clinical outcome is driven by emergent properties of neuronal circuits , rather than by a single target .
complex translational problems that preclude simple animal model extrapolation in cns r&d include ( i ) fundamental differences in neurotransmitter circuitry between rodents and humans , ( ii ) the incomplete representation of the full human pathology , ( iii ) the absence of important functional genotypes in animal models , and ( iv ) the existence of unique pharmacologically active human metabolites .
many of these problems can in principle be reduced using humanized computer - based qsp , as suggested by a white paper ( http://isp.hms.harvard.edu/wordpress/wp-content/uploads/2011/10/
nih-systems-pharma-whitepaper-sorger-et-al-2011.pdf ) .
application of qsp in cns disorders is particularly appealing because of the large academic expertise in computational neuroscience since the seminal paper of hodgkin and huxley , and due to the availability of the specialized software languages and the sharing of software modules ( http://senselab.med.yale.edu/modeldb/ ) .
we will argue with a few examples that computer - based qsp ( figure 1 ) could be a powerful additional tool to reduce clinical attrition in psychiatry and alzheimer 's disease , although in principle , this approach can be applied to other cns diseases , such as parkinson 's disease , huntington 's disease , and cognitive and negative symptoms in schizophrenia .
there appears to be a fatalistic perception that we need to base our decisions for cns projects solely on animal models even realizing that their predictability is limited .
in other successful industries with shorter cycle times and higher success rates such as microelectronics , aerodynamics , and petrochemistry , computer models are extensively used to test the effects of different experimental designs before actually building a prototype , saving significant amounts of money and time .
of course , data in biology are inherently noisier because of intrinsic variability in the human patient population and the limited accessibility and readout of specific experiments .
however , the addition of a simple , yet incomplete computer model that is able to integrate many facts into a single predictive , quantifiable effect estimate will essentially include already a great deal of information that is not currently and easily accessible in any drug pipeline decision process .
current neuropharmacological treatment strategies and decisions are also based on a rational qualitative understanding of human biology and the impact of drugs and pathology on these interactions , usually using cartoon - based schematic drawings .
we would therefore argue that a more quantitative computer - based mechanistic modeling and simulation , even with the current limited knowledge is a possible solution to increase the chances of clinical success .
a possible detailed implementation of such a qsp platform for schizophrenia is shown in figure 2 and is described in detail . with this model ,
the correlation between the simulated outcome of 76 different drug dose combinations and their corresponding effects on clinical scales , such as positive and negative scales in schizophrenia total , is 0.56 as compared with a correlation coefficient of 0.18 with the simple d2r occupancy calculation , the current gold standard for predictive pharmacokinetics/ parkinson 's disease modeling .
this suggests the computer model can explain much more of the biological variance than the correlation with target engagement at one receptor .
this is because the computer model simulates the off - target effects of antipsychotics at other receptors in a physiologically relevant way .
there appears to be a fatalistic perception that we need to base our decisions for cns projects solely on animal models even realizing that their predictability is limited . in other successful industries with shorter cycle times and higher success rates such as microelectronics , aerodynamics , and petrochemistry ,
computer models are extensively used to test the effects of different experimental designs before actually building a prototype , saving significant amounts of money and time .
of course , data in biology are inherently noisier because of intrinsic variability in the human patient population and the limited accessibility and readout of specific experiments .
however , the addition of a simple , yet incomplete computer model that is able to integrate many facts into a single predictive , quantifiable effect estimate will essentially include already a great deal of information that is not currently and easily accessible in any drug pipeline decision process .
current neuropharmacological treatment strategies and decisions are also based on a rational qualitative understanding of human biology and the impact of drugs and pathology on these interactions , usually using cartoon - based schematic drawings .
we would therefore argue that a more quantitative computer - based mechanistic modeling and simulation , even with the current limited knowledge is a possible solution to increase the chances of clinical success .
a possible detailed implementation of such a qsp platform for schizophrenia is shown in figure 2 and is described in detail . with this model ,
the correlation between the simulated outcome of 76 different drug dose combinations and their corresponding effects on clinical scales , such as positive and negative scales in schizophrenia total , is 0.56 as compared with a correlation coefficient of 0.18 with the simple d2r occupancy calculation , the current gold standard for predictive pharmacokinetics/ parkinson 's disease modeling .
this suggests the computer model can explain much more of the biological variance than the correlation with target engagement at one receptor .
this is because the computer model simulates the off - target effects of antipsychotics at other receptors in a physiologically relevant way .
a quantitative simulation study on aripiprazole and bifeprunox found that the specific differences between primate and rodent striatal dopaminergic physiology and aripiprazole 's unique human metabolite opc1485 can account for a large part of their difference in clinical trials .
the clinical development of bifeprunox was halted after 15 years of study and at an enormous financial and resource cost . using the complete human pharmacology of dimebon , an antihistaminergic compound with mitochondrial membrane protecting properties , a qsp failure analysis in alzheimer 's disease suggests strongly that the off - target effect at hd1r significantly reduced cognitive benefits and that this effect is genotype - dependent .
a similar computational approach has been proposed on the effect of ampa modulation in complex neuronal networks in which changes in the kinetics of a ligand - based voltage channel after drug action have a major influence on the network behavior at different time scales .
currently , pharmacometric modeling and simulation becomes involved only in early clinical development with a compound and target that has been selected many years before .
if either the target fails the drug or the drug fails the target , then the best that computational modeling and simulation can do is to find clinical trial solutions to mitigate these problems , which is often insufficient to rescue the project . of note ,
dosing mismatch was the only area in clinical drug development that has significantly improved between 1995 and 2004 likely as a consequence of improved pharmacometric modeling and simulation .
the qsp computational approach as described here was inspired by the seminal work of the late leif finkel and further developed as computational neuropharmacology . taking into account the limitations of this model , proper use of these humanized computer models could significantly reduce the dependence of the go / no - go decisions on traditional and less - predictive animal models and substantially de - risk cns projects .
in fact , the approach lends itself very naturally to the concept of learn - and - confirm ; when the prediction of the model is not confirmed in a clinical trial , we can identify what assumptions are needed to be changed and the model parameters that can be improved so as to better reflect the actual clinical outcomes . the mechanistic disease
computer - based model can further function as a useful biological knowledge repository with a common universal language ( mathematics ) .
the availability of a uniform and well - defined mathematics - based language will naturally improve communication between scientists active in cns drug r&d and make comparisons between different laboratoria and experimental conditions much easier .
a computer model explicitly lists the biological assumptions in a decision process , and provides a framework to discuss the validity of those assumptions . in r&d , such a model can act as a bridge and discussion platform between groups and opinions within the decision process and is helpful in situations where different preclinical models have different outcomes for the investigative drug .
a computational model can then provide an independent test for each opinion in the process by quantifying specific mechanisms and pathways that lead to the specific result and then provide an integrative tool for decision - making process .
the mathematical approach emphasizing neuronal networks and circuits also forces the scientists to explicitly formulate their assumptions in a quantitative way , leading to a better understanding of the human brain biology , often going beyond the single target they are interested in .
technological barriers include the limit on the complexity of the neuronal networks we can simulate ; however , hardware and software advantages together with the availability of cloud computing allow these boundaries to be pushed out considerably .
big - science initiatives such as the human connectome and the human brain atlas from the allen institute can help build increasingly complex computer models using the human neurophysiology and neuroanatomy information .
qsp will never be able to simulate the complete human brain in detail , but even with incomplete biological implementations , we have shown that this approach can already be successful . due to the top down nature , processes with the biggest impact on the clinical outcomes tend to be selected .
after all , engineers did not wait for the grand unified theory to be completely solved to develop the transistor .
other more conceptual limitations include the defeatist nature of the industry ( failure is inherent to the business ) , the risk aversiveness of introducing new , untested , and unfamiliar technologies ( why should we embrace new technologies with steep learning curves ? ) , the milestone - driven nature of drug discovery and limited time horizon of r&d projects ( we have to get a clinical candidate within xx months ) .
full acceptance and understanding of this technology necessitates also a new generation of scientists ; mathematical - savvy individuals with a clear broad understanding of network physiology , pharmacology , biology , and drug discovery , as opposed to the individuals trained in reductionist - focused educational programs such as molecular biology and genetic analysis .
the qsp as presented here is slightly different from the more traditional systems biology approach .
the latter is aimed at interrogating large , omics databases using statistical data mining techniques in a target - agnostic way and uses generic techniques , i.e. , the same approach can be used in different indications .
both approaches are complementary , in that systems biology can help identify potential new pathways , that can then be implemented in a more physiological and anatomical rational context on a qsp platform . in summary , because of the unsustainable failure rate of drugs in clinical development , the pharma industry is forced to embrace new out - of - the - box processes .
we believe the time has come to introduce computer - based mechanistic qsp more into the early stages of drug discovery and development to ensure that the best clinical candidate can be advanced using the most efficient clinical trial design for the right patient population .
the qsp computational approach as described here was inspired by the seminal work of the late leif finkel and further developed as computational neuropharmacology . taking into account the limitations of this model , proper use of these humanized computer models could significantly reduce the dependence of the go / no - go decisions on traditional and less - predictive animal models and substantially de - risk cns projects .
in fact , the approach lends itself very naturally to the concept of learn - and - confirm ; when the prediction of the model is not confirmed in a clinical trial , we can identify what assumptions are needed to be changed and the model parameters that can be improved so as to better reflect the actual clinical outcomes . the mechanistic disease computer - based model can further function as a useful biological knowledge repository with a common universal language ( mathematics ) .
the availability of a uniform and well - defined mathematics - based language will naturally improve communication between scientists active in cns drug r&d and make comparisons between different laboratoria and experimental conditions much easier .
a computer model explicitly lists the biological assumptions in a decision process , and provides a framework to discuss the validity of those assumptions . in r&d , such a model can act as a bridge and discussion platform between groups and opinions within the decision process and is helpful in situations where different preclinical models have different outcomes for the investigative drug .
a computational model can then provide an independent test for each opinion in the process by quantifying specific mechanisms and pathways that lead to the specific result and then provide an integrative tool for decision - making process .
the mathematical approach emphasizing neuronal networks and circuits also forces the scientists to explicitly formulate their assumptions in a quantitative way , leading to a better understanding of the human brain biology , often going beyond the single target they are interested in .
technological barriers include the limit on the complexity of the neuronal networks we can simulate ; however , hardware and software advantages together with the availability of cloud computing allow these boundaries to be pushed out considerably .
initiatives such as the human connectome and the human brain atlas from the allen institute can help build increasingly complex computer models using the human neurophysiology and neuroanatomy information .
qsp will never be able to simulate the complete human brain in detail , but even with incomplete biological implementations , we have shown that this approach can already be successful . due to the top down nature , processes with the biggest impact on the clinical outcomes tend to be selected .
after all , engineers did not wait for the grand unified theory to be completely solved to develop the transistor .
other more conceptual limitations include the defeatist nature of the industry ( failure is inherent to the business ) , the risk aversiveness of introducing new , untested , and unfamiliar technologies ( why should we embrace new technologies with steep learning curves ? ) , the milestone - driven nature of drug discovery and limited time horizon of r&d projects ( we have to get a clinical candidate within xx months ) .
full acceptance and understanding of this technology necessitates also a new generation of scientists ; mathematical - savvy individuals with a clear broad understanding of network physiology , pharmacology , biology , and drug discovery , as opposed to the individuals trained in reductionist - focused educational programs such as molecular biology and genetic analysis .
the qsp as presented here is slightly different from the more traditional systems biology approach .
the latter is aimed at interrogating large , omics databases using statistical data mining techniques in a target - agnostic way and uses generic techniques , i.e. , the same approach can be used in different indications . both approaches
are complementary , in that systems biology can help identify potential new pathways , that can then be implemented in a more physiological and anatomical rational context on a qsp platform . in summary , because of the unsustainable failure rate of drugs in clinical development , the pharma industry is forced to embrace new out - of - the - box processes .
we believe the time has come to introduce computer - based mechanistic qsp more into the early stages of drug discovery and development to ensure that the best clinical candidate can be advanced using the most efficient clinical trial design for the right patient population .
| we discuss whether a new paradigm , quantitative systems pharmacology ( qsp ) , based on computational neuroscience modeling combined with proper drug target engagement and pharmacology , human pathology , imaging studies , and calibration and validation using clinical studies in human subjects might improve the success rate of central nervous systems research and development ( cns r&d ) projects .
we suggest that an improved understanding of neuronal circuit interactions using a humanized computer - based integration of physiology and pharmacology knowledge can substantially de - risk new cns projects . |
the oec tissue samples were obtained during surgical operations performed at pusan national university hospital , korea , from 1999 to 2008 . a total of 123 patients ( mean age , 49.5 years ; range , 15 to 82 years ) who underwent surgical resection for oec were examined .
pathological data , such as pathologic stage , tumor grading , tumor size , mitosis , and histological tumor type , were obtained from the primary pathology reports , and patient chart reviews were performed retrospectively .
the criteria of the international federation of gynecology and obstetrics ( figo ) were used to classify cases as stages i - iv . all patients , except those with grade 1 , stage ia , were given adjuvant chemotherapy of platinum / taxol - containing drugs .
the patients were followed up from the date of surgery until death or the last visit to the outpatient department .
the follow - up period ranged from one to 175 months ( median , 49 months ) .
overall survival ( os ) was calculated from the date of surgery to the date of death or last followup visit .
progression - free survival ( pfs ) was calculated from the date of surgery to the date of tumor progression or relapse , including local recurrence and/or metastasis .
this study was approved by the ethics committee of the pusan national university hospital , and informed consent was obtained from all the patients .
the slides were deparaffinized in xylene and rehydrated through four decreasing grades of ethanol ( 100% , 95% , 80% , and 70% ) for 2 minutes each .
endogenous peroxidase activity was blocked by immersing the slides in 3% hydrogen peroxide in methanol for 15 minutes at room temperature .
heat - induced antigen retrieval was performed for 5 minutes with 1 citrate buffer ( ph 6.3 ) using a microwave and then cooled for 5 minutes .
this process was performed three times . in order to reduce the nonspecific binding of antibodies to the tissues
, the slides were preincubated with blocking serum in 1% bovine serum albumin for 30 minutes at room temperature .
the slides were left to incubate with rabbit polyclonal anti - twist antibody ( 1:200 , santa cruz biotechnology , san diego , ca , usa ) , mouse monoclonal anti - e - cadherin antibody ( 1:400 , invitrogen , carlsbad , ca , usa ) , mouse monoclonal anti - hif1 antibody ( 1:1,000 , novus biologicals , littleton , co , usa ) , and rabbit monoclonal anti - p53 antibody ( 1:200 , thermo scientific , san jose , ca , usa ) overnight at 4. after incubation with the primary antibodies , the slides were rinsed with phosphate - buffered saline for 10 minutes . thereafter , for the detection of the antibody reactions , the envision detection system ( dako , carpinteria , ca , usa ) was used .
finally , the sections were counterstained with mayer 's hematoxylin solution for 1 minute , and dehydrated with graded alcohols , dipped in two changes of xylene , and mounted .
the immunohistochemical staining was assessed with a light microscope by two pathologists who had no prior knowledge of the clinical data . for twist and hif1 ,
only nuclear staining was considered with a 10% cut - off value for positivity.7 for e - cadherin , membrane staining was graded from 0 - 3 : 0 , no staining or weak intensity in 10% of the tumor cells ; 1 , low intensity in 10% of the tumor cells ; 2 , moderate intensity in 10% of the tumor cells ; and 3 , strong intensity in 10% of the tumor cells . the cases were divided into two groups : preserved e - cadherin expression ( score 3 ) or reduced e - cadherin expression ( scores 0 , 1 , and 2).21 p53 was recognized through nuclear staining of the positive cells .
the results were divided into two groups : positive ( 30% positive tumor cells ) and negative ( < 30% positive tumor cells).25 all statistical analyses were performed using spss ver . 19.0 ( ibm co. , armonk , ny , usa ) .
pearson 's chi - squared test was used to study the associations between the clinicopathological factors and immunohistochemical markers .
os was calculated from the date of surgery to the date of death or last follow - up visit to the outpatient department .
pfs was calculated from the date of surgery to the date of tumor relapse or progression .
data on patients who had survived until the end of the observation period were censored at their last follow - up visit .
death from a cause other than ovarian cancer and cases lost to follow - up were considered censoring events .
the effect of immunohistochemical markers on survival was assessed using kaplan - meier survival curves and a log - rank test .
for all the tests , a p - value .05 was considered to be significant .
the oec tissue samples were obtained during surgical operations performed at pusan national university hospital , korea , from 1999 to 2008 . a total of 123 patients ( mean age , 49.5 years ; range , 15 to 82 years ) who underwent surgical resection for oec were examined .
pathological data , such as pathologic stage , tumor grading , tumor size , mitosis , and histological tumor type , were obtained from the primary pathology reports , and patient chart reviews were performed retrospectively .
the criteria of the international federation of gynecology and obstetrics ( figo ) were used to classify cases as stages i - iv . all patients , except those with grade 1 , stage ia , were given adjuvant chemotherapy of platinum / taxol - containing drugs .
the patients were followed up from the date of surgery until death or the last visit to the outpatient department .
the follow - up period ranged from one to 175 months ( median , 49 months ) .
overall survival ( os ) was calculated from the date of surgery to the date of death or last followup visit .
progression - free survival ( pfs ) was calculated from the date of surgery to the date of tumor progression or relapse , including local recurrence and/or metastasis .
this study was approved by the ethics committee of the pusan national university hospital , and informed consent was obtained from all the patients .
the slides were deparaffinized in xylene and rehydrated through four decreasing grades of ethanol ( 100% , 95% , 80% , and 70% ) for 2 minutes each .
endogenous peroxidase activity was blocked by immersing the slides in 3% hydrogen peroxide in methanol for 15 minutes at room temperature .
heat - induced antigen retrieval was performed for 5 minutes with 1 citrate buffer ( ph 6.3 ) using a microwave and then cooled for 5 minutes .
this process was performed three times . in order to reduce the nonspecific binding of antibodies to the tissues
, the slides were preincubated with blocking serum in 1% bovine serum albumin for 30 minutes at room temperature .
the slides were left to incubate with rabbit polyclonal anti - twist antibody ( 1:200 , santa cruz biotechnology , san diego , ca , usa ) , mouse monoclonal anti - e - cadherin antibody ( 1:400 , invitrogen , carlsbad , ca , usa ) , mouse monoclonal anti - hif1 antibody ( 1:1,000 , novus biologicals , littleton , co , usa ) , and rabbit monoclonal anti - p53 antibody ( 1:200 , thermo scientific , san jose , ca , usa ) overnight at 4. after incubation with the primary antibodies , the slides were rinsed with phosphate - buffered saline for 10 minutes . thereafter , for the detection of the antibody reactions , the envision detection system ( dako , carpinteria , ca , usa ) was used .
finally , the sections were counterstained with mayer 's hematoxylin solution for 1 minute , and dehydrated with graded alcohols , dipped in two changes of xylene , and mounted .
the immunohistochemical staining was assessed with a light microscope by two pathologists who had no prior knowledge of the clinical data . for twist and hif1 ,
only nuclear staining was considered with a 10% cut - off value for positivity.7 for e - cadherin , membrane staining was graded from 0 - 3 : 0 , no staining or weak intensity in 10% of the tumor cells ; 1 , low intensity in 10% of the tumor cells ; 2 , moderate intensity in 10% of the tumor cells ; and 3 , strong intensity in 10% of the tumor cells .
the cases were divided into two groups : preserved e - cadherin expression ( score 3 ) or reduced e - cadherin expression ( scores 0 , 1 , and 2).21 p53 was recognized through nuclear staining of the positive cells .
the results were divided into two groups : positive ( 30% positive tumor cells ) and negative ( < 30% positive tumor cells).25
all statistical analyses were performed using spss ver . 19.0 ( ibm co. , armonk , ny , usa ) .
pearson 's chi - squared test was used to study the associations between the clinicopathological factors and immunohistochemical markers .
os was calculated from the date of surgery to the date of death or last follow - up visit to the outpatient department .
pfs was calculated from the date of surgery to the date of tumor relapse or progression .
data on patients who had survived until the end of the observation period were censored at their last follow - up visit .
death from a cause other than ovarian cancer and cases lost to follow - up were considered censoring events .
the effect of immunohistochemical markers on survival was assessed using kaplan - meier survival curves and a log - rank test .
for all the tests , a p - value .05 was considered to be significant .
expression of hif1 , twist , and p53 proteins was detected in 35 ( 28.5% ) , 43 ( 35.0% ) , and 36 ( 29.3% ) of the 123 oec cases , respectively . reduced expression of e - cadherin was observed in 60 cases ( 48.8% ) .
specific staining for twist was observed in the nucleus of tumor cells , but in some cases , both nuclear and cytoplasmic staining was observed .
clear cell types exhibited twist expression more frequently than other histologic types , but this was not statistically significant ( p=.240 ) . reduced e - cadherin expression was more commonly found in the advanced stages of oec ( 38 of 60 cases , 63.3% ) than the early stages ( 22 of 63 case , 34.9% ) ( p=.002 ) .
hif1 showed different expression according to histologic tumor type but was not associated with any clinicopathological parameters .
there was a statistically significant increase in the expression of p53 in serous - type oec compared with other types ( p=.000 ) .
there were also positive correlations between p53 and the histologic tumor grade , figo stage , and mitotic rate ( table 2 ) .
twist expression was significantly correlated with reduced e - cadherin expression ( p=.000 , correlation coefficient [ c.c.]=-0.309 ) and increased hif1 expression ( p=.000 , c.c.=0.532 ) .
twist expression was not correlated with the expression of p53 ( p=.142 , c.c.=0.168 ) .
there was a negative correlation between p53 expression and the reduced expression of e - cadherin ( p=.041 , c.c.=-0.219 ) ( table 3 ) .
follow - up data were available for 123 patients ( median , 49 months ; range , 1 to 175 months ) . fifty patients ( 40.7% of 123 patients ) died from the disease during the follow - up period .
sixty patients ( 48.8% ) developed either recurrence or metastasis ( progression group ) , whereas 63 patients ( 51.2% ) remained free of progression ( progression - free group ) .
the univariate analysis showed that tumor histologic grade , tumor stage , mitotic rate , positive expression of twist and p53 , and reduced expression of e - cadherin were associated with poor os and pfs .
serous type and tumor size were also correlated with poor pfs ( table 4 , fig .
2 ) . we tried to explore whether the combined expression of these protein markers had any prognostic value .
eighteen of 27 patients ( 66.7% ) who had a pattern of positive twist expression and reduced expression of e - cadherin [ twist(+)/e - cadherin(- ) group ] died of disease , compared to 32 of 96 patients ( 33.3% ) without a pattern of twist(+)/e - cadherin(- ) ( p=.003 ) .
of 19 patients showing an altered hif1/twist / e - cadherin pathway with positive expression of hif1 and twist , and reduced e - cadherin expression , 13 ( 68.4% ) died of disease , whereas 37 of 104 patients ( 35.6% ) without an altered hif1/twist / e - cadherin pathway died of disease ( p=.011 ) .
thirteen of 14 patients ( 92.9% ) who displayed expression of both twist and p53 showed disease progression compared to 47 of 109 patients ( 43.1% ) who had no expression of twist or p53 .
this difference was statistically significant ( p=.000 ) . in a multivariate analysis with the variables defined in table 5 , positive expression of twist
was associated with lower os after adjustment for histologic grade , tumor stage , mitotic rate , p53 expression , and altered expression of the hif1/twist / e - cadherin pathway , which were significant parameters in the univariate analysis .
expression of hif1 , twist , and p53 proteins was detected in 35 ( 28.5% ) , 43 ( 35.0% ) , and 36 ( 29.3% ) of the 123 oec cases , respectively . reduced expression of e - cadherin was observed in 60 cases ( 48.8% ) .
specific staining for twist was observed in the nucleus of tumor cells , but in some cases , both nuclear and cytoplasmic staining was observed .
clear cell types exhibited twist expression more frequently than other histologic types , but this was not statistically significant ( p=.240 ) . reduced e - cadherin expression was more commonly found in the advanced stages of oec ( 38 of 60 cases , 63.3% ) than the early stages ( 22 of 63 case , 34.9% ) ( p=.002 ) .
hif1 showed different expression according to histologic tumor type but was not associated with any clinicopathological parameters .
there was a statistically significant increase in the expression of p53 in serous - type oec compared with other types ( p=.000 ) .
there were also positive correlations between p53 and the histologic tumor grade , figo stage , and mitotic rate ( table 2 ) .
twist expression was significantly correlated with reduced e - cadherin expression ( p=.000 , correlation coefficient [ c.c.]=-0.309 ) and increased hif1 expression ( p=.000 , c.c.=0.532 ) .
twist expression was not correlated with the expression of p53 ( p=.142 , c.c.=0.168 ) .
there was a negative correlation between p53 expression and the reduced expression of e - cadherin ( p=.041 , c.c.=-0.219 ) ( table 3 ) .
follow - up data were available for 123 patients ( median , 49 months ; range , 1 to 175 months ) .
fifty patients ( 40.7% of 123 patients ) died from the disease during the follow - up period .
sixty patients ( 48.8% ) developed either recurrence or metastasis ( progression group ) , whereas 63 patients ( 51.2% ) remained free of progression ( progression - free group ) .
the univariate analysis showed that tumor histologic grade , tumor stage , mitotic rate , positive expression of twist and p53 , and reduced expression of e - cadherin were associated with poor os and pfs .
serous type and tumor size were also correlated with poor pfs ( table 4 , fig .
2 ) . we tried to explore whether the combined expression of these protein markers had any prognostic value .
eighteen of 27 patients ( 66.7% ) who had a pattern of positive twist expression and reduced expression of e - cadherin [ twist(+)/e - cadherin(- ) group ] died of disease , compared to 32 of 96 patients ( 33.3% ) without a pattern of twist(+)/e - cadherin(- ) ( p=.003 ) .
of 19 patients showing an altered hif1/twist / e - cadherin pathway with positive expression of hif1 and twist , and reduced e - cadherin expression , 13 ( 68.4% ) died of disease , whereas 37 of 104 patients ( 35.6% ) without an altered hif1/twist / e - cadherin pathway died of disease ( p=.011 ) .
thirteen of 14 patients ( 92.9% ) who displayed expression of both twist and p53 showed disease progression compared to 47 of 109 patients ( 43.1% ) who had no expression of twist or p53 .
this difference was statistically significant ( p=.000 ) . in a multivariate analysis with the variables defined in table 5 , positive expression of twist
was associated with lower os after adjustment for histologic grade , tumor stage , mitotic rate , p53 expression , and altered expression of the hif1/twist / e - cadherin pathway , which were significant parameters in the univariate analysis .
the emt , which is considered to be one of the major mechanisms underlying tumor invasion and metastasis , has been studied to understand how epithelial cancer cells can acquire the ability to migrate and metastasize .
emt is triggered by a set of stimuli , which include hypoxia and growth factors , and tumor stromal cell interactions with transcription factors such as twist , snai1 , slug , and zwb1.4 twist overexpression has also been identified in various kinds of epithelial tumors and plays a distinct role in tumor progression via the loss of e - cadherin expression.6,7,8,9,10,11,12,13,14,15,16 there have been a few other reports showing a correlation between twist expression and clinical significance in patients with oec.8,9 these previous studies have examined the expression of twist in oec cases , reporting immunohistochemical detection of twist expression in about 30 - 40% of cases .
twist expression was correlated with clinicopathological parameters and found to be a predictor of poor survival .
hosono et al.8 also reported that positive twist expression was an independent prognostic factor for survival in their multivariate analyses . in this study of 123 oec patients with follow - up ,
we found that twist expression was correlated with higher histologic grades and shorter os and pfs in oec patients , which is consistent with results from previous studies of other malignant tumors , including oec . in the analysis of the histological type - stratified data , twist expression tended to be higher in the clear cell type of oec ( 44.0% ) , although the difference was not significant ( p=.240 ) .
kajiyama et al.26 reported that twist expression was not only detected more frequently , but was also an independent prognostic factor in the clear cell type of oec .
they suggested that twist might play a role in the mechanisms for poor prognosis and chemoresistance in the clear cell type of oec .
p53 is a tumor suppressor gene that is involved in the regulation of cell growth .
mutations in the p53 gene are among the most commonly detected genetic abnormalities in human neoplasia .
mutations result in a conformational change in the protein , which becomes stabilized , thus usually , but not always , allowing for immunohistochemical detection . although not fully defined , recent evidence has indicated that twist may interfere with the p53 tumor suppressor pathway.23,24 twist has previously been shown to reduce the activity of the p53 promoter as well as mediate suppression on p53 target sequences . wang et al.18 demonstrated that increased twist was responsible for the development of acquired resistance to chemotherapeutic drugs and microtubule - disrupting agents , including paclitaxel .
twist may protect from apoptosis mediated by p53 in response to dna damage induced by radiation and drugs .
chemoresistance , as well as the facilitation of metastasis by twist , may be the underlying mechanism linking twist expression with an unfavorable prognosis . in this study , we found that p53 expression showed no correlation with twist expression , but an inverse correlation with e - cadherin expression was established .
interestingly , co - expression of twist and p53 ( 13/14 cases , 92.9% ) showed unfavorable pfs compared with no co - expression ( 47/109 cases , 43.1% ) ( p=.000 ) .
these results suggest that twist and p53 could cooperate with each other to facilitate cancer progression , and p53 could have an important role in emt .
e - cadherin plays a critical role in regulating the balance of cell - cell adhesion and cell motility , placing it in a significant position to regulate tumor cell proliferation and invasion .
some studies have identified that reduced e - cadherin expression is associated with more aggressive epithelial tumors.9,12,17,21 twist binds the e - cadherin promoter and inhibits e - cadherin transcription levels , which modulate emt in cancer progression . an inverse correlation between twist upregulation and e - cadherin downregulation was also observed in several types of malignancies.5,10 however , yoshida et al.27 demonstrated that there was not an inverse correlation between the expression of e - cadherin and twist in oec . in this study ,
reduced e - cadherin expression was associated with advanced figo stage and poor os and pfs .
our results are consistent with the observation that e - cadherin has been described in patients with advanced stage oec and is associated with poor prognosis.9 moreover , a significant inverse relationship was observed between these two markers in this study , and we showed that twist(+)/e - cadherin(- ) cases are also associated with poor prognosis . for cancer progression ,
intratumoral hypoxia followed by the activation of hif1 is one of the most important mechanisms promoting tumor aggressiveness , leading to metastasis and patient mortality .
recent results have shown the upregulation of mrna and protein levels of twist in different cell lines under hypoxia and constitutive expression of hif1.19,20 twist can be a direct target of hif1 due to the functional presence of a hypoxia - related element located in the proximal promoter of twist.20 the survival analysis in the aforesaid report demonstrated the worst prognosis and shortest metastasis - free period in hnscc patients with hif1 , twist , and snail co - expression , indicating that the co - expression of these markers in tumor tissues could be used as a valuable marker to predict metastasis and prognosis in hnscc patients . in a study by feng et al.,21 the overexpression of hif1 and twist
was frequently detected in endometrial endometrioid carcinoma ( eec ) samples , and they were associated with aggressive tumor characteristics , such as higher histologic grades , deeper myometrial invasion , and lymph node metastasis .
the interesting findings of the study were that twist - positive cases exhibited increased expression of hif1 and reduced expression of e - cadherin in eec .
the researchers suggested that the hif1/twist / e - cadherin pathway may play a critical role in the progression of eec .
our study showed a positive association between hif1 and twist expression ( =0.407 , p=.000 ) and a negative association between twist and e - cadherin expression ( =-0.358 , p=.000 ) .
moreover , altered expression of the hif1/twist / e - cadherin pathway was associated with poor os in oec in the univariate analysis . in summary , we show that increased expression of twist , hif1 , and p53 and reduced expression of e - cadherin are frequently observed in oec cases .
therefore , twist may serve as a useful marker in patients with oec who are likely to have an unfavorable clinical outcome .
our results support the critical role of the hif1/twist / e - cadherin pathway in cancer progression and the acquisition of the metastatic phenotype in oec .
therefore , pathways involved in hypoxia - mediated emt , such as the hif1/twist / e - cadherin pathway , may be possible candidates for molecular targeting in the future treatment of oec .
further studies are needed to clarify the role and molecular mechanisms underlying of the hif1/twist / e - cadherin pathway in tumor progression . | backgroundepithelial - mesenchymal transition ( emt ) is associated with tumor hypoxia .
emt is regulated , in part , by the action of twist , which inhibits of e - cadherin expression and may interfere with the p53 tumor - suppressor pathway.methodswe examined the expression of twist , e - cadherin , hypoxia - inducible factor 1 ( hif1 ) , and p53 by immunohistochemistry in 123 cases of ovarian epithelial cancers ( oec ) to evaluate the role of twist in oec .
we assessed the association between protein expression and clinicopathologic parameters.resultsthe expression of twist , e - cadherin , hif1 , and p53 proteins was found in 28.5% , 51.2% , 35.0% , and 29.3% of cases , respectively .
twist expression was associated with higher histologic grade and unfavorable survival .
twist expression was correlated with hif1 expression and reduced e - cadherin expression .
the altered hif1/twist / e - cadherin pathway was associated with lower overall survival ( os ) , while the co - expression of twist and p53 was correlated with lower progression - free survival . in the multivariate analyses ,
twist expression was an independent prognostic factor for os.conclusionsour data imply that twist expression could be a useful predictor of unfavorable prognosis for oec .
twist may affect the p53 tumor - suppressor pathway .
moreover , hypoxia - mediated emt , which involves the hif1/twist / e - cadherin pathway may play an important role in the progression of oec . |
fluoride is an ubiquitous element in the environment and has a remarkable prophylactic effect at low concentrations by inhibiting dental caries , while at higher concentrations it causes dental and skeletal fluorosis .
endemic fluorosis is prevalent in many parts of the world and causes damage not only to hard tissues of teeth and skeleton , but also to soft tissues , such as brain , liver , kidney , and spinal cord .
epidemiological investigations reveal that intelligence quotient ( iq ) of children living in endemic fluorosis areas is lower than that of children living in low fluoride areas [ 37 ] .
it has been demonstrated that high concentrations of fluoride can decrease learning ability and memory in some animal experiments [ 8 , 9 ] and result in dysfunctions of the central nervous system ( cns ) [ 10 , 11 ] . as the cases of many chronic degenerative diseases , the increase of reactive oxygen species ( ros ) and lipid peroxidation ( lpo ) has been considered to play an important role in the pathogenesis of chronic fluoride toxicity [ 1214 ] .
fluoride administration significantly increases brain lpo level compared with control group in rat , while reduced glutathione ( gsh ) content and superoxide dismutase ( sod ) , glutathione peroxidase ( gpx ) , and glutathione reductase ( gr ) activities decrease markedly in fluoride - treated groups [ 15 , 16 ] .
there are significantly negative correlations between fluoride concentrations in brain and gpx activity , gsh level , and positive correlations between fluoride concentrations and thiobarbituric acid reactive substances ( tbarss ) and carbonyl groups .
the cns is especially sensitive to free radical oxidative damage as it contains more easily oxidizable fatty acids [ 18 , 19 ] .
ros is produced during the respiratory burst of phagocytes , and the regulated generation of ros plays an important role in host defense , oxygen sensing , and signal transduction [ 20 , 21 ] , while excessive production ros promotes cellular injure and tissue damage .
macrophages are sources of free radicals , including ros and reactive nitrogen species ( rns ) .
microglia are a kind of resident macrophage of the cns and play a vital role in immune surveillance and injury repair [ 22 , 23 ] .
microglia activation is a common phenomenon in response to exposure to toxicants , and activated microglia are both phagocytic and potent sources of reactive oxygen and nitrogen intermediates [ 2426 ] .
microglia excessive activation also can trigger or exacerbate neurotoxicity by inducing oxidative stress of neurons .
nitric oxide ( no ) production results from nitric oxide synthase ( nos ) that catalyze the conversion of l - arginine to l - citrulline and no . at high concentrations ,
no readily reacts with superoxide anion ( o2 ) , a kind of ros derived from nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase ( nox ) , to produce peroxynitrite ( onoo ) .
onoo is able to irreversibly inhibit mitochondrial respiration , react with proteins , lipids , carbohydrates and dna , and cause dna fragmentation and lipid oxidation .
a growing number of studies have shown that fluoride can increase the generation of ros and lpo in brain , but it is not known if ros increasing in brain is related with activated microglia at fluoride exposure . in the present study , we treated bv-2 microglia cell line with different concentrations of fluoride and found that bv-2 microglia cells were activated .
the results indicated that activating bv-2 microglia cells by fluoride induced oxidative stress , which provides a potential oxidative stress mechanism for fluoride - related brain damage .
sodium fluoride ( naf , molecular weight 41.99 ) was procured from sigma chemical ( st . louis , mo , usa ) . all
other analytical laboratory chemicals and reagents were obtained from sigma , invitrogen ( carlsbad , ca , usa ) , hyclone ( logan , ut , usa ) . the immortalized murine microglia cell line , bv-2 , was provided by cell culture center , school of basic medicine , peking union medical college .
the bv-2 cells were maintained in dulbecco 's modified eagles medium ( dmem ) that contained 10% fetal bovine serum and antibiotics at 37c in a 5% co2 humified incubator . exponentially growing bv-2 cells at density of 5000 cells in 100 l medium
were treated with 0.5120 mg / l naf respectively for 24 , 48 , and 72 h for cell viability assay .
/ l ) were selected for intracellular sod , gsh , malondialdehyde ( mda ) , ros , o2 , nos , nitrotyrosine ( nt ) measurement and cell medium was collected for no , nos assay . viability and growth patterns of bv-2 cells based on mitochondrial enzyme functions in 96-well plates were determined under naf - treated for 24 h , 48 h , and 72 h by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ( mtt ) conversion to formazan .
briefly , 100 l of mtt solution ( 0.5 mg / ml in the medium ) was added to each well , and the plates were incubated at 37c for additional 4 h. afterwards , the medium containing mtt was removed , and the crystals were dissolved in 150 l of 100% dimethyl sulfoxide ( dmso ) .
the cell viability was quantified using a microplate reader ( multiscan ascent , labsystems , finland ) at 570 nm .
following naf treatment , cultured microglia were fixed with 4% paraformaldehyde and incubated with the diluted primary antibody ( 1 : 200 ox-42 , microglia marker ) for 20 min at 37c , and secondary antibody sheep anti - mouse igg was added for 20 min at 37c .
after washing , the cells were incubated with dab dilution until an observed color change .
staining of ox-42 was examined at high - power fields ( 400 ) under a standard light microscope .
the number of cells as identified by ox-42 antibody was counted in five different fields on each microscope slide .
bv-2 cells were cultured in 10 cm diameter dishes and treated with naf as indicated . at the end of treatment , cells were washed 3 times with ice - cold phosphate - buffered saline ( pbs ) , scraped off the dishes with a silicone policeman and harvested into eppendorf tubes .
then cells were lyzed in pbs by sonication and centrifuged at 15000 g for 5 min at 4c .
the intracellular gsh levels and sod activity were measured with improved 5,5-dithiobis-(2-nitrobenzoic acid ) ( dtnb ) method and hydroxylamine assay , respectively , provided by commercial test kits ( keygen biotech .
the quantification was based on measuring formation of thiobarbituric acid reactive substances ( tbars ) according to the manufacturer 's protocol .
protein concentrations were determined by the method of bradford to normalize the levels of gsh , sod , and mda .
the results were expressed as mg / g protein for gsh , u / mg protein for sod , and nmol / ml for mda .
the production of ros was determined by measuring dichlorofluorescein ( dcf ) fluorescence as described by rothe and valet .
briefly , bv-2 cells in 24-well culture plates , treated with naf for 24 h , were rinsed and resuspended in serum - free medium containing 10 m dcfh - da . after a further 30 min of incubations at 37c , cells were rinsed twice with ice - cold pbs and harvested by trypsin , and were measured via a facscan flow cytometer ( becton dickinson , usa ) with excitation wave length at 488 nm and emission wave length at 525 nm for ros generation . the fluorescence intensity parallels to the amount of ros formed .
briefly , bv-2 cells were harvested after treated with naf for 24 h , washed with serum - free culture medium and incubated with 5 m dhe at 37c for 30 min
. then the cells were harvested , washed and resuspended , and were measured via a facscan flow cytometer ( becton dickinson , usa ) with excitation wave length at 300 nm and emission wave length at 610 nm for o2 generation .
bv-2 cells were treated with various concentrations of naf for 24 h. the cell culture medium was collected , then centrifuged 1 min at 13000 rpm to remove floating cells and cell debris , and analyzed immediately for nitrite content .
the production of no was determined by measurement the nitrite accumulation in the medium using colorimetric assay with griess reagent .
nitrite concentrations were determined by comparison with standard solutions of sodium nitrite at optical density 550 nm .
then cells were lyzed in pbs by sonication and centrifuged at 15000 g for 5 min at 4c , the resulting supernatants and medium were used as sources of nos , respectively .
nos activity was determined by monitoring the formation of [ 3h ] l - citrulline from [ 3h ] l - arginine as the method described previously .
the commercial test kit of nos was provided by nanjing jiancheng biological corporation ( nanjing , china ) .
bv-2 cells were treated with various concentrations of naf for 24 h , and the intracellular nt was assessed by elisa using monoclonal antibodies and the procedure recommended by the supplier ( abway antibody technology co. , ltd . ,
the concentration of nt was calculated according to the standard curve of the elisa kits .
all experiments were performed at least in triplicate and the values represent mean sd .
differences between groups were statistically analyzed by one - way analysis of variance ( anova ) and positive rates of ox-42 expression were analyzed by the fisher exact test .
as shown in figure 1 , fluoride reduced the bv-2 cell viability in 50 , 100 , or 120 mg / l naf groups . at concentrations of 50 , 100 , or 120
mg / l , sodium fluoride caused a significant decrease in cell viability of 57% , 70% , and 72% , respectively , versus control at naf - treated 24 h , 50% , 69% , and 86% , respectively , versus control at naf - treated 48 h , and 51% , 78% , and 92% , respectively , versus control at naf - treated 72 h. integrin m ( ox-42 ) is a cell adhesion molecule that acts as a receptor for cell surface ligands .
immunocytochemistry localization with an ox-42 antibody was used as a microglial marker . to confirm the activation of bv-2 in response to naf , ox-42 expression was determined by immunocytochemistry after 24 h incubation with different naf concentrations ( 120 mg / l ) .
naf of concentrations from 5 to 20 mg / l can stimulate bv-2 cells to change into activated microglia displaying upregulated ox-42 expression and large bodies compared with control microglia with small bodies and long branch ( figure 2 ) .
the number of cells showing ox-42 positive expression markedly increased in 5 , 10 , and 20 mg / l naf - treated bv-2 cells compared with the control . as shown in figure 3 , a marked decrease in sod activities was observed in fluoride - treated bv-2 cells .
sod activities decreased significantly by 22.3% , 25.6% , and 34.4% in 1 , 10 , and 50
mg / l naf - treated cells , respectively , compared to the control cells , and there was a significant negative dose - effect relationship ( r = 0.804 , p < 0.01 ) .
dcf fluorescence intensities in bv-2 cells increased with fluoride concentrations , with a significant dose - dependent manner ( r = 0.851 , p < 0.01 ) , and the dcf fluorescence intensities in the 50
mg / l naf - treated cells were significantly higher than the control cells ( p < 0.01 ) .
mda concentrations also increased in naf - treated cells , and there was a significant positive dose - effect relationship ( r = 0.5 , p < 0.05 ) although there were no statistical differences in naf - treated cells compared with the control cells .
however , there was an increase tendency in naf - treated cells compared to the control for the intracellular gsh levels .
in addition to ros , rns from activated microglia can be a major cause of oxidative stress .
we therefore measured no and nos in bv-2 cells treated with various concentrations of fluoride for 24 h. no level in the culture medium did not increase with fluoride concentrations .
however , activities of nos , synthesizing no , significantly increased in 1 , 10 , and 50 mg / l naf - treated bv-2 cell culture medium compared with the control , in a dose - dependent manner ( r = 0.96 , p < 0.01 ) .
intracellular nos activities only increased in 10 mg / l naf - treated bv-2 cells compared with the control , see figure 4 .
dhe fluorescence intensities in bv-2 cells increased with fluoride concentrations , with a significant dose - dependent manner ( r = 0.943 , p < 0.01 ) , and the dhe fluorescence intensities in the 1 , 10 , and 50
peroxynitrite , the reaction product of no and o2 , can lead to macromolecule damage and formation of nt , a protein adduct , and nt has been as a marker of free radical oxidation of nitric oxide .
our study showed that intracellular nt concentrations in bv-2 cells increased significantly following fluoride exposure in 10 and 50 mg / l naf - treated groups compared with the control group , and there also existed a dose - dependent enhancement ( r = 0.603 , p < 0.05 ) , see figure 5 .
the redox balance of the cell maintained by oxidants and antioxidants is important to physiological activity and function .
disturbance of the natural equilibrium of free radical over generation causes dysfunction and promotes oxidative damage to tissue .
although ros have some essential roles in normal cell functions , they are more associated with their pathological effects that ultimately lead to protein and cellular damage as well as cell death [ 32 , 33 ] .
ros could attack numerous cell components like membrane lipids , resulting in enhanced lipid peroxidation and cellular toxicity .
many studies [ 34 , 35 ] have reported that excessive fluoride exposure can damage the redox balance of the cells in tissues , decrease antioxidant defense capacity in brain , and increase the toxic effects on visceral organs mediated by generation of ros and lipid peroxidation .
a close association between chronic fluoride toxicity and increased oxidative stress has been reported in humans and in experimental animals [ 37 , 38 ] .
neurotoxicity induced by fluoride had been linked with oxidative stress [ 11 , 16 ] .
microglia are the resident immune cells in the brain and readily activated by a variety of foreign substances including environmental toxicants .
the beneficial roles of microglia activation include clearance of toxic cell debris and pathogens , induction of innate immune responses , and enhancement of neuronal survival .
however , an array of ros and rns produced and secreted from activated microglia has been believed to be involved with neurotoxicity in brain disorders [ 4042 ] . in the present study
several in vivo studies have reported microglial proliferation and activation early after various brain insults .
thus , it can be hypothesized that oxidative stress of microglia could be one of the causative factors for fluoride induced brain toxicity .
once activated , microglia undergo morphological changes as well as phenotypic alterations in some molecules .
microglial activation is generally measured by a gradual change in morphology from a quiescent ramified form ( resting state ) to an amoeboid form ( activated state ) , and upregulation of cell surface receptors , such as cd11b ( ox-42 ) . in this study
, the majority of naf - treated bv-2 microglial cells displayed an activated morphology and increased the expression of ox-42 receptor , an abundance surface receptor in activated microglia .
the increased number of ox-42 microglia observed in this study may be due to the reactive response of microglia to fluoride treatment . to our knowledge , this is the first time to verify that microglia are activated by fluoride .
nox is a superoxide producing enzyme system , the major source of ros in microglia [ 4648 ] .
superoxide anions ( o2 ) derivated from nox can be catalyzed into the hydrogen peroxide ( h2o2 ) and molecular oxygen by sod .
sod accelerates o2 to h2o2 , which can be said as primary defence , as it prevents further generation of free radicals . in the present study ,
the sod activity significantly decreased in dose - dependent manner and o2 content increased in fluoride - treated bv-2 microglia .
superoxide radicals especially cause oxidation of sh groups in protein , and it has been demonstrated that thiolate radicals are removed by gsh .
gsh also participates in the reaction that destroys hydrogen peroxide , or organic peroxide , free radicals , and certain foreign compounds .
gsh is as a direct free radical scavenger , as a cosubstrate for gpx activity and as a cofactor for many enzymes and forms conjugates in endo- and xeno - biotic reactions .
the decrease in gsh level is often observed due to increased utilization in oxidative stress . in contrast , gsh levels in bv-2 microglial cells increased with fluoride concentrations in our present study .
oxidative stress is defined as a disturbance in the balance between the production of free radicals and antioxidant defense , and the later is more compensatory .
our observed increase of gsh may be a compensation increase to scavenge excessive superoxides and protect cells against oxidative stress .
the production of lipid peroxidation , such as mda , was accumulated as a result of membrane disruption , elicited by the oxidation of poly unsaturated fatty acids of the bilayer .
the present study found that the mda levels showed an increasing tendency in naf - treated bv-2 cells although there were no significant differences between naf - treated bv-2 cells and control cells .
however , ros level in 50 mg / l naf - treated group increased significantly , which indicated that ros could attack membrane lipids and result in lipid peroxidation .
no is a compound of special interest released by activated microglia and has been described in vitro situations to be an important mechanism by which microglia cause neuronal death [ 5153 ] .
no is produced from l - arginine and molecular oxygen by the action of nos [ 54 , 55 ] in activated microglia and contribute to the oxidative stress accompanying the inflammatory process .
nos is the key enzyme for no production and is quantitatively induced in activated glial cells after exposure to stimulators .
the neurotoxic effects of no are generally attributed to its reaction with o2 to form onoo , an extremely active oxidizing agent , which can lead to macromolecule damage and form the protein adduct , such as nt , and nt has been as a marker of free radical oxidation of nitric oxide . in the present study ,
fluoride significantly increased the nos activity in activated bv-2 cells , and which should produce more no .
interestingly , in our experiment , no level did not increase after bv-2 cells were exposed to fluoride , that could due to produced no reacted rapidly with o2 to form onoo .
indeed , we have observed a significant increase of nt level at 10 and 50 mg / l naf - treated cells , suggesting that neurotoxicity of fluoride is associated with overproduction of nitric oxide and peroxynitrite .
therefore , rns , together with ros produced by activated microglia was involved in oxidative stress induced by fluoride in activated bv-2 cells . in conclusion ,
a main finding of this study was that microglia bv-2 cells were activated by fluoride , and the increase of ros and rns in microglia was associated with the activation of microglia .
the novelty of this work is related to the active role of microglia treated with fluoride .
we provided new evidence from in vitro model indicating that fluoride exerts its toxic effects in cns possibly partly ascribed to activating of microglia in vitro , which enhanced oxidative stress induced by ros and rns . | the generation of ros and lipid peroxidation has been considered to play an important role in the pathogenesis of chronic fluoride toxicity . in the present study
, we observed that fluoride activated bv-2 microglia cell line by observing ox-42 expression in immunocytochemistry .
intracellular superoxide dismutase ( sod ) , glutathione ( gsh ) , malondialdehyde ( mda ) , reactive oxygen species ( ros ) , superoxide anions ( o2 ) , nitric oxide synthase ( nos ) , nitrotyrosine ( nt ) and nitric oxide ( no ) , nos in cell medium were determined for oxidative stress assessment .
our study found that naf of concentration from 5 to 20 mg / l can stimuli bv-2 cells to change into activated microglia displaying upregulated ox-42 expression .
sod activities significantly decreased in fluoride - treated bv-2 cells as compared with control , and mda concentrations and contents of ros and o2 increased in naf - treated cells .
activities of nos in cells and medium significantly increased with fluoride concentrations in a dose - dependent manner .
nt concentrations also increased significantly in 10 and 50 mg / l naf - treated cells compared with the control cells .
our present study demonstrated that toxic effects of fluoride on the central nervous system possibly partly ascribed to activiting of microglia , which enhanced oxidative stress induced by ros and reactive nitrogen species . |
malaria is caused by one or a combination of four species of plasmodia and leads to over one million deaths , of which over 75% occur in african children under 5 years infected mainly with plasmodium falciparum ( pf ) .
pf and plasmodium vivax ( pv ) are identified as the plasmodium species responsible for causing the most severe form of malaria . in epidemiological studies , intervention studies , and clinical trials ,
malaria microscopy is routinely relied upon as a primary endpoint measurement of the level of malaria infection .
this is expressed as parasite density and is classically defined as the number of asexual forms of parasite relative to a blood volume ( e.g. , microliter ) . in the four basic counting techniques using microscopy , white blood cells ( wbcs )
are relatively used in estimating plasmodium parasitaemia by counting the number of parasites against a predetermined number of wbcs on giemsa stained blood smears .
complete blood counts , particularly wbcs count , can be performed with new generation automated haematology analysers and/or manually using stained microscope smears and the neubauer chamber and counters [ 5 , 6 ] .
some health facilities frequently use manual convention methods to determine the complete blood counts of a patient for management as a result of a high cost of purchasing and maintaining fully automated or semiautomated haematology analyzers .
another cost burden using an automated system includes the ability to ensure prompt validation , maintenance , and implementation of rigorous quality systems .
automated machines may therefore not be the preferred choice to quantify wbcs in resource poor areas . due to the frequent lack of facilities in some malaria endemic countries to quantify wbcs ,
an assumed wbcs count of 8000/l of blood has been accepted by world health organization as reasonably accurate to estimate malaria parasite densities . assumed wbcs count of blood may generate systematic errors which could produce incorrect conclusions in patient management or during clinical research that uses malaria parasite counts as an end point [ 3 , 8 ] .
medical laboratories need to set relevant reference ranges for wbcs and other common laboratory parameters for clinical management of patients or during malaria clinical research .
kintampo health research centre has established reference values for common haematological and biochemical laboratory parameters for its study area covering an area of about 7200 km .
the team compared the geometric mean parasite densities ( gmpd ) calculated using absolute wbcs , assumed wbcs , and wbcs reference values of the participants enrolled in the malaria studies in middle ghana to assess the impact of using these as malaria parasite density estimators .
the studies used for this data analysis were approved by the ethical review committees of the ghana health service , london school of hygiene and tropical medicine , kintampo health research centre institutional ethics committee , noguchi memorial institute of medical research , and the ghana food and drugs authority . written informed consent was sought from all mothers whose children participated in the studies .
the data analysis was carried out on laboratory data from a study area with a profile presented by adu - gyasi et al . , 2012 .
briefly , the area is located within the forest - savannah transitional ecological zone in ghana with perennial malaria transmission .
pf is the predominating plasmodium species while p. malariae and p. ovale are in the minority .
the data was obtained from children under 5 years who were recruited into malarial studies carried out by kintampo health research centre between the periods of october 2008 and march 2011 . in the first study , children were enrolled and followed actively every month for two years .
the children were referred to the hospital for care whenever they were unwell to determine the incidence of malaria .
the second study which was a clinical trial was to determine the effect of providing micronutrient powder with or without iron on the incidence of malaria among children under three years living in a high malaria - burden area in ghana . in each of the studies ,
blood samples were collected by finger - prick into 0.5 ml microtainers containing ethylenediaminetetraacetic acid ( k2edta - bd , usa ) from the participants on their scheduled visit days and on any other day participants visited the health facilities with illness .
samples were collected and transported according to the standard operating procedures ( sops ) established by the kintampo health research centre .
briefly , participant 's finger was rubbed , and the tip was cleaned with disposable alcohol swab and allowed to dry .
safety lancet was used to prick and drops of blood were collected into labelled test - tubes to the required volume . collected samples were used for full blood count analysis and for making blood smears .
examination of thick and thin smears was carried out as described by adu - gyasi et al . , 2012 .
parasite densities for all participants were calculated using assumed wbcs count of 5000/l , 6000/l , 8000/l , and 10000/l of blood . in addition , we used the wbcs reference values established for children aged less than one year ( 10000/l of blood ) and also for children less than five years ( 9200/l ) .
data obtained from the database of the malarial studies was checked for completeness and consistency and all queries were resolved .
analyses were done using stata ( version 12 ; stata corp . , tx , usa ) and graphpad prism version 5.0 ( graphpad software , inc . ) particularly for geometric mean and intervals .
of the 39851 results received over the study period , data of 4944 participant results were consistent and complete for the purpose of our analysis . of the total , 50.6% ( 2498/4944 ) were males and 49.4% ( 2443/4944 ) were females .
the mean ( sd ) wbcs and gmpd of the 4944 positive samples were 10500/l ( 4.1 ) and 10644/l ( 95% ci 9986/l to 11347/l ) of blood , respectively .
there was no significant difference between the mean ( sd ) wbcs 10600/l ( 4.0 ) for males and 10500/l ( 4.1 ) for females ( p = 0.255 ) .
neither was there a significant difference between the gmpd 11058/l ( 95% ci 10114/l to 12091/l ) of blood for males ( table 1 ) and 10217/l ( 95% ci 9324/l to 11197/l ) of blood for females ( table 2 ) . with an assumed wbcs count of 8000/l ,
a gmpd of 8679/l ( 95% ci , 8140/l to 9253/l ) of blood was estimated ( table 3 ) . in using the wbcs reference values established among children in the study area , ( 10400/l ) ,
a gmpd of 11282/l ( 95% ci , 10581/l to 12029/l ) of blood and a wbcs count of 9200/l produced a gmpd of 9981/l ( 95% ci , 9361/l to 10641/l ) of blood .
the difference in the gmpd calculated using absolute wbcs compared to densities estimated with assumed wbcs was significantly lower for 8000/l . however , gmpd for assumed wbcs count of 10.0 10/l , wbcs reference values of 10400/l , and 9200/l estimated a geometric mean parasite density of 10848/l ( 95% ci , 10175/l to 11567/l ) , 11282/l ( 95% ci , 10581/l to 12029/l ) , and 9981/l ( 95% ci , 9361/l to 10641/l ) of blood that was not significantly different from estimates obtained with the absolute wbcs ( table 3 ) .
the interval of gmpd obtained when an assumed wbcs of 8000/l , 6000/l and 5000/l were used and that from absolute wbcs count did not overlap .
however , the intervals of gmpd from absolute wbcs count and that obtained with assumed wbcs counts of 9200/l , 10000/l and 10400/l overlapped showing no significant difference in the gmpd ( table 3 ) . a correlation analysis of the malaria parasite densities with the wbcs reference value and assumed wbcs compared to the absolute wbcs showed a significant level of correlation ( p < 0.0001 ) with all the assumed wbcs count ( table 4 ) .
this has become dominated by the convenient but inaccurate assumption of a constant wbcs count of 8000/l of peripheral blood [ 3 , 15 ] due to lack of capacity to measure patients absolute wbcs .
the mean wbcs count from the participants with malaria infection compares with the wbcs reference values , 10400/l for children less than 1 year and 9200/l for children up to 5 years .
the parasite densities from the wbcs reference values agree with the density obtained from using participant 's absolute wbcs .
it is agreed that the best solution in estimating parasite densities would be to use the corresponding absolute wbcs count for each age group .
though there is a significant level of correlation , estimating the parasite density of plasmodium species with the assumed wbcs count of 8000/l of blood , compared to the absolute and reference value wbcs count , would mean underestimating significantly the parasite density of plasmodium species infections for patients in the study area .
therefore , establishing regional based reference wbcs as suggested to estimate parasite densities in malarial infections will be appropriate .
this was evident in the fact that the geometric mean parasite density , 11282/l ( 95% ci , 10581/l to 12029/l ) and 9981/l ( 95% ci , 9361/l to 10641/l ) of blood , estimated by using the established wbcs standard values of 10,400/l and 9200/l of blood for children less than 1 year and up to 5 years old , respectively , overlaps with the geometric mean parasite density obtained by using the mean absolute wbcs count , 10644/l ( 95% ci 9986/l to 11347/l ) of blood , of the participants .
results obtained when an assumed wbcs count of 10000/l of blood was used to estimate the parasite density were also consistent with that from the absolute wbcs count of participants .
the parasitaemia obtained by the use of the other assumed wbcs count was significantly lower ( table 3 ) .
since lack of resources in some settings in africa makes it difficult to estimate malaria parasite density based on actual wbcs count of patients , where available , the study recommends the use of an established wbcs reference value for a known population and where the reference value could be implied . the reference value established in kintampo has been predicted as a malaria density estimator when compared with the absolute wbcs of participants . in environments
where reference values have not been established , the study affirms the use of an assumed wbcs count of 10000/l of blood to estimate malaria parasite density as documented . | introduction . the estimation of malaria parasite density using a microscope heavily relies on white blood cells ( wbcs ) counts
. an assumed wbcs count of 8000/l has been accepted as reasonably accurate in estimating malaria parasite densities due to the challenge to accurately determine wbcs count . method .
the study used 4944 pieces of laboratory data of consented participants of age group less than 5 years .
the study compared parasite densities of absolute wbcs , assumed wbcs , and the wbcs reference values in central ghana .
ethical approvals were given by three ethics committees .
results .
the mean ( sd ) wbcs and geometric mean parasite density ( gmpd ) were 10500/l ( 4.1 ) and 10644/l ( 95% ci 9986/l to 11346/l ) , respectively .
the difference in the gmpd compared using absolute wbcs and densities of assumed wbcs was significantly lower . the difference in gmpd obtained with an assumed wbcs count and that of the wbcs reference values for the study area , 10400/l and 9200/l for children in different age groups , were not significant .
discussion .
significant errors could result when assumed wbcs count is used to estimate malaria parasite density in children .
gmpd generated with wbcs reference values statistically agreed with density from the absolute wbcs .
when obtaining absolute wbc is not possible , the reference value can be used to estimate parasite density . |
anatomical cadaver studies have shown that vastus medialis oblique ( vmo ) fibers originate
from the distal part of the adductor magnus .
thus , vmo fibers provide stability during the
contraction that occurs in hip adduction1 .
hip adduction also stretches the vmo fibers , adjusting the length and tension properties of
the muscle and increasing the contraction force2 .
based on anatomical analyses , previous research has shown that hip
adductor contraction during squat exercises results in selective strengthening of the
vmo3 .
another study reported that the
hip adductor muscle is connected to the internal abdominal muscle through the iliacus , psoas
major , and quadratus lumborum muscles4 .
kim and yoo5 reported that the muscle
activities of the external oblique , internal oblique , and l5 paraspinal muscles increased
during hip adduction using a visual feedback device .
hip adductor muscle contraction also
synergistically facilitates the pelvic floor muscle activity that occurs with the
contraction of the abdominal muscle6 .
therefore , many clinicians have used various hip adduction exercises for low back pain , or
patellofemoral pain or pelvic muscle weakness patients1 , 5 , 6 .
the selection of the exercise load is a very important factor in
the effectiveness of the exercise3 .
therefore , in the present study ,
using regression analysis , we determined the optimal and maximal loads during hip adduction
exercise by asymptomatic subjects .
the purpose of this study was to provide data for
decision making regarding the optimal and maximal hip adduction loads for clinical and
fitness purposes , respectively .
forty - eight ( 24 males , 24 females ) asymptomatic subjects with no known surgical ,
musculoskeletal , or neurological history of any pathological condition in the lower
extremities participated in this study . the mean age of the participants was 24.3
4.4 years , their mean height was 169.5 8.4 cm , and their mean weight was 61.2 12.7 kg .
this study was approved by the inje university faculty of health science human ethics
committee , and all subjects provided their written informed consent to participation before
commencing the study .
subjects were instructed to bend their knees to 45 in a
height - adjustable chair and adduct their knees to their hip .
the optimal hip adduction load was defined as sustainable load
holding for 20 s in isometric hip adduction isometric exercise .
the maximal hip adduction
load was defined as sustainable load holding for 3 s with maximal effort hip adduction .
the
hip adduction load was measured by powertrack ii ( jtech medical , salt lake city , ut , usa ) .
hip adduction load measures were performed using an air cushion with the powertrack ii
placed between the medial joint lines of the knees .
the individual optimal and maximal
hip adduction load data were collected from five repeated tests .
a resting time of 3 min was
given between each test to prevent muscle fatigue .
stepwise multiple regression analysis was
used to determine the optimal and maximal loads during the hip adduction exercise .
a
stepwise multiple regression analysis was performed using gender , body weight and height
variables to identify the most informative variables for predicting the optimal and maximal
loads during the hip adduction exercise .
the coefficients for the regression analysis and
the regression model for the optimal and maximal loads during the hip adduction exercise
were determined .
to control for the gender , body weight , and height variables , stepwise multiple regression
was performed for the optimal and maximal hip adduction loads .
the regression model for
optimal hip adduction load ( kg ) was : 34.3 + 0.4 weight 0.27 height ( r=
0.77 , p<0.05 ) ; and the regression model for maximal hip adduction load ( kg ) was : 39.5 +
0.5 weight 0.3 height ( r=0 0.75 , p<0.05 ) .
this study measured the optimal and maximal loads during hip adduction exercise in
asymptomatic subjects .
the most informative variables for predicting the optimal and maximal
loads during the hip adduction exercise were determined on the basis of statistical
regression .
the results show that weight and height were the most informative variables for
both optimal and maximal hip adduction loads .
using information from these models can aid in
deciding the optimal and maximal hip adduction loads for clinical and fitness purposes ,
respectively .
the selection of the exercise load is a very important factor for effective
exercise .
a previous study investigated the influence of a resistive band on the muscles
with leg adduction performed in a variety of positions7 .
core stability of the trunk can be viewed
as a box , with , the diaphragm as the roof and the pelvic floor and hip muscles as the
bottom8 .
the abdominals comprise the
anterior part of the box and the paraspinals and gluteals the posterior part8 .
this co - activation of the hip adductor ,
pelvic floor , and internal abdominal muscles is necessary for the occurrence of
intra - abdominal pressure9 .
thus , the optimal hip adduction load model can be used to
strengthen the hip adductor muscles or enhance core stability in the clinical setting .
although the independent variables of this study used simple general characteristics , the
regression coefficients of r - squared over 0.7 indicate highly reliability in predicting the
dependent variables .
the variables selected by regression analysis in this study were weight
and height , which have the advantage of being measured easily in the clinic by physical
therapists .
therefore , our study results can help with the selection of the ideal hip
adduction loads in clinical situations . | [ purpose ] the purpose of this study was to provide data for decision making regarding
the optimal and maximal hip adduction loads for clinical and fitness purposes ,
respectively .
[ subjects ] forty - eight ( 24 males , 24 females ) asymptomatic adults
participated in this study . [ methods ]
subjects performed optimal and maximal hip adduction
loads . regarding the gender , body weight and height variables , a stepwise multiple
regression analysis was used to identify the most informative variables for predicting the
optimal and maximal loads during the hip adduction exercise .
[ results ] the regression
model for optimal hip adduction load ( kg ) was : 34.3 + 0.4 weight 0.27 height
( r2= 0.77 ) ; and the regression model for maximal hip adduction load ( kg ) was :
39.5 + 0.5 weight 0.3 height ( r2= 0.75 ) . [ conclusion ]
these models can
aid in deciding the optimal and maximal hip adduction loads for clinical and fitness
purposes , respectively .
thus , the optimal hip adduction load model can be used to
strengthen the hip adductor muscles or enhance core stability in clinical settings . |
the technology
opens new exciting research and technological possibilities in dissipative
systems out of equilibrium .
a plethora of applications , such as lab - on - a - chip
analysis , diagnostics , and particle synthesis , can be accessed , to
mention a few examples .
one particularly important highlight is the
promise of new cheap technologies for health care , which are critical
for underdeveloped countries .
importantly ,
microfluidics can also be used as a probe for more fundamental studies .
this is true not only to study phenomena where the micrometer - length
scales are a relevant part of the phenomenon itself ( e.g. , blood rheology ) but also because many systems can be better
controlled on micrometer - length scales where laminar flow regimes
dominate , allowing the simultaneous control of experimental parameters
that were previously hard to achieve ( e.g. , rate of mixing , shear
rate , concentration gradients , confinement , and geometry ) .
the advantages of using such a platform for small - angle scattering
are also enormous .
first , sample consumption is reduced to the microliter scale , allowing
experiments with expensive and rare materials .
second , the constant
flow of material prevents radiation damage ( critical for x - ray synchrotron
radiation ) .
third , flow - alignment effects can be used to our advantage ,
allowing a gain in resolution and the possibility to probe material
properties and dynamic behavior that are usually not visible in nonoriented
samples .
given the small length scales , confinement ,
and far - from - equilibrium
conditions , unexpected phenomena can also arise . in some cases ,
very
complex phenomena such as folding instabilities can be simplified
by the confinement of one of the dimensions / degrees of freedom to
which the system could randomly evolve , allowing systematic studies
to be performed .
liquid crystals ( lcs )
have an intermediate degree of order between liquids and crystals , and earlier work has shown that under flow ,
shear effects can induce unexpected out - of - equilibrium structural
changes .
more recent studies of lcs under flow but now in
microfluidic devices have also started to increase considerably .
, the phase has only short - range positional order such
as that in a regular liquid , but because of some specific molecular
features ( e.g. , anisotropy , amphiphilicity ) , molecules exhibit a long - range
orientational order , specified by a unit vector n , the nematic director .
this long - range
orientational order gives rise to a wealth of interesting phenomena ,
the most famous being the application in liquid - crystal displays ( lcds ) .
the orientation state of the nematic director n under
flow is determined by the ericksen
parodi ( elp )
theory , which considers the effects of the viscous v and elastic el torques on n. according to the elp theory , the elastic torque vanishes at high
shear rates ( and hence flow rates ) , and only v is
significant .
when the ratio between the shear ( 2 ) and rotational ( 1 ) viscosities (
= 2/1 ) meets the condition
|| > 1 ( the most common situation for nematics , including
5cb ) , the stable solution of the equation
of motion
( v = 0 ) is then achieved when n aligns at a critical angle s with the flow direction
given by1 the presence
of an interface may also lead to a preferred orientation .
if the orientation imposed by the interface is different from that
imposed by flow , then conflicting alignments may give rise to rich
flow behavior .
these effects can be greatly enhanced in microfluidic
devices , where the small geometric length scales can accentuate the
effects of conflicting surfaces , leading to , for example , director
reorientations over smaller length scales than usual .
this has been
recently explored by sengupta et al . , who observed the emergence of
different flow regimes resulting from the viscoelasticity of a nematic
phase ( 5cb ) accentuated at smaller channel gaps .
in a related approach , although without microconfinement ,
the combination between capillary flow alignment and saxs was successfully
employed to study the nematic smectic transition . in this work , we are interested in the competition between
flow
and boundary conditions with respect to the orientation of nematic
liquid crystals . by combining small - angle x - ray scattering ( saxs )
with a microfluidic device , we describe an approach to measure the
orientation of a flowing nematic liquid crystal directly under confinement
near solid or liquid surfaces . for the solid - wall confinement ,
,
we use the hydrodynamic focusing technique , where a sheet of liquid is narrowed by a surrounding fluid . in the
case of immiscible fluids , much of the attention
has been placed on
droplet formation under different regimes , including nematic droplets . here
, however , we aim to create
stratified flows and inspect the nematic director orientation , with
particular emphasis on the role of viscous forces from flow and surface
anchoring forces imposed by the solid and liquid interfaces .
the microfluidic chips , made
of cyclic olefin copolymer ( coc or topas ) were purchased from microfluidic
chipshop ( catalog no .
02 - 0757 - 0166 - 02 ) and used without modification
of the hydrophobic surface .
the channel consists of four inlets meeting
at a cross , with inlets 14 having lengths of 80 , 5 , 5 , and
6 mm , respectively ( figure 1a ) .
the microchannel
cross section is square , with a width of 100 m . in this work ,
contrary to what is more conventional , the main fluid flows in the
reverse mode from what is usually the outlet along the main channel ,
meeting the cross at the end ( figure 1a ) .
this
method allows the study of pure 5cb in simple flow ( without hydrodynamic
focusing ) along the main channel without perturbations from the cross .
more importantly , in the hydrodynamic focusing experiments , allowing
the nematic to flow in this direction helps to stabilize the nematic
sheet and avoid the displacement of the lc into side channels 2 and
3 .
( a ) the flow is along the + y direction ( inlet
1 to outlets 24 ) .
( b ) the channel cross section is square
with a width of 100 m .
the velocity profile can be described
with some level of approximation by a poiseuille flow being maximized
at the center of the channel and fading to zero at the surfaces as
a result of the no - slip boundary condition .
( c ) far from the edges ,
the nematic director n aligns preferentially along
the velocity direction ( y ) , making an angle
in the velocity gradient direction ( along x and z ) .
is the angle between the velocity gradient and
the z direction and defines the nematic director
cone around the velocity direction y. yz is the angle between y and n projected onto the y z plane ( nyz ) .
yx is the angle between y ( velocity direction ) and n projected
onto the y x plane ( nyx ) .
( d ) alternative visualization
of yz , the angle measured in the
2d saxs detector ( y z plane ) ,
and yx . in the simple flow experiments , a nemesys syringe pump ( cetoni ,
korbuen , germany ) was used . in the hydrodynamic focusing experiments ,
a custom - built step - motor - driven syringe pump system with three independently
controlled pumps was used to drive fluid flow in the main channel
and both side inlets . in both experiments ,
tygon tubing ( formulation
s-54-hl ) with a 250 m inner diameter was used .
the tubing is
connected to the syringes via a needle ( gauge 29 ) with a suitable
grip and connected to the microfluidic chip using custom - built connectors . for the hydrodynamic
focusing experiments ,
the side fluid is a
2 wt % solution of triton x-100 , well above the cmc ( 0.019 wt % ) . for the fluorescence microscopy observations ,
0.1 mm fluorescein is added .
for each flow rate , a stability
test is performed to make sure the flow regime is stable .
the stable
flow rates are also approached from different paths to determine if
they are robust or history - dependent ( cf .
after the initial flow mapping through
microscopy and the identification
of stable flow regions , selected flow regimes are probed by saxs .
for the transmission saxs measurements , the chip is mounted on a custom - built
device holder , with translation capacity in the x , y , and z directions , and rotation
of the ( about y ) and ( about x ) angles ( figure 2a , bottom ) . before
the measurements
the alignment is more complicated , and a maximum tilt angle
of 2 can occur .
the sample is placed normal to the x - ray
beam and scanned in the z and y directions .
the
simple flow experiments were performed on the csaxs beamline at the
swiss light source ( sls ) , paul scherrer institute ( psi ) , villigen ,
switzerland .
the hydrodynamic focusing experiments were performed
on saxs / d beamline 4 - 2 at the stanford synchrotron light source ( ssrl ) ,
menlo park , ca , usa . for the csaxs instrument ,
the x - ray wavelength
( ) used was 1.11 , and the sample - to - detector
distance was 2.16 m. the data was collected on a pilatus 2 m detector .
the beamline optics allowed a very small beam size of fwhm
6 m in the z direction and 40 m in
the y direction .
for the saxs / d ( bl 4 - 2 ) instrument ,
the x - ray wavelength ( ) used was 1.03 , and the sample - to - detector distance was 1.1 m. data was collected
on a mx-225 rayonix ccd detector .
the x - ray beam had a size of 30
m ( fwhm ) in the z direction and 300 m
in the y direction .
for both instruments , the obtained
2d scattering patterns were corrected for background by subtracting
the 2d pattern of the chip outside of the microchannel corrected for
the different thicknesses .
the resulting 2d background - corrected patterns
are subsequently analyzed with matlab routines to extract structural
information through qh and ql scans or the nematic director orientation through
scans ( figure 2a , top ) .
( a ) typical saxs
pattern ( top ) and schematic of the microfluidic device ( bottom )
. the
device holder ( shown at the bottom ) can be translated in the x , y , and z directions
and rotated in ( about y ) and ( about x ) .
the x - ray beam is along the x direction ,
and the saxs pattern ( in the qy , qz plane ) is
collected in a detector behind the device . in both the chip and detector ,
the flow is along the + y direction .
a typical saxs
pattern ( flow rate q = 3 l / min , z = 30 m ) at small q is dominated by the nematic
phase correlation peak , which is inversely proportional to the nearest - neighbor
distance between the nematogens along their main axis .
therefore ,
the peak maximum q = 2n / d is along the nematic director n projected
onto the y z plane ( nyz ) , and yz is the angle between nyz and the flow direction ( y )
. the
direction qh is defined as being parallel
to nyz , and ql is perpendicular to nyz . in our convention , = 0 is the negative qz axis , and the positive rotation is counterclockwise .
( b ) snapshot of 5cb flowing slowly ( approaching 0 l / min ) before
data acquisition at the csaxs beamline .
the green box represents
the x - ray beam s fwhm ( 6 m along z and 40 m along y ) and hence the
area probed by each x - ray measurement .
the orange box represents a
typical area probed in a vertical scan ( z intervals
of 4.85 m ) .
( c ) representative scattering patterns of a flowing
nematic phase recorded along the width ( z direction )
of the microchannel at a flow rate of 3 l / min ( left ) and at
rest ( right ) .
the patterns rotate with roughly mirror symmetry with
respect to the center of the channel ( clockwise in the upper part
of the channel ( + z ) and counterclockwise in the lower
half ( z ) ) .
close to the edges , the magnitude
of the rotation is larger at rest . at the edges of the microchannel ,
the scattering intensity is also lower because approximately half
of the x - ray beam is outside of the microchannel and may have a contribution
from the y x plane due to
a small tilt ( < 2 ) of the device in .
figures 1a , b show a simplified
picture of the microchannel geometry , and figures 1b d show the coordinate system used in this work .
the
channel cross section ( x z plane ) is square with a width of 100 m ( figure 1b ) . because of the micrometer - sized dimensions in both the z and x directions
, the velocity gradient
( or shear rate ) extends in both of these directions ( i.e. , vy/x 0 and vy/z 0 ) , and therefore there
is no neutral direction . the velocity profile can be described with
some level of approximation by a 2d poiseuille flow , which is maximized
at the center of the channel and fades to zero at the surfaces ( no - slip
boundary condition assumed , cf .
importantly , throughout this work , the flow ( velocity v ) in the microchannel is always along the + y direction .
the average orientation of a nematic liquid crystal is given by the
director n , and far from the walls , n tends to align along the velocity direction ( y axis )
but with an angle with v ( figure 1c , d ) .
because of the square geometry , the velocity gradient
extends over both the x and z directions
( cf .
figure s3 ) ; therefore , the director
can rotate about a cone determined by a second angle ( i.e. ,
rotation about the y axis ) in which not changing
the magnitude of changes its projection in the y z plane ( containing v and zv ) and y x plane ( containing v and xv ) ( figure 1c ) .
= 0 in the vicinity of the horizontal walls ( z = zwall = 50 m ) ,
and = 90 in the vicinity of the vertical walls ( x = xwall = 50 m ) .
anywhere in the channel ( away from the walls ) , (x , z ) can be calculated from the velocity gradient
profile in the x z plane ,
according to = tan(xv/zv ) .
importantly , yz = tan(cos tan ) is the projection
of in the y z plane
( the 2d - detector plane in the saxs regime ) and is equal to
when = 0 .
yz is the orientation
angle measured in our microfluidic saxs setup ( figures 1c and 2a ) .
yx = tan(sin tan ) is the
projection of in the y x plane and is equal to when = 90. the
flow rate q is varied from 3 to 0 l / min ,
covering a range of shear rates = 200 to 0
s ( table s1 ) .
after
we switch to a new flow condition , 1030 min of waiting is
allowed before the next measurement starts . below 0.1 l / min ,
the pump is switched off , and after 10 min , a new scan is started ,
which we denote as q approaching 0 l / min .
throughout the duration of the scan ( 10 min ) , the phase continued
flowing slowly , as observed with the video camera .
after this measurement ,
we waited 30 min more before a new scan was started , which we denote
as q 0 l / min . in this last scan
figure 2 shows the experimental setup used
for saxs data collection and an example of a 2d saxs pattern in reciprocal
( qh , ql ) space
( a ) , a snapshot of 5cb in the microfluidic chamber at a low flow rate
( b ) , and a series of 2d saxs patterns with the x - ray beam going through
different positions ( z ) across the width of the microfluidic
channel ( c ) .
each saxs pattern corresponds to a displacement in the z direction ( across the width of the channel ) with a step
size of 4.85 m .
the saxs pattern collected by a 2d detector
is displayed with the same orientation as for the microfluidic device .
the saxs pattern is dominated by a broad peak centered at qh = 0.254 ( figure 2a , c ) .
this peak results from the short - range positional
liquid order between nearest neighbors along the direction parallel
to n. hence , the azimuthal ( in plane ) orientation
of the peak directly gives the orientation of n projected
onto the y z plane ( i.e. , nyz ) . in other words , the saxs
patterns directly give the director angle projection onto the y z plane : yz .
the peak is not perfectly radially symmetric but is somewhat
extended in the ql direction . as such ,
normal azimuthal integration smears the pattern to some extent . for
this reason
, we choose to integrate the intensity in projections along
the ql and qh directions ( figure 2a , top ) .
the most striking
effect that can be seen in figure 2c is the
variation in the peak orientation as we scan along z. the rotation in the 2d saxs patterns exhibits roughly mirror symmetry
with respect to the center of the channel ( z = 0
m ) i.e. yz ( the angle between
the axis connecting both peaks and the y axis , figure 2 ) , is approximately zero in the middle of the channel ,
and rotates clockwise in the upper half of the channel ( + z ) and counterclockwise in the lower half ( z ) .
this effect also seems to be pronounced at rest ( q 0 l / min ) because the tilt in yz seems to be larger when compared to q = 3 l / min .
figure 3a shows qh scans through the peak maximum ( qh =
0.254 ) of 5cb at q = 3
l / min for the 2d saxs patterns at z = 0 m
( figure 2c ) averaged over different ql intervals .
1.4 times the
size of a 5cb molecule , in good agreement with the suggestion that
5cb molecules are predominantly in a dimeric form , with their benzene
rings forming a pair , and the alkyl chains protruding outward .
the half - width at half - maximum ( hwhm ) of the peak is 0.057 .
this corresponds to a correlation domain with 1/0.057
= 17.5 , which is expected for liquids
with near - neighbor short - range positional order .
one - dimensional scattering
profiles of the 5cb nematic confined
in the microfluidic device .
the coordinates ( qh and ql ) in reciprocal space are
defined in figure 2a ( top ) .
( a ) qh scans integrated over different ql intervals in the center of the channel ( z 0 m ) at a flow rate of q = 3 l / min .
( b ) qh scans ( integrated over a ql range from 0.036 to 0.036 ) at q = 3 l / min and at different z positions along the channel ( cf .
figure 2c , which shows that the z range goes from
50 to +50 m ) .
the proximity to the
microchannel boundaries does not modify or distort the 1d profiles
integrated over ql .
( c ) qh scans ( integrated over the ql range from 0.036 to 0.036 ) at
different flow rates .
the 1d profiles are not modified or distorted
in the investigated flow rates . in both ( b ) and ( c )
, the curves are
normalized and slightly offset for ease of visualization . in all panels
( a c ) , the circles constitute scattering data and the lines
constitute lorentzian fits . in figure 3b ,
qh scans of the central region of the pattern ( ql interval of 0.036 to 0.036 ) are shown for z at the center and close to the
edges of the channels .
as shown , no noticeable differences can be
spotted between the curves , and the peak positions remain the same .
this extends also to the case of no shear , in which the peak position
and width are essentially the same ( figure 3c ) .
this indicates that flow effects are not distorting the short - range
positional order of the nematic liquid crystal . instead , these effects
result in changes in the orientation of n , which
is the most common situation for nematics under flow .
the
shift in the director orientation is more clearly seen in azimuthal
scans in the ( qh , ql ) plane ( figure 2a , top ) . by fitting
the peak positions in
we fit the peaks with
a double lorentzian equation of the form2where we restrict the position 0 of peak 2 to exactly 180 away from peak 1 because both
peaks are related through a two - fold rotational axis . a1 and a2 are proportional
to the heights of peaks 1 and 2 , b1 and b2
are the hwhm s of the peaks , and c 0 is a constant related to the background . in figure 4 ,
normalized scans and
respective fits are shown for q 0 l / min .
as can be seen , when going from the top to the bottom of the channel
( bottom to top in figure 4a ) ,
0 90 at z = 0 m .
because the flow direction is along the y axis and
is at an angle of = 90 , the director angle with the
flow direction yz is directly extracted
through yz = 0 90. scans and respective double - lorentzian fittings
( cf .
figure 2a , top , for the definition of
angle in the
( qh , ql ) plane )
measuring the orientational width of the nematic correlation peak
for 5cb in a microfluidic device at q 0
l / min .
the quality of the data ( and hence
the fitting ) is reduced at the edges of the microfluidic device ( z 53 m ) , as a result of part of the
x - ray beam being outside the microchannel and hence not hitting the
5cb nematic . in figure 5a , b
, we show the dependence of
yz on z for flow
rates of 0.2 l / min q 3 l / min
and q < 0.1 l / min .
note that to facilitate
the observation of symmetry ( or lack thereof ) of yz across z with a mirror plane in
the middle of the channel ( z = 0 m ) , we display
the absolute value |yz| instead .
also , for easier association of the yz dependence with the poiseuille profile
immediately
evident is the fact that for q 0.2 l / min
all of the curves overlap very well .
likewise , q approaching
0 and q 0 l / min also display very
similar profiles to each other .
more striking is the obvious difference
in curve profiles between the q 0.2 and q < 0.1 l / min groups . in all cases ,
yz is negative ( clockwise rotation ) in the
upper half of the microchannel ( z > 0 ) and is
positive
( counterclockwise rotation ) in the lower half ( z <
0 ) .
the same information can be seen in figure 5c for flow rates of q = 3 l / min and q 0 l / min and where the nematic director
projection nyz orientation
is represented in the form of an orientation field as a function of z and y. figure 5d shows a schematic of the 5cb molecules and nematic director orientation
and corresponding 2d saxs patterns .
fitting results for the nematic director
angle projection yz at different z positions
along the microchannel ( cf .
results for
the ( a ) 0.2 l / min q 3 l / min
and ( b ) q < 0.1 l / min regimes .
the z position is rescaled by centering = 0 at zrescaled = 0 m .
note that the abscissa
represents |yz| in reverse order
for ease of visualization because the resulting z yz dependence
resembles the poiseuille flow profile .
solid data points result from
x - ray patterns where the beam is mostly inside the microchannel in
the y z plane .
open data
points result from patterns where a significant fraction of the x - ray
beam was hitting outside of the y z plane , catching part of the y x plane because of a small rotation of the microfluidic
device holder about the y axis parallel to the flow
( i.e. , the angle shown in figure 1a ) .
note that some points close to z = 55 m shift
to smaller angles .
a clear distinction in the flow profiles can be
seen for q 0.2 and q <
0.1 l / min . in the first group ( a )
, the director tilt is controlled
by flow ( i.e. , the leslie solution discussed in the text , where the
nematic director assumes a dominant angle s ) .
the red and blue lines represent the leslie solution for s = 11 using the poiseuille and non - newtonian velocity
profiles , respectively ( which are very similar ) . in the second group
the line represents the
escaped director configuration model ( eq 3 )
assuming r = 71 m ( half of the diagonal length
of the square channel ) , resulting in r ( the angle of n at the surface ) = 32.
( c ) schematic representation of the orientation of the nematic director
as a function of microchannel z position for q = 3 and 0 l / min .
each blue bar represents
the average orientation of a rectangle with height z 6 m and length y 40 m ( the fwhm of the beam profile ) .
the director
angle yz is multiplied by 3 to
facilitate visualization of the changes across z and
for the different q values .
( d ) schematic illustration
of the molecular arrangement for selected points along the orientation
field in ( b ) and respective x - ray scattering patterns . under pressure - driven flow ( e.g. , poiseuille flow ) ,
far from the
boundaries , and when the ratio between the rotational ( 1 ) and shear ( 2 ) viscosities ( = 2/1 ) meets the condition || > 1
( which
is the case for 5cb and most known nematics ) ,
the torque on the nematic director usually vanishes when the
director aligns at a critical angle ( eq 1 ) s = cos(/)/2 ( henceforth called the shear angle ) between the flow and gradient
directions .
if the orientation imposed
by the surface is different from the alignment imposed by flow , then
a transition layer of length e1
( k/ ) ( where k is related to the nematic - phase elastic
constant and is an average nematic
lc viscosity , cf .
si ) close to the surface
will occur , where the director gradually shifts from the boundary
orientation to the flow orientation .
in addition , because the velocity
field is symmetric about the center of the channel , switches
signs in the center along a transition region of sizee2 ( he12 ) ( h = 100 m gap size in a microfluidic
device ) .
it is therefore important to determine if the observed variation
in yz with z is
an effect of confinement , where the boundary layers could have a greater
influence on the orientation field far from the walls or they are
simply a consequence of the fact that saxs data measures yz ( the projection of on the y z plane ) rather than directly
( cf .
figure 1d ) . as shown in figure 1c , d , is the
angle between the nematic director n and the velocity
( or flow ) direction y. is the angle measuring
the rotation of n about the y axis .
the projection of in the y z plane ( saxs detector plane ) is given by yz = tan(cos tan ) .
to compare our results with leslie s solution , we assume a
profile equal to s throughout the whole
microchannel except within the boundary layer e2 , where for simplicity decreases linearly from
= s at z and x = e2 to = 0 at z and x = 0 ( figure s2 in si ) .
( e1 is neglected because
our measurements in the vicinity of the channel walls are not accurate . )
to obtain , we require the 2d velocity profile ( v(x , z ) ) from which the velocity
gradients can be calculated ( figures s1 and s2 in si ) . from the velocity gradient components in the x and z directions , is easily obtained
( = tan(xv/zv ) ) .
yz is then computed as a function
of x and z using the respective
(x , z ) and (x , z ) and subsequently averaged over the x direction to obtain yz(z ) . a further step in
the fitting includes finite
beam size effects . in the fitting , we compare two different velocity
profiles .
we use the normal poiseuille flow , which yields a parabolically
shaped velocity profile and is suitable for newtonian fluids under
laminar flow . because the nematics are non - newtonian , the actual flow
profile deviates slightly from the parabolic shape , and we also use
the flow profile determined by jewell et al . for 5cb flowing in a
microchannel with a rectangular geometry .
a more detailed description of the fitting procedure can be found
in the supporting information .
as can be
seen in figure 5a , both models ( poiseuille ,
red curve ; non - newtonian , blue curve ) fit the data reasonably well
for s = 11 , which validates the use of the
poiseuille flow profile as an approximation in this case .
more importantly ,
the goodness of the fits confirms that the leslie solution holds under
these conditions ( q > 0.1 l / min ) .
the variations
in the observed shear angle as a function of z therefore
most likely results from the azimuthal angle , which varies
from 0 to 90 ( as a result of the equivalence of walls y x and y z in the square cross section ) and decreases the projection
of angle in the saxs detector plane ( y z ) . using s = cos(/)/2 ( eq 1 )
, one can
extract from the determined s , obtaining
|| = 1.08 , which is in reasonable agreement with the values
of || = 1.12 and s = 13 reported elsewhere .
one should also note that even though e2 ( and e1 ) depends
on , our data does not change in the range of 0.2 l / min
q 3 l / min ( figure 5a ) .
this is mainly a result of being convolved
with . as we see in figure 5b ,
at a low flow rate
below 0.1 l / min the behavior is very different .
( there is a small lack of symmetry
that is discussed in the si . ) despite the
movement of 5cb in the microchannel , clearly seen in the camera and
microscope at q approaching 0 l / min , yz has reoriented across the whole channel ,
except at z = 0 m where yz is still zero .
even though in our measurements
we can only probe the projections on the y z plane , it is very likely that this relaxation also takes
place across x. hence , the line parallel to y in the center of the channel at x and z = 0 m is almost a smoothed disclination line , where
yz ( and yx ) takes opposite signs on each side of this line .
this configuration
resembles the escaped radial configuration ( typically observed in
tubes of cylindrical and square cross sections at rest , where the nematic director reorients
near the central region of the channel along the channel axis in order
to accommodate competing orientation demands from walls perpendicular
to each other ) . in the cylindrical geometry , at rest or low
flow , where boundary
conditions and elastic energy terms dominate , the angle between the
director and cylinder axis ( approximately equal to our condition with
a square cross section ) ( ) can be described by3where r is the radial distance
from the center of the cylinder , r is the cylinder
radius , and r is the angle of n at the surface ( for strong homeotropic anchoring , r = 90 ) .
this equation , convoluted with
the (x , z ) profile and beam
size , fits our data to some degree ( figure 5b ) , resulting in r = 32
if r is taken to be 71 m ( half of the diagonal
of our square geometry ) .
if r is
fixed to r = 90 ( strong homeotropic
anchoring ) and we fit r instead , then we obtain r = 250 m .
however , one also has to recall
that the above equation was derived for cylindrical geometry . in the
case of square geometry , the elastic deformation that causes the escaped
configuration is already very significant at the corners of the channel ,
which may force the escaped configuration to change more abruptly
than what eq 3 would predict for cylinders .
hence , it is not clear on the basis of these measurements if the anchoring
is strong or weak .
it is our view that the observed behavior is in
better agreement with a situation of strong anchoring .
note that we
started at q = 3 l / min and slowly decreased
the flow rate until q 0 l / min , which
resulted in the strong preorientation of the director in the flow
field .
given the fact that stopping the flow leads to a relaxation
of the preoriented profile , with the director reorienting all of the
remaining , except the disclination line at x and z = 0 , then this should give a hint
that the elasticity emanating from the boundary condition is strong .
in any case ,
regardless of strong or weak anchoring , it is evident
that at low flow rates ( q < 0.1 l / min )
the elasticity ( resulting from competing preferential homeotropic
alignment at the walls ) becomes dominant . to conclude this section
,
we stress that through our microfluidic
saxs measurements we were able to directly probe the nematic director
orientation profile of a nematic liquid crystal flowing in a microchannel
with micrometer resolution in z and identify two
different flow regimes ( one for 0.2 l / min q 3 l / min and the other for q < 0.1 l / min ) .
for the strong flow
regime ( high flow rates ) . by using channels with rectangular cross
section ,
the interesting intermediate and weak flow regimes observed
in that work are also expected to be suitably determined .
one advantage
of our method compared to microscopy is the ability to obtain a more
quantitative description of the director tilt across the channel .
the tilt angle can be accurately determined , with its resolution being
only limited by the averaging effects from the beam size and by the
shift in the angle projection due to the azimuthal orientation .
( note
that the azimuthal shift is caused by the square cross - section .
hence ,
resolution can be significantly improved by using channels with a
rectangular cross section of high aspect ratio . )
another advantage
of this present method is the ability to map changes in the molecular
arrangements of the fluid simultaneously . here
we clearly demonstrated
that despite reorientation the nematic peak position and width remain
the same , indicating that the studied flow conditions do not affect
the local arrangements of the fluid simultaneously with the director
orientation .
this may not always be the case in lyotropic systems
that are more compressible and where concentration fluctuations and
instabilities induced by flow can occur . in section 3.1
, we described the alignment
of 5cb and the interplay between viscous and surface forces in simple
flow , where the boundary conditions are due to the walls of the microchannel
( solid interfaces ) . in this section
, we describe a similar approach ,
but where the nematic liquid crystal is bound by flowing liquid from
above and below ( liquid interfaces ) and where capillary forces arising
from the interfacial tension between the immiscible fluids ( nematic
and water with some dissolved surfactant ) also play an important role .
for this purpose ,
figure 6a , b ) as before , with a flow rate of qm , but now a solution of 2 wt % triton x-100 also flows in channels
2 and 3 at a flow rate of qs ( figure 6a ) .
the ratio of the total flow rate from the side
channels ( 2qs ) to the flow rate of the
middle fluid ( qm ) is rq = 2qs / qm .
the three fluids meet at the cross and flow
together into the outlet ( channel 4 ) . for a suitable range of flow
rates
qm and flow rate ratios rq between 5cb and the triton
solutions , stable sheets of 5cb with fluid on both sides can form
( table s2 and figure 6c , d ) . the nematic sheet was found to be stable for a surfactant solution
total flow rate of 2qs = 24 l / min ,
with 5cb flowing in the range of qm =
1 to 3 l / min ( table s2 ) . at lower qm < 1 l / min ,
jetting occurs , with
a thin thread of 5cb forming for a limited length ( 200300
m ) before rupturing to form droplets . at higher
qm > 3 l / min , displacement instabilities occur , leading to an invasion of middle fluid
5cb into the side channels .
the system is also stable when increasing qm while keeping the same flow rate ratio rq , but the same does not happen
when qm is lowered at constant rq .
instead , it is
surrounded by side fluid along the z direction , in
which the y x interfaces
are now the side fluid , but along the x direction ,
it is still bound by the chip walls ( y z surfaces , figure 6b ) .
( note in
figure 6d that in the nematic region there
is no trace of fluorescence .
furthermore , the relief pattern indicated
by the white arrow suggests a concave shape of the nematic sheet . )
it was found that qm = 3 l / min
with rq = 8 is the most
stable flow condition . for lower qm ( <
1.5 l / min )
, the flow condition becomes history - dependent because
the nematic stream is not able to form by itself but remains stable
if it is formed at higher qm and then
decreased slowly to the final value .
summary of the hydrodynamic focusing experiments
with the nematic
phase of 5cb confined in a microfluidic device by solid and liquid
interfaces .
5cb flows
along channel 1 and is focused into channel 4 by the side fluids ( 2
wt % triton x-100 in water ) coming from channels 2 and 3 .
( b ) schematic
representation of the 5cb nematic sheet focused by the triton solution .
( c ) micrograph of a typical hydrodynamic focusing experiment . here
the side fluids contain 0.1 mm fluorescein to facilitate the visualization
of the nematic sheet ( black ) .
5cb flows at 3 l / min , and each
side fluid flows at 12 l / min ( rq = 2qs / qm = 8) .
the green
box represents the x - ray beam s fwhm ( 30 m along z and 3000 m along y ) and
hence the area probed by each x - ray measurement .
the orange box represents
a typical area probed in a vertical scan ( z intervals
of 1020 m ) .
( d ) the same as in ( c ) but with a 5cb flow
rate of 1 l / min and rq = 24 .
the focused nematic sheet seems to have a concave shape
along the x direction ( note the relief pattern indicated
by the white arrow compared to the dark arrow for the formation of
the sheet ) , with its width being ca .
22 m on the top and ca .
8 m in the middle of the channel .
( e ) saxs along the width
of the channel for qm = 3 l / min , rq = 8 . as in the simple flow
experiments
, here also the nematic director rotates from a negative
yz on the top to a positive yz on the bottom side of the sheet .
have studied
the coflow of 5cb and silicon oil .
although the goal was the formation of nematic droplets , stable stratified
flow regimes were also identified .
in addition to capillary and viscous
forces , wetting of the device walls by 5cb was identified as an important
parameter stabilizing the nematic sheet . the non - newtonian nature
of 5cb and defect structures , however , cause instabilities that promote
droplet formation . in a more general
study
, although restricted to newtonian fluids , cubaud and mason have
studied different flow regimes between coflowing immiscible liquids
with different viscosities and interfacial tension contrasts , with
the flow regimes being mapped into a flow map of capillary number ca in middle fluid cam versus
side fluid cas .
the interfacial tension between 5cb and water has been reported
to be approximately 26 mn / m .
this leads
to a predominance of capillary over viscous forces , which does not
allow the formation of a stable nematic sheet of liquid .
instead ,
the system drips , with the immediate formation of droplets ( i.e. ,
to reduce the surface energy cost ) , in agreement with the flow map .
the addition of 2 wt % triton x-100 reduces
the interfacial tension , allowing the formation of nematic sheets
sandwiched between two aqueous surfactant solutions ( figure 6c , d ) . also important for the stabilization of the
sheets against breakup
is the poor wettability of the microchannel
walls by the side fluid when compared to that by the nematic fluid .
( note that the hydrophobic channel walls should
have more affinity for 5cb than for water . )
the nuances of the
flow map and the role of capillary and viscous
forces coupled with the non - newtonian behavior will be the subject
of a more detailed study elsewhere . in this work ,
we are mainly interested
in the effect of the liquid boundaries on the nematic director .
note
that despite the nematic sheet being bound by liquid interfaces at
wm/2 m and solid surfaces
at 50 m , 5cb is squeezed along the z direction , reducing the contact with the solid walls .
also , as noted previously , in these saxs measurements , what
is measured is yz , which is the
projection influenced by the liquid boundaries . at the synchrotron
,
two flow conditions were studied : qm =
3 l / min , rq =
8 ; and qm = 1 l / min , rq = 24 . in figure 6e , 2d saxs profiles due to scattering from the hydrodynamically
focused 5cb at different positions along the z axis
are shown .
as in the simple flow experiment , the same rotation in
the nematic director is observed , with yz being negative in the upper half of 5cb and positive in the
lower half . in this case , the beam size is much larger ( fwhm 30
m ) , which introduces much stronger averaging effects but is
still suitable for an overall determination of the focused nematic
stream
. the larger scanning step size ( 10 and 20 m ) also needs
to be taken into account .
figure 7a shows qh scans averaged over different ql intervals at qm = 3 l / min , rq = 8 , and z = 0 m . in figure 7b ,
qh scans of the central region of the pattern ( ql interval of 0.036 to 0.036 ) are shown across the nematic stream , in contact
with the triton solutions , and compared to the pure system .
all hydrodynamic
focusing patterns show a slight shift in the peak position to lower
angles ( q 0.006 ) , which may indicate a slight penetration / contamination of triton
or triton / water particles within the 5cb sheet . even though this shift
in the peak is very small , this phenomenon is occurring mainly on
the interface , which makes the signal weaker compared to that of the
remaining nematic sheet .
we also note that although the residence time of 5cb
in the channel is small ( 0.1 s ) , the surfactant concentration
is two orders of magnitude above the cmc ( 0.019 wt % ) , which could lead to some solubilization of 5cb at the
interface and lead to some instabilities that could affect the observed
results .
the coordinates ( qh , ql ) and angle in reciprocal space are
defined in figure 2a .
( a ) qh scans integrated over different ql intervals in the center of the channel ( z 0 m ) for a hydrodynamic focusing experiment .
the
flow rate of 5cb ( middle channel ) is qm = 3 l / min , and the ratio rq = 2qs / qm of the combined side fluids (
triton x-100 2% solutions ) with the
inner fluid is rq = 8 .
( b ) comparison between qh scans ( integrated
over ql = 0.036 to 0.036 ) in the system with no hydrodynamic focusing ( black , q = 3 l / min ) and the hydrodynamic focusing system
( q5cb = 3 l / min , rq = 8) .
all hydrodynamic focusing patterns
show a slight shift in the peak position to lower angles ( q 0.006 ) , which may
indicate a slight penetration / contamination of triton or triton / water
particles within the 5cb sheet .
nonetheless , given the very small
shift in the peak , both the contamination and resulting structural
changes should be minimal .
( c ) scans and respective double - lorentzian
fittings for a hydrodynamic focusing run with qm = 3 l / min and rq = 8 .
data is normalized and displaced along the ordinate axis
for ease of visualization . in all panels ( a c ) ,
circles constitute
scattering data and lines are lorentzian fits . as with the simple flow case ( cf .
section 3.1 ) , also here it is convenient to extract yz from the scans .
figure 7c shows
the resulting fits of scans with the double - lorentzian expression
( eq 2 ) .
figure 8 shows
a plot of the measured yz at different z positions along the channel .
the data extends over a wider z range than the widths of the nematic sheets wm .
this results from the fact that the beam size ( fwhm
30 m ) is larger than the nematic stream , which enlarges
the range in z over which the nematic phase gives
a signal .
more importantly , the averaging effects due to beam size
also modify the observed values of yz . in figure 8a , a comparison of the
simple flow system ( i.e.
, no hydrodynamic focusing ) with the small
( fwhm 6 m , blue circles ) and large ( fwhm
30 m , green squares ) beams can be seen .
the curves are slightly
different , with the data from the larger beam showing a shift to smaller
yz angles . despite this ,
the same
overall trend is still observed , which allows the characterization
of the system .
the shift in yz to
smaller angles is easily understood if one recalls that yz has opposite signs in the upper and lower
halves of the microchannel .
if the beam is large enough that it partially
overlaps both halves , then the opposite signs of yz partially cancel each other , making the observed
angle yz smaller .
this effect is
more significant for narrower 5cb streams because the yz variations are more compressed in space ,
accentuating the smearing from the larger beam size . in figure 8b , |yz| versus z curves are shown for different rq .
even with the finite beam size effects ,
clear differences in the slopes of the different curves are easily
observed .
it can be clearly seen that as rq increases from rq = 0 ( simple flow experiments , green squares ) to rq = 8 ( blue circles ) and to rq = 24 ( red diamonds ) , the variation
of |yz| with z also increases .
results for the nematic director angle projection yz at different z positions
along
the microchannel in the hydrodynamic focusing experiments .
( a ) comparison
of measured yz for the simple flow
experiments ( no hydrodynamic focusing ) at q = 3 l / min
using the ssrl ( green ) and csaxs ( blue , finer beam size ) beamlines
.
even with the loss in spatial resolution due to the ca . 5-fold - larger
x - ray beam at ssrl , which also shifts yz to lower values , the same overall evolution in director rotation
is observed .
the z position is rescaled by centering the director angle
= 0 at zrescaled = 0 m .
compared to the simple
flow experiments , fitting the hydrodynamic
focusing data is now significantly more complex .
now , parameters such
as the viscosity mismatch , nematic sheet width , and interfacial curvature ,
among others , should clearly play a role in the velocity profile across
the nematic sheet , which in turn should reflect on the measured yz profile .
in addition , the nonideal resolution
along z ( combination of large beam size and large
step size ) smears the measured yz profiles significantly . as such , rather than fitting the data
lockwood et al . have shown that surfactants with branched chains ( as is
the case for triton x-100 ) have a tendency to induce planar alignment
in nematics . here
we clearly observe a tilt in the director , which
is also seen to increase as rq is increased .
we note that the observed shift in yz to larger angles as rq is increased ( figure 8b ) should be even larger in reality because of the above - mentioned
averaging effects due to the large beam size ( that reduce the measured
yz to lower values ) . hence ,
as
the nematic stream gets narrower , we can conclude that the average
angle yz increases substantially .
what is not clear is whether this angle is approximately constant
or varies smoothly across the stream width . in either case , these
observations seem more consistent with a picture where triton x-100
imposes an angle / tilt at the interface ( i.e. , homeotropic anchoring
or some angle intermediate between 90 and 0 ) rather than planar
alignment .
we note that if triton was imposing planar alignment under
these ( flow ) conditions , then it should be imposing the same orientation
as flow .
then , at most ( as the elp theory predicts ) , a shear angle
of s could be observed across the nematic
sheet as a result of flow , but this angle should not increase when
the nematic stream becomes narrower at higher rq , as we observe in the results .
this
work is the first step toward more detailed explorations and
characterizations of the formation of nematic sheets and flowing liquid
interface effects . in future work ,
an improved spatial resolution
( achieved with smaller beam sizes ) will be employed , along with a
systematic variation of other surfactants with different anchoring
properties ( e.g. , nonbranched alkyl chain surfactants that are known
to impose homeotropic alignment and bolaamphiphile surfactants , that
are known to impose planar alignment ) ,
to look for their effects on the nematic sheets .
we anticipate that
the coupling of flow with the different anchoring properties of different
materials ( e.g. , surfactants , lipids , polymers , and proteins ) will give rise to a wealth of interesting phenomena
for both fundamental and applied fields .
we also stress that this
method can be applied to many different materials in a high - throughput
fashion , which can be particularly useful for lc displays , where switching
orientations are known to induce local flows , and for boundary lubrication ,
where the orientations of lcs under flow determine the tribological
response of this type of material .
in this work , we investigated the interplay between flow and boundary
condition effects on the orientation field of a thermotropic nematic
liquid crystal under flow and confinement by using a combination of
microfluidics with in situ saxs .
two different experiments were performed .
in the first , a nematic liquid crystal flowed through a square - channel
cross section at varying flow rates , and the projection of the nematic
director angle on the y z shear plane was measured . at moderate - to - high flow rates ,
the director
was found to be predominantly aligned in the flow direction , but with
a small tilt angle of 11 in the velocity gradient
direction , in agreement with the leslie solution for poiseuille flow .
at low flow rates , a flow profile with a resemblance to the escaped
radial director
was observed , with the director varying more smoothly
from the edges to the center of the channel . in the second experiment ,
hydrodynamic focusing was employed to
confine the nematic phase in a thin sheet sandwiched between two triton
x-100 aqueous solutions .
the average director orientation was found
to tilt by some amount away from the flow direction toward the liquid
boundaries , although it remains unclear if one tilt angle is dominant
through most of the nematic sheet or if the tilt angle varies smoothly
between two extreme values ( 90 and 0 ) .
we anticipate
that expanding these studies to materials with different anchoring
properties will give rise to a wealth of new phenomena under flow ,
which is interesting for both fundamental and applied sciences . in
particular
, this method can be used for high - throughput routine measurements
to assess the influence of different surfactants or other additives
on the orientation of nematic phases , which can lead to further improvements
in areas such as boundary lubrication and lcds . | in this work we investigate the interplay
between flow and boundary
condition effects on the orientation field of a thermotropic nematic
liquid crystal under flow and confinement in a microfluidic device .
two types of experiments were performed using synchrotron small - angle
x - ray - scattering ( saxs ) . in the first , a nematic liquid crystal flows
through a square - channel cross section at varying flow rates ,
while
the nematic director orientation projected onto the velocity / velocity
gradient plane is measured using a 2d detector . at moderate - to - high
flow rates ,
the nematic director is predominantly aligned in the flow
direction , but with a small tilt angle of 11 in
the velocity gradient direction .
the director tilt angle is constant
throughout most of the channel width but switches sign when crossing
the center of the channel , in agreement with the ericksen
leslie
parodi
( elp ) theory . at low flow rates ,
boundary conditions begin to dominate ,
and a flow profile resembling the escaped radial director configuration
is observed , where the director is seen to vary more smoothly from
the edges ( with homeotropic alignment ) to the center of the channel .
in the second experiment ,
hydrodynamic focusing is employed to confine
the nematic phase into a sheet of liquid sandwiched between two layers
of triton x-100 aqueous solutions .
the average nematic director orientation
shifts to some extent from the flow direction toward the liquid boundaries ,
although it remains unclear if one tilt angle is dominant through
most of the nematic sheet ( with abrupt jumps near the boundaries )
or if the tilt angle varies smoothly between two extreme values ( 90
and 0 ) .
the technique presented here could be applied to perform
high - throughput measurements for assessing the influence of different
surfactants on the orientation of nematic phases and may lead to further
improvements in areas such as boundary lubrication and clarifying
the nature of defect structures in lc displays . |
postoperative pain is a critical concern affecting the choice between open and endoscopic surgical techniques and it is the main outcome parameter in both techniques .
thus , postoperative pain is frequently assessed in studies comparing the results of open and endoscopic release in carpal tunnel syndrome ( cts ) , a common peripheral nerve entrapment .
while several parameters such as functional outcome , recovery time , scar sensitivity and complication rates are also addressed in these studies , none of them focused on a scheduled and periodical followup of pain intensity in the early postoperative period.1234 the aim of this prospective , randomized , controlled study was to compare the course of pain intensity in the early postoperative period in patients undergoing open carpal tunnel release ( octr ) versus those undergoing endoscopic carpal tunnel release ( ectr ) .
eighty two patients , clinically and electrophysiologically diagnosed with cts , were included in this study .
the type of surgery to be performed was decided using a random number generator software ( research randomizer , version 3.0 .
urbaniak gc , plous s : [ computer software ] . retrieved on april 22 , 2010 , from http://www.randomizer.org ) .
patients were randomized into two groups ; endoscopic surgery group [ ( 21 female , 1 male ; mean age 49 years ( range 3164 years ) ] and open surgery group [ ( 25 female , 3 male ; mean age 45.1 years ( range 2968 years ) ] and received carpal tunnel release [ table 1 ] .
demographics of study population inclusion criteria were complaints of cts for at least 3 months which did not respond to conservative treatment , electrophysiological findings of intermediate to advanced level of isolated median nerve involvement in the carpal tunnel , lack of motor deficit , absence of cervical disc pathology , absence of metabolic problem resulting in peripheral neuropathy , lack of previous upper extremity injury or surgery and lack of movement restriction in the wrist and hand .
preoperative physical examination and boston cts scale of patients was performed by the same surgeon.5 the preoperative electrophysiological assessment was repeated in accordance with the study protocol by the same neurologist .
the electrophysiological evaluation was conducted using a nihon - kohden meb 5504 k device in the electromyography laboratory at the department of neurology in our hospital .
all patients received preoperative regional intravenous anesthesia ( riva ) with the same pharmacological agents ( prilocaine hydrochloride 3 mg / kg citanest ; astrazeneca ; complemented with 0.9% nacl for a 40 cc solution ) .
an incision of 34 cm long , 2 mm ulnar to the thenar crease line and distal to the kaplan oblique line was made .
the superficial palmar fascia and the transverse carpal ligament was cut through the ulnar side while protecting the median nerve and its motor branch .
the skin and subcutaneous tissue was released proximally and distally with a retractor , the opening of the tunnel was confirmed .
a second line was drawn proximally from the end of this first line 0.5 cm .
after that , a third line was drawn from the proximal end of the second line radially 1 cm which determined the entry portal . for exit
a transverse line from the distal end of the abducted thumb and a longitudinal line between the 3 and 4 finger toward the proximally was drawn in the palm surface .
a gap was created between the transverse carpal ligament ( tcl ) and the ulnar bursa with the help of a curved dissector after the proximal border of the tcl was determined .
tcl was visualized with an endoscope and divided with a retrograde knife ( ectra 2 ; smith and nephew ) .
a compression bandage was applied to control postoperative bleeding and released 30 min after surgery in all patients .
patient 's pain level was assessed on the 1 , 2 , 4 and 24 postoperative hours using visual analog scale ( vas ) scores .
pain relief was achieved with orally administered paracetamol 500 mg tablets ( parol tb , atabay ila ) . the total dose of postoperative medication in the first 24 h was noted .
data were analyzed using the spss for windows ( version 15.0 ) , chicago , usa , spss inc .
for the assessment of the study data , beside descriptive statistical methods ( average , standard deviation , median , frequency , ratio , minimum , maximum ) , independent samples t - test was used for comparing the normally distributed parameters between the groups and the mann
whitney u - test was used for comparing the nonnormally distributed parameters between the groups in the comparison of quantitative data .
repeated measures test was used to analyze the change of the vas score according to followup time .
the significance of the difference between pre and postoperative mean values was analyzed with paired samples t - test .
an incision of 34 cm long , 2 mm ulnar to the thenar crease line and distal to the kaplan oblique line was made .
the superficial palmar fascia and the transverse carpal ligament was cut through the ulnar side while protecting the median nerve and its motor branch .
the skin and subcutaneous tissue was released proximally and distally with a retractor , the opening of the tunnel was confirmed .
a second line was drawn proximally from the end of this first line 0.5 cm .
after that , a third line was drawn from the proximal end of the second line radially 1 cm which determined the entry portal . for exit
a transverse line from the distal end of the abducted thumb and a longitudinal line between the 3 and 4 finger toward the proximally was drawn in the palm surface .
a gap was created between the transverse carpal ligament ( tcl ) and the ulnar bursa with the help of a curved dissector after the proximal border of the tcl was determined .
tcl was visualized with an endoscope and divided with a retrograde knife ( ectra 2 ; smith and nephew ) .
a compression bandage was applied to control postoperative bleeding and released 30 min after surgery in all patients .
patient 's pain level was assessed on the 1 , 2 , 4 and 24 postoperative hours using visual analog scale ( vas ) scores .
pain relief was achieved with orally administered paracetamol 500 mg tablets ( parol tb , atabay ila ) . the total dose of postoperative medication in the first 24 h was noted .
data were analyzed using the spss for windows ( version 15.0 ) , chicago , usa , spss inc .
for the assessment of the study data , beside descriptive statistical methods ( average , standard deviation , median , frequency , ratio , minimum , maximum ) , independent samples t - test was used for comparing the normally distributed parameters between the groups and the mann
whitney u - test was used for comparing the nonnormally distributed parameters between the groups in the comparison of quantitative data .
repeated measures test was used to analyze the change of the vas score according to followup time .
the significance of the difference between pre and postoperative mean values was analyzed with paired samples t - test .
an incision of 34 cm long , 2 mm ulnar to the thenar crease line and distal to the kaplan oblique line was made .
the superficial palmar fascia and the transverse carpal ligament was cut through the ulnar side while protecting the median nerve and its motor branch .
the skin and subcutaneous tissue was released proximally and distally with a retractor , the opening of the tunnel was confirmed .
a second line was drawn proximally from the end of this first line 0.5 cm .
after that , a third line was drawn from the proximal end of the second line radially 1 cm which determined the entry portal . for exit
a transverse line from the distal end of the abducted thumb and a longitudinal line between the 3 and 4 finger toward the proximally was drawn in the palm surface .
a line bisecting this right angle was extended 1 cm from the vertex toward the ulna .
a gap was created between the transverse carpal ligament ( tcl ) and the ulnar bursa with the help of a curved dissector after the proximal border of the tcl was determined .
tcl was visualized with an endoscope and divided with a retrograde knife ( ectra 2 ; smith and nephew ) .
a compression bandage was applied to control postoperative bleeding and released 30 min after surgery in all patients .
patient 's pain level was assessed on the 1 , 2 , 4 and 24 postoperative hours using visual analog scale ( vas ) scores .
pain relief was achieved with orally administered paracetamol 500 mg tablets ( parol tb , atabay ila ) . the total dose of postoperative medication in the first 24 h was noted .
data were analyzed using the spss for windows ( version 15.0 ) , chicago , usa , spss inc . for the assessment of the study data , beside descriptive statistical methods ( average , standard deviation , median , frequency , ratio , minimum , maximum ) ,
independent samples t - test was used for comparing the normally distributed parameters between the groups and the mann
whitney u - test was used for comparing the nonnormally distributed parameters between the groups in the comparison of quantitative data .
repeated measures test was used to analyze the change of the vas score according to followup time .
the significance of the difference between pre and postoperative mean values was analyzed with paired samples t - test .
the mean age of the patients was 45.1 8.54 ( range 3168 years ) years in the open surgery and 49 8.21 ( range 3164 years ) years in the endoscopy group ; both groups were similar ( p = 0.11 ) .
there were three ( 10.7% ) male and 25 ( 89.3% ) females in the open surgery group .
the endoscopic surgery group had one male ( 4.5% ) and 21 females ( 95.5% ) patients .
mean preoperative duration of complaint was 47.96 50.43 ( range 3180 months ) months in the open surgery and 59.55 57.18 ( range 4180 months ) months in the endoscopic surgery group .
twenty one patients ( 75% ) in the open surgery group and 17 ( 77.3% ) in the endoscopic surgery group were operated on their right upper extremity .
seven patients ( 25% ) in the open surgery group and 5 ( 22.7% ) in the endoscopic surgery group were operated on their left upper extremity .
the boston questionnaire assesses the severity of the symptoms and functional capacity of the patients . there was no statistical difference between the groups in terms of both pre and postoperative boston scores .
a significant difference was observed between the pre and postoperative 6 week boston scores of the two groups [ tables 2 and 3 ] .
pre and postoperative boston scores of open surgery patients pre and postoperative boston scores of endoscopic surgery patients nocturnal complaints of all patients receded in the early postoperative period .
pain assessment at the postoperative 1 , 2 , 4 and 24 hours revealed significantly low vas scores in the endoscopic surgery group [ table 4 ] .
the changes of vas scores according to followup were found statistically insignificant for both groups ( p = 0.445 , p = 0.264 ; p > 0.05 ) . the first analgesic intake requirement in the open surgery group was at 3.81
cumulative dose of paracetamol intake during the first 24 h postoperative period was 772.72 428.93 mg in the endoscopic group and 1 , 535.71 428.79 mg in the open surgery group .
postoperative pain assessment results with vas one patient in the endoscopic surgery group experienced a flexor digitorum superficialis injury of the 5 finger .
the injury was detected peroperatively and the tendon was primarily repaired by expanding the incision .
examination at the postoperative 6 week revealed scar pain in three patients in the open surgery group .
this prospective , randomized and controlled study was undertaken to compare the course of pain intensity in the early postoperative period in patients undergoing octr and ectr .
both open and endoscopic techniques can be used for carpal tunnel release in refractory cases of carpal tunnel syndrome.1 skin problems are one of the drawbacks of open release and can be reduced with the endoscopic technique.26789 in our study , one patient in the endoscopic surgery group experienced a flexor digitorum superficialis injury of the 5 finger , which was detected peroperatively and was repaired primarily by expanding the incision .
2014 ) reported in his meta - analysis that ectr appears to be associated with fewer minor complications compared to octr , but there was no difference in the rates of major complications.10 in another current meta analysis , the risk of nerve injury was found to be higher in endoscopically treated patients , whereas the scar tenderness was less common.11 the findings were supported by keith 's study.12 to the best of our knowledge , postoperative pain after endoscopic and octrs has not been compared . in their studies , atroshi , chow and hantes and agee et al . emphasized the low postoperative level of pain but reported subjectively.131415 standard pain scoring was not performed in both studies.131415 in our study , postoperative pain was analyzed in detail using the vas score .
the pain level in the endoscopic surgery group during the first 24 h after surgery group was low .
meta analysis did not showed any difference between octr and ectr regarding to functional outcome.101112 however , less postoperative pain and faster recovery have been reported following endoscopic release when compared with open technique,3 disadvantages of ectr , including high surgical complication , inability to perform synovectomy , inability to detect space occupying lesions in the carpal tunnel and high cost have been reported in the literature.16 chow and hantes ( 2002 ) demonstrated that patients required 02 tablets of analgesics after endoscopic carpal tunnel surgery .
however , the type of analgesic or its dosage is not mentioned in their study.15 functional capacity of the patients can also be improved.41718192021 in our study , carpal tunnel symptoms disappeared in both patient groups .
2013 ) reported in his study that the pain relief immediately after surgery and after discharge from hospital was also similar for octr and ectr.22 whereas , our results showed that postoperative pain intensity is significantly less after ectr
however , the pain assessment was subjective , momentary and unscheduled . also , the amount of analgesic medication was used to compare pain intensity , rather than standardizing the analgesic regimen between the groups .
our study highlights the need for a more comprehensive investigation in which large patient groups are used to determine whether there is a statistically significant difference between two groups .
| background : results of open and endoscopic carpal tunnel surgery were compared with many studies done previously . to the best of our knowledge ,
difference in pain after endoscopic carpal tunnel release ( ectr ) and open carpal tunnel release ( octr ) has not been objectively documented in literature .
the aim of the study was to compare the pain intensity in the early postoperative period in patients undergoing octr versus those undergoing ectr.materials and methods : fifty patients diagnosed with carpal tunnel syndrome were randomized into two groups using
random number generator software ( research randomizer , version 3.0 ) ; endoscopic surgery group [ ( 21 female , 1 male ; mean age 49 years ( range 3164 years ) ] and open surgery group [ ( 25 female , 3 male ; mean age 45.1 years ( range 2968 years ) ] and received carpal tunnel release .
surgery was performed under regional intravenous anesthesia .
the patients pain level was assessed at the 1st , 2nd , 4th , and 24th postoperative hours using a visual analog scale ( vas ) score.results:mean age , gender and duration of symptoms were found similar for both groups .
boston functional scores were improved for both groups ( p < 0.001 , p < 0.001 ) . pain assessment at the postoperative 1st , 2nd , 4th and 24th hours revealed significantly low vas scores in the endoscopic surgery group ( p = 0.003 , p < 0.001 , p < 0.001 , p < 0.001 ) .
need for analgesic medication was significantly lower in the endoscopic surgery group ( p < 0.001).conclusion : endoscopic carpal tunnel surgery is an effective treatment method in carpal tunnel release vis - a - vis postoperative pain relief . |
the activation of membrane receptors by hormones and growth factors results in the localized generation of intracellular second messengers .
the hydrolysis of membrane phospholipids and the generation of biologically active products play important roles in the regulation of cell function and cell fate .
well known is the activation of phosphoinositide - specific phospholipase c ( plc ) isoforms , which hydrolyze phosphatidylinositol 4,5-bisphosphate ( pip2 ) , a membrane phospholipid found in all eukaryotic cells ( schmidt et al .
stimulation of plc isoforms plays a major role in many early and late cellular responses to receptor activation , including smooth muscle contraction , secretion and neuronal signaling as well as fertilization , cell growth and differentiation ( berridge 2005 ; nishizuka 2003 ) .
phospholipase d ( pld ) was first described 60 years ago as a distinct , phospholipid - specific phosphodiesterase activity in cabbage leaves ( hanahan and chaikoff 1948 ) .
this pioneering research indicated that pld hydrolyzes phosphatidylcholine to yield phosphatidic acid ( pa ) and choline . the recognition
that pld is rapidly and dramatically activated in response to extracellular stimuli in cultured animal cells , now 20 years ago ( bocckino et al .
1987 ; cockcroft 1984 ) , has brought pld signaling to the very forefront of current biological and biomedical research . meanwhile , phosphatidylcholine - hydrolyzing pld has been identified in bacteria , protozoa , fungi , plants and animals , and , due to this widespread distribution , is assumed to be involved in the regulation of fundamental cellular functions .
indeed , it has now been established that activation of pld and the generation of pa by a vast number of membrane receptors modulate such a wide array of cellular responses as calcium mobilization , secretion , superoxide production , endocytosis , exocytosis , vesicle trafficking , glucose transport , rearrangements of the actin cytoskeleton , mitogenesis and survival ( cockcroft 2001 ; exton 2002b ; jenkins and frohman 2005 ; liscovitch et al . 2000 ) .
pip2 is a critical cofactor for pld , and profoundly affects the activity , membrane localization and receptor activation of both pld isoforms , pld1 and pld2 ( brown et al .
thus , reduction of cellular pip2 levels , for instance via scavenging of pip2 by the actin - binding protein fodrin ( lukowski et al . 1998 ) or via forced pip2 hydrolysis by the phosphatase synaptojanin ( chung et al .
vice versa , the synthesis of pip2 by phosphoinositide 5-kinase ( pip5k ) isoforms can be directly stimulated by the pld product pa ( jenkins et al .
1992 ) , and this regulation has also been confirmed to occur at the whole cell level ( divecha et al .
it is now hypothesized that the reciprocal stimulation of pld and pip5k enzymes enables rapid feed - forward stimulation loops for a localized and explosive generation of pa and pip2 , which may then govern the recruitment and activation of proteins to execute specific cellular tasks , especially membrane trafficking , and changes in the organization of the actin cytoskeleton .
the activity and localization of both pld and pip5k are under control of gtpases of the arf and rho families , which are well - defined regulators of membrane transport and actin - reorganization processes .
the reciprocal stimulation of pip5k and pld , and the regulation of these enzymes by arf and rho gtpases , point to concerted mechanisms in cellular actions , involving acute , localized pip2 and pa synthesis ( fig . 1 ) .
this review will focus on the regulation of pld enzymes by membrane receptors and monomeric gtpases , and on how pld signaling is organized and connected by pip2 metabolism .
regulation of pld and pip5k by arf and rho family gtpases is essentially involved in the regulation of intracellular vesicle trafficking and actin cytoskeleton reorganization .
both pld and pip5k are stimulated by cell surface receptors and by conventional pkc isoforms , and the latter can become activated after receptor - induced hydrolysis of pip2 by plc .
positive feed - forward regulation is achieved by stimulation of pld by pip5k - derived pip2 , and of pip5k by pld - derived pa .
activation of arf - gaps by pip2 accelerates the inactivation of arf proteins , and may terminate a round of pa and pip2 synthesis regulation and cellular roles of pld and pip5k .
regulation of pld and pip5k by arf and rho family gtpases is essentially involved in the regulation of intracellular vesicle trafficking and actin cytoskeleton reorganization .
both pld and pip5k are stimulated by cell surface receptors and by conventional pkc isoforms , and the latter can become activated after receptor - induced hydrolysis of pip2 by plc .
positive feed - forward regulation is achieved by stimulation of pld by pip5k - derived pip2 , and of pip5k by pld - derived pa .
activation of arf - gaps by pip2 accelerates the inactivation of arf proteins , and may terminate a round of pa and pip2 synthesis
most cellular responses following pld activation are probably mediated by the immediate reaction product pa .
pa is a multifunctional lipid that can be further metabolized to the bioactive lipids , lysophosphatidic acid ( lpa ) and diacylglycerol ( dag ) , can by itself alter membrane curvature , and can serve as a protein attachment site and affect both cellular localization and activity of various proteins , including raf-1 kinase , protein phosphatase 1 , sphingosine kinase 1 , and mtor ( mammalian target of rapamycin ) , a key regulator of cell growth and proliferation ( jenkins and frohman 2005 ) .
pld enzymes can catalyze a transphosphatidylation reaction in which the phosphatidyl moiety of phosphatidylcholine is accepted by primary alcohols , thereby producing stable phosphatidylalcohol instead of pa .
this transphosphatidylation reaction is widely applied to measure pld activity in biological samples , and quenching of pa synthesis by primary alcohols has proven extremely helpful to identify the involvement of pld enzymes in cell physiology . in this way , a role for pld has been demonstrated in a variety of signaling processes , such as activation of phosphoinositide ( pi3k , pip5k ) and protein ( akt , erk1/2 ) kinases , calcium mobilization , cytoskeleton remodeling , endocytosis , exocytosis , membrane trafficking , superoxide production , glucose transport , cell migration , cell proliferation , and survival signaling ( exton 2002a ; foster and xu 2003 ) .
pld1 has a low basal activity and is extensively regulated by conventional protein kinase c ( pkc , - , - ) isozymes and small gtpases of the arf ( arf1 - arf6 ) and rho ( rhoa , rac1 , cdc42 ) families ( henage et al .
pld2 has a higher basal activity than pld1 , but has been shown to respond to arf and pkc as well ( chen and exton 2004 ) .
pip2 is recognized to be the most important cofactor for pld , and both pld isoforms are absolutely dependent on pip2 for activity .
experiments utilizing inactive pld mutants and rna interference have discriminated isoform - specific pld functions , and showed that pld1 is involved in agonist - induced secretion , actin organization , and cell adhesion and migration ( exton 2002a ; iyer et al .
2006 ; kim et al . 2006 ; vitale et al . 2001 ) , and pld2 in endocytosis and recycling of membrane receptors ( du et al . 2004 ; koch et al . 2006 ; padrn et al .
the pld isoforms , both with two splice variants , share an ~50% amino - acid sequence identity ( colley et al . 1997 ; hammond et al .
the catalytic core of both pld enzymes are composed of four conserved domains ( domain i - iv ) , and the hkd motifs in the domains ii and iv probably associate together to form a catalytic centre ( xie et al .
pld1 is characterized by a 116-amino acid loop region following domain ii , which has been proposed to function as a negative regulatory element ( sung et al .
pld1 and pld2 further possess n - terminal ph ( pleckstrin homology ) and px ( phox homology ) domains .
2000 ) , but also to a polybasic pip2 binding motif within the catalytic core ( sciorra et al . 1999 ) , and interaction of pip2 with both domains has been suggested to be involved in membrane targeting of pld as well as stimulation of pld catalytic activity ( du et al .
the px domain of pld1 has been reported to preferentially bind to phosphatidylinositol-3,4,5-trisphosphate ( pip3 ) ( lee et al .
2004 ) , but interaction with pi5p has been observed as well ( du et al .
recently , it was shown that the px domain of pld has gtpase - activating protein ( gap ) activity towards dynamin , and that pld supports egf receptor endocytosis ( lee et al .
the ph and px domains probably contribute to the proper localization of the pld enzymes within cells . in line with a role for pld enzymes in different cellular tasks , pld1 and pld2
pld1 is found throughout the cell , but primarily localizes to perinuclear endosomes and the golgi apparatus ( brown et al .
pld2 is almost exclusively present at the plasma membrane in lipid raft fractions ( czarny et al .
the localization of pld1 does not seem to be static , and regulated translocation and recycling of the enzyme between cellular compartments may be crucial to its proper functioning . in an elegant study , coordinated subcellular targeting of the lipid binding motifs has been demonstrated to drive this subcellular cycling of pld1 ( du et al .
, pld1 was found to translocate from the intracellular compartments to the plasma membrane , and this process was probably dependent on the polybasic pip2 binding site .
the ph domain then facilitated entry of pld1 into lipid rafts , a step critical for internalization of the enzyme , whereafter interaction of the px domain with pi5p may control the efficient return of pld1 to the endosomes .
hydrolysis of pip2 by plc into the second messengers , inositol-1,4,5-trisphosphate ( ip3 ) and dag , is a general and well - defined answer of cells in response to stimulation of many membrane receptors ( schmidt et al .
phosphorylation of pip2 by pi3k results in the rapid accumulation of pip3 , which recruits and activates mediators involved in actin remodeling , mitogenesis and survival ( vanhaesebroeck et al .
but it is now recognized that pip2 , as well as other phosphoinositides , are signaling molecules by themselves and can , by binding to unique phosphoinositide - binding sequences , such as the ph and px domains , affect the activity and subcellular localization of many proteins , including many actin regulatory proteins , a wide range of ion channels , and pld ( niggli 2005 ; suh and hille 2005 ; yin and janmey 2003 ) . in this way , pip2 can modulate a remarkable variety of cellular processes , including cortical actin organization , membrane ruffling , vesicle trafficking , gene expression , cell migration and cell survival ( ling et al .
subsequent dephosphorylation of pip2 by inositol polyphosphate 5-phosphatases , such as synaptojanin , is believed to terminate local pip2 signaling , for instance in the process of vesicle trafficking ( majerus et al .
pip2 may be organized in discrete functional pools within cells , but the existence of pip2 clusters in the plasma membrane is currently under debate .
using green fluorescent protein - tagged ph domains or antibodies to visualize pip2 , the lipid was found to concentrate in highly dynamic , actin - rich regions ( tall et al .
2000 ) and lipid rafts ( laux et al . 2000 ; parmryd et al .
2003 ) in the plasma membrane , feeding the idea that spatially organized pip2 synthesis regulates actin polymerization and other cellular processes .
the localization of pip2 in rafts is supported by biochemical data ( pike and casey 1996 ) ; however , specific pip2 clustering has been disputed ( van rheenen et al .
cdnas encoding three isoforms of pip5k ( designated i , i and i ) with alternative splice variants have been cloned and characterized ( ishihara et al .
sequence analysis has shown that pip5k enzymes are related to pip4k enzymes , but that they share no identity with most other lipid ( pi3k and pi4k ) or protein kinases . the sequence similarity between the pip4ks and pip5ks
an activation loop spanning the catalytic domain has been shown to determine both substrate specificity and subcellular targeting of pip5ks , which can be swapped by substitution of a single amino acid within this loop ( kunz et al .
two dimerization domains were identified , which may contribute to the proper subcellular localization and functioning of the enzyme ( galiano et al .
the identification of three pip5k isoforms raised the expectation of a differential regulation of the enzymes by cellular signal transduction components , but up to now the regulatory properties of pip5k - i , i and i appear to be remarkably similar .
all pip5k isoforms are stimulated by pa , are extensively regulated by arf and rho gtpases , and inhibited by protein kinase a ( pka ) and pi - stimulated autophosphorylation ( oude weernink et al .
nevertheless , evidence has been provided that pip5k isoforms may selectively control functional pip2 pools , which may support particular processes in different cell types .
thus , actin reorganization down - stream of rac1 in platelets specifically involves murine pip5k - i ( tolias et al . 2000 ) .
human pip5k - i was found to localize in rac1-induced membrane ruffles , and the lim protein ajuba has been identified to interact with and stimulate pip5k - i in leading - edge membrane ruffles in migrating cells ( kisseleva et al . 2005 ) .
human pipk - i was detected primarily in cytosolic vesicular structures ( doughman et al . 2003 ) and may synthesize the pip2 pool involved in constitutive endocytosis ( padrn et al .
the long - splice variant of pip5k - i , pip5k - i90 , is enriched in neurons and is implicated in the regulation of clathrin coat recruitment , actin dynamics ( wenk et al .
2001 ) and focal adhesion formation ( di paolo et al . 2002 ; ling et al . 2002 ) .
in contrast , short pip5k - i87 seems to be the major producer of the pip2 pool that supports receptor - induced ip3 generation ( wang et al .
the execution of specific pip2-modulated processes is very probably achieved by an orchestration of appropriate signaling partners within discrete subcellular microdomains , and pld - derived pa as well as the pld enzymes by themselves can contribute to this organization .
indeed , both pld1 and pld2 interact with pip5k - i , and pld2 recruits pip5k - i to a submembraneous vesicular compartment ( divecha et al .
pld2-derived pa was shown to stimulate pip5k - i splice variants , and the subsequent formation of pip2 to drive the initial stages of integrin - mediated cellular adhesion ( powner et al . 2005 ) . in many processes , the temporal activation and correct localization of pld and pip5k isoforms by monomeric gtpases appears crucial to achieve the spatially organized production of pip2 and pa ( santarius et al .
although the direct interaction site on pld for arf has not yet been unequivocally defined , it is well established that arf proteins , particularly arf1 and arf6 , activate both pld enzymes , but especially pld1 ( hammond et al .
arf1 is localized to the golgi complex , and is required for proper golgi structure and function .
the use of primary alcohols has also pointed to a role for pld in vesicle transport to golgi ( bi et al . 1997 ; ktistakis et al .
pld activity has been shown to stimulate the release of nascent secretory vesicles from the trans - golgi network ( chen et al .
1997 ) , and to be required for maintaining the structural integrity and function of the golgi apparatus , but the precise role for pld in vesicle formation is still controversial .
pip5k is also a direct effector of arf1 , and an arf1 mutant that selectively activates pip5k , but not pld activity , demonstrated that both pld - derived pa and direct activation of pip5k by arf1 contribute to increased pip2 synthesis ( skippen et al .
in permeabilized cells , arf1 has been shown to restore secretion by promoting pip2 synthesis ( fensome et al .
arf6 activates pld , and pa has been implicated in the mediation of the effects of arf6 in vesicular trafficking events .
a critical role for pld1 in exocytosis has been established in different cell types , including neurons ( humeau et al .
2001 ) , neuroendocrine cells ( vitale et al . 2001 ) and pancreatic cells ( hughes et al . 2004 ) .
pld2 has recently emerged as a mediator of arf - dependent internalization of the -opioid receptor ( koch et al .
2003 ) , and both pld isoforms have been implicated in macrophage phagocytosis ( corrotte et al . 2006 ; iyer et al .
in addition , pip5k colocalizes and interacts with , and is directly activated by arf6 at the plasma membrane ( honda et al .
1999 ) , and arf6 and pip2 colocalize on the plasma membrane and on endosomal structures ( brown et al .
arf6-organized pip2 turnover at the plasma membrane is apparently involved in regulated secretion ( aikawa and martin 2003 ; brown et al .
focal and transient accumulation of pip2 by pip5k is required for phagocytosis as well ( botelho et al . 2000 ; coppolino et al .
2002 ; wong and isberg 2003 ) , and pip2 hydrolysis probably dictates the remodeling of actin necessary for completion of phagocytosis ( scott et al .
the synthesis of pip2 is essential for priming the exocytotic apparatus , and the recruitment and activation of pld1 by pip2 seems the primary mechanism for the functional integration of pld1 into the exocytotic pathway ( vitale et al .
thus , cd16-induced cytolytic granule secretion mediated by arf6 was shown to involve pip5k - i membrane targeting and activation of both pip5k and pld ( galandrini et al .
pip2 also recruits additional proteins for instance the endocytic proteins ap-2 , epsin and ap180to initiate clathrin - coat formation preceding endocytosis ( ford et al . 2001 ; itoh et al . 2001 ; padrn et al . 2003 ) , and caps ( grishanin et al . 2004 ) to initiate dense - core vesicle exocytosis . direct activation of pip5k - i by arf6 has been shown to stimulate clathrin - coat recruitment to synaptic membranes to allow synaptic vesicle recycling ( krauss et al .
pld - derived pa may directly contribute to vesicle fusion in a biophysical manner , as pld cleaves the non - fusogenic lipid , pc , to form the fusogenic lipid , pa .
but pa also takes a function as an essential cofactor for pip5k , and disruption of golgi membranes ( sweeney et al .
2002 ) , blockade of clathrin - coat assembly ( arneson et al . 1999 ) and inhibition of arf1-reconstituted secretion ( way et al . 2000 ) after quenching of pa production could be attributed to inhibited pip2 synthesis .
thus , both pld and pip2 synthesis seem necessary for membrane trafficking aspects in the endo- and exocytotic machinery . but pld and pip5k also mediate other processes down - stream of arf6 .
epidermal growth factor ( egf)-induced membrane ruffling requires arf6-induced pip5k - i translocation to the ruffles and local pip2 production .
this leads to the recruitment of pld2 , and pld - derived pa and arf6 may then synergistically activate pip5k ( honda et al .
the relationship between arf and pip2 is also bidirectional , as phosphoinositides can regulate arf activity by binding and activating both arf - specific guanine nucleotide exchange factors ( arf - gefs ) ( klarlund et al .
1998 ; paris et al . 1997 ) and arf - gtpase - activating proteins ( arf - gaps ) ( kam et al . 2000 ; nie et al . 2002 ) via their ph domains .
the fact that arf - gaps bind pip2 with high affinity and specificity offers an attractive feed - back mechanism for terminating arf activation after a cycle of arf - induced pip2 synthesis .
pa formation , especially by pld1 , has been reported to induce stress fibre formation in specific cell types ( cross et al .
1996 ; ha and exton 1993 ; kam and exton 2001 ; porcelli et al .
rho proteins , in particular rhoa , rac1 and cdc42 , which control actin cytoskeleton reorganization , exclusively activate pld1 by direct interaction with its c - terminus ( exton 2002b ; powner and wakelam 2002 ) .
thus , pld stimulation by rhoa may happen by direct interaction , but may involve indirect , rho - dependent mechanisms as well .
inactivation of rho gtpases , with clostridium difficile toxin b or clostridium botulinum c3 exoenzyme , reduced cellular pip2 levels , resulting in inhibiton of receptor - mediated pip2 hydrolysis by plc ( schmidt et al .
1996a ) as well as diminished pld stimulation ( schmidt et al . 1996d ) . as the inhibition of pld signaling after rho inactivation
could be largely rescued by the addition of pip2 , rho proteins do seem to affect pld via pip5k regulation ( schmidt et al .
pip2 is well - known to associate with and regulate the activity of a plethora of actin - binding proteins that organize actin dynamics ( hilpela et al .
2004 ; yin and janmey 2003 ) , and pa and pip2 may act in concert to mediate rho - dependent actin cytoskeleton remodeling .
pip5k isoforms are markedly stimulated by rhoa , rac1 , and cdc42 ( chong et al .
2004a ) , and physically associate with both rhoa ( ren et al . 1996 ) and rac1 ( tolias et al .
pip5k isoforms are now seen as critical mediators of rhoa- and rac1-induced actin organization and remodeling ( doughman et al . 2003 ; shibasaki et al .
the established rho effector rho - kinase , a serine / threonine kinase , is apparently involved in rho - dependent regulation of both pld ( schmidt et al .
2000 ) , and pip5k was found to play an essential role as down - stream effector of rho and rho - kinase in neurite remodeling ( van horck et al .
2002 ; yamazaki et al . 2002 ) and platelet cytoskeleton assembly ( gratacap et al . 2001 ; yang et al . 2004 ) .
but rho may also directly signal to pip5k independently of rho - kinase , as rhoa - induced activation of erm ( ezrin , radixin , moesin ) proteins , that cross - link actin filaments to plasma membranes , was found to be mediated by pip5k , but not by rho - kinase ( matsui et al .
pld and pip5k were also demonstrated to collectively mediate rho - induced changes in the actin cytoskeleton .
thus , myogenic differentiation induced by arginine - vasopressin , which involves actin fiber formation , is mediated by rho proteins and pld1 , and involves pld - induced pip2 synthesis along the actin fibers ( komati et al .
these findings suggest that pld and pip5k enzymes may co - operate down - stream of rho in processes that depend on actin organization .
another rho effector , pkc - related protein kinase n ( pkn ) , also directly interacts with pld ( oishi et al .
interestingly , components of the actin regulatory machinery , -actin and -actinin , have been found to directly associate with and inhibit the activity of pld isoforms ( lee et al .
pld also binds to and is stimulated by filamentous f - actin , and pld1 in particular may act as a signal transduction component responsive to dynamic changes of the actin cytoskeleton ( kusner et al .
pkn interacts with -actinin , and pkn may modulate pld signaling by reversing the inhibitory effect of -actinin on pld1 , and by direct interaction with pld1 .
in line with the critical role of pa in cellular processes , the enzymatic activity of pld is tightly regulated by a variety of hormones , neurotransmitters , and growth factors .
regulation of pld enzymes by membrane receptors , including g protein - coupled receptors ( gpcrs ) and receptor tyrosine kinases ( rtks ) , is complex and mediated by several cytosolic factors , including pkc as well as arf , rho and ras gtpases ( exton 2002b ; liscovitch et al . 2000 ; lpez de jess et al .
most receptors that stimulate pld also increase plc activity , leading to activation of the pld regulator pkc , and it was assumed that pld activation might be secondary to plc activation .
a physical association between pld with pkc isoforms has been reported , resulting in strong activation of in vitro pld1 activity , and the major interaction site was identified within the n - terminus of pld1 ( park et al .
inhibition of pkc was shown to reduce receptor - induced pld responses , and pld1 mutants unresponsive to pkc did respond poorly to activation of gpcrs ( zhang et al . 1999 ) or to active gq proteins ( xie et al .
however , stimulation of pld in several receptor systems , including m3 muscarinic and 1-adrenergic receptors , was actually pkc - independent ( balboa and insel 1998 ; muthalif et al .
1997 ; schmidt et al . 1994 ) , suggesting that pld stimulation must not necessarily be secondary to plc stimulation .
brefeldin a , an inhibitor of certain arf - gefs , reduced receptor signaling to pld in several cell types , indicating that arf proteins participate in receptor - mediated pld stimulation ( fensome et al .
1998 ; mitchell et al . 1998 ; rmenapp et al . 1995 ; shome et al .
likewise , sequestration of arf - gefs by the arf - related protein arp inhibited m3 muscarinic receptor signaling to pld ( schrmann et al . 1999 ) .
clostridial toxins and enzymes that specifically inactivate rho proteins and expression of inactive rho mutants have been used to identify the role of rho in signaling to pld .
thus , rho proteins were found to be involved in pld stimulation by gpcrs ( m3 muscarinic , bradykinin , sphingosine-1-phosphate and lpa ) , rtks ( pdgf , egf ) , and immunoglobulin ( fcri ) receptors ( hess et al .
stimulation of pld by gpcrs was shown to be mediated by both pertussis toxin ( ptx)-insensitive ( gosau et al . 2002 ; schmidt et al . 1994 ) and ptx - sensitive ( cummings et al .
g12 family proteins can stimulate pld ( plonk et al . 1998 ) , and rgs ( regulators of g protein signaling ) proteins , that act as subunit - specific gaps , have been used to position g12 in pld activation by the m3 muscarinic ( rmenapp et al .
2001 ) , the par1 ( fahimi - vahid et al . 2002 ) , and the ca - sensing receptor ( huang et al . 2004 ) , as well as mechanical force ( ziembicki et al . 2005 ) . as forskolin and camp were shown to cause activation of pld via pka and erk1/2 ( ginsberg et al .
1997 ; yoon et al . 2005 ) or , alternatively , via the camp - activated gef for ras - like gtpases , epac and r - ras ( lpez de jess et al .
. pld activation is also controlled by -subunits , possibly via src and/or arf6 ( le stunff et al .
1999 ) , but g can also directly interact with and inhibit pld ( preininger et al .
2006 ) . as the precise mechanism of pld stimulation in intact cells was only poorly understood , during the last 10 years our laboratory in essen has focused on the regulation of pld activity by membrane receptors . in hek-293 cells , signaling to pld by a typical gpcr , the m3 muscarinic receptor , and an rtk , the egf receptor , was studied and shown to be executed by several distinct pathways ( fig . 2 ) .
in addition , by expressing inactive pld mutants , the m3 muscarinic and the egf receptors were found to signal to individual pld isozymes and to selectively stimulate pld1 and pld2 respectively ( han et al .
the m3 muscarinic receptor stimulates both plc and pld via ptx - insensitive mechanisms ( offermanns et al .
interestingly , stimulation of pld by the agonist carbachol was not affected by pkc inhibitors , suggesting that activation of pld by the m3 muscarinic receptor was rather independent of plc ( rmenapp et al .
expression of -subunits of g proteins and of specific rgs proteins was used to identify the g proteins involved in these pathways , and demonstrated that whereas the m3 receptor signals to plc via gq proteins , activation of pld is mediated by g12 family proteins ( rmenapp et al .
pld activation by the m3 receptor , but not by the egf receptor , was further found to be under control of arf ( rmenapp et al . 1995 , 1997 ) as well as rho proteins , particularly rhoa ( schmidt et al .
likewise , regulation of mtor by lpa , but not pdgf , involved pld1 activation by rho gtpases ( kam and exton 2004 ) . both arf1 and rhoa were found to become activated after m3 receptor activation ( keller et al .
1995 ) , and a role for rho - kinase in rhoa - controlled pld stimulation could be demonstrated ( schmidt et al . 1999 ) .
in further studies , it was shown that activation of pld by rhoa and rho - kinase is mediated by g12 and the tyrosine kinase pyk2 , whereas activation by arf1 is mediated by g13 , pi3k and the arf - gef arno ( han et al .
rho proteins were shown to affect signaling to pld by both endothelin-1 and thrombin , apparently by controlling pip2 synthesis , whereas arf selectively affects signaling by the par1 receptor ( fahimi - vahid et al .
2regulation of pld by the m3 muscarinic receptor and receptor tyrosine kinases in hek-293 cells .
in human embryonic kidney ( hek-293 ) cells , signaling to pld by the m3 muscarinic receptor and by typical rtks ( egf , pdgf , insulin ) is organized into rather discrete pathways and channeled by particular heterotrimeric g proteins and small gtpases ( orange ) , specific gef proteins ( pink ) and further signaling components ( green ) .
ac , adenylyl cyclase ; rock , rho - kinase regulation of pld by the m3 muscarinic receptor and receptor tyrosine kinases in hek-293 cells . in human embryonic kidney ( hek-293 )
cells , signaling to pld by the m3 muscarinic receptor and by typical rtks ( egf , pdgf , insulin ) is organized into rather discrete pathways and channeled by particular heterotrimeric g proteins and small gtpases ( orange ) , specific gef proteins ( pink ) and further signaling components ( green ) .
ac , adenylyl cyclase ; rock , rho - kinase pld can directly interact with rala , and a ras / ral signaling cascade was shown to regulate pld responses . in hek-293 cells , ras and rala but not rho proteins were located in rtk signaling to pld , and this ras / ral - dependent signaling cascade was found to be dependent on pkc- and a ral - specific gef ( fig .
2 ) ( schmidt et al . 1998 ; voss et al . 1999 ) .
rala apparently co - operates with arf ( kim et al . 1998 ; xu et al . 2003 ) and rho proteins ( frankel et al . 1999 ; wilde et al . 2002 ) to achieve full pld activation .
likewise , ras proteins were found to modulate pld responses by pdgf ( lucas et al . 2000 ) , and rala to affect egf receptor signaling to pld ( lu et al . 2000 ) .
it was recently shown that direct activation of ras - related r - ras by epac is involved in pld stimulation by the m3 muscarinic receptor , apparently by coupling to gs proteins ( lpez de jess et al .
2006 ) , but a contribution of ral proteins to gpcr - induced pld activation has not been found ( meacci et al .
collectively , these data demonstrate that heterotrimeric g proteins as well as small gtpases co - ordinate pld activation by specific membrane receptors in particular cell types , and these mechanisms probably contribute to the organization of agonist - induced pa production for the execution of diverse cellular signaling tasks .
in addition , the synthesis of pip2 can be directly stimulated by gpcrs ( thrombin , lpa , m3 muscarinic ) as well as rtks ( cochet et al .
receptor activation leads to increased association of pip5k with the actin cytoskeleton ( grondin et al . 1991 ; payrastre et al .
1991 ) , and receptor - induced stimulation and cytoskeletal association of pip5k may be directly involved in actin cytoskeletal regulation and initialize the assembly of enzymes into signaling complexes .
gpcr - induced stimulation of pip2 synthesis was found to be mediated by pertussis toxin - sensitive gi proteins ( schmidt et al .
1993 ) , but also by g12 and gq proteins ( oude weernink et al .
enhanced pip2 synthesis is also caused by conventional pkc isoforms , which may increase pip5k activity by stimulating pip5k dephosphorylation by the okadaic acid - sensitive protein phosphatase 1 ( park et al .
in the last decade , pld has taken a firm position as all - round player in cellular signaling events .
it is now appreciated that pld and pip5k act together to execute several important cellular functions , including vesicle transport , cytoskeleton dynamics and cell adhesion . because of the reciprocal stimulation of their activities it seems inappropriate to generally assign a conventional upstairs - downstairs relationship to pld and pip5k isozymes .
the localized generation of the lipid messengers by pld and pip5k , pa and pip2 , is clearly co - ordinated by small gtpases of the arf , rho and ras families .
the following picture emerges of how pld and pip5k may co - operate to execute their cellular tasks .
particular small gtpases , activated by membrane receptors or cellular factors , bind to pip5k and recruit the enzyme to specific cellular compartments .
subsequent activation of pip5k catalytic activity triggers the localized generation of pip2 , which now serves as an anchor for specific proteins , including pld enzymes .
the sequestered pld is activated by pip2 and the gtpases , and pld - derived pa now , among other tasks , contributes to the activation of pip5k .
this feed - forward regulation loop depends on both pip5k and pld , and quenching of pa formation ( by primary alcohols ) or reduction of pip2 levels ( by plc - mediated hydrolysis or dephosphorylation by phosphatases ) can interrupt the snowball from rolling .
pip2 dephosphorylation may be important in the cell as a decisive mechanism to terminate the localized reactions before a cellular avalanche develops .
attractive candidates are further specific gefs and gaps for the gtpases , some of which have been shown to be directly regulated by pip2 .
pip2-dependent inactivation of the organizing gtpase may then provide the final turn - off signal . | hydrolysis of phosphatidylcholine by phospholipase d ( pld ) leads to the generation of the versatile lipid second messenger , phosphatidic acid ( pa ) , which is involved in fundamental cellular processes , including membrane trafficking , actin cytoskeleton remodeling , cell proliferation and cell survival .
pld activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors , including protein kinase c isoforms , small gtpases of the arf , rho and ras families and , particularly , by the phosphoinositide , phosphatidylinositol 4,5-bisphosphate ( pip2 ) .
pip2 is well known as substrate for the generation of second messengers by phospholipase c , but is now also understood to recruit and/or activate a variety of actin regulatory proteins , ion channels and other signaling proteins , including pld , by direct interaction .
the synthesis of pip2 by phosphoinositide 5-kinase ( pip5k ) isoforms is tightly regulated by small gtpases and , interestingly , by pa as well , and the concerted formation of pip2 and pa has been shown to mediate receptor - regulated cellular events .
this review highlights the regulation of pld by membrane receptors , and describes how the close encounter of pld and pip5k isoforms with small gtpases permits the execution of specific cellular functions . |
one possible option for radical prostate cancer treatment is the highly efficacious radical prostatectomy ( rp ) .
however , treatment failures still occur in a significant percentage of patients [ 1 , 2 ] .
therefore , methods to improve the treatment results continue to be sought ; one of these methods is adjuvant radiotherapy ( rt ) [ 3 , 4 ] .
the initiation of rt in the prostate cancer treatment is based on the assessment of a number of prognostic factors , such as pt , pn , or post - operative margin status , all included in the histopathology protocol following the rp , and in addition on the determination of psa levels . in the group of patients after rp with high risk of prostate cancer progression , there is a possibility to initiate early rt without signs of biochemical progression , delayed rt initiated upon detection of biochemical progression , or hormonal therapy in case of systemic progression [ 6 , 7 ] . in case when recurrence risk factors are present ( positive post - operative margin , infiltration outside the prostatic capsule
pt3b ) , the preferred treatment method is early rt [ 8 , 9 ] .
the use of salvage rt , reserved for cases of biochemical progression , is a less efficacious method [ 10 , 11 ] .
however , one must assume that the number of patients , in whom early rt was abandoned despite the presence of the disease progression risk factors defined in the pathology report , could be significant . at the same time
, it must be stressed that this group is highly heterogeneous as regards the biological aggressiveness of the neoplastic process .
another subgroup of patients after rp may consist of patients at very high risk of systemic progression and , at the same time , at very low risk of local progression .
and the last group , include patients with biochemical progression after rp with very high risk of local and at the same time very low risk of systemic progression .
therefore , rt in this subgroup ( high risk of cell presence in the surgery site ) is likely to be associated with therapeutic benefit .
however , in clinical practice , it is very difficult to assign a patient to one of these groups , and therefore , additional tools , which would allow to do it in the best possible way , are being sought .
the goal of the surgical treatment is to remove the entire pool of tumor cells present in the prostate gland , in the seminal vesicles , and less commonly in lymph nodes .
however , local efficacy of rp is not always sufficient , leading to biochemical recurrence , preceding or accompanying a simultaneous local recurrence .
postoperative assessment of psa levels is an early measure of rp efficacy , commonly used in clinical practice .
unfortunately , single determination of psa levels does not allow defining the failure site ( two sequential determinations are used to define the biochemical failure ) .
it must also be stressed that non - lesioned fragments of prostate gland may be retained after the surgery in a group of patients .
this may result in the maintenance of psa level above the accepted cut - off levels , which , when exceeded , suggest biochemical failure . in general , documentation of biochemical failure extorts definition of failure geography
firstly , we should define whether the neoplastic process is limited only to post - operative site , or to the site with the accompanying distant metastasis currently beyond the detection capacities of diagnostic methods , or whether it is only a
this list shows that basing the therapeutic decision on the pathology report and single determination of post - operative psa levels suggestive of biochemical failure is still associated with high risk of initiating a suboptimal treatment . in patients after rp
, rt is an established adjuvant treatment method , leading to reduction of biochemical failure incidence by ca .
unfortunately , most randomized clinical trials conducted to date did not bring evidence of a statistically significant effect of early rt on the improvement in total survival rates [ 1418 ] .
an exception is the analysis of survival rates of patients in the swog study , presented at the asco conference in 2008 . despite an overwhelming number of publications suggesting that early rt is more efficacious than salvage rt , many urology centers hold to the belief that since there are no clinical trial results explicitly suggesting an increase in total survival rates thanks to early rt , it should be used only in case of biochemical progression .
unfortunately , diagnostic tools helping to define the source of biochemical failure , and thus helpful in qualifying patients for rt , are imprecise due to their low sensitivity and specificity . the role of the simplest of these tools , i.e. the digital rectal examination ( dre ) , in the diagnosis of local recurrence in patients after rp is very limited ; in case of lack of biochemical recurrence this examination provides no useful information at al . . also , the usefulness of imaging examinations in defining local recurrences is low , even in cases of significant increase in psa levels exceeding 0.2 ng / ml . despite the diagnosed biochemical recurrence , the sensitivity and specificity of trus procedures and , in case of determining dissemination or isolated local recurrence , examinations such as bone scan , ct , or mri with surface or anal coils are of limited importance [ 21 , 22 ] .
hope for more sensitive and specific detection of micrometastases or minute local recurrences lies upon molecular imaging methods , namely [ ( 11)c]choline pet / ct [ 23 , 24 ] recent reports support its use , showing good pathologic correlation with imaging data .
distinguishing of failure source ( isolated local recurrence versus distant metastasis local recurrence ) is important to the extent that it allows to initiate efficacious treatment in the form of salvage rt on one hand and , on the other hand , to avoid unnecessary rt in case of patients with a distant metastasis and to refer such patient to a clinical trial assessing novel systemic treatments .
one of the parameters most commonly used in clinical practice due to its availability and low acquisition costs is determination of the dynamics of psa levels , expressed by psa doubling time ( psadt ) and psa velocity ( psav ) .
therefore , a number of articles were published in recent years with regard to the usefulness of these parameters in the therapeutic decision - making process in patients after rp .
one of the most important research teams is the d'amico team , which assessed the usefulness of measuring the kinetics of psa levels prior to rp for the assessment of patients fates after prostatectomy in a group of 1,095 prostate cancer patients .
the authors pointed that in 28% of patients , in whom the psa velocity ( psav ) exceeded 2 ng / ml / year , a 10-fold increase in the risk of death due to prostate cancer than in the group of patients with psav < 2 ng / ml / year .
what 's interesting , this risk was practically independent of other clinical and pathological parameters describing the prostate cancer .
according to the authors , adjuvant rt in cases when psav exceeds 2 ng / ml / year brings little benefit due to large risk of disease dissemination .
therefore , authors think it would be advisable to consider initiation of systemic treatment in this group of patients ( psav > 2 ng / ml / year ) .
the next study by d'amico et al . was very important in terms of defining the role of prognostic factors in patients after rp and rt . in this study , clinical parameters associated with the risk of death due to prostate cancer in case of biochemical recurrence after radical treatment were singled out . with this purpose ,
an analysis of 8,669 patients ( 5,918 patients after rp , 2,751 patients after radical rp ) was conducted , with mean observation time of 7.1 years after rt and 6.9 years after rp .
the results of the statistical analysis showed that psadt <3 months ( found in 12% of patients in the operative treatment group and in 20% of patients in radiotherapy group ) was an independent prognostic factor of the risk of death due to prostate cancer ( hr = 19.6 , ci 95% ; 12.5 - 30.9 ) .
therefore , the authors claim that documentation of psadt <3 months indicates advisability of considering initiation of systemic treatment . in addition
, the authors stress the potential usefulness of psadt as an early endpoint , which might replace the assessment of prostate cancer - specific survival rates in clinical trials .
the use of this parameter might lead to significant reduction in the waiting time with respect to the summaries of the results of clinical trials evaluating novel treatments .
however , it must be highlighted that the most important premise stemming from this study is the fact that patients with short psadt should constitute a group in which hormonal therapy or participation in clinical trials evaluating novel treatments should be considered .
this is especially important since the probable cause of treatment failure is associated with micrometastases , present even before the radical treatment ( rp ) .
also the study by zhou et al . assessed the usefulness of psadt as a prognostic factor in patients after rp and radical rt . based on observation of 1,159 patients with prostate cancer ( 498 patients after rp , 661 patients after radical rt ) , the psa doubling time shorter than 3 months was associated with relative risk of death due to prostate cancer of 54.9 ( 16.7 - 180.0 ) in rp patients , and 12.8 ( 7.0 - 23.1 ) in radical rt patients .
for analytic purposes , the author differentiated three disease progression risk subgroups : a high risk subgroup , when psadt was shorter than 12 months ; a medium risk subgroup , when psadt was between 1 and 10 years ; and low risk subgroup , when psadt was longer than 10 years .
the relative risk of distant metastases was 21.7 ( 8.0 - 58.6 ) and 6.8 ( 2.3 - 19.8 ) in the high and low risk groups , respectively .
the author suggests that patients from the high risk group should firstly be the potential candidates for initiation of systemic therapies , while patients at medium risk ( psadt between 12 and 120 months ) should be qualified for adjuvant rt , and patients at low risk remain under observation .
analyzed the relationship between psadt and the risk of death due to prostate cancer in a group of 5,096 patients after rp .
the statistical analysis performed by the authors allowed differentiating patients with psadt of less than 3 months , in whom the relative risk of death due to prostate cancer was 27.48 ( 10.66 - 70.85 ) . in the subgroup of patients with psadt ranging from 3.0 to 8.9 months , the risk of death was 8.76 ( 3.74 - 20.50 ) , while in the subgroup of patients with psadt between 9.0 and 14.9 months , the risk of death was 2.44 ( 0.88 - 6.81 ) . thus , a question arises , whether early hormonal therapy may improve the survival of patients in the high failure risk subgroup ?
experience gathered to date suggest a potential possibility of such an effect , but the lack of results of randomized clinical trials assessing this aspect of hormonal therapy does not allow routine recommendation of this treatment in clinical practice .
thus , probably future rcts would evaluate all aspects of early ht in patients at high risk of prostate cancer progression determined on the basis of the psadt .
psadt and psav seem to be attractive parameters that might significantly improve the optimization of treatment selection process in patients after rp .
however , it must be kept in mind that calculation of these parameters requires observation of patients in whom biochemical progression was observed .
on the other hand , it is a commonly held belief that early initiation of salvage rt , i.e. at the lowest possible psa levels , is most efficient .
thus , based on the available clinical data it is impossible to assess how the length of the waiting period required for psa measurements for psadt or psav calculation might negatively affect the results of salvage rt .
it is possible that in the future , the use of determination of psa dynamics based on psa determinations in the range of 0 ng / ml to 0.2 ng / ml would allow early determination of the geography of the biochemical failure .
the goal of the surgical treatment is to remove the entire pool of tumor cells present in the prostate gland , in the seminal vesicles , and less commonly in lymph nodes . however , local efficacy of rp is not always sufficient , leading to biochemical recurrence , preceding or accompanying a simultaneous local recurrence .
postoperative assessment of psa levels is an early measure of rp efficacy , commonly used in clinical practice .
unfortunately , single determination of psa levels does not allow defining the failure site ( two sequential determinations are used to define the biochemical failure ) .
it must also be stressed that non - lesioned fragments of prostate gland may be retained after the surgery in a group of patients .
this may result in the maintenance of psa level above the accepted cut - off levels , which , when exceeded , suggest biochemical failure . in general , documentation of biochemical failure extorts definition of failure geography
firstly , we should define whether the neoplastic process is limited only to post - operative site , or to the site with the accompanying distant metastasis currently beyond the detection capacities of diagnostic methods , or whether it is only a
this list shows that basing the therapeutic decision on the pathology report and single determination of post - operative psa levels suggestive of biochemical failure is still associated with high risk of initiating a suboptimal treatment . in patients after rp
, rt is an established adjuvant treatment method , leading to reduction of biochemical failure incidence by ca .
unfortunately , most randomized clinical trials conducted to date did not bring evidence of a statistically significant effect of early rt on the improvement in total survival rates [ 1418 ] .
an exception is the analysis of survival rates of patients in the swog study , presented at the asco conference in 2008 . despite an overwhelming number of publications suggesting that early rt is more efficacious than salvage rt , many urology centers hold to the belief that since there are no clinical trial results explicitly suggesting an increase in total survival rates thanks to early rt , it should be used only in case of biochemical progression .
unfortunately , diagnostic tools helping to define the source of biochemical failure , and thus helpful in qualifying patients for rt , are imprecise due to their low sensitivity and specificity .
the role of the simplest of these tools , i.e. the digital rectal examination ( dre ) , in the diagnosis of local recurrence in patients after rp is very limited ; in case of lack of biochemical recurrence this examination provides no useful information at al . . also , the usefulness of imaging examinations in defining local recurrences is low , even in cases of significant increase in psa levels exceeding 0.2 ng / ml . despite the diagnosed biochemical recurrence , the sensitivity and specificity of trus procedures and , in case of determining dissemination or isolated local recurrence , examinations such as bone scan , ct , or mri with surface or anal coils are of limited importance [ 21 , 22 ] .
hope for more sensitive and specific detection of micrometastases or minute local recurrences lies upon molecular imaging methods , namely [ ( 11)c]choline pet / ct [ 23 , 24 ] recent reports support its use , showing good pathologic correlation with imaging data .
distinguishing of failure source ( isolated local recurrence versus distant metastasis local recurrence ) is important to the extent that it allows to initiate efficacious treatment in the form of salvage rt on one hand and , on the other hand , to avoid unnecessary rt in case of patients with a distant metastasis and to refer such patient to a clinical trial assessing novel systemic treatments .
one of the parameters most commonly used in clinical practice due to its availability and low acquisition costs is determination of the dynamics of psa levels , expressed by psa doubling time ( psadt ) and psa velocity ( psav ) .
therefore , a number of articles were published in recent years with regard to the usefulness of these parameters in the therapeutic decision - making process in patients after rp .
one of the most important research teams is the d'amico team , which assessed the usefulness of measuring the kinetics of psa levels prior to rp for the assessment of patients fates after prostatectomy in a group of 1,095 prostate cancer patients .
the authors pointed that in 28% of patients , in whom the psa velocity ( psav ) exceeded 2 ng / ml / year , a 10-fold increase in the risk of death due to prostate cancer than in the group of patients with psav < 2 ng / ml / year .
what 's interesting , this risk was practically independent of other clinical and pathological parameters describing the prostate cancer . according to the authors , adjuvant rt in cases when psav exceeds 2 ng / ml / year brings little benefit due to large risk of disease dissemination .
therefore , authors think it would be advisable to consider initiation of systemic treatment in this group of patients ( psav > 2 ng / ml / year ) .
was very important in terms of defining the role of prognostic factors in patients after rp and rt . in this study ,
clinical parameters associated with the risk of death due to prostate cancer in case of biochemical recurrence after radical treatment were singled out . with this purpose , an analysis of 8,669 patients ( 5,918 patients after rp , 2,751 patients after radical rp ) was conducted , with mean observation time of 7.1 years after rt and 6.9 years after rp .
the results of the statistical analysis showed that psadt <3 months ( found in 12% of patients in the operative treatment group and in 20% of patients in radiotherapy group ) was an independent prognostic factor of the risk of death due to prostate cancer ( hr = 19.6 , ci 95% ; 12.5 - 30.9 ) .
therefore , the authors claim that documentation of psadt <3 months indicates advisability of considering initiation of systemic treatment . in addition
, the authors stress the potential usefulness of psadt as an early endpoint , which might replace the assessment of prostate cancer - specific survival rates in clinical trials .
the use of this parameter might lead to significant reduction in the waiting time with respect to the summaries of the results of clinical trials evaluating novel treatments .
however , it must be highlighted that the most important premise stemming from this study is the fact that patients with short psadt should constitute a group in which hormonal therapy or participation in clinical trials evaluating novel treatments should be considered .
this is especially important since the probable cause of treatment failure is associated with micrometastases , present even before the radical treatment ( rp ) .
also the study by zhou et al . assessed the usefulness of psadt as a prognostic factor in patients after rp and radical rt . based on observation of 1,159 patients with prostate cancer ( 498 patients after rp , 661 patients after radical rt ) , the psa doubling time shorter than 3 months was associated with relative risk of death due to prostate cancer of 54.9 ( 16.7 - 180.0 ) in rp patients , and 12.8 ( 7.0 - 23.1 ) in radical rt patients .
for analytic purposes , the author differentiated three disease progression risk subgroups : a high risk subgroup , when psadt was shorter than 12 months ; a medium risk subgroup , when psadt was between 1 and 10 years ; and low risk subgroup , when psadt was longer than 10 years .
the relative risk of distant metastases was 21.7 ( 8.0 - 58.6 ) and 6.8 ( 2.3 - 19.8 ) in the high and low risk groups , respectively .
the author suggests that patients from the high risk group should firstly be the potential candidates for initiation of systemic therapies , while patients at medium risk ( psadt between 12 and 120 months ) should be qualified for adjuvant rt , and patients at low risk remain under observation .
analyzed the relationship between psadt and the risk of death due to prostate cancer in a group of 5,096 patients after rp .
the statistical analysis performed by the authors allowed differentiating patients with psadt of less than 3 months , in whom the relative risk of death due to prostate cancer was 27.48 ( 10.66 - 70.85 ) .
in the subgroup of patients with psadt ranging from 3.0 to 8.9 months , the risk of death was 8.76 ( 3.74 - 20.50 ) , while in the subgroup of patients with psadt between 9.0 and 14.9 months , the risk of death was 2.44 ( 0.88 - 6.81 ) .
thus , a question arises , whether early hormonal therapy may improve the survival of patients in the high failure risk subgroup ?
experience gathered to date suggest a potential possibility of such an effect , but the lack of results of randomized clinical trials assessing this aspect of hormonal therapy does not allow routine recommendation of this treatment in clinical practice .
thus , probably future rcts would evaluate all aspects of early ht in patients at high risk of prostate cancer progression determined on the basis of the psadt .
psadt and psav seem to be attractive parameters that might significantly improve the optimization of treatment selection process in patients after rp .
however , it must be kept in mind that calculation of these parameters requires observation of patients in whom biochemical progression was observed . on the other hand , it is a commonly held belief that early initiation of salvage rt , i.e. at the lowest possible psa levels , is most efficient .
thus , based on the available clinical data it is impossible to assess how the length of the waiting period required for psa measurements for psadt or psav calculation might negatively affect the results of salvage rt .
it is possible that in the future , the use of determination of psa dynamics based on psa determinations in the range of 0 ng / ml to 0.2 ng / ml would allow early determination of the geography of the biochemical failure .
to sum up , it must be stated that psadt is a very useful tool for defining subpopulations of patients after rp in case of biochemical failure .
this endpoint may be used as a potential tool for proposing local or systemic therapy in a subgroup of patients at high risk of distant metastases .
such differentiation would allow abandoning systemic therapy in patients at low risk of systemic progression while proposing salvage rt as highly efficacious treatment method .
based on the review of studies assessing the kinetics of psa level changes , it can be stated that in patients with the presence of biochemical recurrence , psadt may be an early treatment efficacy endpoint , which might potentially replace the assessment of causespecific survival rates , especially in the clinical trials [ 27 , 29 , 30 ] .
the most important premise stemming from this bibliographical review is that patients with short psadt should constitute a group in which hormonal therapy should be considered , while patients with long psadt should be destined for salvage rt . | this article is an attempt to present a contemporary view on the role of the kinetics of psa levels as defined by psa doubling time ( psadt ) and psa velocity ( psav ) in the decision - making process to initiate salvage radiotherapy in patients with prostate cancer after radical prostatectomy ( rp).the dynamics of the rise of psa levels may be an early endpoint parameter , preceding the diagnosis of distant metastasis or death due to prostate cancer based on a single psa determination .
thus , it seems reasonable to include the kinetics of psa levels , apart from single psa determination , in the decision - making algorithm.in a group of patients after rp , psadt might be an early endpoint that could replace cause - specific survival rate as a late endpoint .
psadt allows distinguishing subgroups of patients at high risk of distant metastases and death , which in turn may lead to a change in the further treatment strategy .
therefore , patients with short psa doubling time should become a subgroup , in which hormonal therapy should be considered .
to date , there is no unanimous consent to accept the criteria of assessment of the dynamics of psa levels as determinants of treatment in case of recurrences following rp .
however , a number of non - randomized clinical trials in patients after rp suggest it would be useful to include these parameters in the decision - making process .
for instance , a relationship was found between increased psa velocity ( > 2 ng / ml / year ) before initiation of oncological treatment and increased ( 12-fold ) risk of death .
a number of well - documented retrospective analyses show that psadt is one of the most important parameters to describe the disease aggressiveness .
it has to be stressed that single determination of psa levels is much less precise in terms of describing the biological aggressiveness of prostate cancer than psadt .
of course , the question regarding the need to include the psa levels kinetic parameters as crucial elements of patient management algorithms can be answered in a definitive manner only by randomized clinical trials . |
in sweden , approximately 400 patients per year are newly diagnosed with cancer of the esophagus and an additional 200 with cancer of the cardia .
there are several methods to determine this diagnosis : computed tomography ( ct ) , endoscopic examination of the esophagus ( esophagoscopy ) combined with endoscopic ultrasonography ( eus ) and needle biopsy and positron emission tomography ( pet ) examination with f - fluoro - deoxyglucose ( f - fdg ) .
ct has the disadvantage that it is less able to diagnose overgrowth of the tumor to adjacent organs and to detect small tumors < 1 cm and growth to different layers in the esophageal wall .
pet and the combination of f - fdg - pet / ct are both more accurate than ct alone with respect to diagnosing lymph node metastasis close to the tumor , and therefore staging .
eus is superior to f - fdg - pet / ct with respect to diagnosing tumor growth through different layers in the esophageal wall , overgrowth to adjacent organs , and lymph node metastasis close to the tumor [ 25 ] .
examination with f - fdg - pet is based on the metabolic activity of the tumor , and reflects its pathophysiological characteristics . however , despite their high sensitivity in detecting esophageal cancer , these methods lack specificity ; an elevated f - fdg uptake could be seen in different nonmalignant conditions , such as inflammation and barrett 's esophagus [ 612 ] .
another way to characterize tumors is evaluation of different receptors , such as the somatostatin receptors .
while the surface of a cell usually includes some amount of somatostatin receptors , these receptors are over - expressed to a very high degree in multiple neoplastic diseases such as neuroendocrine tumors and in tumors of the central nervous system , breast , lung , and lymphoid tissue [ 12 , 14 ] . when this study was initiated , there were several radiopharmaceuticals available for somatostatin receptor scintigraphy , including tc - depreotide .
this tracer is tc - labeled , and demonstrates good imaging characteristics with a short investigation protocol .
we have previously used scintigraphy with tc - depreotide for the diagnosis of lung cancer , and showed an accurate discrimination between benign and malignant lesions with conventional gamma cameras [ 6 , 9 , 11 ] .
therefore , visualization of overexpression of somatostatin receptors in cancer types other than lung cancer should , theoretically , be feasible .
moreover , our previous results showed that this tracer accumulates both in squamous cancers and in adenocarcinomas , which is of clinical relevance in view of the almost exponential increase in the incidence of adenocarcinoma in the distal esophagus .
the aim of the present study was to find out whether esophageal cancer can be imaged scintigraphically with tc - depreotide and to determine the uptake characteristics of tc - depreotide in the two main cancer types of the esophagus and relate these to results in patients with benign lesions ( barrett 's esophagus ) .
the study was approved by the regional ethical review board in stockholm , sweden and the radiation safety committee at karolinska university hospital , huddinge .
34 patients with dysphagia were referred to the surgery department at huddinge university hospital and further examined with gastroscopy , eus , and ct .
nine of these were female and 25 male , with a median age of 63 - 64 years ( range : 3385 years ) . among the 34 patients ,
the cancer diagnosis was established by histopathological examination of biopsy specimens in 19 cases and with eus and cytological confirmation of diagnosis in 2 cases .
all patients with barrett 's esophagus were diagnosed via endoscope and subsequent multiple biopsies .
single - photon emission computed tomography ( spect ) of the thorax was performed at 2 and 4 hours after injection , with the arms elevated , using three different gamma cameras .
most of the patients ( 25 of 34 ) were examined with a double - headed gamma camera ( e - cam , siemens , erlangen , germany ) and low - energy high - resolution parallel - hole collimators , using a 128 128 matrix , 64 projections through 360 rotation , and an acquisition time of 40 s per projection .
an additional 5 patients were examined with a double - headed gamma camera ( dst - xl ; sopha medical vision scandinavia ab , gif - sur - yvette , france ) and low - energy ultra - high - resolution parallel - hole collimators , using the same acquisition parameters as above .
finally , 4 patients were examined with a three - headed gamma camera ( picker irix , cleveland , ohio , usa ) and low - energy high - resolution parallel - hole collimators , using a 128 128 matrix , 60 projections through 360 rotation , and an acquisition time of 64 s per projection .
transverse slices were reconstructed with an iterative algorithm ( hosem v 3.5 iterative program ; hermes / nud , stockholm , sweden ) and formatted as a 128 128 matrix without attenuation correction .
images were postfiltered with a three - dimensional fourier filter ( butterworth filter ) with a cut - off frequency of 1.1 cycles / cm ( order 5.00 ) .
the results were evaluated both through visual assessment and through quantitative calculations in the 2-hour and 4-hour images .
ct scans were used for an accurate localization of the tc - depreotide uptake and for placement of the region of interest ( roi ) . on visual assessment , any focal tc - depreotide uptake in the region of the known esophageal lesion was considered pathological . the quantitative evaluation of tc - depreotide uptake was performed retrospectively on spect images in all 34 patients .
first , an roi was drawn manually around the esophageal tumor on each slice , using small margins .
next , a background roi was drawn in healthy lung parenchyma ( figure 1 ) .
in - house software , originally developed for volumetric measurements in magnetic resonance images and implemented on a hermes workstation ( hermes medical solution ab , stockholm , sweden ) , was used to calculate the total counts and volume of the tumor and background vois , thus giving a count density ( counts / cm ) . to produce a normalized tumor uptake ,
each patient was normalized to his or her own normal lung parenchyma using the formula u = ( t b)/b , where u is the normalized uptake , t is the count density in the tumor , and b is the count density in the lung parenchyma . to increase accuracy and to investigate the intraobserver variability ,
evaluations were performed twice , 6 months apart , by the same radiologist and the mean value of the two uptake values was used in further analysis .
in addition , a second radiologist made individual evaluations in order to investigate the interobserver variability of the uptake values at 2-hour images .
for patients with negative uptake ( i.e. , tumor count density lower than the lung background count density ) , the uptake was scored as zero . due to the small number of patients in each group
a two - sided mann - whitney u test was used to investigate the difference in uptake between malign and benign tumors . to assess intraobserver and interobserver variability , intraclass correlation coefficients ( icc )
data were analyzed based on both the 2-hour and the 4-hour postinjection recordings except for the icc which was only determined for the 2-hour recordings . a difference in uptake
among the 21 patients with cancer of the esophagus , 8 had squamous cell carcinoma , 11 had adenocarcinoma , 1 had an undifferentiated cancer , and 1 had an intramucosal cancer .
the position of the tumor was in the proximal esophagus in 2 cases , in the middle part in 5 cases , and in the distal part in the remaining 14 cases . visual assessment revealed a pathological tc - depreotide uptake in 16 of the 21 cancer patients ( true - positive 76% ) and an absence of pathological uptake in the remaining 5 ( false - negative 24% ) .
six of the eight patients with squamous cell carcinoma and nine of the eleven patients with adenocarcinoma showed a pathological tc - depreotide uptake . the remaining patient with pathological uptake had an 11 cm undifferentiated cancer in the mid - esophagus with a very high tc - depreotide uptake . among the false - negative cases ,
one had a small ( 5 mm ) squamous cell cancer located in the middle part of the esophagus .
the remaining four undetectable cancers were above 1 cm in size ( varying from 12 9 mm to 12 38 mm ) and were located in the distal part of the esophagus .
the sensitivity of tc - depreotide scintigraphy in the detection of esophageal cancer was thus 0.76 95% confidence interval 0.55 to 0.89 .
there was no tc - depreotide uptake in the columnar metaplastic mucosa in any of the 13 barrett 's patients , irrespective of the presence of low and high - grade dysplasia in the metaplastic epithelium ( figure 2 ) .
the specificity of tc - depreotide scintigraphy in this cohort of patients was thus 1.00 , 95% confidence interval 0.77 to 1.00 .
there were no significant differences between the roi delineation and quantitative measurement of tc - depreotide performed on the 2-hour acquisitions and those performed on the 4-hour acquisitions .
a corresponding second roi delineation and quantification , performed 6 months later , gave consistent results .
both intraobserver and interobserver variability was low with icc = 0.97 when comparing the evalutions by the same radiologist ( intraobserver ) and icc = 0.96 when comparing the evaluations made by the two radiologists ( interobserver ) . a statistically significant difference ( p <
.005 ) was found between tc - depreotide uptake in malignant lesions compared to that in benign or premalignant lesions ( figure 2 ) , both 2 and 4 hours after injection .
the absolute tc - depreotide uptake value was also higher in all malignant lesions after 2 compared to 4 hours .
there was no difference in uptake between adenocarcinoma and squamous cell carcinoma . in the 13 patients who had lymph node metastases at the final examination of the surgical specimen and with eus only 5 showed tc - depreotide uptake in the area of the lymph nodes .
in this study , we have shown that the imaging of esophageal cancer by means of somatostatin receptor scintigraphy with tc - depreotide is feasible .
our hypothesis was based on two facts : first , that the physiological tc - depreotide uptake in the thorax is low .
therefore , this could be a suitable area for tumor detection in most cases , and , second , that esophageal cancer has the same main histopathological types as lung cancer such as adenocarcinoma and squamous cell carcinoma .
as scintigraphy with tc - depreotide is useful for lung cancer detection , this second fact suggested that it could also be applied in esophageal cancer .
the majority of tumors ( 16/21 ) displayed a significant uptake of the tracer which could be clearly distinguished from that in the surrounding tissue .
it was not unexpected that tumors under or near 10 mm in size were missed on the scintigraphic images .
the detection limit of conventional gamma camera due to poor spatial resolution is well known , and according to widespread consensus scintigraphic methods are not suitable for screening purposes for any cancer types .
another observation is that even larger tumors in the distal part of the esophagus , 4 of 13 in the present study , could be missed with this method .
uptake of tc - depreotide in lung cancers located in the lowest part of the right low lobe and even in esophageal cancers located at the level of the diaphragm and lower in the abdomen could be obscured because of the high physiological tracer uptake in the liver .
our sensitivity figure of 76% is only an approximate value , due to the small number of patients in this study .
still , this is somewhat lower than both the sensitivity for detecting lung cancers [ 2 , 611 ] with the same tracer and that for detecting lung cancer with f - fdg - pet [ 68 ] . while f - fdg uptake reflects a metabolic activity of the lesion and is not specific to tumors [ 68 ] , overexpression of somatostatin receptors on tumor cells could give another valuable piece of information regarding tumor properties . as a control group , we used patients with barrett 's esophagus .
barrett 's esophagus refers to an abnormal change ( metaplasia ) in the cells in the lower end of the esophagus .
it is thought to be caused by damage from chronic acid exposure or reflux oesophagitis .
none of the 13 cancer - free barrett 's esophagus patients in this study showed an increased tc - depreotide uptake .
meanwhile , only 3 of 5 patients with both cancer and barrett 's esophagus showed an increased tc - depreotide uptake , leaving 2 false - negative results .
the specificity of 100% for the applied method is high but should be used with caution , as the number of patients was relatively low and the spectra of different benign conditions in the esophagus was not fully represented in this pilot study .
only 5 of 13 patients with metastases seen with eus and confirmed by histological examination were clearly detected by tc - depreotide scintigraphy .
it is probably caused by the close location to the primary tumor , where a high depreotide uptake can not be separated from the uptake in the metastatic lymph nodes .
it was disappointing to note that very few of the local , metastatic lymph nodes could be detected by this method . through this pilot observation
it can be envisioned that this technology can not add to the available methods , such as eus , in determining the node status in oesophageal cancer during the diagnostic and therapeutic workup . as
this study is the first of its kind , we considered it important to explore whether the quantitative assessment was reliable between different investigators and over time .
both intraobserver and interobserver variability was very low meaning that the applied calculations have good reliability .
we applied a somatostatin receptor scintigraphy with tc - depreotide in a previously nonexplored cancer type where the optimal acquisition time was unknown .
we used the same starting point for the imaging session as for the standard procedure in the detection of lung cancer , that is , 2 hours after injection [ 8 , 10 , 11 ] . during the last decade there has been a trend of performing a double - phase registration in order to increase specificity .
the double - phase registration is based on assumptions that the relative tracer uptake in benign lesions decreases with time , while uptake in malignant lesions remains high or even increases with the time [ 10 , 1720 ] .
this approach is now in routine for parathyroid scintigraphy , and its use has also been suggested for scintimammography , tumor imaging with f - fdg - pet , and somatostatin receptor scintigraphy with tc - depreotide in lung cancer patients [ 1720 ] . in order to optimize the imaging procedure from the very beginning , we performed double - phase registrations with imaging 2 and 4 hours after injection .
our results showed that in the majority of patients the absolute uptake decreased more rapidly in the background ( i.e. , the normal lung parenchyma compared to the malignant lesions in the esophagus ) , resulting in a higher relative uptake in cancer over time .
as both the 2-hour , and the 4-hour quantitative evaluations showed a statistically significant difference ( p < .005 ) between tc - depreotide uptake in malignant lesions and in lesions without cancer , the 4-hour imaging seems unnecessary , and thus could be omitted for practical reasons . although future immunohistochemical studies will be needed to carefully map the density , detailed distribution , and localization of somatostatin receptors in the squamous cell esophageal carcinoma and adenocarcinomas , our data indicate that there is no major difference as reflected by the similarity in tracer accumulation between these two major tumor types .
it is , however , of particular interest that in patients with barrett 's esophagus , no accumulation of tracer was observed either in those with or in those without dysplastic histomorphologic changes in the columnar epithelium .
since there is no corresponding preneoplastic condition , concerning the squamous cell carcinoma development , it can be hypothesized that the somatostatin receptor expression reaches far higher levels in infiltrative neoplastic growth than in the intraepithelial neoplastic disease states .
if so , this observation may be potentially very important and offer unique clinical opportunities , for example , when pet / ct or somatostatin receptor scintigraphy with tc - depreotide technologies are applied .
results of this pilot study showed feasibility of imaging oesophageal carcinoma with labeled somatostatin receptor analogue .
we used a single - photon emitting tracer , but the results should be applicable or even better with positron emitting tracers .
use of pet / ct cameras combines a better spatial resolution of functional pet imaging with detailed anatomical information leading to a higher sensitivity .
a multitude of new pet analogs are applied , whereas [ ga - dota , tyr ] octreotide or [ ga - dota , tyr ] octreotate is likely to become the new standard for somatostatin receptor imaging with pet .
this is because these analogs have a high affinity for the somatostatin receptor subtype-2 and because ga is a generator product with a relatively simple labeling .
the additional reason is that their y- or lu - labeled counterparts are used for peptide receptor radionuclide therapy ( prrt ) and it seems desirable that peptide used in diagnostic imaging mimics the peptide used later for therapy .
after the successful visualization of somatostatin receptor positive tumors , a logical next step would be to use radiolabelled somatostatin analogues as a treatment of these patients .
while the objective responses for chemotherapy with the median time to progression is reported to be less than 18 months , prrt with y - octreotide or lu - octreotate performs considerably better with a median time to progression of 30 and 40 months , respectively , and significantly improved quality of life .
obviously , the method is not suitable neither for screening or primary diagnosis , because of methods modest sensitivity .
could this method be used for the detection of distant metastases expressing somatostatin receptors with somatostatin receptor - mediated radionuclide therapy as a consequent result ? does the uptake of the tc - depreotide could be related to the prognosis of the oesophageal tumor ? may this method be used in evaluation of treatment response in patients with tracer uptake ?
does the natural history of barrett 's esophagus and its malignisation could be predicted by tracer uptake ?
scintigraphic examination with tc - depreotide is feasible for imaging esophageal cancer , but the method is not suitable neither for screening or primary diagnosis , because of methods modest sensitivity .
our first results showed high specificity which should be used with caution , as the number of patients was relatively low .
further studies are needed to explore and determine the role of somatostatin receptor scintigraphy in clinical practice . | background . while the surface of a cell normally has some amount of somatostatin receptors , these receptors are overexpressed to a very high degree in multiple neoplastic diseases .
no data exist for esophageal carcinoma .
purpose . to find out whether esophageal carcinoma could be imaged using somatostatin receptor scintigraphy .
material and methods .
34 patients with esophageal lesions were prospectively examined by 99mtc - depreotide scintigraphy 2 and 4 hours after injection .
quantitative evaluation of 99mtc - depreotide uptake was performed around the lesion ( t ) and in healthy lung parenchyma ( b ) .
the relative uptake was calculated as tb / b .
scintigraphy results were compared with histopathology from surgery or biopsy specimens from endoscopic ultrasonography .
results .
21 patients had esophageal cancer , and 13 lesions were benign .
visual assessment revealed positive 99mtc - depreotide uptake in 16 of the 21 cancers .
the 13 patients without cancer had no depreotide uptake .
the mann - whitney u test showed a statistically significant difference ( p < .005 ) between 99mtc - depreotide uptake in malignant and benign lesions , for both the 2-hour and the 4-hour measurements .
conclusion .
scintigraphic examination with 99mtc - depreotide is feasible for imaging esophageal cancer , but the method is not suitable neither for screening or primary diagnosis , because of methods modest sensitivity .
our first results showed high specificity which should be used with caution , as the number of patients was relatively low .
further studies are needed to determine the role of the method . |
neurodegenerative diseases represent a large group of neurological disorders with heterogeneous clinical and pathological expressions , affecting specific groups of neurons , in specialized functional anatomic systems .
a mixture of environmental and genetic factors seems to engender neurodegenerative diseases , and aging has been found a common risk factor .
neurodegenerative diseases result from the gradual and progressive loss of neuronal cells , leading to nervous system dysfunction .
they are characterized by the formation of distinct pathological changes in the brain , including extracellular protein deposits , cellular inclusions , and remodelling of cell morphology . however , while many different forms of neurodegenerative disease are recognized , the lines that separate one from another are often unclear .
for instance , symptoms such as motor impairment and memory loss may occur in many different types of neurodegenerative disease .
alzheimer 's disease , parkinson 's disease , prion diseases , and polyglutamine disorders , including huntington 's disease and various spinocerebellar ataxias , are well - known neurodegenerative disorders . to date , with few exceptions , no diagnostic laboratory tools exist that can clearly indicate the presence , absence , or category of a neurodegenerative disease .
the microrna-(mirna- ) guided rna silencing pathway is a recently discovered process found to regulate gene expression acting on messenger rna ( mrna ) .
mirna biogenesis is mediated by dicer which catalyzes the processing of double - stranded rnas ( dsrnas ) into 22 nt - long small mirnas .
these small noncoding rna molecules operate as guides for risc ( rna induced silencing complex ) to cleave a target mrna in case of a perfect complementarity ( sirna ) or to block the target mrna translation ( mirna ) when there is an imperfect pairing between mirnas and the targets . in mammalian cells the repression of translation by mirna is mediated by an imperfect pairing with 3utr of the mrna target .
mirnas are conserved throughout the evolution , and their expression may be constitutive or spatially and temporally regulated . increasing efforts to identify the specific targets of mirnas lead to speculate that mirnas can regulate more than 90% of human genes .
specific mirna subsets were expressed in specific brain area and in neuronal and glial cell subtypes .
the studies of micrornas expression profiles in nervous system represent the first step in understanding how , where , and when mirnas are involved in the regulation of neurodevelopment , differentiation , dendritic spine development , local protein synthesis , and synaptic plasticity .
several works have shown spatially and/or temporally restricted distribution of mirnas , suggesting that they might regulate neuronal gene expression . by comparative analysis of mirna expression in the normal and pathologic brain , the microrna signatures in several neurodegenerative diseases , including polyglutamine expansions ,
to date we have few pieces of information about the expression profiles , and the complex composition of the brain , containing several neuronal and glial istotypes , while representing its main biological characteristic , is also the principal obstacle for an accurate analysis .
furthermore in order to interpretate mirna expression data from post - mortem human brains affected by neurodegenerative diseases , the source of neural tissues , together with the rna isolation techniques used , need to be carefully considered .
overall , the identification of mirna 's physiological target genes should be a primary approach to reveal the specific contribution of micrornas to neural function .
recent studies of microrna in nonneuronal cellular systems could drive future research in primary neuronal cells .
in fact , prediction of microrna targets utilizing different algorithms based on the general rule of the seed region has been complemented by an elegant proteomic approach which uses a mass spectrometric method called stable - isotope labelling with aminoacid in cell culture ( silac ) to measure changes in protein levels in response to mirna induction or knockdown .
silac approach showed that individual microrna can reduce the production of hundred proteins [ 5 , 6 ] .
many targets are repressed at both mrna and protein level and others are predominantly regulated at protein level .
although several mirna - induced changes in the proteome correlate with the presence of seeds in the mrna of the affected proteins , some changes remain to be explained . they might be due both to indirect effects and/or to mirna direct targeting mediated by still unknown rules .
remarkably an increasing level of complexity of species - specific mirna expression during evolution emerged .
however , 447 new mirna genes expressed in human fetal and chimpanzee adult brains were identified .
since 8% of mirna were found to be human - specific , they might play a role in the human brain evolution .
however , expression levels of mirnas common to human and chimpanzee were not determined , because different regions were analyzed at various ages . several features in cognitive functions of humans and chimpanzees are probably elucidated by variations in cortical structures .
therefore , the diversity of mirna repertoire in the brain likely contributes to the dissimilarities between human and chimpanzee , arguing for a role of mirna in brain evolution and function . in this review
we discuss the recent studies on the involvement of mirna - mediated gene silencing in neurodegenerative diseases .
alzheimer 's disease ( ad ) is the best known degenerative disease affecting the central nervous system .
ad is a chronic progressive disease characterized by early memory impairments followed by these cognitive deficits : aphasia ( language disturbances ) , agnosia ( failure to recognize people or objects in presence of intact sensory function ) , apraxia ( inability to perform motor acts in presence of intact motor system ) .
neuropathologically the areas of brain most affected are the hippocampus followed by association cortices and subcortical structures .
the neurodegeneration is characterised by synapse and cellular loss , -amyloid plaques , and neurofibrillary lesions .
the major component of plaques is the a peptide which derives from the proteolytic processing of its precursor protein ( app ) .
this histopathological hallmark is used in braak 's alzheimer 's system to describe postmortem ad brain samples in six stages : in the transentorhinal stage ( stages i and ii ) , the neurofibrillary pathology is essentially confined to the transentorhinal and entorhinal cortex and slightly to the ca1/ca2 sections of the hippocampus ; the limbic stage ( stages iii and iv ) frames a severe involvement of the entorhinal areas and a moderate engagement of the hippocampus ; the hallmark of the neocortical stage ( stages v and vi ) was the dismantlement of the neocortex .
about 1% early onset familial form of the disease ( onset before 60 to 65 years of age ) is due to mutations in three genes , app , presenilin 1 ( psen1 ) , and presenilin 2 ( psen2 ) all of which cause a overproduction .
a production is initiated by the processing of app by the -amyloid cleavage enzyme 1 ( bace1 ) which generated a c - terminal fragment of app , labelled as c99 .
this fragment is further cleaved by -secretase complex , which includes the presenilins , and generates the more abundant a40 and the less abundant , but more pathogenic , a42 .
for the majority of ad cases , which shows less obvious familial aggregation ( hence they are also called sporadic ad ) , the molecular bases of the disease are matter of intensive research . a discrete number of studies has suggested that a dysregulated microrna expression could be aging - associated and could contribute to ad .
lukiw 's laboratory evaluated the expression of 12 mirnas in hippocampal region of fetal , adult , and ad brain .
they found that mir-9 was upregulated in both fetal and ad hippocampus , and mir-128 was increased specifically in ad hippocampus .
however , the translational changes induced by these specific mirnas in ad hippocampus remain to be investigated .
more recently espression profiles of 328 micrornas in anterior temporal cortex from five sporadic ad patients and five age - mathced controls showed 13 micrornas significantly downregulated in cortex of sporadic ad . using various prediction algorithms to identify ad - related potential target genes , 7 of the 13 micrornas had candidate binding sites in the 3utr of bace ( mir-15a , -29b-1 , -9 , and -19b ) or app ( let-7 , mir-101 , mir-15a , and mir-106b , ) or in the 3utr of psen1 ( mir-9 ) . on the other hand
, 6 micrornas do not seem to be related to obvious targets ( mir-210 , -181c , -22 , -26b , -363 , -93 ) .
in particular the relationship between bace1 and mir-29 was deeply investigated ( mentioned in what follows ) .
the changes of mirna profiles might be specific for sporadic ad and might cause or exacerbate the neuropathology .
furthermore , linking microrna expression to their specific targets could suggest novel pathways of the disease .
a recent study evaluated the importance of deregulation of mirna expression in brains and cerebrospinal fluid ( csf ) of alzheimer 's patients . over
300 micrornas were determined in the hippocampus , medial frontal gyrus , and cerebellum from early and late stage ad compared to age - matched control .
deregulated micrornas have been associated to known and novel molecular pathways in ad pathogenesis such as neurogenesis , oxidative stress , insulin resistance , and innate immunity .
for example , mir-9 and mir-132 downregulation was correlated to impaired neurogenesis and neuronal differentiation .
the finding that mir-423 was upregulated in hippocampus while mir-98 was decreased in cerebellum was appealing .
in fact both mirnas modulate idh2 ( isocitrate dehydrogenase 2 ) expression and idh2 reduction was described to be involved in oxidative stress in ad prefrontal cortex .
these observations suggest a mechanism for the specific susceptibility of particular ad brain areas such as the hippocampus and the relative sparing of others such as the cerebellum .
sixty mirnas were differentially expressed in the csf of patients between braak stage 5 and stage 1 . among these mirnas
few were brain enriched while several were not correlated to the mirnas changes observed in ad brain regions .
therefore , csf micrornas were suggested to derive from t lymphocytes present in the csf .
the altered expression of mirna in the csf of patients affected by alzheimer 's disease , opens a new scenario on the use of these expression profiles as putative ad biomarkers . to date ,
csf analysis from ad patients produced some of the most reproducible biomarkers , such as decreased a42 , increased total tau ( ttau ) , and increased phosphorylated tau ( p - tau ) .
future work might be to evaluate if the csf mirnas profile correlates with a , total and phosphorylated tau protein presently carried out in the csf of ad patients .
specific molecular mechanisms involving micrornas and expression of bace1 and app are emerging in the ad field . by microarray and in situ hybridization of superior - medial frontal cortex of ad
, nelson 's laboratory showed that the expression of mir-107 decreased during progression of the disease in parallel to bace1 mrna increase .
in addition cell culture experiments showed that the expression of a luciferase reporter gene fused to a 3utr containing bace1 microrna 107 binding site is modulated by mir-107 .
another study , investigating changes in microrna expression profiles of anterior temporal cortex from sporadic ad patients found that the expression of the cluster mir-29a / b-1 was significantly decreased in a subgroup of ad patients in which bace 1 protein was abnormally upregulated while bace 1 mrna levels were unchanged .
consistently , during mouse brain development from e17 to 1 year , bace1 protein level decrease was correlated to mir-29a / b-1 upregulation while bace1 mrna level was stable . in cell culture experiments ,
bace1 target validation was demonstrated by monitoring the effects of mir-29a / b-1 on the translation of a bace1 3utr luciferase reporter carrying wilde - type or mutated mir29a / b-1 responsive site .
finally , upon either overexpression or downregulation of mir-29a / b-1 in human cell culture both bace1 protein levels and app cleavage product a were , respectively , reduced and increased . in both previous studies [ 1114 ] ,
the loss of microrna in ad is not specific for a certain brain area more susceptible to the disease , that is , mir-107 is also downregulated in motor cortex of ad patients and
mir-29a / b-1 expression also decreases in ad cerebellum . therefore , altered bace expression due to microrna deregulation is not responsible for increased sensitivity of particular brain regions .
however also in the ad familial cases , mutations of app and psen are present in all cells of the brain and only specific regions are affected from ad .
interestingly other two micrornas , mir-298 and mir-328 , regulate bace 1 protein expression in cultured neuronal cells .
it is relevant that in appswe / psen1 transgenic mice , an ad mouse model which recapitulates some features of the disease , it was observed that bace1 mrna decreased and protein levels increased in the hippocampus at 19 months of age . in transgenic mice ,
the expression of mir-298 and mir-328 decreased in the granular neurons of the hippocampus during aging .
however , while the mir-328 sequence is perfectly conserved between mouse and human , that of mir-298 is only 72% identical .
clearly , additional work will be needed to determine whether all of these micrornas are really active in human brain and their relative contribution to bace expression in physiological and pathological conditions and in different neuronal populations .
it has been shown that ad can be caused by increased expression of the app gene due to either genomic duplication or regulatory sequence alterations . in c. elegans , app orthologue apl-1 is regulated by developmentally timed microrna . in particular , apl-1 expression in seam cells is indirectly repressed by let-7 family microrna , and apl-1 transcription is regulated by downstream targets of let-7 microrna .
recently , utilizing human hek-293 cells , it has been demonstrated that mir106a and mir-520c negatively regulate expression of reporter genes containing their predicted target sequences present in the app 3utr .
in addition , overexpression of mir-106a or mir-520c ( which is not expressed in brain ) reduces app levels by 50% .
it will be important to translate these results in a cellular context relevant for ad pathology .
interestingly , mir-106b is one of the four microrna that have been predicted to target the 3utr of app ( let-7 , mir-101 , mir-15a , and mir-106b ) and that were found to be downregulated in anterior temporal cortex from five sporadic ad patients .
all these investigations suggest that dysregulation of mirnas , by modulation of app and bace1 expression , might be a cause or a consequence of ad ( figure 1 ) .
parkinson 's disease ( pd ) is associated with progressive neurodegeneration of dopaminergic neurons ( dns ) in the substantia nigra and leads to tremor , rigidity , and bradykinesia .
lewy bodies are a neropathological feature of pd and are cellular inclusions comprising a dense core of filamentous material surrounded by a halo of fibrils , which mainly consists of a - synuclein .
mutations in genes coding for synuclein ( snca ) , parkin , pink1 , dj - i , lrrk2 , can explain only a limited number of familial pd cases , while the molecular bases of vast numbers of non familial cases are not yet understood .
a recent study shed some light on the role of microrna in dns differentiation and raises the question whether microrna are involved in etiology of pd .
since dicer deletion was partially rescued by transfection of small rna derived from embryonic mouse midbrain , it is likely that micrornas are involved in dns differentiation and survival .
in addition , specific deletion of dicer in vivo , in mouse midbrain dopaminergic neurons , leads to cell death in the substantia nigra .
behavioural studies of the animals revealed reduced locomotion in an - open - field assay , reminiscent of the phenotype of human patients with pd .
mirna expression profiles of normal adult midbrain compared with the profiles of midbrain depleted of dns from pd patients revealed alterations of certain midbrain - enriched mirna , in pd brain .
the role of mir-133b , enriched in midbrain and absent in the brains of pd patients , was further investigated .
it was demonstrated that mir-133b constitutes a negative feedback loop with the transcription factor ptx-3 : ptx-3 transcribes mir-133b which in turn represses ptx-3 translation . in vitro experiments
showed that depletion of mir-133b increases the expression of dn markers and depolarization - induced dopamine release while mir-133b overexpression suppresses the full differentiation of dn neurons and produces a significant decrease in dopamine release .
thus , although mir-133b is involved in differentiation and function of dn neurons , additional micrornas should be responsible for dicer deletion phenotype in dn .
polymorphisms affecting the interactions between micrornas and their targets are emerging in various studies on neurodegenerative disease .
genetic analyses showed that relevant polymorphic variations in the fibroblast growth factor 20 gene ( fgf20 ) are associated with the risk of developing pd .
more recently , one snp ( rs 127202208 ) , located within the fgf20 3utr , was strongly associated with pd .
snp rs 127202208 lies within a predicted binding site for microrna 433 which is highly expressed in the brain . through several functional assays , it was demonstrated that the risk allele rs 127202208 damped a binding site for microrna 433 and increased translation of fgf20 . in cell culture experiments and in pd brains
synuclein is included into the genes responsible for familial cases of pd and althought the function of this protein is not yet defined , it has been demonstrated that overexpression and point mutations can cause pd .
polyglutamine ( polyq ) disorders constitute a family of dominantly inherited neurodegenerative diseases caused by the expansion of cag triplet repeats in a specific gene .
to date , ten such neurodegenerative disorders known to be caused by expansion of the cag repeat in the coding region of the respective genes have been identified .
these prototypical protein misfolding disorders include huntington disease ( hd ) , six distinct forms of spinocerebellar ataxia ( sca-1 , 2 , 3 , 6 , 7 and 17 ) , dentatorubropallidoluysian atrophy ( drpla ) , and spinobulbar muscular atrophy ( sbma ) .
huntington 's disease ( hd ) is the most common and well - studied polyglutamine neurodegenerative disorder .
it is an hereditary autosomal dominant disease characterized by motor , cognitive , and psychiatric symptoms .
the onset of symptoms typically occurs between the ages 35 and 50 years , though it may appear at any age .
the molecular basis of the disease is the expansion of the trinucleotide cag in the first exon of a gene on chromosome four ( 4p 16.3 ) .
this cag / polygln expansion has 639 units in normal individuals and 36 to 180 units in hd patients .
huntingtin appears to be associated to protein trafficking , transcriptional regulation , synaptic signalling , vesicle transport , and apoptosis .
hd patients show progressive loss of cortical and striatal neurons associated with choreic movement and dementia .
the neuropathological hallmark is the gradual atrophy of the striatum ( caudate nucleus and putamen ) , observed in 95% of the hd brains . mechanisms of neurodegeneration implicated in hd pathology are excitotoxicity , dopamine toxicity , mithocondrial dysfunction , oxidative stress , apoptosis , and autophagy .
interaction between wild type htt and repressor element1 silencing transcription ( rest ) factor was described . in pathological conditions
htt mutation inhibits its interaction with rest and provokes rest build - up in the nucleus of hd neurons , decreasing neuronal gene expression .
recently , johnson et al . identified mirna regulated by rest in neurons , and measured the expression of these mirnas in the brains of hd mouse models and in postmortem tissue of hd patients .
several changes in micrornas expression profile were allocated to species - specific differences , and others to the comparative analysis of a specific human cortex , area versus whole mouse cortex .
both in hd mouse model and in human hd cortex , mir-132 was downregulated , and its mrna target p250 gap , which modulates dendritic plasticity , was increased .
since rest is highly involved in hd , micrornas are likely expected to play an important role in the disease pathogenesis .
in addition , it was demonstrated that huntingtin protein co - purified with argonaute proteins , fundamental components of risc complex .
htt , colocalized with argonaute2 in p bodies , and depletion of htt showed compromised rna - mediated gene silencing .
thereafter , in mouse striatal neurons expressing htt mutation , p bodies formation and translation mirna - mediated repression were impaired .
the polyglutamine ( polyq ) protein ataxin-3 is mutated in the human polyglutamine disease spinocerebellar ataxia type 3 ( sca3 ) , resulting in a progressive dysfunction of the cerebellum .
sca3 is typically a late - onset fatal autsosomal dominant neurodegenerative disease that , like all ataxias , is characterized by loss of motor coordination and balance . in sca-3 drosophila model ,
the suppression of mirna processing by dicer mutation increases ataxin-3 toxicity , inducing a neurodegenerative phenotype
. moreover , depletion of r3d1 , a dsrna - binding protein , that forms a stable complex with dicer-1 , causes accumulation of precursor mirna , increasing ataxin-3-induced toxicity [ 26 , 27 ] .
in hela cell line , dicer reduction by rnai enhances polyq protein toxicity only in cells expressing pathogenic ataxin-3 , causing loss of 70% of the cultured cells .
spinocerebellar ataxia type 1 ( sca1 ) is a dominant inherited disease caused by expanded trinucleotide repeats resulting in an increased polyglutamine tract in the gene product ataxin-1 ( atxn-1 ) .
sca1 patients loose motor coordination and develop slurred speech , spasticity , and cognitive impairments .
a typical feature of sca1 pathology is the atrophy and loss of purkinje cells from the cerebellar cortex .
purkinje cells are the major integrative neurons of the cerebellar cortex , projecting their axons onto the deep cerebellar nuclei .
a recent study showed that a conditional purkinje ( pk ) cell - specific ablation of dicer leads to pk cell death , cerebellar dysfunction , and ataxia indicating an involvement of mirnas in cerebellar neurodegeneration .
another line of evidence suggesting a role of mirnas in sca1 pathogenesis comes from the observation that mir-19 , mir-101 , and mir-130 cooperatively regulate atxn1 levels . when mir-19 , mir-101 and mir-130 were inhibited by 2-o - methyl oligonucleotides ,
moreover , it was demonstrated that mir-19 , mir-101 , and mir-130 were expressed in mouse cerebellum and purkinje cells by northern blot analysis and in situ hybridization .
these mirnas regulate the cell toxicity of the polyq - expanded atxn1 , suggesting to investigate mirnas - mediated regulation in sca1 neurodegenerative disorder .
frontotemporal dementia ( ftd ) is a neurodegenerative disease representing ~5% of all dementia patients , characterized by the progressive degeneration of the frontal and anterior temporal cortex . considering the involvement of the frontal lobe ,
the clinical picture is cognitive and memory impairment , language dysfunction , and/or changes in personality or behavioural disorders .
ftd can be divided into two main neuropathological subtypes : frontotemporal lobar degeneration ( ftld ) with neuronal and glial tau inclusions ( ftld - tau ) , and ftld with neuronal cytoplasmic inclusions ( ncis ) that are positive for ubiquitin and tar dna - binding protein ( tdp-43 ) ( ftld - u ) . however , 20%30% of cases of ftd follow an autosomal dominant pattern of inheritance , and half of them are caused by defects in microtubule - associated protein tau ( mapt ) , multi - vesicular body protein 2b ( chmp2b ) , and valosin - containing protein ( vcp ) .
mutations in the progranulin gene ( grn ) , encoding a secreted growth factor , on chromosome 17q21 , have recently been identified as a major cause of familial ftld - u .
these cases have a characteristic pattern of neuropathology that is a distinct subtype of frontotemporal lobar degeneration with ubiquitinated inclusions ( ftld - u ) , with ncis in layer ii of the cortex and lentiform neuronal intranuclear inclusions ( niis ) . to date ,
more than 60 different mutations in grn were mapped , and a recent breakthrough was the identification of a genetic variant ( rs5848 ) , located in the 3utr of grn mrna , in a binding site for mir-659 .
this research showed that mir-659 targets grn suppressing its translation , and demonstrated a decrease of grn protein levels of ~30% in ftld - u rs5848 homozygous tt carriers compared to cc carriers .
consistently , in ftld - u patients heterozygous for rs5848 , an intermediate dosage of grn protein was determined .
mir-659 seems to be a specie - specific human microrna , which expressed in brain , including frontal and temporal neocortex .
in addition seed sequence for mir-659 in the grn 3utr is only present in humans , and is not found in other mammals .
although a small number of ftld - u patients were examined , the enhanced binding of mir-659 to the 3utr of the grn gene is an important risk for tdp-43-positive ftld - u .
future studies on specific human cortical mirnas might be relevant to decrypt human neurodegenerative disease .
prion diseases or transmissible spongiform encephalopathies ( tses ) are a family of rare progressive neurodegenerative disorders that affect both humans and animals .
they are distinguished by long incubation periods , characteristic spongiform changes associated with neuronal loss , and a failure to induce inflammatory response .
the causative agent of tses is believed to be a prion , a transmissible agent , which is able to induce abnormal folding of normal cellular prion proteins in the brain . according to the protein - only hypothesis ,
the central event in the pathogenesis of prion diseases is the conversion of a normal cellular protein termed prp(c ) to prp(sc ) , a conformational isoform .
prion diseases impair brain function , causing memory impairment , personality changes , dementia , and movement disorders and the characteristic signs and symptoms of the disease .
tses begin in adulthood are rapidly progressive and lead to death within a few months to several years .
familial prion diseases of humans include classic creutzfeldt - jakob disease ( cjd ) , gerstmann - strussler - scheinker syndrome ( gss ) , and fatal insomnia ( fi ) .
to explain tse pathogenesis , it is important to identify disease - associated alterations in gene expression .
recently , by microarrays and rt - pcr , the analysis of mirna expression was made .
brain mirnas expression of mice infected with mouse - adapted scrapie showed changes of 15 mirnas . among these , only two , mir-338 - 3p and mir337 - 3p , were downregulated , whereas the others were up - regulated .
several predictions of the theoretical mrna targets of changed mirnas during prion disease were performed , using web - based computational algorithms . from this in silico analysis , genes involved both in transcription , cell cycle , ubiquitin - proteasome pathway , and in normal functioning of synapses , neuronal activity , neurogenesis , and neurites growth were identified .
lastly , only one target , the transcriptional regulator egr1 , was experimentally validated by luciferase assay in vitro . in particular
, the authors suggested that the prion disease upregulates mir-191 which represses the egr1 mrna translation .
the transcriptional regulators egr1 and creb1 were already identified as downregulated prion - related genes with a central role in biologically relevant networks in prion infection . as a consequence ,
mirnas mediated regulation of these prion - related genes could contribute to neuronal death and neurodegeneration .
the studies on mirna in neurodegenerative diseases ( table 1 ) are only now coming to light .
until now , both changes of several mirna expression profiles and polymorphisms affecting the interactions between mirnas and their targets are emerging in various studies on neurodegenerative disease .
it is difficult to determine if the changes in mirna expression detected in the brains or csf of patients are primary or secondary events , or both . nevertheless early or late in the evolution of the disease
unique patterns of mirna expression profile in the csf of particular neurodegenerative disease could be useful as molecular biomarkers for disease diagnosis and eventually prediction of therapeutic responses .
the identification of mirna causing a specific pathology could open new therapeutic perspectives to block endogenous mirnas or deliver exogenous mirnas . until now
either antisense oligonucleotides chemically modified or expressed sequences corresponding to multiple mirna seed target ( mirna sponge ) have been used as microrna inhibitors .
delivery of these molecules to the cns , avoiding toxicities , could be the challenge of future research .
furthermore since in several neurodegenerative disorders specific nuclear or cytoplasmic protein accumulation is causative of the neuropathological picture , the identification of micrornas regulating the translation of these targets could represent the first step aimed to therapeutic applications .
the second step might be to evaluate the quantitative effects on the proteome of specific amounts of the | in few years our understanding of microrna ( mirna ) biogenesis , molecular mechanisms by which mirnas regulate gene expression , and the functional roles of mirnas has been expanded .
interestingly , numerous mirnas are expressed in a spatially and temporally controlled manner in the nervous system , suggesting that their posttrascriptional regulation may be particularly relevant in neural development and function .
mirna studies in neurobiology showed their involvement in synaptic plasticity and brain diseases . in this review ,
correlations between mirna - mediated gene silencing and alzheimer 's , parkinson 's , and other neurodegenerative diseases will be discussed .
molecular and cellular neurobiological studies of the mirnas in neurodegeneration represent the exploration of a new frontier of mirnas biology and the potential development of new diagnostic tests and genetic therapies for neurodegenerative diseases . |
approximately 20 million people in the united states are infected with genital human papillomavirus ( hpv ) .
an estimated 5.5 million people will acquire a new genital hpv infection each year , and the incidence of infection is highest among sexually active adolescent girls and young women between the age of 18 and 28 years old .
it has been reported that 37% of males and 28% of females in the ninth grade have had sexual intercourse and 7% of students had sexual intercourse before the age of 13 . therefore , hpv vaccination ideally would be directed toward preadolescents and young adolescents in an effort to provide the greatest public health benefit offered by a prophylactic hpv vaccine .
it is predicted that hpv vaccination will prevent thousands of cases of cervical cancer worldwide .
in addition , the emotional stress and economic burden associated with abnormal papanicolaou test results and the treatment of preinvasive cervical lesions will be greatly reduced .
the quadrivalent hpv vaccine ( gardasil , merck & co. ) was found to have 97% efficacy at preventing hpv 16 and/or 18 related cervical intraepithelial neoplasia 2 or 3 , adenocarcinoma in situ , and cervical cancer .
the quadrivalent hpv is approved for use in males and females between 9 and 26 years of age .
on october 16 , 2009 , the fda approved a recombinant hpv bivalent vaccine ( cervarix , glaxo smith kline biologicals ) to prevent cervical cancer and precancerous lesions caused by hpv types 16 and 18 .
this hpv vaccine is approved for use in females between 10 and 25 years of age and is the second hpv vaccine licensed for use in females in the united states . until recently
, studies of women and young adults have shown poor levels of knowledge about hpv , papanicolaou smear testing , and cervical cancer [ 4 , 5 ] .
a review by brewer and fazekas reported that only 21% of respondents knew that hpv is common , 59% knew the purpose of a papanicolaou smear , and 68% knew that hpv is a sexually transmitted infection .
in addition , awareness of hpv among a racially diverse sample of young adults , aged 1826 years old , was found to be relatively high , with more than 75% of study participants indicating that they had heard of hpv from various sources .
however , another recent study examining the acceptability of the hpv vaccination among latina immigrants and african american women found that 61% of latinas and 78% of african americans had never heard about hpv .
continuing , several studies indicate that most parents , especially with proper knowledge of hpv and the vaccine , are willing to accept the hpv vaccine for their children [ 912 ] .
healthcare providers are an important source of information for parents and children , and studies show that they generally have a positive attitude towards recommending hpv vaccination [ 13 , 14 ] .
the vaccination series can be started beginning at 9 years old , and the advisory committee on immunization practices currently recommends routine vaccination of females aged 11 or 12 years with three doses of hpv vaccine .
in addition , the quadrivalent hpv vaccination has been approved in males aged 9 through 26 years to reduce their likelihood of acquiring genital warts .
ideally , vaccination should be administered before potential exposure to hpv through sexual contact .
the purpose of this study was to evaluate the understanding and potential acceptance of the hpv vaccine by adolescents aged 13 to 18 years at adolescent health clinics affiliated with new york presbyterian hospital .
although previous research shows that most adolescents , parents , and health care providers are willing to accept universal vaccination for themselves , their attitudes and knowledge regarding the vaccine have yet to be evaluated . improved knowledge of adolescent understanding and acceptance of the hpv vaccine would help practitioners provide appropriate and useful information to parents , providers , and adolescents when making the decision to vaccinate .
we administered a questionnaire to all adolescent patients ( defined as between 13 and 18 years of age ) awaiting annual well - patient medical examinations in adolescent health clinics affiliated with weill cornell medical college and columbia university medical center .
these health clinics provide affordable , comprehensive services to a diverse patient population including underserved populations from each of the five boroughs in the new york city area .
we could not predict the number of adolescent patients who would be interested in participating in this study , and therefore prior to study initiation we defined the survey collection period as september 1 , 2007 through february 1 , 2008 with the goal of collecting the maximum number of surveys possible .
the research personnel emphasized that participation was voluntary , anonymous , and without incentive for questionnaire completion .
the questionnaire consisted of two parts ; the first part elicited the adolescent 's demographic information and knowledge pertaining to sexually transmitted diseases and vaccinations .
when the first part was completed , the adolescent was provided with a second part that contained current facts about the hpv vaccine and cervical cancer , followed by questions specifically designed to ascertain adolescent acceptance of the vaccination .
adolescents whose primary language was neither english nor spanish were excluded from the study since the questionnaire was self - administered and available only in english and spanish .
data were analyzed using commercially available software ( spss version 16.0 ; spss , inc . ,
a series of 2 and fisher 's exact tests were used for comparison of proportions between hpv vaccine status and 15 other variables of interest ( see table 2 ) and willingness to receive the hpv vaccine and 15 other variables of interest ( see table 3 ) .
a p value of < 0.05 was considered significant for all tests . for the series of 15 analyses ,
the sample sizes of 223 participants ( hpv vaccine status ) and 143 participants ( willingness to be vaccinated ) provided a mean power > 99.9% for detecting a difference between both whether the adolescent had been vaccinated and whether the adolescent was willing to receive the hpv vaccine and the respective variable , using 2-tailed test with statistical significance defined as p < 0.05 .
of the 223 adolescents who completed the survey , 28% were male and 70% were female ( 2% did not respond to the question ) .
the demographics of adolescents including in this study are displayed in table 1 . a majority of adolescents surveyed ( 168 , 75% ) stated that they had prior knowledge of cervical cancer .
however , a smaller percentage of adolescents ( 103 , 46% ) acknowledged prior knowledge of what a pap smear was , and only 57 respondents ( 26% ) were aware that the pap smear was used to screen for cervical cancer .
5% ( 11 ) of respondents reported knowing someone who had / has cervical cancer , and 91% ( 202 ) were familiar with the term sexually transmitted disease or
of the 223 adolescent surveyed , 33% stated that they had received the hpv vaccine , 64% had not had the vaccine , and 3% where not sure . among the 143 adolescents who had not yet been vaccinated ,
52% were willing to get the hpv vaccine , 12% were not willing , and 36% were unsure .
of the 143 adolescents who had not yet been vaccinated , only 4% believed that they were at risk for being exposed to hpv .
of the 73 respondents that stated that they had received the hpv vaccine , the mean age was 16 years old ( range 1318 ) .
female respondents were more likely to have received the hpv vaccine than male respondents ( 45% versus 3% , p < 0.001 ) .
hpv vaccination status was not significantly associated with language , race , religion , or having younger or older siblings who had received the hpv vaccine .
hpv vaccine status was associated with knowledge of cervical cancer ( 41% of those who had heard of cervical cancer were vaccinated ) , knowledge of pap screening ( 43% who had heard of pap screening were vaccinated ) , and having had other vaccines ( 73% of those who had received other vaccines were vaccinated ) ( table 2 ) . among the adolescents who had not yet received the hpv vaccine , 58% of females and 42% of males were willing to be vaccinated ; however this difference was not statistically significant .
in addition , 53% of hispanic adolescents and 56% of african american adolescents were willing to accept vaccination .
there was no association with willingness to receive the vaccine and gender , race , religion , language , having vaccinated siblings , knowledge of cervical cancer , pap screening , sexually transmitted disease , or any other studied variable .
the current study reveals only a marginal willingness of teenagers to accept the hpv vaccine .
the majority of data to date suggest that young women are interested in vaccines that prevent hpv and other sexually transmitted infections .
however , most of these studies have been conducted with women and men of 18 years of age or older .
holcomb et al . found that women are interested in learning about hpv , how the virus is transmitted , and how women can prevent becoming infected . in 2001 , a study of women recruited from community and clinical sites displayed that roughly 85% of participants indicated an intention to receive an hpv vaccine for cervical cancer prevention once it became available .
a study of young , australian , men and women aged 1823 years old showed a high vaccine acceptance despite inadequate knowledge of hpv infection .
another study of male and female college students found an overall acceptance rate of the hpv vaccine of 74% . in this study , although females were more likely to have already been vaccinated , gender did not affect vaccine acceptance among unvaccinated adolescents .
in contrast to most of the data available in the literature , this current review sampled a younger population .
although the majority of adolescents had heard of cervical cancer ( 75% ) , only 46% knew what a pap smear was , and only 26% knew that the pap smear is a screening test for cervical cancer .
furthermore , only 4% of the adolescents surveyed who had not yet received the hpv vaccine believed that they were at risk for hpv infection .
the lack of awareness about cervical cancer screening and underestimated risk of hpv risk likely contributes to the limited willingness of the adolescents surveyed to accept the hpv vaccine for themselves .
many of the adolescents stated that they would seek advice from a parent or physician when deciding whether or not to accept the hpv vaccine .
interestingly , we found no significant association between having had the hpv vaccine or willingness to receive the vaccine and the level of education of the adolescent 's parents .
there was also no association between having younger or older siblings who had been vaccinated and having had the hpv vaccine or willingness to receive the vaccine .
many studies have shown that most parents are willing to accept the hpv vaccine for their children . a recent study , postlicensure of the vaccine , evaluated mothers of children in primary and secondary schools in england and found that 75% of mothers would accept the hpv vaccine for their daughter .
acceptance was higher in mothers who had experienced cancer in their families , had older daughters , perceived approval from their husbands / partners , and believed vaccine acceptance would be more normative .
another postlicensure study used a random - digit - dial telephone survey to examine the likelihood of parental acceptance of the hpv vaccination for young adolescent girls in california . within this sample ,
hispanic parents were more likely to accept vaccination than were non - hispanic parents , and african american and asian american parents were the least likely to accept vaccination .
overall , parental attitude and acceptance studies suggest a great deal of parental interest in hpv vaccination for their adolescent children . healthcare providers may be able to encourage participation in hpv vaccine programs by bolstering parents ' underlying desire to protect their children , as recommendation by a healthcare provider is a crucial prompt for vaccination .
healthcare providers are an important source of information for parents and children , and studies show that they generally have a positive attitude towards recommending an hpv vaccination .
one study of 207 fellows of the american college of obstetricians and gynecologists found a fairly positive disposition towards recommending the hpv vaccine , with a mean rating for vaccine recommendation of 79 out of 100 .
similarly , 224 nurse practitioners had a favorable attitude about hpv vaccine recommendation , with a mean rating of 72 out of 100 .
our study reveals that knowledge of cervical cancer and pap screening is associated with having had the hpv vaccine , however interestingly not with willingness to accept the vaccine among those not yet vaccinated .
previous studies have shown that knowledge of personal risk is generally limited with regard to sexually transmitted infections .
a study of sexually active heterosexual college students found that personal risk of contracting acquires immune deficiency syndrome was estimated to be significantly lower than risk for each of a set of hypothetical persons who varied in degree of similarity to respondents . in a study of university students in the united kingdom , screening for chlamydia was limited , and perception of personal risk was poor . in a pilot study among australian university students , even though the majority of students were comfortable with opportunistic testing for chlamydia by their general practitioner , the likelihood of being tested in the upcoming year for most students was low , as was personal concern about chlamydia infection . with regard to hpv , a recent study of australian women with diverse sexual orientation , found that the majority of women had risk factors for hpv but few felt personally at risk of acquiring infection .
among the 143 adolescents in our study who had not yet received the hpv vaccine , only 4% believed that they were at risk for being exposed to hpv .
the very low personal risk perception for hpv suggests the need for targeted education for this group regarding hpv transmission and prevention .
participants were recruited from a hospital - based adolescent health clinic serving a racially and ethnically diverse , predominantly low - income , population . as a result , the findings may not be applicable to all adolescents in the united states . in order to include 13 and 14 years old in this study at an adolescent health clinic , we were unable to ask certain direct questions , including
the answer to this question may have provided more information regarding age of sexual activity in the current study population and would allow direct comparison of these results with a personal risk perception question .
in addition , because the time frame was not stated explicitly in the question regarding personal risk , it is unclear whether participants were rating their risk of hpv infection at present , in the immediate future , or over their lifetime , which would also affect the accuracy of their ratings .
it is clear that education will play an important role in the implementation of an hpv vaccination program . in this study , only 53% of adolescents who had not yet received the hpv vaccine were willing to be vaccinated , and lack of education likely contributes to this hesitancy .
healthcare providers must be proactive in educating and discussing benefits of vaccination . based on factors affecting vaccine acceptability ,
hpv vaccine programs in the united states should emphasize high vaccine effectiveness , the high likelihood of hpv infection , physician recommendations , and address barriers to vaccination .
it is important to educate patients that the vaccine is most effective prior to sexual activity and hpv exposure .
however , the vaccine does not replace routine screening for cervical cancer or the need for education about continued cervical cancer screening .
thus , it is essential for healthcare providers to offer guidance about the ongoing need for screening .
future studies will be necessary to evaluate public health issues that arise once the hpv vaccination programs have been established more extensively . | background . hpv vaccination may prevent thousands of cases of cervical cancer . we aimed to evaluate the understanding and acceptance of the hpv vaccine among adolescents
. methods .
a questionnaire was distributed to adolescents at health clinics affiliated with a large urban hospital system to determine knowledge pertaining to sexually transmitted diseases and acceptance of the hpv vaccine .
results .
223 adolescents completed the survey .
28% were male , and 70% were female .
the mean age for respondents was 16 years old .
adolescents who had received the hpv vaccine were more likely to be female and to have heard of cervical cancer and pap testing .
of the 143 adolescents who had not yet been vaccinated , only 4% believed that they were at risk of hpv infection and 52% were willing to be vaccinated .
conclusions . surveyed adolescents demonstrated a marginal willingness to receive the hpv vaccine and a lack of awareness of personal risk for acquiring hpv . |
low back pain ( lbp ) is thought to have originated from intervertebral lesion ( 739% ) .
most patients with discogenic lbp respond well to conservative care , but 5% of patients suffer from constant pain313 ) .
various treatment modalities , such as injection therapy , fusion surgery , total disc replacement , and thermal annular procedure , are used for discogenic lbp , and yet clinical efficacy is controversial125678111925 ) . until now
, there were some thermal therapies for discogenic lbp , namely , intradiscal electrothermal therapy ( idet ) using thermocoil located at the nucleo - annular junction , disctrode using monopolar radiofrequency to apply ionic heat , and biacuplasty using cooled bipolar radiofrequency81624 ) . according to lee et al.17 ) , annuloplasty was performed using laser as a heat source and a 90% success rate was reported .
first , using heat causes denervation of the nociceptor in the annulus , and ingrowth of the unmyelinated nerve ending that causes pain .
second , enhancing stability is achieved through the stiffening of the annular fiber from denaturation and shrinkage of the collagen .
however , their mechanism is still unclear12 ) . in order to satisfy the aforementioned rationale ,
the temperature must reach 45 in order to destroy the nociceptors and 60 to alter and shrink the collagen fiber9 ) . however , there are some problems and disadvantages that are associated with this modality
. it can induce degenerated intervertebral disc and disc herniation from the puncture site after the annulus puncture .
moreover , a probe tip that focuses heat was placed on the intra - annular area or nucleo - annular junction portion instead of the outer annulus with an ingrowth sensory - free nerve ending . in our experiment , we decided to apply heat to the posterior annulus of the intervertebral disc of a cadaveric lumbar spine model by using a 1414-nm nd : yag laser , which was developed recently .
a 1414-nm nd : yag laser , compared with ho : yag laser , is easier laser control , a smaller manufacturing fee , and equivocal effectiveness . also compared with conventional ng : yag laser , it is more safe because of shallow tissue penetration .
for this study , five discs from t12l1 to l45 were obtained from 4 fresh adult human cadavers .
the donor criteria excluded subjects with severe degeneration , such as fissuring , bridging osteophytes , history of spine trauma , infection , tumor , and any other bony diseases . the specimen was initially prepared by isolating the vertebral column from t12 to l5 and by cutting at t1112 and l5s1 disc level .
it was prepared as en bloc , and the adjacent muscles and soft tissues were removed .
then , we removed the posterior elements of the spine by electric saw at the mid aspect of the pedicles .
the cut vertebral column was placed in the water bath and maintained at a constant temperature of 371 by heat probe . in order to correct the temperature of the discs that are affected by the cerebrospinal fluid and blood circulation
, we made some continuous artificial stir approximately 300 rpm via input and output circuit system of water bath ( fig .
1 ) . for the measurement of the disc temperature distribution around the laser probe , 5 thermocouple probes [ hyp1 ( hyp0 - 30 - 1/2-t - g60-smpw - m ) , omega , stamford ,
each probe was placed at 3 mm , 6 mm , and 9 mm laterally , and at 3 mm and 6 mm by depth as shown in fig .
once all of the 5 probes constantly reached 37 , we decided to apply heat at 600 m fiber diameter of 1414 nm nd : yag laser system ( accuplasti , lutronic , goyang , korea ) placed at the midpoint of the posterior surface of the disc for 10 seconds .
the pulse energy was kept at 200 mj , while the pulse rate was set at 20 hz .
this recently developed 1414 nm nd : yag laser 's maximal pulse energy and rate was 600 mj and 40 hz . we used the selected parameter ( 200 mj and 20 hz ) for our experiment dose without causing carbonization and vaporization of the cadaveric disc , and also observed gross shrinkage and hyalinization in the water bath during the preliminary study .
we recorded and analyzed the temperature by using the multichannel data acquisition system ( mv1000 , yokogawa electric corporation , tokyo , japan ) .
the average temperature recordings of the 5 probes from 20 specimens , according to time , can be observed from fig .
3 . the temperature would rise upon irradiation and fall slowly with the cessation of irradiation .
the most distinctive elevation of the temperature was observed from probes located 3 mm away from the laser fiber tip , both in depth and lateral positions .
the distribution curves , as measured across time , were quite varied depending on the location of the probes .
the temperatures that were collected from the probes , which were closer to the laser tip , were higher than the temperatures from the probes that were located further away , while the probes that were placed laterally showed higher temperatures than the probes that were placed deeper .
the average maximum temperature recorded from the probes 3-mm and 6-mm deep from the laser tip were 47.04.3 and 38.70.9 , and each measured at 13.60.8 seconds and 24.83.0 seconds , respectively .
the average maximum temperature recorded from the probes 3 mm , 6 mm , and 9 mm lateral to the laser tip were 69.513.3 , 37.80.7 , and 37.20.3 , and each recorded at 10.60.5 seconds , 26.63.4 seconds , and 38.84.4 seconds , respectively ( fig .
the combination of the laser tip , 3-mm depth , and 16 levels ( 80% ) showed a temperature above 45 , which would result in a nociceptor ablation .
meanwhile , the relationship between the laser tip and laterality showed all levels at temperatures above 45 , and 16 levels showed temperatures above 60 , where collagen denaturation can be achieved .
the temperatures from the lateral probes showed higher temperatures than the depth probes , which could be related to the characteristic of 1414 nm nd : yag laser , where the water showed much greater heat absorption potential than the human tissue .
the thermal procedures for discogenic lbp have been performed since the late 1990s , and the theoretical background was that the sources of discogenic pain were the nerve ingrowth and tissue regeneration into the annulus21 ) .
therefore , achieving 45 or 60 through idet or biacuplasty , where nociceptive ablation and collagen denaturation were possible , was the key point to cadaveric and animal experiments for thermal procedures4141518202224 ) .
bono et al.4 ) reported that upon using idet , the temperature above 60 was achieved within a 2-mm distance from the catheter , and the temperature above 45 was achieved at 9-mm to 14-mm distance away from the device .
shah et al.22 ) reported that by using idet on the cadaveric model , they have managed to reach 55 in the outer annulus , while denaturation , shrinkage , and coalescence of the annular collagen were demonstrated on the histologic finding .
however , kleinstueck et al.15 ) , reported that during their cadaveric study of idet , temperatures above 60 were only achieved at approximately 1 mm to 2 mm around the catheter tip , while the temperatures adequate to damage nociceptor fibers were observed at 6 mm around the catheter tip without affecting the posterior annulus , where it is clinically relevant .
these results have shown that the idet , where the catheter is inserted and positioned in the inner border of the posterior annulus and then heated , has its limits in giving a direct thermal control on the free nerve endings from gray rami communicans and sinuvertebral nerves that innervated to the disc in cases of disc degeneration .
we have performed annuloplasty and used a laser for minimally invasive endoscopic procedures at the same time as one of thermal energy sources , and chose 1414 nm pulse wave nd : yag laser to evaluate the temperature distribution . for laser irradiation ,
an nd : yag laser with a wavelength of 1414 nm operating in the 200-mj to 600-mj range was used .
we can observe the epidural space by epiduroscope and remove lesions , such as herniated disc , fibrosis , and adhesion , by laser .
the laser power level was set to 200 mj to 300 mj ( 10 hz to 20 hz ) for denervation and coagulation , and 300 mj to 600 mj ( 10 hz to 20 hz ) for vaporization and ablation .
the adequate temperature for treatment in thermal therapy is considered to be above 60 at minimum , where collagen fibril shrinkage can be managed23 ) .
there have been a few studies describing the effects of thermal therapy on collagen . in their research on the effect of nonablative laser energy on in vitro rabbit femoropatellar joint capsule , hayashi et al.10 )
observed the fusion of collagen and pyknotic nuclear changes in a fibroblast , and reported that there was no evidence of charring or removal of tissue .
high - power energy usage will result in laser that can induce tissue coagulation and charring , thereby leading to destruction .
therefore , it is essential to select a proper energy setting to ensure that the temperature , albeit above 60 , is not high enough to damage the tissue .
jayasree et al.12 ) reported that when using a 1064-nm nd : yag laser for a disc decompression , it is ideal to use an initial delivery of 40 w laser and a reduced power of 10 w to 15 w thereafter .
however , this setting is a parameter used in disc ablation , and it is too high to apply in laser annuloplasty . there were no investigations on specific parameters for such purpose , hence the pre - research was done to find the parameter that selectively induces shrinkage without gross annular charring or coagulation , as well as a pulse energy of 200 mj , a pulse rate of 20 hz , and a duration of 10 seconds .
the fact that the lateral surface of annulus have shown a higher temperature than the deeper portion of the annulus was quite a surprise , considering the laser directionality .
although the thermal properties of the tissue is determined by its water content , the spread of energy via thermal conductivity is influenced by the structure and complicated geometry of the tissue16 ) .
also , transforaminal epidural lqser annuloplasty ( tela ) actually is outside- in technique under epiduroscopic vision for the ablation of pain sources from the degenerative disc , while our experiment is close to idet , which is actually inside posterior annulus - out ablating procedure without visionary assistance . that might be the reason why proper high temperature was conducted within 3 mm of laser tip .
the temperature increase and distribution within a laser - irradiated tissue depends on the volume and thermal properties of the tissue25 ) . in comparison to the former nd : yag laser , the 1414-nm wave nd : yag laser has a higher absorption rate of water .
furthermore , there was a higher heat conduction within the water bath than the annulus tissue , thereby showing a higher conduction laterally than longitudinally . since it is a newly developed laser with a new wave length , further research about thermal distribution and histological change of tissue will be required . in this study ,
the thermal distribution after laser irradiation in the annulus has shown a temperature increase in the relatively smaller radius , as compared to the previous studies on idet and its temperatures4141520 ) .
similar to the idet , it is hard to apply heat to the entire annulus .
however , it seems suitable for the treatment of focal discogenic pain source , such as annular tear or high intensity zone , by endoscopy or fluoroscopy . unlike idet
, it will provide an extradiscal approach , thereby preserving a wide extent of healthy annulus without secondary damage due to a disc puncture .
furthermore , it can be applied to an endoscopic device that is compatible with percutaneous laser disc decompression or percutaneous endoscopic laser discectomy .
but our study has a several limitation . even though depth of tissue penetration is 0.4 mm , by using a 1414-nm nd : yag laser , we did n't measured the temperature of surrounding root and dural sac which severe complication was arisen , if damaged . as one solution to this , there is a tela procedure .
the tela has recently been introduced and considered as a new method for the treatment of discogenic lbp .
the advantages of tela as compared with back surgery , include relatively non - invasiveness , short operating time , reduction of risks related to general anesthesia , possibility of communication with the patient during the procedure ( leading to reduced risks of accidental nerve damage ) , and easier access into ventral epidural space . and
the results of this study are limited through the use of cadaveric spine . although our experiments were performed in a water bath to make closely similar situation of human spine as possible . however
, this does not reflect the impact of soft tissue such as muscles and connective tissue around disc .
when measuring the temperature distribution of the annulus during laser annuloplasty with 1414-nm nd : yag laser , it could reach a sufficient temperature to ablate the nociceptor and induce collagen denaturation within a relatively narrow radius of 3 mm from the laser tip .
it is also thought to be one of the most useful thermal procedures in clinical applications . | objectivelow back pain , caused intervertebral disc degeneration has been treated by thermal annuloplasty procedure , which is a non - surgical treatement .
the theoretical backgrounds of the annuloplasty are thermal destruct of nociceptor and denaturization of collagen fiber to induce contraction , to shrink annulus and thus enhancing stability .
this study is about temperature and its distribution during thermal annuloplasty using 1414 nm nd : yag laser.methodsthermal annuloplasty was performed on fresh human cadaveric lumbar spine with 20 intact intervertebral discs in a 37 circulating water bath using newly developed 1414 nm nd : yag laser .
five thermocouples were attached to different locations on the disc , and at the same time , temperature during annuloplasty was measured and analyzed.resultsthermal probe 's temperature was higher in locations closer to laser fiber tip and on lateral locations , rather than the in depth locations . in accordance with the laser fiber tip and the depth , temperatures above 45.0 was measured in 3.0 mm depth which trigger nociceptive ablation in 16 levels ( 80% ) , in accordance with the laser fiber end tip and laterality , every measurement had above 45.0 , and also was measured temperature over 60.0 , which can trigger collagen denaturation at 16 levels ( 80%).conclusionwhen thermal annuloplasty is needed in a selective lesion , annuloplasty using a 1414 nm nd : yag laser can be one of the treatment options . |
facial angiofibromas seen in tuberous sclerosis are usually bilateral.unilateral facial angiofibromas are seen rarely .
unilateral facial angiofibromas , however , represent a rare variant of tsc , as only few cases are reported in the literature .
we describe a case of a 30-year - old male who presented with unilateral facial angiofibromas without other features of tsc .
a-30-year - old unmarried male born of non - consanguinous marriage presented for evaluation of right - sided facial papules that were first noted at 15 years ago .
the lesions reportedly began as flat - topped , skin - colored , and erythematous papules involving the right side of his face .
he denied history of any previous treatment . during last five years , gradually increasing numbers of new and similar lesions have appeared and spread to involve the right cheek , right nasofacial sulcus , and the right side of the nose . on examination , the patient has multiple , discrete , 2 - 5 mm , flat , skin - colored and hyper - pigmented papules involving the right cheek and right side of the nose [ figure 1 ] .
there were no periungual fibromas , shagreen patches , caf - au - lait macules , forehead plaques or hypopigmented patches .
the patient has no evidence of mental retardation and no history of seizures , cardiopulmonary lesions , renal lesions , or retinal abnormalities .
multiple skins colored to hyperpigmented papules of various size distributed unilaterally on right side of face our clinical differential diagnosis included angiofibromas , verruca plana , syringoma and trichoepitheliomas . as there were no other cutaneous signs of tsc , the patient denied further evaluation and imaging studies to rule out internal manifestations of tsc .
skin biopsy specimens of the facial papules revealed characteristic findings of angiofibromas , namely telangiectases , perivascular and periadnexal concentric dermal fibrosis , and large stellate fibroblastic cells scattered in the upper dermis .
[ figures 2 and 3 ] based on above observations a final diagnosis of unilateral facial angiofibromas was made .
patient was advised cryotherapy for his lesions , but we lost the follow - up with patient .
( h and e , 50 ) high power view showing dense collagen thickening , dilated vessels and few melanophages ( h and e , 200 )
the major impact of the disorder is on the skin , brain , and kidneys , though it also affects the eyes and heart .
a major point regarding symptoms and signs of tsc is the variable clinical expression of the disorder .
the major manifestations of tsc include skin lesions in more than 95% , mental retardation in approximately 50% , autism , seizures in approximately 85% . the classic triad of mental retardation , seizures and angiofibromas occur in only 29% of patients and 6% of them lack all three manifestations .
facial angiofibromas occur in 80% to 90% of patients with tsc , typically presenting after 5 years of age .
they typically occur as bilateral and symmetric , small , tan to erythematous papules on the cheeks , chin , and nose .
our patient has multiple angiofibromas affecting only one side of the face but no other characteristic features of tsc were present , which is a rare presentation of tsc .
mcgrae and hashimoto reported a case of unilateral facial angiofibromas as a segmental form of tsc . in 1997 , anliker , et al . and
in 1998 , garcia , et al . reported similar cases . in 2000 , silvestre , et al . reported two patients with similar findings as a mosaic form of tsc .
trauner , et al . in 2003 , reported similar case with poliosis of occipital area in a 53-year old man . in 2006 ,
camprubi , et al . reported a similar case in 13-year - old male and reviewed previous cases .
hall , et al . in 2007 reported a case similar to our present case without any other manifestations of tsc . in 2008 , bordel - gmez reported a 28-year - old male with unilateral angiofibromas .
lopez , et al . in 2009 , reported a case of unilateral eyelid angiofibroma with complete blepharoptosis as a presenting sign of tsc .
our case of unilateral facial angiofibromas and without any other manifestation represents a rare clinical presentation of tsc .
however , the possibility that this case represents a distinct clinical disorder can not be ruled out completely .
this case highlights a rare occurrence of unilateral multiple facial angiofibromas without any other manifestations of tuberous sclerosis . | tuberous sclerosis ( tsc ) is an autosomal dominant hereditary condition with many varied forms of clinical presentation .
the most frequent cutaneous findings in tsc include multiple angiofibromas , hypopigmented macules , periungual fibromas , and shagreen patch . rarely , unilateral multiple facial angiofibromas have been reported .
we report a case of unilateral multiple facial angiofibromas without any other manifestations of tsc .
although rare , unilateral multiple facial angiofibromas may be a mosaic form of tsc . |
anaphylaxis is a rapid , potentially fatal , multiorgan system , allergic reaction caused by the excessive release of mediators including histamine and cytokines / chemokines from mast cells and basophils upon allergen / ige stimulation .
it typically causes a number of symptoms including an itchy rash , throat swelling , and low blood pressure associated with low body temperature . a recent nationwide cross - sectional telephone survey in usa estimated that the prevalence of anaphylaxis in the general adult population is at least 1.6% and medications were the most common trigger ( 35% ) , followed by foods ( 32% ) and insect stings ( 19% ) .
it is also recently reported that an estimated 1 in 300 of the european population at some time in their lives is affected . given the increased prevalence of anaphylaxis in industrialized countries , more basic research is urgently needed for better understanding of anaphylaxis that leads to efficient prevention of the disease .
however , the pathophysiology of anaphylaxis is not fully understood . in mammals , the internal time keeping system circadian clock drives the daily rhythms in behavior and physiology ( e.g. , sleep - wake cycles , body temperature , blood pressure , and hormonal secretions ) that enable the organisms to keep track of the time of day according to daily changes in light intensity [ 4 , 5 ] .
mammalian circadian clock system consists of the central oscillator located in the suprachiasmatic nucleus ( scn ) of the hypothalamus and peripheral oscillators present in virtually all cell types [ 4 , 5 ] .
light activates a specific group of photoreceptors in the retina connected to the central scn clock which synchronizes and entrains peripheral circadian clock via neural and endocrine pathways .
the molecular mechanisms of rhythm generation are cell autonomous , highly conserved in the scn and peripheral cells , and created and maintained by interlocked transcriptional - translational feedback loops consisting of several clock genes and their protein products [ 4 , 5 ] .
we have recently shown that a passive systemic anaphylactic ( psa ) reaction in mice exhibits a time of day - dependent variation , relying on the normal activity of a key clock gene , period2 ( per2 ) , suggesting that the canonical clock gene is required for the daily rhythm generation observed in psa reaction . however
, it remains obscure whether the daily rhythms are generated precisely by the circadian clock system or not , since per2 has nonclock functions and may regulate the daily rhythms independently of the circadian clock system [ 4 , 5 ] .
therefore , this short study aimed to clarify the precise role of the circadian clock system in the generation of daily rhythms in anaphylaxis .
for this purpose , we examined the effects of scn ablation on the time of day - dependent variations in psa reaction since scn ablation reliably eliminates the normal activity of the circadian clock system [ 4 , 5 ] .
male 812-week - old icr mice ( japan slc , tokyo , japan ) were bred under specific pathogen - free conditions and 12-hour light/12-hour dark conditions ( l / d cycles ; the light was turned on at 6:00 a.m. , zeitgeber time ( zt ) 0 , and the light was turned off at 6:00 p.m. , zt12 ) with ad libitum access to food and water , for at least 2 weeks .
all animal experiments were approved by the institutional review board of university of yamanashi and waseda university .
bilateral thermal lesions of the scn were obtained stereotactically ( narishige co. , tokyo , japan ) under anaesthesia as described previously .
one month after the surgery , we selected animals with complete lesioning of the scn after confirmation of arrhythmic general locomotor activity recorded with an infrared radiation sensor ( f5b , omron , tokyo , japan ) and analyzed with clocklab software ( actimetrics , wilmette , il , usa ) as described previously .
a histological check of lesion sites was also performed after finishing the psa experiments using nissl staining .
a passive systemic anaphylactic ( psa ) reaction was induced in mice as previously described with some modifications .
briefly , mice were sensitized with an intravenous injection of 20 g with mouse anti - tnp ige in 0.2 ml pbs .
the mice were intravenously challenged with 1000 g dnp - bsa in 0.2 ml pbs 24 hours later .
their rectal temperature was measured with a digital thermometer ( shibaura electronics , tokyo , japan ) every 5 minutes from 20 minutes before dnp - bsa challenge and thereafter until 120 minutes after the start of the examination .
the rectal temperature was also measured at 180 minutes after the start of the examination .
serum samples were collected from the mice at 10 minutes or 180 minutes after induction of psa reactions .
the amounts of mcp-1 ( ccl2 ) and il-6 in the serum at 180 minutes after induction of psa reaction were measured using the mouse mcp-1 and il-6 elisa kits ( r&d , minneapolis , mn ) .
the amounts of histamine in the serum at 10 minutes and 180 minutes after induction of psa were measured using histamine eia kit ( oxford biomedical research , inc . , oxford , mi , usa ) .
serum levels of total ige and corticosterone were determined by using the mouse ige elisa kit ( r&d , minneapolis , mn , usa , or morinaga institute of biological science , kanagawa , japan ) or assaymax corticosterone elisa kit ( assaypro , charles , mo , usa ) , respectively .
statistical analysis was performed using an unpaired student 's t - test to compare data in different groups .
in order to clarify the precise role of the circadian clock system in the generation of daily rhythms in systemic anaphylactic reaction , we examined the effects of scn ablation on a time of day - dependent variations in passive systemic anaphylactic ( psa ) reaction in mice , which is a representative model of anaphylaxis .
we confirmed complete disruption of the scn in mice by a histological check of lesion sites by nissl staining after finishing all the experiments ( figure 1(a ) ) and by examining behavioral patterns ( general locomotor activity with arrhythmicity ) ( figure 1(b ) ) .
the kinetics of psa reaction was then compared in sham - operated mice and mice with disrupted scn ( figure 2(a ) ) .
sham - operated mice challenged with the antigen at 10:00 pm ( zt16 ) showed a smaller drop in the extent of rectal temperatures than the mice challenged at 10:00 am ( zt4 ) .
however , such a time of day - dependent variation in psa reaction was absent in mice with disrupted scn .
consistently , serum histamine , mcp-1 ( ccl2 ) , and il-6 levels following the induction of the psa reactions showed similar time of day - dependent variations to the psa reactions in sham - operated mice , but the variations were absent in mice with disrupted scn ( figures 2(b)2(d ) ) .
the daily variations in serum corticosterone levels observed in sham - operated mice were also absent in mice with disrupted scn ( figure 2(e ) ) .
in contrast , serum total ige levels were comparable between sham - operated mice and mice with disrupted scn ( data not shown ) .
these results indicated that disruption of the central clock scn blunted a time of a day - dependent variation in psa reaction in association with a loss of rhythmic secretion of corticosterone .
these findings suggest that the circadian clock system may contribute to the generation of daily rhythms in anaphylaxis .
however , it appears that the basal body temperatures at zt4 and zt16 before induction of psa ( 020 minutes before the antigen challenge ) were comparable in control sham - operated mice ( figure 2(a ) ) .
we speculate that this discrepancy might be caused , at least in part , by the experimental manipulation for measuring body temperature .
direct insertion of a digital thermometer into the rectum every 5 minutes likely affects mouse activity in the resting phase and might upregulate the body temperature .
in contrast , a time of day - dependent variation in psa reaction was clearly observed at zt4 and zt16 in control sham - operated mice ( figure 2(a ) ) .
thus , it appears that the extent of a time of day - dependent systemic mast cell responses overrides the possible effects of the experimental manipulation on body temperature .
the surgery for scn ablation may destroy other important nuclei located in this brain area such as temperature center and corticosterone releasing hormone ( crh ) neurons .
we observed that body temperatures at 180 minutes after the examination were comparable between sham - operated mice and mice with disrupted scn ( figure 2(a ) ) , suggesting that the brain area for temperature regulation was functional in mice with disrupted scn .
we also observed that basal levels of serum corticosterone were comparable between sham - operated mice and mice with disrupted scn ( figure 2(a ) ) , suggesting that the brain area for crh regulation was functional in mice with disrupted scn ( figure 2(e ) ) . these findings may support the specificity of the surgery to the scn
although we can not completely exclude some of the nonspecific influences of the scn ablation on the current results .
it remains to be determined precisely how the dysfunction of the circadian clock ( disruption of the scn ) affects the time of day - dependent variation in psa reaction .
we have previously shown that daily rhythms observed in passive cutaneous anaphylactic ( pca ) reaction were absent in adrenalectomized mice , suggesting that humoral factors from the adrenal gland are necessary to maintain the daily rhythms .
the central scn pacemaker primarily governs the daily rhythmic secretions of humoral factors such as corticosterone from the adrenal gland .
therefore , the scn may control the time of day - dependent variation in psa ( and also pca ) reaction via regulation of the rhythmic secretion of humoral factors ( possibly corticosterone ) from the adrenal gland .
the current findings that the time of day - dependent variation in psa reaction in sham - operated mice showed an inverse association with the serum corticosterone levels and the extent of psa reaction was severe in mice with disrupted scn in association with low serum corticosterone levels may support this hypothesis . in summary ,
the current findings provide additional support for the notion that the circadian clock system times ige - mediated systemic anaphylactic reaction .
the findings may provide a novel insight into the pathophysiology of anaphylaxis and also implicate that environmental and intrinsic factors disrupting the normal activity of the circadian clock system , such as sleep disturbance and mental stress [ 5 , 6 , 10 ] , might predispose some types of allergic patients ( e.g. , patients with drug and food allergy ) to severe anaphylaxis .
the circadian clock may be an important regulator of anaphylaxis , which will provide a novel insight into the pathophysiology of the disease . | anaphylaxis is a severe systemic allergic reaction which is rapid in onset and potentially fatal , caused by excessive release of mediators including histamine and cytokines / chemokines from mast cells and basophils upon allergen / ige stimulation .
increased prevalence of anaphylaxis in industrialized countries requires urgent needs for better understanding of anaphylaxis .
however , the pathophysiology of the disease is not fully understood . here
we report that the circadian clock may be an important regulator of anaphylaxis . in mammals ,
the central clock located in the suprachiasmatic nucleus ( scn ) of the hypothalamus synchronizes and entrains peripheral circadian clock present in virtually all cell types via neural and endocrine pathways , thereby driving the daily rhythms in behavior and physiology .
we found that mechanical disruption of the scn resulted in the absence of a time of day - dependent variation in passive systemic anaphylactic ( psa ) reaction in mice , associated with loss of daily variations in serum histamine , mcp-1 ( ccl2 ) , and il-6 levels .
these results suggest that the central scn clock controls the time of day - dependent variation in ige - mediated systemic anaphylactic reaction , which may provide a novel insight into the pathophysiology of anaphylaxis . |
today , cancer is one of the important , fundamental problems in health and treatment worldwide , and its importance is increasing in countries . colorectal cancer ( crc ) is the most prevalent gastrointestinal cancer and the cause of 10% of the cancer mortalities . in iran
in addition , lifestyle is an important risk factor for the increased risk of crc development .
low activity , inappropriate diet , smoking , suffering from metabolic syndrome ( obesity , diabetes , hypertension , and dyslipidemia ) alongside past medical and family history of cancer development , and polyp are among the risk factors of crc occurrence . in recent years ,
survival of crc patients has enhanced in some regions of the world , and it is not clear which factors have contributed to this . according to the previous literature , body mass index ( bmi ) , inflammatory bowel disease , alcoholism , tumor grade , lymph nodes metastasis , the degree of intestinal wall metastasis , metastasis of other organs , and tumor stage affect the survival of the patients while bmi , type of the first treatment , and metastasis of lymph nodes and other organs contribute to the time of death from crc and hence it could be said that the effect of risk factors on different sections of colon is different and should be investigated separately . an increasing number of epidemiologic researches have found that diabetes mellitus ( dm ) may change the risk of developing a variety of cancers .
mean survival time of patients with colon cancer was estimated 7.75 1.118 years and that of patients with rectum cancer 3.917 0.26 . in a research conducted on female patients with crc ,
longer survival was more likely in patients with no history of inflammatory bowel disease , distinguished , weak tumor grade , and tumor pathologic with stage one .
worldwide , approximately 1 billion people have hypertension , contributing to > 7.1 million deaths per year .
the increase in the rate of hypertension and type 2 dm could be attributed to the aging of the population , obesity prevalence , salt intake , sedentary life , urbanization , and socioeconomic changes .
patients with diabetes and hypertension are at a higher risk than the general population of developing cancer .
there is a growing body of evidence published in recent years that suggest a substantial increase in cancer incidence in diabetic patients .
the worldwide prevalence of diabetes was estimated to rise from 171 million in 2000 to 366 million in 2030 .
the present study was aimed to investigate the prevalence of hypertension and type 2 dm and their effect on median survival time in patients with crc .
in this study we analyzed the data of a prospective , cohort study conducted by gastroenterology and liver disease research center affiliated to shahid beheshti university of medical sciences .
informed , written consent was obtained from all the patients and the design of the study was approved by shahid beheshti university ethics committee ( approval no . 676 ) .
the patients were followed - up from definite diagnosis of crc till death or completion of the study or their censoring once each 6 months .
overall , 2570 individual - year follow - ups were conducted for 1127 patients with crc .
the study was initiated at september 23 , 2006 , and the patients were followed - up until october 23 , 2011 .
crc diagnosis was determined according to criteria of international classification of pathologies and diseases , the pathology report , and criteria of american commission on cancer . the final outcome in patients
the test for diagnosing type 2 diabetes and hypertension were done at enrollment of the participants .
for the diagnosis of type 2 dm , fasting blood sugar test and glycosylated hemoglobin test were used and for that of hypertension , blood pressure ( bp ) was measured in two turns .
hba1c was measured using column chromatography method with nyco card reader ii made in norway .
fetal bovine serum was measured using a spectrophotometer by erba - xl 300 made in germany .
we estimated and graphed survival curves using the kaplan - mier method for crc patients with dm , hypertension and both of them separately .
we also compared survival curves of these groups of patients with log - rank test . to calculated prevalence and
mean age of the patients at diagnosis was 53.38 ( standard deviation [ sd ] : 14.3 ) years and the mean tumor size was 53.35 ( sd : 31.9 ) mm .
30.2% of the patients were under 45-year - old , 45.5% between 45 and 65-year - old , and 24.2% over 65-year - old .
according to the histopathological diagnosis 55.5% of the patients were in grade one , 35.7% in grade two , 8.8% in grade three and four of the disease .
patients characteristics for demographic variables and mortality rate due to crc are shown in table 1 .
the prevalence of hypertension and type 2 dm in the patients with crc were respectively 13.38% and 8.69% .
the prevalence and confidence interval ( ci ) of hypertension and type 2 dm are shown in table 2 .
mean and median survival time with ci is shown in table 3 . according to log - rank test
, no significant difference was observed between the survival time of crc patients suffering from hypertension and diabetes type 2 ( p = 0.8188 ) .
patients characteristics for demographic variables and mortality rate due to crc the prevalence ( p ) of hypertension and type 2 dm in first stage of crc patients mean and median survival time with ci in the patients suffering from hypertension and type 2 dm
colorectal cancer is ranked as the fourth cause of death from cancers with 320,600 mortalities in men after lung cancer , liver cancer , and gastric cancer and as the third cause of death with 288,100 mortalities in women after lung cancer and breast cancer in the world . in iran , this cancer has been reported to occur in seven per 100,000 individuals .
it seems that rapid growth of crc in our society could be derived from the contribution of environmental factors including particular nutritional habits such as increase in carbohydrates and fat consumption and decrease in fiber intake especially among youth , change of lifestyle , lack of sufficient activity and obesity , large numbers of youth in society , and possibly involvement of genetic issues .
income status , alcohol consumption history , metastasis , and the approach to diagnosis and treatment are among the factors effective on survival rate .
diabetes and hypertension are among risk factors for crc , in this regard , there are theories stating that the individuals with diabetes are at a higher risk of developing crc compared with healthy individuals and it occurs in men more frequently compared with women . in addition decreased short - term survival rates were noted among diabetics undergoing surgical resection of crc .
the association between glucose - level and cancer was approximately linear across full range of fasting glucose - levels .
an increase in 1 mmol / l of fasting plasma glucose - levels was associated with risk ratio ( rr ) 1.05 ( 95% ci ; 1.01 - 1.10 ) and 1.11 ( 1.05 - 1.16 ) for incidental cancer in men and women , respectively .
rr was 1.37 ( 1.14 - 1.64 , for trend = 0.002 ) for all - site cancer incidence in men ( excluding prostate cancer ) and 1.42 ( 1.18 - 1.74 , p for trend < 0.001 ) in women .
several case - control studies have also confirmed the positive association of diabetes with crc occurrence in male patients , which is not the case for women .
most recent meta - analysis comprising 24 studies ( 8 case control and 16 cohorts ) linked diabetes with modest increase in risk of crc , the most common cancer of digestive tract in western societies , with rrs 1.26 ( 95% ci ; 1.20 - 1.31 ) .
some mechanisms are suggested for the increased crc occurrence in the patients with diabetes in comparison to healthy individuals .
diabetes cause slow transmission of substance throughout bowel which results in retention of intraintestinal toxic substances .
diabetes causes production of carcinogenic , biliary acids , the increase in blood insulin level causes an increase in tumor growth , and delayed transmission of carcinogenic substance exacerbates the damage to dna and other cells .
a study of diabetic and nondiabetic individuals reported that substance transmission in intestine of diabetic individuals is 70 - 80% longer compared with nondiabetic .
in other studies , the symptoms of constipation in the patients with diabetes were more prevalent compared with nondiabetic individuals . in a study conducted on women with diabetes , these women were found to have a higher risk of developing crc ; but the survival of the patients with crc was not affected by diabetes , in a way that a same survival of crc has been found for diabetic and nondiabetic individuals .
this study is consistent with the findings of the present study because diabetes had no effect on survival of crc patients . in some studies
the effect of hypertension on crc has been measured , showing that the foods causing decline in bp , if consumed daily , contribute greatly to decrease in crc occurrence . in many studies ,
the patients with crc and hypertension treated with bevacizumab had a higher likelihood of survival compared with the group not suffering from hypertension . in the present study ,
hypertension had no effect on the survival of the patients with crc and the prevalence of hypertension and diabetes type 2 in population under study was estimated 13.38% and 8.69% , respectively , while in a study in malaysia on 138 patients with crc , the prevalence of diabetes type 2 and hypertension was obtained 13% and 34.8% , respectively , reporting that these two metabolic diseases were associated with crc .
the researchers found that among patients with early stage disease , patients with diabetes or high bp had a significantly greater risk of cancer recurrence rate and death after treatment .
for example , 47.7% of patients who did not have diabetes were still alive 5 years after diagnosis compared to only 41.3% of patients with diabetes .
when the researchers looked more closely , they also found that cancer recurrence rates at 5 years were approximately 8% higher in patients with diabetes or hypertension .
diabetes and hypertension is a high - risk state for several diseases , such as crc .
the obtained findings in this study indicate that survival time in patients with type 2 dm less than hypertension , but two metabolic diseases ( type 2 dm and hypertension ) have no effect on survival rate of the patients with crc and the survival of these patients may be dependent on other factors including the site of cancer , the stage when the disease is diagnosed to treatment commencement , type of treatment , nutrition status , etc .
| background : type 2 diabetes mellitus ( dm ) and hypertension are worldwide epidemic .
association between dm and colon cancer was obtained in previous studies .
prevalence of dm and hypertension in the patients with colorectal cancer ( crc ) has not been reported in iran .
the present study was aimed to investigate the prevalence of hypertension and type 2 dm and their effect on median survival time in patients with crc.materials and methods : overall , 2570 individual - year follow - ups were conducted for 1127 patients with crc . for the diagnosis of type 2 dm , fasting blood sugar test and glycosylated hemoglobin test were used and for hypertension , blood pressure was measured in two turns .
the descriptive indices were calculated , and the mean and median survival from crc diagnosis time was calculated using survival analysis and a comparison among survival times was done through log - rank test .
stata software 12 ( stata corp .
2011 .
stata statistical software : release 12 .
college station , tx : stata corp lp ) was used for data analysis.results:the prevalence of hypertension and type 2 dm in the patients with crc was respectively 13.38% ( 95% confidence interval [ ci ] : 11.1 - 15.8 ) and 8.69% ( 95% ci : 7 - 10.7 ) .
median survival time in patients with hypertension and dm were 8.52 and 4.9 years .
according to log - rank test , no significant difference was observed between the survival time of crc patients suffering from hypertension and diabetes type 2.conclusion:the obtained findings in this study indicate that survival time in patients with type 2 dm less than hypertension but two metabolic diseases have the same effect on survival rate of the patients with crc .
understanding the risk factors for crc may guide the development of strategies targeted toward its prevention . |
twenty - four different integrins are expressed in humans , each composed of non - covalently associated and chains ( hynes , 2002 ) .
integrins have large extracellular domains , single - pass transmembrane segments , and short intracellular tails . lacking enzymatic activity ,
the short integrin cytoplasmic domains serve as scaffolds for signaling and structural proteins that allow integrins to be a conduit of bidirectional communication between the cytoplasm and extracellular ligands ( legate and fassler , 2009 ; moser et al . , 2009b ) [ box 1 ] .
the integrin ligand recognition site spans the subunit inserted - like ( i ) domain and subunit -propeller domain or , for about half of the integrin family , resides entirely within the subunit i ( i ) domain ( lu et al . , 2001 ; luo et al . , 2007 ) .
all 2 integrins , including lfa-1 ( l2 or cd11a / cd18 ) [ box 2 ] , contain this extra i domain within the headpiece .
the structural features of the various domains of lfa-1 have been reviewed in great detail elsewhere ( luo et al . , 2007 ) .
inside - out signaling refers to a process by which a cellular stimulus , for example , a chemokine binding to its receptor , leads to integrin activation by intracellular signaling pathways .
inside - out signaling leads to vast conformational changes in integrins , but not directly to clustering ( redistribution of integrin heterodimers in the plane of the plasma membrane ) .
by contrast , outside - in activation refers to integrin activation induced by ligand binding .
although the terminology is often confused , outside - in signaling , distinct from outside - in activation , refers to a process whereby the cell receives signals from the extracellular environment through integrins that have already been activated and are now bound to immobilized ligands on another cell or in the extracellular matrix .
adhesion strengthening , or stabilization , is a process following integrin activation and initial ligand binding .
adhesion strengthening most likely involves integrin clustering and is required to keep arrested leukocytes from being washed away .
l2 ( lfa-1 , cd11a / cd18 ) is expressed on all leukocytes ; m2 ( mac-1 , cd11b / cd18 ) is expressed on granulocytes , monocytes , macrophages , and subsets of activated lymphocytes ; x2 ( p150,95 , cd11c / cd18 ) is expressed on dendritic cells , macrophages , and small subsets of blood monocytes ; and d2 ( cd11d / cd18 ) is expressed on tissue - specific subsets of macrophages . the macromolecular structure of integrins is coupled to the accessibility of the ligand - binding pocket and to its ligand - binding affinity .
therefore , the regulation of integrin conformation is critical for their adhesive and signaling function ( luo et al . , 2007 ) .
studies employing crystallography ( shimaoka et al . , 2003b ) , nuclear magnetic resonance ( huth et al . , 2000
; legge et al . , 2000 ) , electron microscopy ( nishida et al . , 2006 ) , and molecular dynamics simulation ( jin et al . , 2004 )
have suggested that lfa-1 can assume at least three distinct conformational states ( figure 1 ) . in its inactive state , the lfa-1 extracellular domain has a bent structure shaped like an inverted v ( nishida et al . , 2006 ) with the low affinity headpiece closely approaching the plasma membrane ( larson et al .
, 2005 ; nishida et al . , 2006 ) , similar to what has been shown for other integrins ( xiong et al . , 2001 ; chigaev et al . , 2003 ; zhu et al . , 2008 ; xie et al . , 2010 ) . the inactive state is also characterized by inter - domain contacts between the n - terminal headpiece and membrane - proximal lower legs , between the lower legs of the and chains , and between the and transmembrane domains ( tmds ; takagi et al .
, 2002 ; li et al . , 2005 ; luo et al . , 2005 ; partridge et al .
based primarily on mutational studies , it is thought that close association of tmds stabilizes the inactive state and disruption of this association leads to integrin activation ( hughes et al . , 1996 ;
lu and springer , 1997 ; vinogradova et al . , 2002 ; luo et al . , 2005 ) .
integrin function is structurally regulated and three distinct conformations have been demonstrated . in its low affinity state (
left ) , lfa-1 is compact with a sharp bend at the genu and headpiece closely approaching the plasma membrane .
extended conformations ( middle ) differ in the orientation of their hybrid domain ( purple ) , with swingout of this domain representing a conversion from intermediate to high affinity state ( right ) .
data suggest that the extended / closed conformation ( middle ) can have either a low or intermediate affinity i domain .
in contrast to the compact structure of inactive lfa-1 , active lfa-1 conformations exhibit an extended extracellular domain with the ligand - binding headpiece situated more than 20 nm above the membrane ( nishida et al . , 2006 ) . extended structures of lfa-1 ( figure 1 ) differ in the conformation of the headpiece and i domain , the angle between the i domain and hybrid domain into which the i is inserted , and the distance between the l and 2 tmds in the plane of the plasma membrane .
the extended ectodomain with a closed headpiece has also been termed the intermediate affinity state
it is thought that the spatial orientation of the headpiece alone in the extended intermediate affinity structure may account for enhanced recognition of ligands , as steric hinderance may prevent large , immobilized ligands from accessing the i domain in the bent , inactive lfa-1 conformer .
this idea is supported by data showing that the isolated lfa-1 i domain alone , in its basal state , can mediate transient interactions with intercellular adhesion molecule-1 ( icam-1 ) that result in a rolling phenotype ( knorr and dustin , 1997 ; eniola et al . , 2005 ) , whereas the full - length lfa-1 molecule does not support interaction under flow conditions ( salas et al . , 2002 ) .
however , in a series of disulfide - stabilized crystal structures of the l i domain , distinct conformations with low , intermediate , and high affinity were identified , where the transition from low to intermediate affinity was mainly driven by an increase in the bimolecular association rate ( kon ) and the transition from intermediate to high affinity was mainly driven by lower koff ( slower release of bound ligand ; shimaoka et al . , 2003b ) .
notably , the i metal ion - dependent adhesion site ( midas ) that recognizes ligand retains the same coordination and conformation in the putative i low and intermediate affinity conformations . therefore , in addition to the macromolecular integrin structures , it is apparent that the i domain may exist in three distinct conformations regulated by the relative position of the 7 helix .
there is ample evidence , as will be discussed below , that extended lfa-1 mediates neutrophil slow rolling by transiently binding to icam-1 . during slow rolling ,
the l i domain is most likely not in the high affinity conformation . in the high affinity state
structure , swingout of the hybrid domain away from the subunit by approximately 60 is coupled to opening of the headpiece through the downward movement of the i domain 7 helix that connects to the i domain as an internal ligand for the i midas ( alonso et al . , 2002 ; xiao et al . , 2004 ; luo et al . , 2007
this shift in the 7 helix is coupled to rearrangement of the i domain , enhancing its affinity for ligand ( alonso et al . , 2002 ; yang et al . , 2004
it is the high affinity lfa-1 conformer that mediates arrest of leukocytes on the endothelium ( constantin et al .
2004 ) , and in vitro studies demonstrate that the open headpiece of lfa-1 is necessary and sufficient to mediate cell arrest under flow conditions ( salas et al . , 2002 , 2004 ) .
the structure and affinity of integrin receptors is thought to be regulated by cells in an inside - out manner [ box 1 ] through signaling cascades that impinge upon the integrin cytoplasmic tails ( dustin and springer , 1989 ; otoole et al . , 1990 ; sims et al . , 1991 ) .
talins and kindlins both contain a band 4.1/ezrin / radixin / moesin ( ferm ) domain with four independently folded subdomains ( f0f3 ; moser et al . , 2009b )
while the ferm domain comprises the talin head that is sufficient for activating integrins ( calderwood et al . , 1999
2010 ) , talins also have a flexible rod domain that contains actin - binding sequences and mediates cytoskeletal association ( critchley , 2009 ) .
although many of the structural and functional studies that have dissected the role of talin in integrin activation have employed experimental systems where the talin head domain alone is used , the full - length molecule exists in a basal autoinhibited state that is mediated by contact between the integrin - binding f3 subdomain and rod domain ( goksoy et al . , 2008 ) .
binding to phospholipids , such as phosphatidylinositol 4,5-bisphosphate ( pip2 ) , relieves this autoinhibition and activates talin for integrin binding ( martel et al . , 2001 ;
the ability of talins and kindlins to activate integrins depends on their binding to membrane phospholipids . for talin
, this occurs through positively charged surfaces within the f1 , f2 , and f3 subdomains ( anthis et al . , 2009 ; goult et al . ,
kindlins have an analogous surface within the f1 subdomain ( bouaouina et al . , 2012 ) as well as an additional pleckstrin homology domain that is inserted within its f2 subdomain ( tu et al . , 2003 ; qu et al . , 2011
) , both of which bind to membrane phospholipids and are important for integrin activation . talins and kindlins mediate integrin activation by binding directly to separate nxx(y / f ) motifs within the integrin chain short cytoplasmic tail ( tadokoro et al . , 2003 ; harburger et al . , 2009 )
multiple lines of evidence demonstrate that talins contribute to the separation of the integrin tmds ( kim et al .
, 2003 ; wegener et al . , 2007 ) , but the mechanisms by which kindlins are involved in regulating integrin conformation are largely unknown ( moser et al . , 2009b ; shattil et al . , 2010 )
. mutations of kindlin-3 , the kindlin isoform expressed in hematopoietic cells , were found to underlie the leukocyte adhesion deficiency type iii ( lad - iii ) pathology ( malinin et al .
, 2009 ; moser et al . , 2009a ; svensson et al . , 2009 ) [ box 3 ] .
kindlin-3-deficient leukocytes are unable to arrest on inflamed endothelium , but their selectin - mediated rolling capacity is normal , indicating an important role for kindlin-3 in lfa-1 activation ( moser et al . , 2009a ) .
the roles of talin-1 and kindlin-3 in regulating lfa-1 in neutrophils are discussed in further detail below . in the
human disease leukocyte adhesion deficiency type iii ( lad - iii ) , activation of 2 , 3 , and some 1 integrins is defective .
lad - iii was discovered in 1997 and initially called lad - i variant ( kuijpers et al . , 1997 ) .
kuijpers and colleagues recognized that expression of 2 integrins was normal but their activation was defective in these patients ( alon et al .
, 2003 ; alon and etzioni , 2003 ; malinin et al . , 2009 ; manevich - mendelson et al .
alon and colleagues ( pasvolsky et al . , 2007 ) reported a point mutation in rasgrp2 , the gene encoding caldag - gefi , and thought that defective caldag - gefi caused lad - iii .
this idea was supported by the leukocyte and platelet adhesion deficiencies exhibited by caldag - gefi knockout mice ( bergmeier et al . , 2007 ) .
however , it was later found that mutation of the fermt3 gene encoding kindlin-3 that causes the pathology of lad - iii ( kuijpers et al .
, 2009 ; svensson et al . , 2009 ) , dominates the phenotype of the subset of lad - iii patients with mutations in both fermt3 and rasgrp2 .
humans deficient in caldag - gefi , but not kindlin-3 , have not been described .
there is now consensus that mutations in the fermt3 gene encoding kindlin-3 cause all known lad - iii cases ( abram and lowell , 2009 ) .
the dynamic equilibrium that exists between the various integrin conformational states ( figure 1 ) appears to vary between the 24 different integrin subtypes and even amongst the four different 2 integrins ( nishida et al . , 2006 ) .
therefore , aside from the most basic structural commonalities , integrin activation mechanisms may exhibit significant variability .
the switchblade model of integrin activation stems from the delineation of the three distinct affinity states described above , and was formulated based on crystallography ( xiong et al . , 2001 ;
xiao et al . , 2004 ) and studies of isolated integrins by electron microscopy ( takagi et al . , 2002 ;
the switchblade model postulates that only the closed , unliganded integrin headpiece can stably exist in the overall bent structure .
the switchblade - like extension of the extracellular domain occurs as a result of disruption of headpiece - lower leg and / lower leg stabilizing contacts , either by separation of the tmds or by ligand - induced hybrid domain swingout ( luo et al . , 2007 ) .
the switchblade model received support from the recent finding that single , full - length iib3 integrin embedded in membrane has the same compact structure observed in isolated extracellular domains and can undergo ectodomain extension in the presence of talin head domain bound to the integrin cytoplasmic tail ( ye et al . , 2010 ) .
most of the activated structures observed in this study , however , resembled the intermediate rather than the high affinity conformation .
as discussed below , an important unresolved question is whether inside - out signaling alone ( that is , in the absence of ligand binding ) induces the high affinity structure with an open headpiece .
the deadbolt model of integrin activation was proposed ( xiong et al . , 2003 ) based on the observation that crystals of the unbound ( xiong et al . , 2001 ) and ligand - occupied ( xiong et al . , 2002 )
the crystal structure of ligand - occupied , bent v3 was further supported by electron microscopy studies ( adair et al . , 2005 ) .
in addition , fret studies have shown that small ligand binding to 41 integrin ( vla-4 ) on the living cell surface can occur in the absence of ectodomain extension ( chigaev et al . , 2007 ) .
the deadbolt model posits that a loop within the subunit extracellular membrane - proximal -tail domain contacts the 7 helix of the i domain that regulates its conformation and affinity ( xiong et al . , 2003 ) . during activation , movement of the tmds
would disrupt the deadbolt and allow headpiece opening in the bent structure ( xiong et al . , 2003 ) .
however , elimination of the -tail domain loop that was proposed to form the deadbolt did not enhance ligand binding ( zhu et al . , 2007 ) .
more recent work suggests that inside - out integrin activation is more complicated than the switchblade and deadbolt models imply .
for example , in kinetic studies of vla-4 on live cells , binding of antibodies that recognize epitopes selectively exposed in extended integrin structures was minimal after triggering two different physiologic inside - out activation pathways , but was robust in the presence of a small ligand even without cell stimulation ( chigaev et al . , 2009 ) .
the same group recently performed similar experiments with a fluorescent ligand probe of lfa-1 and found that small ligand binding to lfa-1 in the absence of inside - out activation was very slow ( chigaev et al . , 2011 ) .
these findings indicate a greater restraint on inactive lfa-1 compared to vla-4 with respect to ligand binding .
inside - out stimulation via chemokine or formyl peptide receptors [ both g - protein coupled receptors ( gpcrs ) ] rapidly enhanced lfa-1 small ligand binding , suggesting affinity upregulation of the headpiece .
these studies suggest that mechanisms of integrin conformational regulation , including inside - out and ligand - induced outside - in activation , have both shared and distinct components between integrin subtypes and activating stimuli .
it is clear that inside - out signal transduction pathways , such as those elicited by chemokine receptor engagement , stimulate an increase in the affinity of integrins .
the ability of lfa-1 to bind icam-1 in its soluble , rather than immobilized , form has been utilized as a sensitive assay that specifically reports the high affinity state ( constantin et al .
further support for this assay as a means to discriminate between extended / closed and extended / open conformations of lfa-1 was provided by laudanna and colleagues ( bolomini - vittori et al . , 2009 ) by showing that a signaling defect in headpiece opening due to silencing of phosphatidylinositol-4-phosphate 5-kinase type i gamma ( pip5kc ) blocks chemokine - induced soluble icam-1 binding to t lymphocytes despite the ability of lfa-1 to become extended ( as indicated by a reporter mab ) under the same conditions .
many studies have thus demonstrated that chemokines induce a high affinity state of lfa-1 that is competent for soluble icam-1 binding and that mediates t lymphocyte arrest ( constantin et al . , 2000 ; giagulli et al .
, 2004 ; bolomini - vittori et al . , 2009 ) . despite these extensive studies ,
it is not clear whether chemokine stimulation alone , in the absence of ligand , triggers opening of the lfa-1 headpiece .
chemokines induce the binding of epitope - specific reporter antibodies , such as mab 24 ( dransfield et al . , 1992 ) and 327c mab ( beals et al . , 2001 ) , that specifically recognize the extended / open high affinity conformation of lfa-1 ( bolomini - vittori et al . , 2009 ) .
however , these reporter antibodies also recognize icam-1-bound lfa-1 in the absence of cellular stimulation ( beals et al . , 2001 ) .
therefore , it remains possible that chemokines enhance the affinity of lfa-1 for icam-1 and permit a transition to the high affinity state , but do not directly induce opening of the headpiece associated with the high affinity i domain .
rather , ligand engagement and a force pulling on the engaged integrin may subsequently be needed for lfa-1 to achieve its high affinity state .
affinity measurements of lfa-1 on the cell surface show that inside - out signaling in response to stromal cell - derived factor-1 ( sdf-1 , also known as chemokine cxcl12 ) or pma , an activator of protein kinase c , enhanced binding of soluble monovalent icam-1 , indicating affinity upregulation ( schurpf and springer , 2011 ) .
however , artificially stabilizing specific lfa-1 structures resulted in icam-1 binding affinities of the intermediate affinity state that were similar to those achieved by inside - out activation , and of the high affinity state that were much higher . in a novel assay
in which antibodies that specifically recognize extended lfa-1 conformers ( kim127 mab and nki - l16 mab ) or the putative high affinity state of lfa-1 ( 327c mab ) were co - immobilized with the chemokine cxcl12 , it was found that peripheral blood lymphocytes bound only kim127 and nki - l16 , but not 327c ( shamri et al . , 2005 ) .
these data all suggest that chemokines trigger lfa-1 extension , but can not induce the full affinity of the ligand - binding headpiece .
if inside - out activation does not shift integrin affinity to its highest state , how then does ligand binding do so ?
the demonstration of a catch bond between lfa-1 and icam-1 provides insight into a possible mechanism and also suggests an important role for force in affinity maturation of the i domain ( chen et al . , 2010 ) . using a biomembrane probe to ligate single lfa-1 molecules and measure bond kinetics in the absence or presence of a pulling force ,
zhu and colleagues ( chen et al . , 2010 ) demonstrate that icam-1-occupied lfa-1 passes through three distinct bond lifetime regimes with increasing force .
pulling on the bond first enhanced lfa-1 affinity ( catch bond ) and then , for larger forces , resulted in a decrease in bond lifetime ( slip bond ) .
they propose that pulling on the i domain results in its movement away from the i domain , causing the anchored ( to the i midas ) 7 helix to experience a relative shift downward ( as discussed above ) and resulting in affinity upregulation of the i domain ( chen et al . , 2010 ) .
thus , in the context of physiologic leukocyte endothelial interactions in which lfa-1 bonds experience a force from blood flow acting on rolling or adherent cells , chemokines may stimulate lfa-1 extension while enhancing affinity of the headpiece only moderately . upon icam-1 engagement
, the bond would then undergo further affinity maturation and transition into the high affinity conformation supporting long - lived bonds .
this model of force - mediated lfa-1 affinity maturation also suggests a role for cytoskeletal anchorage of the integrin tail ( alon and dustin , 2007 ; zhu et al . , 2008 ) .
talin represents the best candidate for mediating such actin linkage through sequences in its rod domain ( critchley , 2009 ) , though a specific role for talin - mediated actin filament association in lfa-1 activation is yet to be described . a potential role for kindlins in mediating indirect cytoskeletal anchorage
neutrophils play a central role in immunity as the first leukocyte subset to enter tissues in response to infection or injury .
inflammatory cues produced locally in the tissue are relayed to neutrophils through the expression of adhesion molecules , such as e - selectin and icam-1 , on the luminal surface of endothelial cells .
this induced expression of adhesion molecules initiates a cascade of progressive interactions between leukocytes and the vascular wall that precede transmigration across the endothelial barrier and into the affected tissue ( ley et al . , 2007 ) .
members of the 2 integrin family [ box 2 ] , whose expression is restricted to leukocytes and leukocyte - derived tissue cells , are involved at several steps of the leukocyte adhesion cascade .
initial capture and rolling of neutrophils is mediated primarily by endothelial e- and p - selectins binding to p - selectin glycoprotein ligand-1 ( psgl-1 ) and other selectin ligands on the surface of neutrophils .
in addition to its function as an adhesion receptor , psgl-1 transduces an intracellular signal that partially activates lfa-1 ( zarbock et al . , 2007b ; miner et al . , 2008
although other leukocyte subsets , including some memory t lymphocytes , express functional psgl-1 , it seems that ligation of psgl-1 triggers lfa-1 extension only in myeloid cells .
it may be that the lymphoid homologs of one or more molecules downstream of psgl-1 do not participate in this signaling pathway ( alon and ley , 2008 ) .
the molecular details of the signal transduction pathway triggered by psgl-1 engagement and leading to lfa-1 activation in neutrophils has been the subject of recent reviews ( zarbock et al . , 2009 , 2011 ) , and new details on this signaling cascade
, 2012 ; shao et al . , 2012 ; spertini et al . , 2012 ) . during neutrophil
rolling interactions with p- and e - selectin , psgl-1 signaling results in the conversion of lfa-1 from an inactive state to an extended conformation that then interacts with icam-1 to reduce neutrophil rolling velocity ( chesnutt et al .
, 2006 ; zarbock et al . , 2007b ; kuwano et al . , 2010 ) .
two main pieces of evidence demonstrate that the extended lfa-1 conformer with a closed headpiece mediates slow rolling in both murine and human neutrophils .
first , an lfa-1 allosteric antagonist that binds to the i domain and blocks structural communication with the i domain , thus preventing headpiece opening , completely abrogates soluble icam-1 binding ( shimaoka et al . , 2003a ) and neutrophil arrest , but does not impair lfa-1-dependent slow rolling ( zarbock et al . , 2007b )
second , the kim127 mab ( robinson et al . , 1992 ) and nki - l16 mab ( van kooyk et al . , 1991 )
epitopes that report extension of the 2 and l legs , respectively , are exposed in human neutrophils rolling on e - selectin in flow chambers ( kuwano et al . , 2010 ) .
in the same assay , the mab 24 ( dransfield et al . , 1992 ) recognition site that is only accessible in the high affinity state remains buried unless neutrophils are activated by mn ( kuwano et al . , 2010 ) .
thus , ligation of psgl-1 induces lfa-1 extension , but not ( full ) opening of the headpiece .
two distinct signal transduction pathways modulate lfa-1 activation in neutrophils : ( 1 ) selectin binding to psgl-1 , and ( 2 ) chemokine receptor engagement .
psgl-1 signals talin-1-dependent lfa-1 extension , whereas chemokine gpcrs signal lfa-1 activation to a high affinity state that requires both talin-1 and kindlin-3 .
some of these signaling molecules may exist in preformed modules to facilitate rapid integrin activation .
abbreviations : fgr , feline gardner rasheed sarcoma kinase ; fcr , immunoglobulin gamma fc receptor ; dap12 , dnax activating protein of 12 kda ; syk , spleen tyrosine kinase ; adap , adhesion and degranulation promoting adaptor protein ; slp-76 , sh2 domain - containing leukocyte phosphoprotein of 76 kda ; btk , bruton tyrosine kinase ; pi3k , phosphoinositide 3-kinase ; plc , phospholipase c ; p38 , p38 mitogen activated protein kinase ; ip3 , inositol trisphosphate ; dag , diacylglycerol ; caldag - gefi , calcium- and diacylglycerol - regulated guanine nucleotide exchange factor i ; psgl-1 , p - selectin glycoprotein ligand-1 ; icam-1 , intercellular adhesion molecule-1 ; gag , glycosaminoglycan . during inflammation , neutrophils encounter immobilized chemokines as they roll on the vascular wall .
lfa-1 is the primary integrin receptor involved in leukocyte arrest on inflamed endothelium , with little or no detectable contribution of mac-1 ( ding et al . , 1999 ;
sdf-1 ( a ligand for cxcr4 ) and chemokine ligands for cxcr2 trigger soluble icam-1 binding and rapid lymphocyte arrest on immobilized icam-1 ( constantin et al . , 2000 ;
, 2004 ; shamri et al . , 2005 ; bolomini - vittori et al . ,
this has been interpreted to mean that high affinity lfa-1 is required to mediate leukocyte arrest .
intravital microscopy analysis of the mouse cremaster muscle , a thin tissue that envelops the testes , after intravenous injection of cxcl1 provides a sensitive arrest assay , as neutrophils rapidly ( within 15 s ) transition from rolling to arrest on the endothelium .
this assay provides the ability to distinguish the arrest step from adhesion stabilization , as neutrophils lacking signaling molecules involved in the latter process , such as pi3k ( smith et al .
, 2006 ) and pkc ( bertram et al . , 2012 ) , quickly detach and return to the bulk flow . since chemokine signaling also contributes to the stabilization and strengthening of leukocyte adhesion following arrest by enhancing lfa-1 mobility and allowing clustering to occur ( constantin et al .
the analysis of rapid arrest in vivo is critical for assessing the functional role of molecules in inside - out activation of lfa-1 .
cxcl1 ( also known as keratinocyte - derived chemokine ) is the primary chemokine involved in murine neutrophil arrest through cxcr2 and activation of the gi2 signaling cascade ( ley , 2003 ; smith et al . , 2004 ; zarbock et al . , 2007a )
in the context of inflammation , both the psgl-1 and cxcr2 signaling pathways that regulate lfa-1 activation contribute to neutrophil adhesion and recruitment ( smith et al .
psgl-1 signaling seems to induce only the extended conformation of lfa-1 , and even pulling on lfa-1 as it transiently engages with icam-1 in slow rolling is not sufficient to induce rapid neutrophil arrest mediated by the high affinity conformation .
by contrast , in the presence of chemokines , neutrophil lfa-1 reaches its high affinity state and mediates arrest .
another potential difference between these two signaling pathways is that chemokine receptors trigger rapid and locally restricted lfa-1 activation ( shamri et al . , 2005 ) , whereas psgl-1 signals to lfa-1 may not be as spatially confined ( alon and ley , 2008 ; kuwano et al . , 2010 ) .
it has been proposed that the downstream signaling proteins involved in integrin activation may be pre - assembled into complexes , thus allowing for leukocyte arrest to occur on the timescales observed experimentally ( alon and ley , 2008 ) .
consistent with this idea , a signaling module containing skap55 ( src kinase - associated phosphoprotein of 55 kda ) , adap ( adhesion and degranulation promoting adaptor protein ) , riam ( rap1-interacting adaptor molecule ) , and kindlin-3 exists constitutively in unstimulated human t lymphocytes , and inducibly associates with the common integrin activator rap1 gtpase ( kliche et al . , 2012 ) .
the role of inside - out signaling in regulating lfa-1 structure and affinity is a topic of great interest not only in the fields of immunology and leukocyte adhesion , but also among integrin biologists . until recently ,
the roles of two families of common integrin co - activators , talins and kindlins , in regulating lfa-1 conformational activation had not been studied . using mixed chimeric mice with genetic deletion of either fermt3 encoding kindlin-3 or tln1 encoding talin-1
, we tested the individual roles of these two proteins in neutrophil slow rolling and arrest mediated by lfa-1 ( lefort et al . , 2012 ) .
we reasoned that since different conformations of lfa-1 are induced by stimulating either the psgl-1 or cxcr2 signaling cascades , it would be possible to separately test whether talin-1 and kindlin-3 were involved in lfa-1 extension versus headpiece opening .
we observed that lfa-1-dependent neutrophil slow rolling on e - selectin / icam-1 substrates in flow chambers and on inflamed endothelium in vivo was impaired in talin-1-deficient neutrophils , but was unaffected by knockout of kindlin-3 .
in contrast , both talin-1 and kindlin-3 were required for cxcl1-stimulated rapid neutrophil arrest and soluble icam-1 binding to lfa-1 , indicating an important role for these co - activators in reaching the high affinity state of lfa-1 .
our findings in neutrophils were corroborated by studies measuring lfa-1 affinity states using the reporter antibodies kim127 ( robinson et al . , 1992 ) , nki - l16 ( van kooyk et al . , 1991 ) ,
mab 24 ( dransfield et al . , 1992 ) , and 2e8 ( carreno et al . ,
together , these results show that talin-1 is needed for lfa-1 extension , while kindlin-3 is involved in transition of the lfa-1 headpiece to its high affinity state .
these data are the first to demonstrate distinct functions of talin-1 and kindlin-3 in inducing specific conformers of lfa-1 .
it remains to be shown whether talin-1 plays an additional role in lfa-1 headpiece opening to reach the high affinity state , or whether lfa-1 extension is a prerequisite for headpiece opening in the context of chemokine - induced neutrophil arrest .
furthermore , previous studies of cultured effector t lymphocytes derived from lad - iii patients suggest that kindlin-3 is needed for lfa-1 extension in the context of chemokine stimulation ( manevich - mendelson et al . , 2009 ) .
it is apparent that there are diverse mechanisms among the various modalities of integrin activation in leukocytes .
what do these new insights into talin-1 and kindlin-3 regulation of lfa-1 structure tell us about the mechanisms of inside - out activation ?
as shown for iib3 in a reconstituted system , the talin head domain ( but not intact , autoinhibited talin ) is sufficient to induce extension of the integrin extracellular domain ( ye et al . , 2010 ) .
several studies have shown that talin directly disrupts the basal association of integrin tmds by contacting the salt bridge between the and chains close to the inner membrane leaflet ( wegener et al . , 2007 ; anthis et al . , 2009 ) .
talin is likely involved in further rearrangement of the tmds , as mutations that disrupt the / salt bridge do not fully overcome the requirement for talin in integrin activation ( tadokoro et al .
, 2003 ; wegener et al . , 2007 ) . that kindlin-3 was not required for neutrophil slow rolling mediated by the extended / closed conformer of lfa-1 ( lefort et al . , 2012 )
. however , structural studies of lfa-1 suggest that the tmds and lower legs are separated even further in high affinity than in intermediate affinity lfa-1 ( nishida et al . , 2006 ) . thus , kindlin-3 may be involved in inducing swingout of the hybrid domain by causing a further separation of the tmds .
it is also possible that kindlin-3 mediates the indirect linkage of the 2 integrin tail to the actin cytoskeleton through a scaffolding function , thus providing an anchor for force - induced conversion of ligated integrins to the high affinity state .
it was recently found that lfa-1-mediated neutrophil slow rolling is not dependent on the linkage of lfa-1 to the actin cytoskeleton ( shao et al . , 2012 ) , but chemokine - stimulated arrest of lymphocytes ( shamri et al . , 2005 ) and neutrophils ( shao et al . , 2012 )
these studies support the idea that lfa-1 extension and headpiece opening are distinctly regulated processes , and that kindlin-3 may be involved in the transition to a high affinity state through an indirect interaction with the actin cytoskeleton .
kindlins have been shown to bind to several actin - binding proteins and complexes , including integrin - linked kinase ( mackinnon et al . , 2002 ) , migfilin ( tu et al . , 2003 ) , focal adhesion kinase , and -actinin ( has et al . , 2009 ) .
the expression and role of these adaptor molecules in lfa-1 activation will need to be tested in leukocyte arrest under flow conditions .
the distinct roles of talin-1 and kindlin-3 in neutrophil slow rolling and arrest raise several questions about the two signaling pathways that stimulate these separate behaviors .
clearly , lfa-1 engages icam-1 during slow rolling interactions induced by psgl-1 signaling . if chemokines directly stimulate only lfa-1 extension and not conversion to the high affinity state , why does cxcr2 engagement lead to neutrophil behavior qualitatively distinct from the psgl-1 pathway that has also been shown to induce lfa-1 extension ? likewise , if force on the lfa-1/icam-1 bond results in affinity maturation to the long - lived state , how are neutrophils able to use lfa-1 for rolling interactions that require a transient bond with a fast off - rate ?
structural communication between the i and i domains through the 7 helix is needed for headpiece opening ( shimaoka et al .
perhaps kindlin-3 provides a permissive signal or induces a structural rearrangement that allows coupling of the i and i domains , such as swingout of the hybrid domain .
we speculate that kindlin-3 may be actively excluded from binding to the 2 cytoplasmic tail after inside - out activation of lfa-1 by the psgl-1 signaling pathway and this prevents the transition to the open headpiece and neutrophil arrest .
this could occur by promoting the binding of a competing molecule to the kindlin-3 binding npkf site on the 2 tail , or by regulating phosphorylation of threonine residues that also contribute to kindlin-3 binding ( ma et al .
, 2008 ) . indeed , t cell receptor signaling affects the phosphorylation of 2t758 and promotes binding of 14 - 3 - 3 proteins and lfa-1 activation ( fagerholm et al . , 2005 ; gronholm et al . ,
a potential alternative mechanism for lfa-1 extension resulting in distinct slow rolling and arrest behaviors after psgl-1 and cxcr2 signaling , respectively , may be that chemokines also stimulate an increase in lfa-1 mobility in the membrane ( constantin et al . , 2000 ) that could lead to ligand - driven lfa-1 clustering and subsequent firm adhesion .
this mechanism would require that lfa-1 ligands like icam-1 are pre - clustered on endothelial cells ( barreiro et al . , 2008 ) , because integrin clustering is most likely a post - ligand binding event ( kim et al . , 2004 ) . in the case of slow rolling , if lfa-1 is immobile following psgl-1 engagement then clustering may be disfavored and the force on individual lfa-1/icam-1 bonds could be sufficiently high so that lfa-1 resides in the slip bond regime with short enough lifetimes ( high koff ) to support rolling behavior .
clearly , much remains to be tested to uncover the mechanisms regulating lfa-1 structure and affinity on the surface of leukocytes .
lfa-1 plays important roles in the trafficking of multiple leukocyte subsets during the immune response .
at least two distinct signaling pathways impact the adhesive function of lfa-1 by altering its structure , and thus affinity for extracellular ligands .
our understanding of how cellular factors regulate lfa-1 conformation by binding to its short cytoplasmic tails has evolved as kindlin-3 and talin-1 have been identified as major and distinct players .
the concepts explaining how structural rearrangements are propagated through the tmds and integrin legs to the ligand - binding headpiece are being refined .
it has recently become clear that force plays an integral role in these processes , and future work will be aimed at determining the molecular requirements and mechanisms of lfa-1 affinity regulation by force in rolling and arresting leukocytes .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | lymphocyte function - associated antigen-1 ( lfa-1 ) is a heterodimeric integrin consisting of l ( gene name , itgal ) and 2 ( gene name , itgb2 ) subunits expressed in all leukocytes .
lfa-1 is essential for neutrophil recruitment to inflamed tissue .
activation of lfa-1 by chemokines allows neutrophils and other leukocytes to undergo arrest , resulting in firm adhesion on endothelia expressing intercellular adhesion molecules ( icams ) . in mice ,
cxcr2 is the primary chemokine receptor involved in triggering neutrophil arrest , and it does so through inside - out activation of lfa-1 .
cxcr2 signaling induces changes in lfa-1 conformation that are coupled to affinity upregulation of the ligand - binding headpiece ( extended with open i domain ) .
unlike nave lymphocytes , engagement of p - selectin glycoprotein ligand-1 ( psgl-1 ) on neutrophils stimulates a slow rolling behavior that is mediated by lfa-1 in a distinct activation state ( extended with closed i domain ) . how inside - out signaling cascades
regulate the structure and function of lfa-1 is being studied using flow chambers , intravital microscopy , and flow cytometry for ligand and reporter antibody binding . here , we review how lfa-1 activation is regulated by cellular signaling and ligand binding .
two ferm domain - containing proteins , talin-1 and kindlin-3 , are critical integrin co - activators and have distinct roles in the induction of lfa-1 conformational rearrangements .
this review integrates these new results into existing models of lfa-1 activation . |
airway acidification has been shown to be attained by either the exogenous way , that is , the microaspiration of acid contents into the airway during gastroesophageal reflux and inhalation of low ph pollutant aerosol , or the endogenous way , that is , ischemia and inflammation of the airways in inflammatory diseases , such as asthma , cystic fibrosis , and chronic obstructive pulmonary disease ( copd ) [ 13 ] . in ischemic and inflammatory situations ,
, it has been reported that airway ph reaches 5.2 to 7.1 , depending on the severity : ph is normalized with corticosteroid therapy . although alteration of airway ph may serve an innate host defense capacity , that is , inhibiting the survival of bacteria in an acidic environment , it is also implicated in the pathophysiology of obstructive airway diseases .
thus , exposure to acids evokes a cough , bronchoconstriction , airway hyperreactivity ( ahr ) , and microvascular leakage and stimulates mucus production .
however , molecular mechanisms underlying the extracellular acidic ph - induced actions in the airways have not been fully understood . in the present review ,
we discuss the proton - sensing mechanisms , focusing on proton - sensing ionotropic receptors , such as transient receptor potential vanilloid-1 ( trpv1 ) and acid - sensing ion channels ( asics ) , and metabotropic ovarian cancer g protein - coupled receptor 1 ( ogr1)-family g protein - coupled receptors ( gpcrs ) , in the airway inflammation and ahr in asthma and respiratory diseases .
the mammalian transient receptor potential ( trp ) superfamily of nonselective cation channels encompasses 28 isotypes and is divided into six subfamilies , that is , trpc , trpv , trpm , trpa , trpp , and trpml .
these channels are expressed in neurons and a wide range of cell types in many biological systems [ 46 ] .
trp channels have six putative transmembrane domains and a pore - forming loop between the fifth and sixth segments .
they are thought to be composed of homo- or heterotetramers . among them , capsaicin - sensitive trpv1 is activated by a diverse range of chemical and noxious stimuli , including protons [ 57 ] .
trpv1 senses relatively strong acidic ph of 4 to 5 through glutamic acid in the extracellular domain of the channel ( figure 1 ) .
trp channels other than trpv1 , including trpa1 , trpv4 , and trpm8 , are expressed in the respiratory system and involved in the regulation of airway functions [ 810 ] ; however , whether protons practically trigger their channel activation remains unknown .
asics are proposed to assemble as tetramers with homomeric or heteromeric subunits ; each subunit consists of two transmembrane domains ( figure 1 ) .
six asic subunit proteins , encoded by four genes , have been identified : asic1a , asic1b , asic2a , asic2b , asic3 , and asic4 .
recent studies have demonstrated that asics activated by acidic ph play an important role in a wide range of physiological and pathophysiological processes such as nociception , mechanosensation , synaptic plasticity , and acidosis - mediated neuronal injury .
histidine , glutamic acid , and aspartic acid may determine a broad range of optical ph of 4 to 7 for activation , depending on the subtypes [ 4 , 13 , 14 ] .
in addition to trpv1 and asics , there is increasing evidence that further acid - sensitive ion channels are involved in monitoring acidosis .
these include trp family ion channels other than trpv1 , including trpv4 , trpc4 , trpc5 , and trpp2 ( pkd2l1 ) ; two - pore domain k ( k2p ) channels ; ionotropic purinoceptors ( p2x ) ; inward rectifier k channels ; voltage - activated k channels ; l - type ca channels ; hyperpolarization - activated cyclic nucleotide gated channels ; gap junction channels ; and cl channels .
although most of these channels seem to be expressed and play important roles in the respiratory system , their extracellular proton - sensitivity has not , to our knowledge , been demonstrated for specific airway responses [ 10 , 1517 ] .
recent studies suggest that ogr1-family gpcrs , including ogr1 ( gpr68 ) , gpr4 , and t cell - death associated gene 8 ( tdag8 or gpr65 ) , also sense extracellular protons and , thereby , stimulates a variety of cellular activities through several types of g proteins [ 1821 ] ( figure 1 ) .
ogr1-family gpcrs were previously described as gpcrs for lysolipids , such as sphingosylphosphorylcholine [ 1921 ] .
, however , first discovered that ogr1 and gpr4 sense extracellular protons and are coupled to gq and gs , leading to activation of the phospholipase c / ca signaling pathway and the adenylyl cyclase / camp signaling pathway , respectively .
later it was found that tdag8 similarly senses extracellular protons , leading to the activation of the camp signaling pathway [ 22 , 23 ] .
ogr1-family gpcrs sense weak acidic to weak alkaline ph of 6 to 8 through histidine residues [ 18 , 19 , 24 , 25 ] .
g2a is also classified in this receptor family and is expressed by a broad range of immunoregulatory cell types , including macrophages , dendritic cells , neutrophils , mast cells , and t and b cells , and it is suggested to play an important role in innate and adaptive immunity . however , the proton sensitivity of the receptor is very small and its role as a proton sensor has been questioned . in the present review , therefore , we do not focus further on g2a .
ogr1-family gpcrs are expressed in many cell types localized in the airways , and the cases in which their roles are demonstrated are summarized in figure 2 .
the proton - sensing trpv1 channel and/or asics in sensory nerves have first been proposed to be involved in acidic ph - induced airway responses [ 2 , 28 , 29 ] .
it has been established that trpv1 on capsaicin - sensitive primary sensory neurons plays an important role in nociception and transmission of pain as a sensor of noxious stimuli [ 2 , 4 , 30 ] .
the activation of trpv1 on the sensory neurons by irritant compounds , including capsaicin and citric acid , generates reflex responses that , in turn , stimulate the release of tachykinins from the terminals of the sensory nerves , causing cough and bronchoconstriction [ 2 , 5 , 29 ] . the possibility that the afferent signals and neuropeptide release are coupled at the same nerve endings has also been proposed .
the neurogenic role of trpv1 was based mainly upon experiments with selective agonists and antagonists .
for example , capsazepine , a trpv1-selective antagonist , and sr48968 , a selective nk2 receptor antagonist , blocked citric acid inhalation - induced bronchoconstriction in guinea pig airways in vivo .
the role of trpv1 in ahr has recently been demonstrated by the finding that oral administration of trpv1-specific antagonists significantly attenuated ahr in the asthma model of ova - sensitized guinea pigs .
in addition to trpv1 , involvement of asics in acidic ph - induced ahr was suggested by using selective asic inhibitors in the guinea pig tracheal rings ex vivo .
these results suggest that bronchoconstriction and ahr under acidic environments are indirectly mediated by sensory neurons through proton - sensing channels , such as trpv1 and asics [ 6 , 30 ] .
in fact , no report has shown that trpv1 and asics are expressed and are directly functioning in asm cells , although vascular smooth muscle cells seem to express trpv1 and asics .
the previous studies , however , did not rule out the direct actions of acidic ph on asm cells .
. showed that extracellular acidification stimulates mrna expression and protein production of il-6 , a proinflammatory cytokine , in association with the phosphorylation of extracellular signal - regulated kinase ( erk ) and p38mapk , in human asm cells .
they also showed that extracellular acidification induced an increase in intracellular ca concentration ( [ ca]i ) , which was accompanied by asm cell contraction .
acidification also induced expression of connective tissue growth factor ( ctgf ) , a critical factor involved in the formation of extracellular matrix proteins and , hence , airway remodeling .
tgf--induced ctgf expression was also enhanced by acidic ph . in asm cells , ogr1 is expressed at by far the highest levels among proton - sensing gpcrs .
the knockdown of ogr1 and gq with their specific small interfering rnas and an inhibition of gq with ym-254890 attenuated the acidification - induced actions [ 35 , 37 ] .
these results suggest that extracellular acidification stimulates ca mobilization , inflammatory cytokine il-6 and ctgf production , and contraction through ogr1/gq in human asm cells ( figure 2 ) .
the role of ogr1 in ahr is supported by recent findings with rgs2- and rgs5-deficient mice .
rgs2 and rgs5 are gtpase - activating proteins that inactivate gq stimulated by gq - coupled receptors , including ogr1 .
. showed that rgs2 deficiency caused spontaneous ahr in response to methacholine without any prior antigen sensitization / challenge .
they also showed that the loss of rgs2 augmented ca mobilization and the contraction of asm cells , increased asm mass , and stimulated asm cell growth via erk and phosphatidylinositol 3-kinase ( pi3k ) pathways .
these results are highly consistent with those of in vitro studies with human asm cells [ 35 , 36 ] .
interestingly , asthma patients display lower expression of rgs2 in epithelial cells and asm cells in lung and circulating monocytes .
rgs5-deficient mice also showed spontaneous ahr , even without antigen sensitization in association with enhanced ca mobilization , erk activation , and asm cell contraction . in both cases , however , appreciable infiltration of inflammatory cells in the lung and production of cytokines , such as il-4 and il-5 , was not observed [ 38 , 39 ] .
these results suggest that t - cell activation is not always required for gpcr - mediated asthma pathophysiology .
liu et al . have recently shown that an acidic ph of 6.4 stimulates mucin5ac ( muc5ac ) secretion in the human bronchial epithelial cell line ( 16hbe ) through the ogr1/gq / phospholipase c pathway ( figure 2 ) .
knockdown of ogr1 and gq expression with small interfering rnas inhibited acidification - induced increases in [ ca]i and mucin production .
similarly , overexpression of rgs2 protein attenuated acidic ph - induced cellular responses , whereas knockdown of rgs2 slightly but significantly enhanced these responses .
these results suggest that airway acidification induces mucin production through the ogr1 and gq , of which activation can be regulated by rgs2 .
the same group has also shown that trpv1 is expressed and citric acid ( ph 5.0)-induced mucin secretion is mediated by ca influx via trpv1 in the same cells .
these results suggest that epithelial cells utilize different proton - sensing machineries depending on the acidity of microenvironments ; ogr1-family gpcrs sense mild alkaline to acidic ph of 8 to 6 , whereas trpv1 senses more acidic ph of 6 to 4 , as described above .
gpr4 has been shown to be expressed in endothelial cells and to be activated by extracellular acidification , leading to accumulation of camp , which may be relevant to normal blood vessel formation .
chen et al . have recently shown that activation of gpr4 by acidosis increases endothelial cell adhesion of monocytes in association with the expression of adhesion molecules , such as vcam-1 and icam-1 , through the camp / epac pathway . since vcam-1 on the endothelium
has been shown to mediate eosinophil adhesion and transmigration , this suggests that gpr4 is involved in the perivascular accumulation of eosinophils in the lung with inflammatory asthma ( figure 2 ) .
however , it remains unknown whether gpr4 deficiency affects eosinophilia in the airways with asthma .
have reported that acidic ph increased eosinophil viability due to an inhibition of apoptosis , which was associated with an increase in camp accumulation .
the acidic ph - induced increase in cell viability was attenuated by the adenylyl cyclase inhibitor and , in contrast , the phosphodiesterase inhibitor , the cell - permeable camp analog , and forskolin mimicked the acidic ph effect . the acidification - induced increase in eosinophil viability and camp accumulation
these results suggest that eosinophil viability is increased in acidic microenvironments through the tdag8/camp pathway ( figure 2 ) .
however , the mechanism by which camp inhibits eosinophil apoptosis remains unknown . in macrophages , tdag8 and ogr1 are expressed .
extracellular acidification inhibited lipopolysaccharide ( lps)-induced tnf- and il-6 protein and mrna production in mouse peritoneal macrophages .
the inhibitory action on cytokine production by acidic ph was significantly attenuated in macrophages from tdag8-deficient mice but not in those from ogr1-deficient mice .
further characterization revealed that acidic ph inhibited proinflammatory cytokine production through the tdag8/gs / camp / pka signaling pathway in mouse macrophages .
the expression of tdag8 was increased by glucocorticoid in the macrophage , which was associated with the enhancement of acidification - induced inhibition of tnf- production .
thus , tdag8 seems to be indirectly involved in glucocorticoid - induced anti - inflammatory actions .
extracellular acidification has been shown to induce human neutrophil activation , inducing an increase in [ ca]i , a shape - changing response , upregulation of the expression of cd18 , an inhibition of apoptosis , and enhancement of agonist - induced h2o2 production .
these acidic ph - induced responses are accompanied by the activation of akt and erk . on the other hand ,
although intracellular acidification was supposed to , in part , explain the acidic ph - induced actions [ 48 , 50 ] , the precise proton - sensing mechanisms remained largely unknown .
murata et al . have shown that acidic ph inhibited fmlp- and c5a - induced superoxide anion production .
the acidic ph effect was mimicked by camp increasing agents and attenuated by a pka inhibitor . moreover ,
these data suggest that tdag8 may mediate extracellular acidification - induced inhibition of o2 production through camp .
thus , tdag8 is coupled to anti - inflammatory camp signaling pathways in macrophages and neutrophils .
consistently with an anti - inflammatory role , tdag8-deficient mice showed exacerbation of anti - type ii collagen antibody - induced arthritis , in which macrophages and neutrophils have been shown to play a critical role .
however , it remains uncharacterized whether the anti - inflammatory actions of tdag8 in macrophages and neutrophils are involved in the pathophysiology of airway inflammation of asthma .
geffner and his colleagues have shown that extracellular acidification ( ph 6.5 ) stimulates internalization of antigens ; upregulated the expression of cell surface proteins , including cd11c , mhc class ii , cd40 , and cd86 , involved in antigen presentation ; and promoted an efficient mhc class i - restricted presentation of antigen peptides in dendritic cells ( dcs ) of c57bl/6 mice .
antigen - pulsed dcs under acidic ph showed an improved efficacy for inducing both specific cd8 cytotoxic t lymphocytes and specific antibody responses in vivo .
they further characterized acidic ph effects using human dcs and found that the transient exposure of human dcs to ph of 6.5 markedly increases several costimulatory proteins and improves the t - cell priming ability of dcs , which was associated with a dramatic increase in p38mak - dependent il-12 production .
dc maturation by acidic ph stimulated the production of ifn- , but not of il-4 , by antigen - specific cd4 t cells .
these results suggest that extracellular acidification may contribute to the initiation of adaptive immune responses by dcs , favoring the development of the th1 phenotype in humans .
basu and srivastava suggested that trpv1 is expressed in mouse dcs and involved in their maturation .
trpv1 expression was not confirmed and either capsaicin or acidic ph failed to elicit a change in [ ca]i or the membrane current in mouse dcs .
recent studies have shown that trpv1 is expressed in human dcs ; however , in this case , the channel seems to inactivate rather than activate their maturation .
asic1 , asic2 , and asic3 are expressed in mouse dcs , and selective inhibitors for asics , such as amiloride and nonsteroidal anti - inflammatory drugs ( nsaids ) , inhibited acidic ph 6.5-induced expression of cell - surface molecules cd11c , mhc class ii , cd80 , and cd86 .
mouse dcs express ogr1-family receptors , including ogr1 ; the functional expression of ogr1 was confirmed by the extracellular acidic ph- and ogr1-dependent increase in [ ca]i .
ova - sensitized dcs from ogr1-deficient mice showed the reduction in the expression of ccr7 , a chemokine receptor for mature dcs , and the migration responses to ccl19 and ccl21 , ligands for ccr7 , as compared with those from wild - type mice .
thus , ogr1 seems to be functioning in the migratory process of dcs to draining lymph nodes ( see the next section ) .
although the contribution of trpv1 to acidic ph - induced cough and bronchoconstriction is well demonstrated as discussed , it remains to be proven whether trpv1-mediated neurogenic inflammation plays a central role in asthma and other respiratory diseases .
the inarticulate conclusion is based on the lack of obvious effects by gene targeting experiments ; trpv1 deficiency did not attenuate or rather enhanced the airway inflammation and ahr as induced by lps , antigens [ 61 , 62 ] , and cigarette smoke in trpv1 knockout mice .
the anti - inflammatory role of trpv1 might be , in part , explained by the release of anti - inflammatory somatostatin from the sensory nerve terminals in response to trpv1 stimulation .
regardless , the role of trpv1 in the airway inflammation and ahr remains to be established in mice .
however , uncertain results with mice may be partly explained by the strain difference : the previous studies [ 6063 ] used c57bl/6 .
have recently shown that trpv1 knockdown with sirna attenuates il-13- and antigen - induced asthmatic features including airway inflammation and ahr in balb / c mice .
in addition to proton - sensing channels , proton - sensing gpcrs also play an important role in the pathophysiology of asthma . as described above , kottyan et al .
have shown that tdag8 deficiency causes the stimulation of eosinophil apoptosis and , thereby , reduces airway eosinophilia in ova- and aspergillus fumigatus - sensitization models of mice in vivo .
they have also shown increased expression of tdag8 in lungs from ova - sensitized mice and in nasal brushing samples from pediatric asthma patients .
unfortunately , whether ahr and airway inflammation as cardinal features of asthma are modulated by tdag8 in vivo has not been examined ; however , it is noted that il-13 production , which plays a role in these processes , was not affected by tdag8 deficiency .
the findings of the expression of proton - sensing ogr1 in dc , a critical cell for antigen recognition and its presentation to t cells , and the involvement of ogr1 in the dc migration process suggest participation of ogr1 in the pathophysiology of allergic asthma . indeed , ogr1-deficient mice are resistant to the cardinal features of asthma , including airway eosinophilia , ahr , and goblet cell metaplasia , in association with a remarkable inhibition of the production of th2 cytokines , including il-4 , il-5 , and il-13 , and ova - specific ige in an ova - induced asthma model . intratracheal transfer to wild - type mice of ova - primed bone marrow - derived dcs from ogr1-deficient mice developed lower ahr and eosinophilia as compared with the transfer of those from wild - type mice , which was associated with lower migratory activity to the peribronchial lymph nodes in ogr1-deficient dcs than in wild - type dcs .
these results suggest that stimulation of ogr1 on dcs is critical for the early processes , that is , migration to lymph nodes and initiation of th2 polarization , and , thereby , induces eosinophilia , airway inflammation , and ahr .
since ogr1 is expressed in structural cells , including asm cells and epithelial cells , and functions in a variety of cell - specific responses , the reduction of ahr and goblet hyperplasia in ogr1-deficient mice may be partly attributed to the reduction of acidification - induced constriction of asm cells [ 35 , 36 ] and mucin production in epithelial cells .
finally , it should be noted that acidic ph modulates dcs leading to th1 polarization in humans as described , which contrasts with the role of ogr1 in th2 polarization in mice .
the reason for the difference in the fate of t cells , that is , th1 or th2 , is currently unknown . dcs express proton - sensing gpcrs other than ogr1 , and ogr1-family gpcrs are expressed in neutrophils and macrophages as well .
therefore , differences in experimental conditions , for example , species , stimulants , antigens , and ph , may modify the state of dc activation and dc - t cell interaction , making nave t cells polarize to th1 , th2 , th17 , or other phenotypes
. how change in the ph microenvironment modulates dc function and t cell polarization warrants further study .
proton - sensing channels , such as trpv1 and asics , and ogr1-family gpcrs are expressed in structural cells , including asm cells and epithelial cells , and inflammatory and immune cells , including eosinophils and dcs , and play a variety of roles in airway responses , depending on the optimum ph of proton - sensing channels and gpcrs .
cough and bronchoconstriction are activated by severe acidic ph of 4 to 5 and are mainly mediated by ph - sensing channels through sensory neurons , although ph - sensing ogr1 on asm cells may also be involved in the cell constriction . at mild alkaline or mild acidic ph of more than 6
, however , ogr1-family gpcrs may be the main receptors involved in the regulation of airway responses under pathophysiological situations , such as allergic asthma .
thus , ionotropic and metabotropic proton - sensing receptors may be therapeutic targets for inflammatory and ischemic diseases , such as asthma , for which drugs that are more specific and have fewer side effects are still required . | an acidic microenvironment has been shown to evoke a variety of airway responses , including cough , bronchoconstriction , airway hyperresponsiveness ( ahr ) , infiltration of inflammatory cells in the lung , and stimulation of mucus hyperproduction . except for the participation of transient receptor potential vanilloid-1 ( trpv1 ) and acid - sensing ion channels ( asics ) in severe acidic ph ( of less than 6.0)-induced cough and bronchoconstriction through sensory neurons ,
the molecular mechanisms underlying extracellular acidic ph - induced actions in the airways have not been fully understood .
recent studies have revealed that ovarian cancer g protein - coupled receptor 1 ( ogr1)-family g protein - coupled receptors , which sense ph of more than 6.0 , are expressed in structural cells , such as airway smooth muscle cells and epithelial cells , and in inflammatory and immune cells , such as eosinophils and dendritic cells .
they function in a variety of airway responses related to the pathophysiology of inflammatory diseases , including allergic asthma . in the present review ,
we discuss the roles of ionotropic trpv1 and asics and metabotropic ogr1-family g protein - coupled receptors in the airway inflammation and ahr in asthma and respiratory diseases . |
healthcare expenditure is substantial since syncope is a common symptom with a variety of potential underlying causes leading to patient visits to physicians , the emergency department , and to hospitalizations after syncope with or without associated trauma .
syncope was the fifth most common cause for an emergency department visit in the uk according to the hospital episode statistics 201112 .
the diagnostic tests undertaken to find the cause of syncope are important drivers of the cost .
updated guidelines and/or consensus documents present evidence and recommendations on which investigations are most relevant and likely to lead to a diagnosis while at the same time less effective measures can be replaced .
guidelines are dynamic documents that are updated as new research produces evidence that justifies changes in recommendations .
however , the dissemination and penetration of the messages of guidelines and expert consensus documents are far from satisfactory and clinical practice adapts slowly .
previous studies have reported substantial costs of evaluating syncope patients and have suggested the adoption of a more systematic patient care pathway . the observational picture ( place of
reveal in the care pathway and treatment of patients with unexplained recurrent syncope ) registry reported that an apparent lack of structured care pathways leads to significant over - investigation of unexplained syncope before patients received an implantable loop recorder ( ilr ) .
reasonably , this influenced the cost of evaluation of the patients , partly because tests could be repeated many times and also because patients undergo various expensive diagnostic tests with a low diagnostic yield .
an earlier rather than a later ilr implant reduced the number of preimplant tests without reducing the diagnostic yield .
the aims of the present microeconomic analysis based on the large , international , picture registry were to provide costs of diagnostic tests .
the design , methods , and main results of the picture registry have been previously reported .
briefly , 570 patients with recurrent , unexplained syncope were enrolled and the interpretation of the term
their median number of syncope episodes was 4 ( inter - quartile range , iqr , 25 ) , and 3 ( iqr 24 ) of them occurred during the 2 years preceding the ilr implant .
the median time interval between the first and last episode was 2 years ( iqr 04 ) .
the purpose was to document the care pathway in patients eventually implanted with an ilr as well as to examine the diagnostic yield of the ilr .
the study protocol complied with the declaration of helsinki and was approved by the relevant locally appointed ethics committee .
an important finding of picture was that a large number of diagnostic tests were performed , many of them despite a low probability of providing a diagnosis .
the present analysis focused on the preimplant diagnostic evaluation , while other costs , such as specialist visits in relation to syncope , admissions to the emergency room , and/or hospitalizations or those caused by severe trauma associated with syncope ( defined as fracture or injury with bleeding ) , were not assessed .
the costs of diagnostic tests were collected from a medium - sized uk university hospital since only few costs of diagnostic tests were available from data sets such as the uk nhs reference costs .
the costs of diagnostic tests from the micro - costing study and the nhs reference costs 2009/10 , when available , were similar , as summarized in table 1 .
the median and mean costs of diagnostic tests per patient were calculated as the total number of tests recorded in the picture registry multiplied by the estimated cost of the test in the micro - costing study .
table 1costs of teststestcostspatients tested ( % ) picture studysyncope costing studynational reference costs 2009/10standard electrocardiogram373398echocardiography473286basic laboratory tests2486ambulatory ecg monitoring1186067in - hospital ecg monitoring426055exercise testing787552orthostatic blood pressure movements1148neurological or psychiatric evaluation11147mri or ct scan1497724447carotid sinus massage5536eeg18039tilt test10035electrophysiology testing139225coronary angiography128523external loop recording14712other tests = outpatient consultation1639adenosine triphosphate ( atp ) test703 the micro - costing study of each individual test was based on a combination of existing hospital costs and original data collection . for each test , direct and indirect costs as well as overhead costs were calculated .
this involved building an understanding of the typical process of conducting a diagnostic test as well as the associated level of direct resource input regarding the level of direct staff input based on the staff grade and time spent on the key tasks within a standard procedure , any relevant consumables , e.g. drugs and ecg electrodes , and the equipment used .
five cost components were identified ( figure 1 ) : ( i ) labour ( direct pay costs ) . a 21% on - cost was added to reflect national insurance and superannuation contributions , unless otherwise stated .
( iv ) capital equipment costs including direct clinical equipment such as machinery ( e.g. the ecg machine , ct scan , etc . ) . to understand depreciation and maintenance costs ,
the following formula was used : average cost per test = annual depreciation or maintenance cost / total no . of tests per annum .
( v ) corporate overhead costs were added based on the current level of contribution required within the hospital , which according to the selected hospital finance department was 30% .
this contribution covered the rates , rents , and other corporate functions such as finance , human resources , and information technology , and was comparable to the percentage of other nhs hospitals .
the cost of mri / ct was calculated as half the sum of an mri and a ct , since the study data did not differentiate between the two and we do not know the proportion of mri and ct .
figure 1the five cost components of a diagnostic test are shown using the example of a standard ecg .
the five cost components of a diagnostic test are shown using the example of a standard ecg .
patient demographics , concomitant diseases , and the first and last specialist who were visited before the ilr implant are presented in table 2 .
as many as 70% of the patients had been hospitalized for syncope , 36% had suffered severe trauma defined as fracture and/or bleeding .
table 2patient demographicsall patients ( n = 570)early implant ( n = 128)late implant ( n = 386)clinical features of syncope hospitalized because of syncope370 ( 70.0%)68 ( 53.1%)291 ( 75.4% ) any severe trauma ( fractures , haemorrhage , etc.)204 ( 35.8%)29 ( 22.7%)151 ( 39.1%)clinical features of last episode position at the beginning of the episode supine52 ( 9.0%)12 ( 9.4%)37 ( 9.6% ) sitting154 ( 27.0%)28 ( 21.9%)108 ( 28.0% ) standing274 ( 48.0%)56 ( 43.8%)192 ( 49.7% ) unknown83 ( 15.0%)29 ( 22.7%)45 ( 11.7% ) activity at the beginning of the episode rest294 ( 52.0%)42 ( 32.8%)223 ( 57.8% ) during effort144 ( 25.0%)44 ( 34.4%)89 ( 23.1% ) after effort28 ( 5.0%)7 ( 5.5%)15 ( 3.9% ) unknown97 ( 17.0%)34 ( 26.6%)53 ( 13.7% ) symptoms during the episode muscle spasms ( one sided)8 ( 1.4%)4 ( 3.1%)2 ( 0.5% ) muscle spasms ( two sided)19 ( 3.3%)4 ( 3.1%)15 ( 3.9% ) grand mal10 ( 1.8%)2 ( 1.6%)2 ( 1.3% ) other muscle spasms14 ( 2.5%)2 ( 1.6%)10 ( 2.6% ) transpiration73 ( 12.8%)19 ( 14.8%)47 ( 12.2% ) cyanosis19 ( 3.3%)1 ( 0.8%)13 ( 3.4% ) angina pectoris23 ( 4.0%)5 ( 3.9%)14 ( 3.6% ) palpitations76 ( 13.3%)21 ( 16.4%)49 ( 12.7% ) dizziness163 ( 28.6%)56 ( 43.8%)93 ( 24.1% ) dyspnoea33 ( 5.8%)6 ( 4.7%)24 ( 6.2% ) fatigue95 ( 16.7%)21 ( 16.4%)65 ( 16.8%)comorbidity hypertension277 ( 48.6%)57 ( 44.5%)187 ( 48.4% ) diabetes84 ( 14.7%)16 ( 12.5%)61 ( 15.8% ) parkinson disease2 ( 0.4%)1 ( 0.8%)1 ( 0.3% ) transient ischaemic attack ( tia)20 ( 3.5%)6 ( 4.7%)11 ( 2.8% ) stroke37(6.5%)5 ( 3.9%)25 ( 6.5% ) other neurological disorder36 ( 6.3%)4 ( 3.1%)16 ( 4.1% ) structural heart disease cardiomyopathy18 ( 3.2%)2 ( 1.6%)12 ( 3.1% ) valvular heart disease30 ( 5.2%)5 ( 3.9%)24 ( 6.2% ) coronary artery disease84 ( 14.7%)16 ( 12.5%)62 ( 16.1% ) other29 ( 5.1%)4 ( 3.1%)18 ( 4.7%)care pathway profession of first consulted specialist hospital ( n , % ) cardiologist232 ( 41.0%)67 ( 52.3%)151 ( 39.1% ) electrophysiologist12 ( 2.0%)3 ( 2.3%)9 ( 2.3% ) cardiothoracic surgeon1 ( 0%)0 ( 0.0%)1 ( 0.3% ) specialist for internal diseases100 ( 18.0%)12 ( 9.4%)78 ( 20.2% ) emergency medicine133 ( 23%)24 ( 18.8%)95 ( 24.6% ) imaging and radiologist3 ( 1%)1 ( 0.8%)2 ( 0.5% ) neurologist63 ( 11.0%)14 ( 10.9%)35 ( 9.1% ) all specialists seen in relation to syncope general practitioner357 ( 62.6%)68 ( 53.1%)255 ( 66.1% ) cardiologist521 ( 91.4%)121 ( 94.5%)348 ( 90.2% ) electrophysiologist166 ( 29.1%)25 ( 19.5%)132 ( 34.2% ) cardiothoracic surgeon3 ( 0.5%)0 ( 0%)3 ( 0.8% ) specialist for internal diseases214 ( 37.5%)27 ( 21.1%)159 ( 41.2% ) emergency medicine207 ( 36.3%)40 ( 31.3%)145 ( 37.6% ) imaging and radiologist104 ( 18.2%)14 ( 10.9%)72 ( 18.7% ) neurologist270 ( 47.7%)41 ( 32.0%)192 ( 49.7% ) last referral general practitioner47 ( 8.2%)12 ( 9.4%)29 ( 7.5% ) cardiologist346 ( 60.7%)90 ( 70.3%)225 ( 58.3% ) electrophysiologist64 ( 11.2%)13 ( 10.2%)49 ( 12.7% ) cardiothoracic surgeon1 ( 0.2%)0 ( 0.0%)1 ( 0.3% ) specialist for internal diseases50 ( 8.8%)5 ( 3.9%)41 ( 10.6% ) emergency medicine27 ( 4.7%)8 ( 6.3%)13 ( 3.4% ) imaging and radiologist2 ( 0.4%)0 ( 0%)1 ( 0.3% ) neurologist23 ( 4.0%)0 ( 0%)18 ( 4.7%)the classification
early implant vs. late implant was according to the investigators assessment . in 56 patients , these data were missing . the classification
the total number of tests performed per patient is shown in figure 2a and b. as shown in the box plot and histogram , there is a lot of variation in the number of tests per patient and some patients received a large number of tests .
in fact , as many as 25% of the patients underwent more than 20 tests , whereas 10% had more than 31 tests done .
( a ) the box shows the iqr and that the middle 50% of the patients had between 9 and 20 tests .
the line is below the middle of the box illustrating that the median is smaller than the mean ( 17 ) .
the whiskers around the box are drawn at 1.5 times the iqr , with the upper whisker at 36.5 tests .
the box plot illustrates that many patients received far more than the median number of tests .
note that patients who had more than 100 tests are only shown in the box plot .
( a ) the box shows the iqr and that the middle 50% of the patients had between 9 and 20 tests .
the line is below the middle of the box illustrating that the median is smaller than the mean ( 17 ) .
the whiskers around the box are drawn at 1.5 times the iqr , with the upper whisker at 36.5 tests .
the box plot illustrates that many patients received far more than the median number of tests .
( b ) the histogram shows the tests performed by patient in more detail . note that patients who had more than 100 tests are only shown in the box plot .
the distribution of the costs per patient mirrors the findings from the distribution of tests performed ( figure 3 ) .
analogously to the test data , the costs per patient varies a lot , and the medial 50% of the data is in the range of 5692246 .
the median costs of diagnostic tests per patient were 1114 ( 95% ci 9951233 ) , while the mean expenditure per patient was substantially higher with 1613 ( 95% ci 14941732 ) .
the costs for the most expensive 10% of the patients exceeded 3539 per patient .
( a ) the distribution of the total costs shows high costs for a substantial share of patients .
the median ( 1114 ) is much smaller than the mean costs of tests ( 1613 ) .
( b ) the histogram shows the distribution of total costs per patient in more detail and illustrates the significant costs incurred for patients in whom a large number of tests were done . in the most expensive ,
( a ) the distribution of the total costs shows high costs for a substantial share of patients .
the median ( 1114 ) is much smaller than the mean costs of tests ( 1613 ) .
( b ) the histogram shows the distribution of total costs per patient in more detail and illustrates the significant costs incurred for patients in whom a large number of tests were done . in the most expensive ,
since all patients had unexplained syncope at the time of the ilr implant , the diagnostic yield of any test performed before that was zero .
table 1 summarizes the percentage of syncope patients who underwent the different diagnostic tests at least once .
the most commonly prescribed tests were a 12-lead ecg 98% , echocardiography 86% , and basic laboratory tests 86% .
the five most commonly performed tests are all included in the initial evaluation according to the guidelines . other tests included in the initial evaluation were orthostatic blood pressure movements ( 48% ) , carotid sinus massage ( 36% ) , tilt test ( 35% ) , and external loop recording ( 12% ) .
however , tests that did not belong to the initial evaluation were also commonly used , e.g. exercise test ( 52% ) , mri or ct scan ( 47% ) , neurological or psychiatric evaluation ( 47% ) , and eeg ( 39% ) . the highest costs per test were related to electrophysiology testing with 1391 per test ( the weighted cost in picture was 348 , since 25% of patients underwent the test ) , followed by coronary angiography with 1285 ( weighted cost 296 , 23% ) and electroencephalography with 180 ( weighted cost 70 , 39% ) .
neurological investigations were common , which may be explained by the fact that a neurologist or a psychiatrist was frequently consulted as the first specialist .
eleven per cent of patients were referred to a neurologist as their first specialist , and in total 47% had seen a neurologist before the ilr implant .
probably as a consequence , neurological tests , such as eeg ( 39% ) and mri / ct ( 47% ) , were commonly prescribed , also in what the investigators described as early in the investigation . in the current guidelines , such investigations
are only recommended when non - syncopal transient loss of consciousness ( t - loc ) is suspected , most importantly when epilepsy is a likely or possible reason .
similarly , the number of patients undergoing more than one eeg and/or mri / ct was high ( figure 4 ) .
figure 4the pyramide plots demonstrate that tests were often repeated and show how often they were prescribed early ( to the left of the middle line ) and late ( to the right of the middle line ) evaluation as judged by the investigators .
( a ) exercise test ; ( b ) neurological and/or psychiatric evaluation ; ( c ) electroencephalography ; and ( d ) mri or ct scan .
the pyramide plots demonstrate that tests were often repeated and show how often they were prescribed early ( to the left of the middle line ) and late ( to the right of the middle line ) evaluation as judged by the investigators .
( a ) exercise test ; ( b ) neurological and/or psychiatric evaluation ; ( c ) electroencephalography ; and ( d ) mri or ct scan .
some tests were repeated many times , meaning that sometimes even inexpensive tests , such as an ecg , contributed significantly to the costs .
for example , a standard ecg and a holter recording were very frequently repeated , and this was also the case with more expensive tests , e.g. exercise testing and tests that could be related to the frequent visits to neurologists ( figure 4 ) .
the pyramide plots in figure 4 show how often the tests were performed and whether they were performed early or late . considering the complicated care pathway for many patients
since the study was entirely observational , there was nothing in the protocol or the case record forms to inform or guide the investigator about the current ( 2004 ) guidelines .
however , the investigator was asked to indicate , by ticking a box , whether the implant , in his / her view , was performed in an initial phase of diagnostic work - up of syncope , i.e. early , or after full evaluation of the mechanism of syncope , i.e. late .
the investigators classified 22.5% of the patients as having an early implant , whereas 67.7% had a late implant and the time - point was missing in 9.8% of the 570 patients .
after the completion of the study , the early implants were compared with what the current guidelines included in the initial evaluation .
we found that the proportion of patients who only had tests within the recommended initial evaluation was low , 12% , while there were many examples of over - investigation ( figure 5 ) .
the mean cost of diagnostic tests per patient in these 12% of patients was 709 compared with 1113 ( 95% ci 9951232 ) in the entire study population .
figure 5some of the tests that are recommended in the guidelines in the ( a ) initial and the ( b ) extended evaluation of syncope .
while several tests of the initial evaluation were performed much less often than recommended , some more specific tests were performed more often and more early than recommended .
some of the tests that are recommended in the guidelines in the ( a ) initial and the ( b ) extended evaluation of syncope .
while several tests of the initial evaluation were performed much less often than recommended , some more specific tests were performed more often and more early than recommended .
the uk reference costs are the best available proxy of the true costs of an ilr implant , and in 200910 the associated reference costs were 2606 ( hrg ea03z ) , including the device and all procedure costs and overheads such as staffing , catheterization laboratory utilization , disposable material , and hospital bed costs .
the total number of tests performed per patient is shown in figure 2a and b. as shown in the box plot and histogram , there is a lot of variation in the number of tests per patient and some patients received a large number of tests .
in fact , as many as 25% of the patients underwent more than 20 tests , whereas 10% had more than 31 tests done .
( a ) the box shows the iqr and that the middle 50% of the patients had between 9 and 20 tests .
the line is below the middle of the box illustrating that the median is smaller than the mean ( 17 ) .
the whiskers around the box are drawn at 1.5 times the iqr , with the upper whisker at 36.5 tests .
the box plot illustrates that many patients received far more than the median number of tests .
note that patients who had more than 100 tests are only shown in the box plot .
the box shows the iqr and that the middle 50% of the patients had between 9 and 20 tests .
the line is below the middle of the box illustrating that the median is smaller than the mean ( 17 ) .
the whiskers around the box are drawn at 1.5 times the iqr , with the upper whisker at 36.5 tests .
the box plot illustrates that many patients received far more than the median number of tests .
note that patients who had more than 100 tests are only shown in the box plot .
the distribution of the costs per patient mirrors the findings from the distribution of tests performed ( figure 3 ) .
analogously to the test data , the costs per patient varies a lot , and the medial 50% of the data is in the range of 5692246 .
the median costs of diagnostic tests per patient were 1114 ( 95% ci 9951233 ) , while the mean expenditure per patient was substantially higher with 1613 ( 95% ci 14941732 ) .
the costs for the most expensive 10% of the patients exceeded 3539 per patient .
figure 3the total costs of diagnostic tests per patient . ( a ) the distribution of the total costs shows high costs for a substantial share of patients .
the median ( 1114 ) is much smaller than the mean costs of tests ( 1613 ) .
( b ) the histogram shows the distribution of total costs per patient in more detail and illustrates the significant costs incurred for patients in whom a large number of tests were done . in the most expensive , 10% of patients the cost exceeded 3539 per patient .
( a ) the distribution of the total costs shows high costs for a substantial share of patients .
the median ( 1114 ) is much smaller than the mean costs of tests ( 1613 ) .
( b ) the histogram shows the distribution of total costs per patient in more detail and illustrates the significant costs incurred for patients in whom a large number of tests were done . in the most expensive ,
since all patients had unexplained syncope at the time of the ilr implant , the diagnostic yield of any test performed before that was zero .
table 1 summarizes the percentage of syncope patients who underwent the different diagnostic tests at least once .
the most commonly prescribed tests were a 12-lead ecg 98% , echocardiography 86% , and basic laboratory tests 86% .
the five most commonly performed tests are all included in the initial evaluation according to the guidelines .
other tests included in the initial evaluation were orthostatic blood pressure movements ( 48% ) , carotid sinus massage ( 36% ) , tilt test ( 35% ) , and external loop recording ( 12% ) .
however , tests that did not belong to the initial evaluation were also commonly used , e.g. exercise test ( 52% ) , mri or ct scan ( 47% ) , neurological or psychiatric evaluation ( 47% ) , and eeg ( 39% ) .
the highest costs per test were related to electrophysiology testing with 1391 per test ( the weighted cost in picture was 348 , since 25% of patients underwent the test ) , followed by coronary angiography with 1285 ( weighted cost 296 , 23% ) and electroencephalography with 180 ( weighted cost 70 , 39% ) .
neurological investigations were common , which may be explained by the fact that a neurologist or a psychiatrist was frequently consulted as the first specialist .
eleven per cent of patients were referred to a neurologist as their first specialist , and in total 47% had seen a neurologist before the ilr implant .
probably as a consequence , neurological tests , such as eeg ( 39% ) and mri / ct ( 47% ) , were commonly prescribed , also in what the investigators described as early in the investigation . in the current guidelines , such investigations
are only recommended when non - syncopal transient loss of consciousness ( t - loc ) is suspected , most importantly when epilepsy is a likely or possible reason .
similarly , the number of patients undergoing more than one eeg and/or mri / ct was high ( figure 4 ) .
figure 4the pyramide plots demonstrate that tests were often repeated and show how often they were prescribed early ( to the left of the middle line ) and late ( to the right of the middle line ) evaluation as judged by the investigators .
( a ) exercise test ; ( b ) neurological and/or psychiatric evaluation ; ( c ) electroencephalography ; and ( d ) mri or ct scan .
the pyramide plots demonstrate that tests were often repeated and show how often they were prescribed early ( to the left of the middle line ) and late ( to the right of the middle line ) evaluation as judged by the investigators .
( a ) exercise test ; ( b ) neurological and/or psychiatric evaluation ; ( c ) electroencephalography ; and ( d ) mri or ct scan .
some tests were repeated many times , meaning that sometimes even inexpensive tests , such as an ecg , contributed significantly to the costs .
for example , a standard ecg and a holter recording were very frequently repeated , and this was also the case with more expensive tests , e.g. exercise testing and tests that could be related to the frequent visits to neurologists ( figure 4 ) .
the pyramide plots in figure 4 show how often the tests were performed and whether they were performed early or late . considering the complicated care pathway for many patients
since the study was entirely observational , there was nothing in the protocol or the case record forms to inform or guide the investigator about the current ( 2004 ) guidelines .
however , the investigator was asked to indicate , by ticking a box , whether the implant , in his / her view , was performed in an initial phase of diagnostic work - up of syncope , i.e. early , or after full evaluation of the mechanism of syncope , i.e. late .
the investigators classified 22.5% of the patients as having an early implant , whereas 67.7% had a late implant and the time - point was missing in 9.8% of the 570 patients .
after the completion of the study , the early implants were compared with what the current guidelines included in the initial evaluation .
we found that the proportion of patients who only had tests within the recommended initial evaluation was low , 12% , while there were many examples of over - investigation ( figure 5 ) .
the mean cost of diagnostic tests per patient in these 12% of patients was 709 compared with 1113 ( 95% ci 9951232 ) in the entire study population .
figure 5some of the tests that are recommended in the guidelines in the ( a ) initial and the ( b ) extended evaluation of syncope .
while several tests of the initial evaluation were performed much less often than recommended , some more specific tests were performed more often and more early than recommended . some of the tests that are recommended in the guidelines in the ( a ) initial and the ( b ) extended evaluation of syncope .
while several tests of the initial evaluation were performed much less often than recommended , some more specific tests were performed more often and more early than recommended .
the uk reference costs are the best available proxy of the true costs of an ilr implant , and in 200910 the associated reference costs were 2606 ( hrg ea03z ) , including the device and all procedure costs and overheads such as staffing , catheterization laboratory utilization , disposable material , and hospital bed costs .
the cost of investigation in patients with unexplained syncope varied greatly due to the number and type of tests performed per patient before the ilr implant .
while some patients underwent many tests , which were often repeated , other patients received relatively little testing .
the expenditure for some patients seems far too high , which most probably mainly reflects the lack of a structured care pathway . in this strictly observational registry
, only 12% of the patients received tests entirely within the guideline recommendations for initial investigation before receiving the ilr , which is a very important finding , since it implies that the messages and recommendations of current guidelines are not known or at least not followed .
although several previous reports agree that the economic impact of syncope management is substantial , the approaches and suggested solutions for reducing costs have varied .
the creation of structured care pathways , inside or outside specific multidisciplinary syncope units , may lead to a uniform management according to pre - set guidelines and/or improved diagnostic and treatment algorithms .
specifically , approaches leading to fewer hospitalizations may be cost - effective as well as those leading to the appropriate use of available tests , avoiding both over- and under - investigations .
overuse of diagnostic tests puts a burden on healthcare costs , but a prolonged and unsuccessful series of visits and diagnostic tests also cause patient frustration . considering that arrhythmias are common causes in patients with unexplained syncope , an ilr implant early after the initial evaluation
continuous ecg monitoring has a high chance of providing a symptom vs. rhythm correlation during the next syncope recurrence , which is crucial for determining the mechanism and guide subsequent appropriate treatment .
the diagnostic tests of the initial evaluation of unselected syncope , including a standard ecg , echocardiography , carotid sinus massage in patients older than 40 years , and orthostatic blood pressure challenge , aim at identifying the most common causes of syncope , while those who remain undiagnosed after the initial evaluation may be defined as unexplained . in a clinical study , based on guidelines only 12% of unexplained syncope patients received an eeg and 14% a head ct . while patients with unexplained syncope are more likely to have an underlying arrhythmia mechanism than unselected patients with syncope , arrhythmias causing syncope may occur at varying and often long intervals , giving standard ecgs and short - lasting ecg monitoring little chance of providing symptoms vs. ecg correlation when compared with continuous long - term ecg monitoring .
nevertheless , holter monitoring was a very frequently used and often repeated test in picture patients before the decision to implant an ilr , thus representing an inadequate use of available diagnostic tests .
an earlier ilr implant rather than repetition of already non - diagnostic tests would seem more reasonable and is consistent with the current guidelines , with a good chance of being cost - effective in comparison with unstructured conventional testing .
tests such as neurological evaluation or blood tests are only indicated when there is suspicion of non - syncope t - loc , but are nevertheless often prescribed , also very early in the evaluation , the former probably because the symptoms frequently include various signs of convulsions . when described to the physician after the event
, these symptoms might sound alarming enough to lead the early evaluation in a neurological direction , perhaps aiming to exclude or in some cases with the hope of confirming a neurological cause , e.g. epilepsy .
however , convulsions are common in syncope and the underlying mechanism is more likely to be clarified by symptom vs. ecg monitoring than with neurological tests .
this likeliness increases in patients defined as having unexplained syncope . in studies using an ilr in unexplained syncope , 80% of the diagnostic yield
was based on arrhythmias , including both brady- and tachycardias , that needed very different subsequent treatments .
in contrast , the diagnostic yield using conventional diagnostic tests was only 12.5% . in the 2004 version of the guidelines on the management of syncope
, the implant of an ilr was presented as an option after an initial evaluation .
patients who had an early implant as defined by the investigators were most often more extensively investigated than recommended in the guidelines , but there was a significant difference as opposed to what the investigators regarded as a late implant .
the observation that the diagnostic yield was similar supports that less investigation and lower costs for diagnostic tests before an earlier ilr implant can be achieved without losing diagnostic yield .
patients in picture had had a median of four syncope events during a median of 2 years before their ilr implant . during this period ,
70% were at some time hospitalized for syncope and 36% suffered severe trauma with fracture and/or bleeding .
a long time between the first healthcare contact and diagnosis may reasonably increase the risk of recurrent syncope and syncope - related hospitalization and trauma .
while brady- and tachyarrhythmias have their specific treatment , patients with syncope during sinus rhythm , e.g. with psychogenic syncope , are also an important subgroup that will benefit from a correct diagnosis and information , which can subsequently help them to manage without further visits to healthcare facilities .
as shown in our analysis of the picture study , the frequent repetition of tests and the use of specific tests early in the investigation was a poor use of healthcare resources as they did not result in a diagnosis .
at the same time , ilrs have been shown to be cost - effective in patients with unexplained syncope , implying that the higher initial costs should not discourage physicians from using an ilr when it is an option according to the current guidelines .
in addition , a miniaturized ilr has recently become available , the reveal linq , and due to its much smaller size a new insertion technique has made the implant procedure minimally invasive . as a consequence , this minimally invasive procedure can be performed also in settings outside the catheterization laboratory , thus offering possibilities to reduce the procedure costs .
although several previous reports agree that the economic impact of syncope management is substantial , the approaches and suggested solutions for reducing costs have varied .
the creation of structured care pathways , inside or outside specific multidisciplinary syncope units , may lead to a uniform management according to pre - set guidelines and/or improved diagnostic and treatment algorithms .
specifically , approaches leading to fewer hospitalizations may be cost - effective as well as those leading to the appropriate use of available tests , avoiding both over- and under - investigations .
overuse of diagnostic tests puts a burden on healthcare costs , but a prolonged and unsuccessful series of visits and diagnostic tests also cause patient frustration . considering that arrhythmias are common causes in patients with unexplained syncope , an ilr implant early after the initial evaluation has been found a reasonable approach .
continuous ecg monitoring has a high chance of providing a symptom vs. rhythm correlation during the next syncope recurrence , which is crucial for determining the mechanism and guide subsequent appropriate treatment .
the diagnostic tests of the initial evaluation of unselected syncope , including a standard ecg , echocardiography , carotid sinus massage in patients older than 40 years , and orthostatic blood pressure challenge , aim at identifying the most common causes of syncope , while those who remain undiagnosed after the initial evaluation may be defined as unexplained . in a clinical study , based on guidelines only 12% of unexplained syncope patients received an eeg and 14% a head ct .
while patients with unexplained syncope are more likely to have an underlying arrhythmia mechanism than unselected patients with syncope , arrhythmias causing syncope may occur at varying and often long intervals , giving standard ecgs and short - lasting ecg monitoring little chance of providing symptoms vs. ecg correlation when compared with continuous long - term ecg monitoring .
nevertheless , holter monitoring was a very frequently used and often repeated test in picture patients before the decision to implant an ilr , thus representing an inadequate use of available diagnostic tests .
an earlier ilr implant rather than repetition of already non - diagnostic tests would seem more reasonable and is consistent with the current guidelines , with a good chance of being cost - effective in comparison with unstructured conventional testing .
tests such as neurological evaluation or blood tests are only indicated when there is suspicion of non - syncope t - loc , but are nevertheless often prescribed , also very early in the evaluation , the former probably because the symptoms frequently include various signs of convulsions . when described to the physician after the event
, these symptoms might sound alarming enough to lead the early evaluation in a neurological direction , perhaps aiming to exclude or in some cases with the hope of confirming a neurological cause , e.g. epilepsy .
however , convulsions are common in syncope and the underlying mechanism is more likely to be clarified by symptom vs. ecg monitoring than with neurological tests .
this likeliness increases in patients defined as having unexplained syncope . in studies using an ilr in unexplained syncope , 80% of the diagnostic yield
was based on arrhythmias , including both brady- and tachycardias , that needed very different subsequent treatments .
in the 2004 version of the guidelines on the management of syncope , the implant of an ilr was presented as an option after an initial evaluation .
patients who had an early implant as defined by the investigators were most often more extensively investigated than recommended in the guidelines , but there was a significant difference as opposed to what the investigators regarded as a late implant .
the observation that the diagnostic yield was similar supports that less investigation and lower costs for diagnostic tests before an earlier ilr implant can be achieved without losing diagnostic yield .
patients in picture had had a median of four syncope events during a median of 2 years before their ilr implant . during this period ,
70% were at some time hospitalized for syncope and 36% suffered severe trauma with fracture and/or bleeding .
a long time between the first healthcare contact and diagnosis may reasonably increase the risk of recurrent syncope and syncope - related hospitalization and trauma .
while brady- and tachyarrhythmias have their specific treatment , patients with syncope during sinus rhythm , e.g. with psychogenic syncope , are also an important subgroup that will benefit from a correct diagnosis and information , which can subsequently help them to manage without further visits to healthcare facilities .
as shown in our analysis of the picture study , the frequent repetition of tests and the use of specific tests early in the investigation was a poor use of healthcare resources as they did not result in a diagnosis .
at the same time , ilrs have been shown to be cost - effective in patients with unexplained syncope , implying that the higher initial costs should not discourage physicians from using an ilr when it is an option according to the current guidelines .
in addition , a miniaturized ilr has recently become available , the reveal linq , and due to its much smaller size a new insertion technique has made the implant procedure minimally invasive . as a consequence , this minimally invasive procedure can be performed also in settings outside the catheterization laboratory , thus offering possibilities to reduce the procedure costs .
the cost level and reimbursement system may vary between countries and regions , but the relative difference in cost between tests is likely to be similar , which should make it possible to substitute our cost figures for other currencies to get valid estimations .
the selected university hospital may not reflect other hospitals in all test procedures for unexplained syncope .
in addition , there is significant variation in the costs of treating individual patients , which is not reflected in this study .
the times required for procedures , the grade of staff that usually administers the procedure , and the usual pathway were overall averages , gained from speaking to clinical staff either at the selected hospital , or where not available , from other uk hospitals . again , these are likely to vary from patient to patient and from physician to physician .
this analysis of the microeconomics of the use of diagnostic tests in the picture registry identified significant over - investigation in terms of both number and types of tests , especially in the initial phase of the evaluation .
as a consequence , the analysis identified important opportunities to reduce test - related costs before an ilr implant by more appropriate use of tests recommended in the initial evaluation , by reducing frequent repetition of the same tests , and by avoiding early use of specific and expensive tests usually performed only on specific suspicions about the underlying mechanism .
funding to pay the open access publication charges for this article was provided by medtronic . | aimsthe observational picture ( place of reveal in the care pathway and treatment of patients with unexplained recurrent syncope ) registry enrolled 570 patients with unexplained syncope , documented their care pathway and the various tests they underwent before the insertion of an implantable loop recorder ( ilr ) .
the aims were to describe the extent and cost of diagnostic tests performed before the implant.methods and resultsactual costs of 17 predefined diagnostic tests were characterized based on a combination of data from picture and a micro - costing study performed at a medium - sized uk university hospital in the uk .
the median cost of diagnostic tests per patient was 1114 ( 95% ci 9951233 ) .
as many patients received more than the median number of tests , the mean expenditure per patient was higher with 1613 ( 95% ci 14941732 ) , and for 10% of the patients the cost exceeded 3539 .
tests were frequently repeated , and early use of specific and expensive tests was common . in the 12% of patients with types of tests entirely within the recommendations for an initial evaluation before ilr implant , the mean cost was 710.conclusionimportant opportunities to reduce test - related costs before an ilr implant were identified , e.g. by more appropriate use of tests recommended in the initial evaluation , by decreasing repetition of tests , and by avoiding early use of specialized and expensive tests .
a structured multidisciplinary approach would be the best model to achieve an optimal outcome . |
the competition for solar photons among
aquatic photosynthetic
organisms striving to maintain viability at various depths in the
water column is fierce , often requiring adaptation of the species
for survival .
a prime example of the development of adaptive traits
is found in the purple photosynthetic bacterium , rhodoblastus ( rbl . ) acidophilus ( formerly rhodopseudomonas acidophila ) strain 7050 .
this bacterium
is able to alter its number and size of photosynthetic units as well
as its pigment composition and light absorption properties of the
major light harvesting ii ( lh2 ) antenna pigment protein complex
in response to changes in illumination conditions .
if the bacterium is grown under high light , the well - characterized
b800 - 850 lh2 complex is formed having bacteriochlorophyll ( bchl ) absorption
bands near 800 and 850 nm , and rhodopin and rhodopin glucoside are
the primary carotenoid pigments . under low - light conditions , genes
that code for a variant lh2 denoted b800 - 820 ( also sometimes referred
to as lh3 in the literature )
are activated , the bchl qy absorption band near 850 nm shifts to 820 nm , and
the organism accumulates rhodopinal and rhodopinal glucoside in the
lh2 complex as primary carotenoid pigments ( figures 1a and 1b ) .
this response to changes in ambient light is controlled
by a combination of a classical two component regulatory system and
bacteriophytochromes that regulate the synthesis of the photosynthetic
apparatus .
structures of ( a ) all - trans - rhodopin
glucoside
and 13-cis - rhodopinal glucoside and ( b ) one - third
portion of the lh2 b800 - 820 ring complex from rbl .
acidophilus strain 7050 ( pdb 1ijd ) showing the protein - bound bchls ( green ) and carotenoids ( purple ) .
the shift of the bchl qy band from 850 nm to
820 nm that occurs at low light is due to alterations in the
amino acid sequence of the apoproteins that are assembled in the variant
lh2 pigment protein complex .
results from x - ray crystallography ( figure 1b ) , site - directed mutagenesis , and resonance raman spectroscopy indicate that h - bonding residues 44 ( tyr )
and 45 ( trp ) in the b800 - 850 lh2 prevent rotation of the c3-acetyl
group of the b850 bchl and fix the functional group so that its c = o
-electron bond resides in a planar orientation relative to
the porphyrin macrocycle .
the conversion
of these h - bonding residues to non - h - bonding 44 ( phe ) and 45
( leu ) in the b800 - 820 lh2 leads to a rotation of the c3-acetyl group
out of the plane of the porphyrin ring , thereby inhibiting delocalization
of the -electron conjugation to the acetyl carbonyl , resulting
in more restricted -electron delocalization and consequently
a blue shift of the qy band from 850 nm to 820
nm . accompanying the shift of the bchl qy absorption
band
in the lh2 complex is a change in the absorption spectrum of the carotenoid .
under low - light growth conditions , rhodopin and rhodopin glucoside
are enzymatically converted to rhodopinal and rhodopinal glucoside
as an aldehyde group replaces the methyl group at carbon c20 in the
carotenoid structures ( figure 1a ) . the spectral origin ( 00 ) vibronic band of rhodopin glucoside
in methanol appears at 500 nm , whereas for rhodopinal glucoside ,
the band is less resolved spectrally , and it is located at 540
nm in the same solvent ( figure 2 ) .
previous
workers compared the carotenoid - to - bchl energy transfer properties
of lh2 complexes isolated from cells of rbl .
acidophilus strain 7050 grown under different illumination conditions and found
that there was an increase in the energy transfer efficiency from
between 50 and 55% for the b800 - 850 complex to between 70 and 75%
for the b800 - 820 complex . however
, the
previous investigation did not address the specific reasons for the
increase , i.e. , whether changes in the bchl absorption spectra , or
the conversion of rhodopin to rhodopinal in the protein complex , or
both factors , were responsible for the enhanced ability of the lh2
complex to effectively harvest photons in the region of carotenoid
absorption .
moreover , the previous work and subsequent ultrafast spectroscopic
experiments carried out on the b800 - 820 lh2 complex from rbl .
acidophilus strain 7050 did
not assign specific values to the energy transfer efficiencies of
the individual carotenoids bound in the complexes , nor has there been
any direct comparison of the spectra and dynamics of the excited states
of rhodopin and rhodopinal either in solution or in the lh2 complexes .
these data are important for addressing the specific mechanism of
how these alterations in bchl and carotenoid structures and spectra
increase the carotenoid - to - bchl energy transfer efficiency and , as
a consequence , enhance the viability of the photosynthetic bacterial
organism .
normalized steady - state absorption spectra of ( a ) rhodopin glucoside
and ( b ) rhodopinal glucoside in carbon disulfide , benzyl alcohol ,
methanol , and acetonitrile recorded in 2 mm path length cuvettes at
room temperature .
energy transfer from
carotenoids involves at least two excited
singlet states that can act as donors of absorbed light energy to
bchl .
these are the s1 ( 2ag ) and s2 ( 1bu ) states whose properties are strikingly distinct . a one - photon
transition from the ground s0 ( 1ag ) state to the s1
( 2ag ) state is forbidden by symmetry , whereas
a transition to the s2 ( 1bu ) state is strongly allowed .
the s0 ( 1ag ) s2 ( 1bu )
transition is responsible for the vibrant coloration of carotenoids
in nature .
motivated by the landmark
report of the x - ray crystal structure of the lh2 complex from rbl .
acidophilus strain 10050 , several investigators sought to understand the role of the s1 ( 2ag ) and s2 ( 1bu ) excited singlet
states in the mechanism of energy transfer to bchl in this pigment
used
ultrafast time - resolved optical spectroscopy applied to the lh2 complex
prepared from rbl .
acidophilus strain 10050 and reported
that the s2 ( 1bu ) state
of rhodopin glucoside dominated the pathway for energy transfer to
bchl despite its extremely short intrinsic lifetime of 120
fs in solution .
the s1 ( 2ag ) state of rhodopin glucoside , which has a much longer
lifetime of 4 ps in solution , was reported to make only a
minor contribution to the overall energy transfer efficiency . in another
study using steady - state and ultrafast time - resolved spectroscopy
to elucidate the carotenoid - to - bchl energy transfer mechanism in the
lh2 complex from rbl .
acidophilus 10050 , cong et
al . reported the partitioning of energy
transfer to be 23 7% from the s1 ( 2ag ) state and 63 10% from the s2 ( 1bu ) state . a recent
broadband 2d electronic spectroscopic investigation of lh2 complexes
from rbl .
acidophilus 10050 and rhodobacter
sphaeroides strain 2.4.1 provided convincing evidence for
the additional involvement of a dark intermediate state in the carotenoid - to - bchl
energy transfer pathway . the present
work provides a detailed experimental and computational
comparison of the excited state energy levels , spectra , and dynamics
of rhodopin , rhodopinal , and their associated glucosides in various
solvents and in their respective lh2 complexes isolated from rbl .
the use of steady - state absorption , fluorescence and fluorescence
excitation spectroscopy and ultrafast time - resolved transient absorption
spectroscopy in the visible spectral region have revealed the rates
and efficiencies of carotenoid - to - bchl energy transfer for the individual
carotenoids .
the data address the questions of how and why photosynthetic
organisms alter their pigment composition and light - harvesting characteristics
to ensure survival under the challenging , light - deprived environmental
conditions in which they are sometimes found .
acidophilus 10050 and 7050 cultures were
grown anaerobically in the light using
pfennig s medium .
normal growth
conditions ( hereafter referred to as high - light ( hl ) conditions ) used
continuous illumination at an intensity of 30 mol s m. the rbl .
acidophilus 7050
culture was also grown at a lower light intensity ( hereafter denoted
low - light ( ll ) ) ranging from 3.7 to 5 mol s m. cells were harvested by centrifugation at
4000 g in a beckman model j-6b centrifuge .
the resulting
pellets were resuspended in 1 l of 20 mm mes ph 6.8 buffer , containing
100 mm kcl , and centrifuged again to remove any residual media .
extraction of the carotenoids
from whole cells of the bacteria was accomplished by mixing 24
g of thawed cells with 30 ml of methanol at room temperature and stirring
in the dark for 15 min .
the mixture was then centrifuged at 3000 g using an ss-34 rotor at 4 c in a sorvall rc-5b centrifuge .
the pellet was then mixed with 30 ml of fresh methanol and centrifuged
repeatedly until the cells appeared gray , signifying that all the
pigments had been extracted .
the supernatant from the second and subsequent
extractions contained primarily carotenoids as evidenced by absorption
spectra recorded using a varian cary 50 uv / visible spectrometer .
the dried extracts were dissolved in acetonitrile / methanol ( 6:4 ,
v / v ) and analyzed using a waters 600e/600s hplc system equipped with
a waters atlantis t3 obd preparative column having dimensions of 19
100 mm . the mobile phase consisted of acetonitrile / methanol
( 6:4 ,
v / v ) delivered isocratically at a flow rate of 7.0 ml / min .
individual
peaks were collected and identified by mass spectrometry using a fisons
quattro ii instrument employing atmospheric pressure chemical ionization
( apci ) in negative mode with the following conditions : corona voltage ,
2.5 v ; cone voltage , 25 v ; source temperature , 120 c ; probe
temperature , 300 c , mobile phase , acetonitrile .
all solvents
were hplc - grade and were purchased from sigma - aldrich corp .
briefly , 5 g of pelleted whole cells
of the bacteria were suspended in 30 ml of 20 mm tris buffer
adjusted to ph 8.0 using 6 m hcl ( hereafter referred to as tris buffer ) ,
and 2050 mg of dnase and a few grains of mgcl2 were added to degrade the released dna during cellular disruption .
the sample was briefly homogenized using a glass tissue homogenizer
to ensure smooth passage through the french press operating at 15,000
psi .
cells were passed through the press three times to ensure complete
disruption , and the resulting suspension was centrifuged for 2 h at
4 c in a type 70 ti rotor spinning at 180000 g in a beckman l8 - 55 m ultracentrifuge .
the resultant pellet
containing membrane fragments was diluted using tris buffer to an
od of 50 measured in a 1 cm cuvette at the bchl qy absorption
band maximum ( 800 nm for ll grown rbl .
the membranes
were then solubilized by adding 30% lauryldimethylamine oxide ( ldao )
dropwise to a final concentration of 1.0% and allowing the sample
to incubate for 60 min while being stirred at room temperature in
the dark .
the sample was then centrifuged for 30 min at 27000 g in the sorvall rc-5b centrifuge to remove denatured protein
and any nonsolubilized material .
the supernatant containing the solubilized
proteins was then collected for further treatment using sucrose density
gradient ultracentrifugation .
solutions of 0.2 , 0.4 , 0.6 , and 0.8 m sucrose
were prepared using tris buffer containing 0.1% ldao and were carefully
layered to form discontinuous gradients in the tubes by using the
following amounts : 0.8 m , 5.0 ml ; 0.6 m , 6.5 ml ; 0.4 m , 6.5 ml ; and
0.2 m , 5.0 ml .
the tubes were then placed in a ti70 rotor and spun at 160000 g for 12 h at 4 c in the beckman l8 - 55 m ultracentrifuge .
the procedure effectively separates any free pigments present in the
0.2 m sucrose layer , from the lh2 complex appearing in the 0.4 m sucrose
layer , from the lh1-rc core complexes that appear
at the interface of the 0.6 and 0.8 m sucrose solutions .
the
0.4 m sucrose solution layer containing the lh2 complex was
carefully removed from each tube and pooled .
the complex was then
purified further by column chromatography using de52 anion exchange
resin ( 15 g , whatman scientific ) packed into a 5 cm diameter
30 cm long solid phase , sintered glass column pre - equilibrated with
several bed volumes of tris buffer .
after loading the sample onto
the column , several bed volumes of tl buffer ( 0.1% ldao , 20 mm tris
ph 8.0 ) were applied to remove the sucrose .
the lh2 complex was then
eluted using tl buffer containing increasing concentrations of nacl
in 30 mm increments starting with 10 mm .
acidophilus 10050 and 7050 cells eluted between 10
and 20 mm nacl , whereas that isolated from ll rbl .
the purity of the eluting
fractions was monitored by absorption spectroscopy using a shimadzu
uv-1700 pharmaspec spectrometer .
fractions from hl samples exhibiting
spectra with an 850 nm to 280 nm ratio of > 3.0 were pooled for
further
purification . for the ll sample ,
fractions exhibiting spectra with
an 800 nm to 280 nm absorbance ratio of > 2.5 were pooled for further
purification .
the pooled sample was reduced to a volume of < 1
ml by centrifugation
using vivaspin 4 50 k mw cutoff concentrators ( sartorius stedim biotech )
placed in an 11390 rotor and spun at 3250 g in a sigma
3k30 centrifuge .
the concentrated lh2 was further purified using an
akta primeplus ( ge healthcare ) automated chromatography system equipped
with an xk-16 long gel filtration column filled with superdex-200 .
0.5 ml fractions were collected of the lh2 band and subsequently assayed
using the ratio of the absorbance maximum of the bchl qy band to the protein absorbance at 280 nm .
fractions having an 800
nm to 280 nm ( from the ll - grown rbl .
acidophilus 7050 )
ratio of > 2.7 or an 850 nm to 280 nm ( from the hl - grown rbl .
acidophilus cells ) ratio of 3.3 were pooled and concentrated
using the vivaspin 4 50 k mw cutoff concentrators to an od of 100
measured at the absorbance maximum of the bchl qy band
in a 1 cm cuvette .
the sample was then divided into 30 l aliquots ,
placed in pcr tubes , flash frozen in liquid nitrogen , and stored in
a 80 c freezer . a 10 l aliquot
of the frozen rbl .
acidophilus 7050 lh2 complex was
thawed on ice , and the liquid was evaporated
to dryness using nitrogen gas .
the remaining residue was then redissolved
in 2 ml of methanol to release the pigments .
this extract was then
centrifuged at 13600 g for 2 min at room temperature
in a fisher scientific 235c benchtop microcentrifuge .
the supernatant
was then analyzed using a waters 600e/600s hplc system equipped with
a waters atlantis t3 5 m analytical column having dimensions
of 4.6 250 mm .
the mobile phase consisted of an isocratic delivery
of acetonitrile / methanol ( 6:4 , v / v ) at a rate of 2.0 ml / min .
acidophilus 10050
lh2 complex was dried under nitrogen gas and denatured with 2 ml of
acetone .
following centrifugation , the supernatant was removed and
2 ml of fresh acetone was added to the remaining pellet , and the mixture
was centrifuged again , this time resulting in a colorless pellet .
the sample was taken up in 1 ml of acetonitrile / methanol
( 6:4 , v / v ) and analyzed on the same hplc system with a waters atlantis
t3 obd 5 m preparative column .
the mobile phase was the same
as described above , but with the flow rate increased to 7.0 ml / min .
the molar percentages of the carotenoids were calculated using
the area of each hplc peak detected at the wavelength of maximum absorption
divided by the extinction coefficient of the carotenoid , and then determining the percentage of each pigment
relative to the total carotenoid content .
peaks that could not be
conclusively identified by mass spectrometry remain unidentified ,
but are presumed to be isomers formed during the extraction procedure .
all steady - state absorption and
fluorescence emission and excitation spectroscopic measurements were
carried out in 1 cm square cuvettes at room temperature unless otherwise
stated .
rhodopin glucoside and rhodopinal glucoside were dissolved
in spectroscopic grade carbon disulfide ( acros organics ) , benzyl alcohol
( sigma - aldrich ) , methanol ( sigma - aldrich ) , or acetonitrile ( sigma - aldrich ) .
steady - state absorption spectra of the carotenoids in various
solvents and in the lh2 complexes were obtained using either a varian
cary 50 or a cary 5000 uv visible spectrophotometer .
fluorescence
emission and excitation spectra were recorded using a jobin - yvon horiba
fl3 - 22 fluorimeter equipped with double excitation and emission monochromators
having 1200 grooves / mm gratings , a 450 w osram xbo xenon arc lamp ,
and a hamamatsu r928p photomultiplier tube detector .
emission
spectra of the lh2 complexes were recorded using samples having an
od between 0.025 and 0.1 in a 1 cm path cuvette at the bchl qx band at 591 nm , which was also the excitation wavelength .
the excitation and emission slit widths corresponded to bandpasses
of 6 and 3 nm , respectively for experiments on the lh2 complex from rbl .
acidophilus 7050 ( hl and ll ) lh2 complexes were obtained
using excitation and emission slit widths corresponding to bandpasses
of 12 and 6 nm , respectively .
all emission spectra were corrected
using an emission correction factor file generated by taking the ratio
of the spectral response of a calibrated 200 w quartz tungsten - halogen
filament lamp and the instrument detection system .
fluorescence
excitation spectra were recorded by monitoring the
bchl emission at its maximum wavelength ( 870 nm for the b800 - 850 complexes
and 860 nm for the b800 - 820 complex ) using samples with an od of 0.025
in a 1 cm path cuvette at the bchl qx band .
emission was
detected at a right angle relative to the excitation with bandpasses
corresponding to 6 nm ( 10050 lh2 ) , 7 nm ( 7050 hl ) , and 4 nm ( 7050
ll ) for the excitation monochromator , and 12 nm ( 10050 lh2 ) and 14
nm ( 7050 hl and ll ) for the emission monochromator .
an excitation
correction factor file was used to correct for the wavelength variability
of the source lamp and excitation monochromator .
this file was generated
using a photodiode calibration kit consisting of a photodiode assembly
and a dm303-p module that was rented from horiba .
probe
ultrafast transient absorption spectroscopy was
carried out using a helios femtosecond transient absorption spectrometer
( ultrafast systems llc , sarasota , fl , usa ) coupled to a laser setup
that has been previously described .
surface xplorer
pro 1.2.2.26 ( ultrafast systems llc , sarasota , fl , usa ) was used to
correct for the dispersion in the transient absorption spectra .
samples
having an od between 0.2 and 0.5 in a 2 mm path cuvette at the carotenoid
spectral origin ( 00 ) band were mixed continuously using a
magnetic microstirrer to avoid photodegradation .
the pump laser had
an energy of 1 j / pulse focused on a 1 mm diameter spot , which
corresponds to a laser intensity between 3.2 and 3.9 10 photons / cm .
the integrity of the samples was
assayed by taking steady - state absorption spectra before and after
laser excitation .
global fitting of the transient absorption
data sets was performed
in asufit 3.0 provided by dr .
evaldas katilius at arizona state university
and carried out according to a sequential excited state decay model
which yielded evolution associated difference spectra ( eads ) .
reconstruction of the 1-t and fluorescence excitation
spectra was done using origin software version 9 .
ground state geometries were
generated using b3lyp/6 - 31g(d ) methods as implemented
within gaussian 09 .
excited state geometries
were generated using single - configuration interaction ( cis ) methods
and full single ci .
the effect of the
solvent environment was simulated by using the polarizable continuum
model ( pcm ) .
spectroscopic
properties were calculated using mndo - psdci and equation
of motion coupled - cluster singles and doubles ( eom - ccsd ) methods .
the mndo - psdci methods are semiempirical and have been
used successfully to study long - chain polyenes and carotenoids .
the eom - ccsd calculations were carried out using
a d95 double - zeta basis set , but the size of the target systems limited
the use of this method to only rhodopin .
the steady - state absorption spectra of rhodopin
glucoside and rhodopinal
glucoside recorded in carbon disulfide , benzyl alcohol , methanol ,
and acetonitrile are shown in figure 2 .
the
primary bands in these spectra represent an electronic transition
from the ground s0 ( 1ag ) state to the s2 ( 1bu ) state . in any particular solvent ,
the spectrum of rhodopinal glucoside
is shifted to longer wavelength by 3040 nm compared to that
of rhodopin glucoside .
the spectrum of rhodopin glucoside displays
well - resolved vibronic bands in all of the solvents , whereas the spectrum
of rhodopinal glucoside is much less structured , except in carbon
disulfide , where a shoulder on the long - wavelength side of the primary
absorption band is observed . also , the high polarizability of carbon
disulfide ( p( ) = 0.354 ) results in a 3040
nm red shift of the spectra of rhodopin glucoside and rhodopinal glucoside
compared to their spectra recorded in the less polarizable solvents ,
acetonitrile ( p( ) = 0.210 ) and methanol ( p( ) = 0.202 ) . benzyl alcohol , which has a polarizability
value ( p( ) = 0.314 ) between those of carbon
disulfide and methanol or acetonitrile , red shifts the spectrum of
rhodopinal glucoside more than for rhodopin glucoside ( figure 2 ) .
this is undoubtedly due to the presence of the
aldehyde group on rhodopinal glucoside , which interacts more strongly
with this solvent than the methyl group in the same position on rhodopin
glucoside . except for a small change in relative intensities of the
vibronic bands of rhodopin glucoside ,
the spectra are not significantly
affected by changing the solvent from a protic ( methanol ) to a nonprotic
( acetonitrile ) polar solvent .
it should be mentioned that the glucoside
moiety has no effect on the positions and intensities of the absorption
bands .
the spectra of rhodopin glucoside and rhodopinal glucoside
are indistinguishable from the corresponding spectra of rhodopin and
rhodopinal .
acidophilus 10050 ,
7050 hl , and 7050 ll recorded at room temperature are shown in figure 3 .
all spectra show the strong s0 ( 1ag ) s2 ( 1bu ) transition characteristic of carotenoids
in the 400550 nm region . also , the positions of the bchl soret
band at 375 nm and the bchl qx band at 590
nm are nearly identical for all of the complexes . however , one bchl
qy band of the lh2 complex is located at 800 nm , and the
other is at 859 nm for strain 10050 , at 855 nm for 7050 hl , and at
823 nm for 7050 ll .
in addition , compared to the spectrum of the lh2
complex from strain 10050 , the spectra of the lh2 complexes from 7050
hl and 7050 ll show more absorption in the region between 550 and
600 nm where the spectrum of the carotenoid partially overlaps with
the bchl qx band at 590 nm .
this is due to the
presence of rhodopinal and rhodopinal glucoside in the lh2 complexes
from strain 7050 .
acidophilus 7050 will always result in a very small amount of the b800 - 820 lh2
complex in hl cells due to the effect of light shading in the culture
media .
likewise , a small amount of b800 - 850 lh2 complex will be present
in the ll cells due to the fact that there is a limit to how low the
light intensity can be adjusted to ensure a realistic amount of bacterial
growth .
a spectral analysis of the bchl absorption bands in the qy region ( figure s1 in the supporting information ) shows that this amount is less than 20% .
moreover , it should be
noted that the regulatory pathways for the switch from rhodopin glucoside
to rhodopinal glucoside and from b800 - 850 to b800 - 820 are independent
of each other . during growth at progressively decreasing light intensity ,
the carotenoid pathway switch is activated before , i.e. , at a higher
light intensity than the switch that controls the type of complex
present .
therefore , it is possible to
obtain a b800 - 850 complex that contains a significant amount of rhodopinal
glucoside as is the case for lh2 complex isolated from 7050 hl cells
( table 1 ) .
the pronounced similarity in the
structures of the b800 - 850 and b800 - 820 protein complexes precludes
complete separation by chromatographic techniques .
however , by a judicious
choice of excitation and detection wavelengths in the steady - state
and transient absorption spectroscopic experiments , the properties
of the individual pigment
acidophilus 10050 , 7050 ll , and 7050 hl
recorded in 2 mm path length cuvettes at room temperature .
uncertainties in the values ,
based on standard deviations from the mean , were equal to or less
than two percentage points .
the carotenoid
composition of the different lh2 complexes was determined
by hplc analyses carried out as illustrated in figure 4 .
the molar percentages of the carotenoids
in the different lh2 complexes are given in table 1 .
acidophilus 7050 cells display a significant
increase in total rhodopinal ( defined in this context as rhodopinal
plus rhodopinal glucoside ) concurrent with a decrease in total rhodopin
( defined here as rhodopin plus rhodopin glucoside ) compared to that
found in the bacterium grown under hl conditions .
total
rhodopinal in the lh2 complexes increased from 32% in the 7050 hl
sample to 65% ( 58% + 7% ) in the 7050 ll sample ( table 1 ) .
concurrently , total rhodopin in the lh2 complexes decreased
from 56% ( 28% + 28% ) to 15% ( 5% + 10% ) when cells were grown using
ll ( table 1 ) . as previously reported and confirmed by the present work , the lh2 complex from rbl .
acidophilus 7050 lh2 complexes prepared from cells
grown under ll ( top trace )
and hl ( bottom trace ) conditions .
the major pigments were identified as follows : 1 , rhodopinal
glucoside ; 2 , rhodopinal ; 3 , rhodopin glucoside ; 4 , bchl a ; 5 , rhodopin ; and 6 , lycopene .
the fluorescence spectra of the lh2 complexes recorded using
591
nm excitation , which excites the bchl qx band , are shown
in figure 5 ( blue traces ) .
the maximum wavelength
of emission occurs at 870 nm for the lh2 complexes from 10050 and
7050 hl , and at 860 nm for the lh2 complex from 7050 ll .
fluorescence
spectra were also recorded using excitation wavelengths of 560 , 570 ,
580 , and 590 nm , but no changes in the position or shape of the resulting
emission spectra were observed .
fluorescence excitation spectra of
the lh2 complexes are also shown in figure 5 ( red traces ) .
these spectra were recorded by detecting the fluorescence
from the samples at the maximum wavelength of bchl emission , but identical
lineshapes were observed using any detection wavelength between 820
and 880 nm ( figure s2 in the supporting information ) .
emission ( blue ) , excitation ( red ) , and 1-t ( black ) spectra of lh2
complexes obtained from rbl .
the green line shows the ratio of the normalized
excitation and 1-t spectra and in the region of carotenoid absorption
gives a quantitative measurement of the carotenoid - to - bchl energy
transfer efficiency .
it is somewhat surprising that the fluorescence maximum of
the
b800 - 820 complex occurs at 860 nm rather than closer to the 820 nm
qy band .
however , as mentioned above , previous work detailing
the effect of light intensity and temperature on cell growth and the
formation of the b800 - 820 complex indicated that the conversion of
rhodopin to rhodopinal occurs prior to the conversion of b800 - 850
to b800 - 820 .
therefore , it is likely that
the fraction of b800 - 850 complex in our ll sample ( see figure s1 in
the supporting information ) contains rhodopinal
and represents the main emission component .
this interpretation rationalizes
the appearance of the fluorescence maximum at 860 nm and the fluorescence
excitation spectra not being affected by changing the detection wavelength .
t is transmittance
( black traces ) , and the excitation spectra ( red traces ) were normalized
at the bchl qx and qy bands , and the ratio of
the excitation and 1-t spectral amplitudes were determined in the
region from 300 to 650 nm ( green traces ) .
these traces represent the
efficiency of excitation energy transfer ( eet ) to bchl , which was
found in the carotenoid absorption region between 425 and 550 nm to
be in the range of 55% to 61% for the 10050 lh2 complex consistent
with previously published results .
the lh2 complexes
from 7050 hl and 7050 ll displayed higher carotenoid - to - bchl eet efficiencies
and were in the range of 63% to 71% and 76% to 86% , respectively . hplc analysis ( table 1 ) revealed that the
lh2 samples from the 7050 samples contain both rhodopin and rhodopinal
( and their associated glucosides ) in different amounts .
therefore ,
in order to examine the contributions of these individual chromophores
to the spectra of the pigment protein complexes , reconstructions
of the 1-t ( left panel , figure 6 ) and fluorescence
excitation ( right panel , figure 6 ) spectra
were carried out based on the absorption spectra of the hplc - purified
carotenoids recorded in benzyl alcohol .
the spectra of the carotenoids
in the lh2 protein ( figure 3 ) are well - reproduced
by their spectra in benzyl alcohol ( figure 2 ) due to the fact that this solvent has a polarity and polarizability
similar to the average of those of the protein in the binding environment
of the carotenoid .
the
spectral reconstructions yielded the specific carotenoid - to - bchl eet
efficiencies for rhodopin and rhodopinal from the ratios of the individual
fluorescence excitation and 1-t bands in the profiles .
( because rhodopin
and rhodopinal have identical absorption ( and 1-t ) spectra as their
corresponding glucoside derivatives , in
this context and unless explicitly noted otherwise , any statements
about the spectra of rhodopin or rhodopinal should be taken to mean
the combined contribution from the chromophores associated with both
the glucoside and non - glucoside molecules . )
the eet efficiency for
rhodopin in the lh2 complex from the 10050 sample becomes evident
from a side - by - side , horizontal comparison of the amplitudes of the
1-t and fluorescence excitation spectra ( figures 6a and 6b ) in the region of carotenoid
absorption , and was found to be 56 1% , consistent with previous
reports .
a similar analysis was carried out to obtain
the values for the individual carotenoid - to - bchl eet efficiencies
in the lh2 complex from the 7050 ll sample ( figures 6c and 6d ) , which were found from the
spectral reconstruction to be 54 5% for rhodopin ( orange traces )
and 97 2% for rhodopinal ( purple traces ) .
the computed individual
carotenoid - to - bchl eet efficiencies for the lh2 complex prepared from
7050 hl cells ( figures 6e and 6f ) were 50 3% for rhodopin ( orange traces ) and 98
2% for rhodopinal ( purple traces ) , consistent with the values determined
for the lh2 complex from 7050 ll .
these results
are summarized in table 2 and reveal the remarkable
finding that rhodopinal in the lh2 complex from rbl .
acidophilus 7050 transfers essentially all of its excited state energy to bchl
with minimal loss through internal conversion to the ground state .
this stands in striking contrast to rhodopin in the lh2 complexes
from all rbl .
reconstruction of the
( a , c , e ) 1-t and ( b , d , f ) fluorescence
excitation spectra ( black traces ) of the lh2 complexes from rbl .
the 1-t spectra
of purified rhodopin glucoside ( orange traces ) and rhodopinal glucoside
( purple traces ) were recorded in benzyl alcohol and summed to generate
the reconstructed spectra ( red traces ) .
the bchl bands in the soret
region between 300 and 400 nm and in the qx region near
600 nm were modeled using gaussian functions ( green lines ) for simplicity .
the uncertainties
in the amplitudes
were obtained by comparing the reconstructed spectra with those experimentally
recorded . those values
figure 7 shows ultrafast
time - resolved transient
absorption spectra of rhodopin glucoside ( left panels ) and rhodopinal
glucoside ( right panels ) recorded in carbon disulfide , benzyl alcohol ,
methanol , or acetonitrile at various delay times after laser excitation .
upon excitation , an immediate onset of bleaching of the strongly allowed
s0 ( 1ag )
s2 ( 1bu ) transition
occurs , resulting in a negative signal in the 450550 nm region .
in addition , a strong positive signal in the 520700 nm region
appears that can be attributed to excited state absorption ( esa ) associated
with the s1 ( 2ag ) sn transition .
this suggests that the decay
of the s2 state via internal conversion to populate the
s1 state is occurring on the same time scale as the instrument
response time of 100 fs . similar to the steady - state absorption
spectra of the carotenoids , the excited state absorption bands shift
to longer wavelength and are broader as the polarizability of the
solvent increases .
the spectra are broadest and most red - shifted in
the highly polarizable solvent , carbon disulfide , compared to the
spectra recorded in the other solvents .
a similar effect is evident
in the spectrum of rhodopinal glucoside in carbon disulfide and benzyl
alcohol ( figure 7 , right panels ) , where it
is also clear that the spectral bands are broader overall than the
spectra of rhodopin glucoside in the same solvents ( figure 7 , left panels ) .
also , an additional peak at 740
nm , which is not found in the spectra of rhodopin glucoside , is seen
in the transient absorption spectra from rhodopinal glucoside .
unlike
the main s1 ( 2ag ) sn transient absorption band , this feature
is insensitive to the polarizability of the solvent , and therefore
appears more separated from the main esa feature in methanol and acetonitrile
compared to in carbon disulfide .
transient absorption spectra of rhodopin
glucoside and rhodopinal
glucoside in carbon disulfide , benzyl alcohol , methanol , and acetonitrile
recorded at room temperature using the indicated excitation wavelengths . in order to obtain detailed information
regarding the dynamics
of the excited states of the carotenoids ,
global fitting according
to a sequential decay model , resulting in evolution associated difference
spectra ( eads ) , was carried out on the transient absorption data sets .
for both rhodopin glucoside and rhodopinal glucoside
in all four solvents , the first eads has a very short lifetime ranging
from < 100 to 120 fs , and the profile of this component shows bleaching
of the ground state s0 ( 1ag ) s2 ( 1bu ) absorption and stimulated emission from the s2 ( 1bu ) state .
it is important
to point out here that although the best global fitting results are
achieved in some cases using time constants smaller than 100 fs , these
very small values can not be taken factually because the instrument
response time of the laser spectrometer is on the order of 100
fs .
hence , these values are specified as < 100 fs . more precise
values in this time domain were reported by macpherson et al . who used fluorescence upconversion spectroscopy
and found the s2 ( 1bu ) lifetime of rhodopin glucoside in benzyl alcohol to be 124
8 fs .
the second eads component for both molecules has a time constant
that ranges from 330 to 530 fs in the different solvents .
this component
can be assigned to a transition from a vibronically hot s1 ( 2ag ) sn excited singlet state due to the fact that its broad esa peak narrows
and shifts to shorter wavelength upon decaying to form the third eads
component .
the second and third eads for rhodopinal glucoside in all solvents
also show the additional peak at 740 nm alluded to above ,
which is not found in the spectra of rhodopin glucoside .
the fact
that this feature does not depend on solvent polarity indicates that
it is not associated with the formation of an intramolecular charge
transfer ( ict ) state in rhodopinal glucoside .
evolution
associated difference spectra ( eads ) obtained from globally
fitting the transient absorption data sets from rhodopin glucoside
and rhodopinal glucoside in carbon disulfide , benzyl alcohol , methanol ,
and acetonitrile given in figure 7 .
the band profile of the third eads component for
both molecules
is very strong and well - known to be associated with esa from the relaxed
s1 ( 2ag ) state
making a transition to a higher excited singlet state .
the lifetime
of this component for rhodopin glucoside in all solvents was found
to be in the narrow range of 3.8 to 4.3 ps with no obvious effect
of solvent polarity on the value . however , the s1 ( 2ag ) lifetime for rhodopinal
glucoside in the polar solvents methanol and acetonitrile was 3.7
and 3.9 ps respectively , which are slightly shorter than the 4.4 and
4.7 ps values found for the molecule in the less polar solvents , benzyl
alcohol and carbon disulfide , respectively .
the fact that the lifetime
of this component is reasonably similar for both rhodopin glucoside
and rhodopinal glucoside indicates that neither the configuration ,
which is 13-cis for rhodopinal glucoside in solution
compared to all - trans for rhodopin glucoside ( figure 1a ) , nor the presence of the aldehyde group at carbon
c20 on rhodopinal glucoside compared to the methyl group at the same
position on rhodopin glucoside results in any significant change in
the dynamics of the s1 state that would impact its role
in light - harvesting .
the fourth eads component of both molecules
in all solvents has
a lifetime that ranges from 5.9 to 15.3 ps .
this component is rather
weak , but displays negative features associated with ground state
bleaching as well as a positive feature on the short wavelength side
of the main s1 sn absorption profile .
the negative features indicate that some fraction of the carotenoid
population is still in an excited state .
the short wavelength band
is reminiscent of the s * state initially proposed to be an intermediate
state between the s1 ( 2ag ) and s2 ( 1bu ) excited
singlet states that is involved in both s2 ( 1bu ) depopulation and carotenoid triplet state
formation in light harvesting complexes .
subsequent
work on several open - chain carotenoids
has shown that the s * yield is larger for molecules with longer -electron
conjugation suggestive of it being associated with a twisted molecular
conformation of the carotenoid in the s1 ( 2ag ) state .
finally , a very weak , infinitely long - lived component was necessary
to obtain a completely satisfactory fit to the data sets .
the lh2 complexes
were excited in the region of carotenoid absorption at either 525
or 570 nm , and then transient absorption spectra were recorded at
various delay times after the pump laser pulse ( figure 9 ) .
acidophilus 10050
displayed transient absorption profiles ( top panel of figure 9 ) that were similar to previously reported spectra
and include the instantaneous onset of bleaching of the carotenoid
ground state spectrum and broad positive esa at intermediate times ,
followed by the appearance of a strong narrow s1
sn transition at 580 nm at later times .
the hplc pigment analysis of the lh2 complex
from strain 10050 ( table 1 ) revealed that the
carotenoid composition consists of roughly equal amounts of rhodopin
and rhodopin glucoside with a small amount of lycopene , all of which
have identical absorption spectra . using this information regarding
the spectra of the carotenoids in the lh2 complex from rbl .
acidophilus 10050 as a control , the features attributable
to rhodopin and rhodopinal can be distinguished in the transient spectra
of the lh2 complexes prepared from 7050 ll and 7050 hl cells , which
contain a mixture of these carotenoids ( table 1 ) .
( recall that any reference to rhodopin or rhodopinal in this context
should be taken to mean the combined spectral properties of the molecules
and their respective glucosides , which are indistinguishable . )
laser
excitation at 525 nm of the 7050 ll or 7050 hl lh2 complexes results
in a combination of transient absorption signals from rhodopin and
rhodopinal because both molecules absorb at this wavelength .
selective
excitation of rhodopinal can be achieved by tuning the pump laser
to 570 nm , which is a wavelength where rhodopin does not absorb .
acidophilus 10050 , 7050 ll , and 7050 hl recorded at room
temperature using the indicated excitation wavelengths .
transient absorption spectra from the 7050 ll and
hl lh2 complexes
excited at 525 nm ( combined rhodopin plus rhodopinal excitation ) or
at 570 nm ( selective rhodopinal excitation ) are shown in figure 9 .
similar to the transient absorption spectra from
strain 10050 , the transient spectra recorded for these lh2 complexes
at a 50 fs delay time ( black traces in figure 9 ) have negative features corresponding to the bleaching of the carotenoid
s0 ( 1ag )
s2 ( 1bu ) ground state
absorption bands .
in addition , there is a positive signal in the 540700
nm region which represents an s1 ( 2ag ) sn transition indicating
very fast decay of the s2 ( 1bu ) state to populate the s1 ( 2ag ) state of the carotenoid . in
the 200 fs
delay time spectra ( red traces ) , the ground state bleaching has partially
recovered , indicative of energy transfer to bchl , but the strong positive
s1 ( 2ag )
sn absorption band has gained amplitude and is broader
compared to the subsequent 1 ps delay time spectrum ( green traces ) .
the gain in amplitude of the feature associated with the s1 ( 2ag ) sn transition indicates that internal conversion from s2 to populate the s1 state is competing effectively with
eet from s2 to bchl .
a close examination of the 1
ps time delay ( green trace ) spectrum
recorded for the 7050 ll lh2 excited at 525 nm ( second panel in figure 9 ) shows a narrow positive feature at 580
nm and a broader one at 630 nm . comparison of this spectrum
with those taken at the same time delay from the 10050 lh2 excited
at 525 nm ( selective rhodopin excitation , upper panel of figure 9 ) and the 7050 ll lh2 sample excited at 570 nm ( selective
rhodopinal excitation , third panel of figure 9 ) reveals that the narrow peak at 580 nm in the spectrum
from the 7050 ll lh2 excited at 525 nm is due to the s1 sn transition of rhodopin whereas the 630
nm peak is from rhodopinal .
this is expected because excitation of
the 7050 ll sample using 525 nm light excites both carotenoids which
are present in this lh2 sample ( table 1 ) .
this
interpretation is supported by the transient absorption spectra resulting
from 525 nm excitation of the 7050 hl lh2 complex ( fourth panel of
figure 9 ) .
note that the narrow positive feature
at 580 nm in the 1 ps time delay spectrum ( green trace ) attributable
to the s1 sn transition of rhodopin
is much more pronounced relative to the broader band at 630
nm associated with rhodopinal .
this is because the lh2 complex from
7050 hl contains 56% total rhodopin compared to the 7050 ll lh2 sample ,
which has only 15% rhodopin ( table 1 ) .
as expected ,
selective excitation of rhodopinal at 570 nm in the 7050 hl lh2 sample
( bottom panel of figure 9 ) shows no sign of
the narrow s1 sn feature belonging
to rhodopin .
the major positive band in the 1 ps time delay spectrum
( green trace ) is that of the s1 sn esa
of rhodopinal .
the 5 ps delay time spectra of the 10050 and
7050 ll lh2 samples
( blue traces in figure 9 ) show significantly
diminished carotenoid ground state bleaching and reduced s1 sn esa indicating that a substantial amount
of the carotenoid s1 state excited state population either
has been transferred to bchl or has decayed via internal conversion
back to the ground state .
also in this time frame , an additional peak
appears at 560 nm on the short wavelength side of the main
esa peak and persists longer than 100 ps ( magenta trace ) .
the eads
components of the lh2 complexes obtained from a global fitting of
the transient absorption data sets are shown in figure 10 .
the fitting of the transient absorption spectra required
five components , the first of which has a lifetime < 100 fs for
all the lh2 complexes and has features associated with the bleaching
of the ground state absorption as well as stimulated emission from
s2 ( 1bu ) .
this is consistent
with the value of 57 fs reported by macpherson et al .
the
second eads component ( red traces in figure 10 ) has a lifetime ranging from 250 to 490 fs and has broad features
in the carotenoid absorption region characteristic of vibronically
hot s1 ( 2ag )
sn excited singlet state transition .
this second
eads has weaker negative bands in the carotenoid ground state absorption
region than the first eads , indicating that some of the carotenoid
molecules have returned to the ground state via eet from the s2 state to bchl .
also evident in the second eads is a small
negative dip at 590 nm appearing on the broad positive esa
spectrum .
this is due to the bleaching of the bchl qx band
brought about by eet to bchl from the carotenoids . as the second eads
component decays into a third ( green lines in figure 10 ) , the line shape narrows considerably for the 10050 lh2 excited
at 525 nm and a strong band associated with the s1 ( 2ag ) sn transition
is evident at 580 nm .
this line narrowing is accompanied by a decrease
in the extent of ground state bleaching indicating that more of the
carotenoid molecules have returned to the ground state , perhaps by
eet from the vibronically hot s1 state .
this third component
decays in 3.0 ps for the 10050 lh2 and represents the s1 lifetime of rhodopin in the lh2 pigment protein complex .
the peak at 580 nm has much less amplitude in the third eads component
from the 7050 ll lh2 sample obtained from data also using 525 nm excitation
( green trace in the second panel of figure 10 ) .
this is due to the fact that this sample has much less total rhodopin
( rhodopin plus rhodopin glucoside ) than the 10050 lh2 .
in fact , the
peak at 580 nm is completely absent in the third eads obtained from
data using 570 nm excitation ( green trace in the third panel of figure 10 ) . instead , a broad line shape peaking at 620
nm and belonging to rhodopinal is observed .
note that the lifetime of
this component is 1.2 ps in the 7050 ll lh2 and 1.3 ps in the 7050
hl lh2 , which is significantly shorter than the average value of 4.1
ps found for rhodopinal in the different solvents and also shorter
than the value of 3.0 ps found for rhodopin in the lh2 complex from
strain 10050 .
this is suggestive of significantly faster eet to bchl
from the s1 state of rhodopinal compared to rhodopin .
evolution
associated difference spectra ( eads ) obtained from globally
fitting the transient absorption data sets of lh2 complexes from rbl .
the uncertainties
in the values
were obtained by exploring the region of solution for each parameter
according to the goodness of fit and minimization of the residuals .
an infinitely long ( on the time scale of the experiment ) component
the uncertainties
were obtained
by exploring the region of solution for each parameter according to
the goodness of fit and minimization of the residuals .
an infinitely
long ( on the time scale of the experiment ) component was required
for a good fit in all cases .
the fourth eads component has a lifetime in the range 13 to 27
ps for all the data sets and a significantly diminished overall amplitude
relative to the preceding eads profiles .
the lifetime of this component ,
along with the clearly evident positive band on the short wavelength
side of the feature attributable to the main s1
sn absorption band , and the wavy features in the region
of the carotenoid ground state absorption are all characteristics
of the s * state in accord with previous reports .
the fifth and final eads component
has an infinitely long lifetime on the time scale of the experiment
and was necessary to obtain a satisfactory fit in all cases .
the primary
spectral feature in this last eads component is the negative amplitude
at 590 nm , which corresponds to the bleaching of the bchl qx band that is expected to persist on the order of nanoseconds until
the s1 state of bchl relaxes back to the ground state .
quantum computations
were carried out to augment the experimental results , but in order
to make them tractable , the majority were carried out on the model
chromophores shown in figures 11c and 11d .
the model chromophores include the entire central
polyene portion of rhodopin and rhodopinal , but replace the glucoside
and aliphatic end groups with methyl groups .
the resulting model polyenes
have cs or c2h symmetry , and this symmetry , in combination
with the smaller size , allows higher quality calculations to be carried
out than would otherwise be possible .
test calculations on the full
and model chromophores indicate that the end groups do not have a
significant impact on the atomic charges other than small changes
in the charges on the carbon atoms at the ends of the polyene chains .
single ci calculations on the full system ( figures 11a and 11b ) and the model systems ( figures 11c and 11d ) demonstrate that
the end groups create less than a 0.02 ev shift in the transition
energies .
this conclusion is consistent with the observation that
the absorption spectra of rhodopin and rhodopin glucoside are indistinguishable .
b3lyp/6 - 31g(d ) calculated structures of ( a ) trans - rhodopin glucoside , ( b ) trans - rhodopinal glucoside ,
( c ) trans - rhodopin , ( d ) trans - rhodopinal ,
( e ) the s1 relaxed excited state of trans - rhodopinal , ( f ) 13-cis - rhodopinal , and 13-cis - rhodopinal model .
the structures given in panels c ,
d , e , and g are simplified , higher - symmetry analogues used in the
mndo - psdci , eom - ccsd and cas - scf theoretical calculations , which retain
the full -system . the calculated vacuum dipole moments ( in
debyes ( d ) ) and the dipole moment vectors ( the length is not relevant )
are shown underneath those structures that have a dipole moment . the
dashed ellipse in panel d shows the primary repulsive atom
atom
interaction responsible for making the cis configuration
more stable than the trans configuration by 8
kj / mol in nonpolar solvent ( n - hexane ) and 8.5
kj / mol in polar solvent ( acetonitrile ) .
although a
primary goal of this work is to understand the photophysical properties
of rhodopinal in lh2 complexes , the process is started by identifying
the geometry of rhodopinal in solution .
previous studies have proposed
that rhodopinal acquires a cis configuration in solution . based on density functional theory and an analysis
of the absorption spectra , it is demonstrated here that rhodopinal
takes on a cis configuration in both nonpolar and
polar solvent .
the all - trans rhodopinal chromophore
exhibits just one significant intramolecular repulsion involving the
interaction of the aldehyde hydrogen with the nearest hydrogen atom
on the main polyene chain .
the repulsion
is both electrostatic ( both atoms have positive charge ) and steric
( the atoms are separated by 2 ) .
. the aldehyde group can
rotate out of plane , or the double bond adjacent to the aldehyde group
can rotate 180 to create a cis linkage
near the center of the polyene chain .
based on b3lyp/6 - 31g(d)/pcm
calculations the resulting 13-cis configuration is
more stable than the all - trans configuration by 8
kj / mol in nonpolar solvent ( n - hexane ) and 8.5
kj / mol in polar solvent ( acetonitrile ) .
this number is invariant to
whether the calculation is done on the smaller model chromophore ( figure 11d ) or the full chromophore ( figure 11b ) .
the value in n - hexane increases to 9.3
kj / mol when a much larger basis set [ 6 - 311+g(2d , p ) ] is used .
experimental support for the 13-cis configuration
of rhodopinal in solution is provided by comparing the excited state
manifolds calculated for both the cis and the trans configurations with the experimental spectra .
note that there is a relatively strong vibronically
resolved band at 28 kk ( 28,000 cm ) observed in
the experimental spectra ( solid yellow ) .
the mndo - psdci calculations
indicate that this band is associated with a ag excited state ( top right - hand panel of figure 12 ) , resulting in transition from the ground state
that is often called the cis - band .
it should be mentioned that the viability of
the mndo - psdci methods was explored by carrying out an eom - ccsd calculation
on rhodopin .
the level ordering of the first eight excited singlet
states was identical with the exception that the eom - ccsd methods
predicted that the bu state
is higher in energy than the bu state .
the high relative intensity of the cis - band band provides spectroscopic evidence for a cis linkage near the center of the polyene chain .
the combination of theory and experiment provide
strong support for rhodopinal having a cis configuration
in both polar and nonpolar solvent .
analysis of the excited state manifold
responsible for the electronic
absorption spectrum of rhodopinal in n - hexane ( solid
yellow spectra ) based on mndo - psdci theory .
four configurations ( figure 11 ) were investigated relative to experimental observations : trans - rhodopinal ( upper left ) , 13-cis - rhodopinal
( upper right ) , trans - rhodopinal in a corkscrew conformation
( lower left ) , and 13-cis - rhodopinal in a corkscrew
conformation ( lower right ) .
the corkscrew conformation was generated
by adjusting the dihedral angles of the single bonds 10 from
planar and the double bonds 5 from planar , with all dihedral
distortions in the same direction .
the cis configuration
involves rotation of the double bond directly connected to the aldehyde
group , identified using the torsional arrow in figure 11d .
the heights of the bars are proportional to the calculated
oscillator strengths of the transitions , and the color reflects the
ionic versus covalent character ( figure 13 ) .
the approximate symmetry is indicated for selected states , and 1 kilokayser
( kk ) = 1000 cm .
the computations suggest , however , that 13-cis - rhodopinal may form a corkscrew conformation to help stabilize the
molecule in solution .
a corkscrew conformation involves clockwise
or counterclockwise dihedral distortion involving both single and
double bonds , with a majority of the distortion in the single bonds . the change in geometry costs very little in
terms of torsional distortion energy but provides intramolecular electrostatic
stabilization in nonpolar solvent and solute solvent stabilization
in polar solvent .
a modest corkscrew rotation of 10 in the single
bonds and 5 in the double bonds generates improved agreement
between experiment and theory ( bottom panels in figure 12 ) .
it should be emphasized that the same improvement in simulating
the observed spectra is obtained by generating modest , random dihedral
distortions , such as would be generated via thermal motions and occupation
of low - frequency torsional modes .
the crystal structure of
the b800 - 820 lh2 complex did not fully
resolve the rhodopinal structure , but provided
ample evidence that the structure is in an all - trans configuration . given the above results that demonstrate that 13-cis - rhodopinal is more stable than trans - rhodopinal in solution
, the question arises as to why an all - trans configuration for rhodopinal exists in the lh2
complex .
first , the
binding site for rhodopinal glucoside , which , as mentioned previously ,
is formed independently of , and prior to , the conversion of b800 - 850
to b800 - 820 , must also serve as the binding site for rhodopin glucoside ,
whose most stable configuration is undisputedly all - trans .
second , the formation of rhodopinal requires enzymatic activity
to attach the aldehyde group .
the enzyme would likely release trans - rhodopinal glucoside into solution for assembly into
the pigment protein complex , and formation of the 13-cis - rhodopinal glucoside would require either an isomerase
or > 30 min for thermal isomerization to generate the equilibrium
structure .
as shown in figure 7 , there
are significant
differences in the transient absorption spectra of rhodopinal compared
to rhodopin .
in particular , the transient absorption maximum is red - shifted
and an additional feature at 740 nm is observed for rhodopinal .
to explore these differences theoretically , mndo - psdci theory was
used to calculate the energies and oscillator strengths for excitations
from the ground state and the relaxed first excited state ( figure 13 ) .
the computations also simulated the transient
absorption spectra based on the assumption that the origin states
are the fully relaxed lowest - excited s1 ( 2ag ) states generated using full single ci
and acetonitrile as the solvent .
however , the mndo - psdci calculations
are for vacuum conditions , and the effect of solvent is limited to
the geometry of the relaxed origin state .
the simulated spectra ( figure 14 ) reproduce the key features shown in figure 7 by predicting both the red shift of the transient
maximum and the farther red - shifted weaker band observed only for
rhodopinal .
the calculations of the s1 sn transitions for rhodopin predict a maximum absorbance at 500
nm , which corresponds to the experimental band observed at 600
nm .
this band is associated with a transition from the relaxed s1 ( 2ag ) excited state
to the 11th excited ( 5bu ) singlet state ( figure 13 ) .
this state also gives rise to a weakly allowed
transition from the ground state calculated to be at 340 nm .
this ensuing discussion of the transient absorption spectrum of rhodopinal
in solution is limited to an analysis of the 13-cis configuration because it was demonstrated above that the 13-cis - isomer dominates in that environment ( figure 12 ) .
the strong band observed spectroscopically for
rhodopinal at 630 nm ( calculated to be at 520 nm )
is associated with a transition from the relaxed s1 ( 2ag ) excited state to the 11th
excited ( 5bu ) singlet state ( figure 13 ) , which is an assignment identical to that for
the intense band of rhodopin .
the major difference is a lower energy
transition calculated to be at 680 nm and experimentally observed
at 740 nm .
this more red - shifted band is associated with a
transition from the 2ag excited
state to the eighth excited ( 5ag ) singlet state ( figure 13 ) .
reported similar red - shifted transient absorption
bands from 8-apo--carotenal and also interpreted the
results in terms of a transition from the s1 ( 2ag ) state to a higher ag state .
moreover , they proposed that the allowedness
for the transition arises due to the carbonyl group introducing asymmetry
into the conjugated polyene system .
the presence of asymmetry in the
molecule would have the effect of relaxing the selection rules that
render the transition forbidden in more symmetric , linear carotenoids ,
e.g. , rhodopin .
excited state * level ordering for trans - rhodopin , trans - rhodopinal , and 13-cis - rhodopinal for excitations from the ground state for
the equilibrium
ground state conformation ( s0 geom ) and the relaxed first excited
singlet conformation ( s1 rlxd ) .
the energies and oscillator strengths
were calculated based on mndo - psdci theory using a ci basis set of
the 10 highest energy filled orbitals and the 10 lowest energy
unfilled orbitals .
the stationary states are represented by
rectangles where the height is proportional to the oscillator strength ,
and the color reflects the ionic versus covalent character of the
state ( see inset ) .
the values
in parentheses index electronic states 8 and 11 discussed in the text .
simulation of the s1 to sn spectra of trans - rhodopin ( top ) , trans - rhodopinal
( middle ) , and 13-cis - rhodopinal ( bottom ) based on
mndo - psdci theory ( full single and double configuration interaction
involving the 11 highest energy filled and 11 lowest energy unfilled
orbitals ) .
the
horizontal axis is linear in energy , where 1 kilokayser ( kk ) = 1000
cm , and the corresponding wavelength is marked
as an inset in green .
although there are higher energy bands calculated to be present
only in 13-cis - rhodopinal ( compare the middle and
bottom spectra in figure 14 ) , the above - mentioned
red - shifted band calculated to be at 680 nm appears to be
present in both cis- and trans - rhodopinal .
this observation can be traced to the fact that , whereas a transition
from the ground s0 ( 1ag ) state to the 5ag state only
has intensity in molecules with cis configurations ,
hence its designation as the cis - band ,
a transition from the s1 ( 2ag ) excited state is strong for both trans- and cis - rhodopinal .
therefore , the additional
red - shifted band seen only in the transient absorption spectrum of
rhodopinal in solution is due to the presence of the aldehyde group
on the molecule , and not to the configuration of the carotenoid .
reconstruction
and comparison of the 1-t and fluorescence excitation spectra from
the lh2 complexes ( figures 5 and 6 ) show clearly that rhodopinal is much more efficient than
rhodopin at transferring excited state energy to bchl .
the precise
factors responsible for this effect can be obtained from a consideration
of the rate constants for de - excitation of the carotenoid excited
states derived from the global fitting analysis .
the s1 and s2 states of carotenoids represent important donor
excited states for eet to bchl .
these routes are represented in figure 15 by the rate constants ket1 and ket2 , respectively .
deactivation
of these two excited states may also occur via internal conversion ,
and these processes are indicated in the figure by the rate constants , k10 and k21 .
it is
important to mention here that broadband 2d electronic spectroscopic
results provide compelling evidence for the participation in eet of
a dark intermediate state of the carotenoid having an energy in the
vicinity of the state associated with the qx transition
of bchl .
this dark state was reported
to be populated in 21 fs and depopulated in 62 fs .
both of these times
are shorter than the 100 fs time resolution of the laser spectrometer
used in the present work .
hence , the rate constant ket2 illustrated in figure 15 should
be interpreted as including the kinetics of this dark state in addition
to those of s2 .
the total eet efficiency , et , resulting from
both the s1 and s2 pathways is given by the
expression1where the
rate constants correspond to the
processes illustrated in figure 15 .
the rate
constants can be computed from the lifetimes of the s1 and
s2 excited states measured in solution , sisoln , and in the lh2 complexes , silh2 .
the relevant expressions are2and3 tables 3 , 4 , and 5 summarize the lifetimes obtained from the global
fits and the rate constants and efficiencies obtained either from
the data presented here and using eqs 13 or from previous work .
rate constants for the lh2 complexes were obtained using laser excitation
at 525 nm ( rhodopin excitation ) for the sample prepared from rbl .
acidophilus strain 10050 ( which contains only the rhodopin
chromophore ) and at 570 nm ( selective rhodopinal excitation ) for the
sample prepared from rbl .
acidophilus strain 7050
ll . determined from the
dynamics ( dyn )
of the excited states according to eq 1 . determined from steady - state fluorescence
excitation spectroscopy ( fl ) as shown in figures 5 and 6 . computed from eq 2 using the values
of 3.0 ps ( 3 in table 4 )
and 4.1 ps measured here for the lifetime of the
s1 state of rhodopin in the lh2 complex and as an average
value in solution ( 3 in table 3 ) , respectively . computed
from eq 2 using the values of 57 and 95 fs for
the lifetime of the
s2 state in the lh2 complex and in solution , respectively .
the 57 fs value was obtained from fluorescence upconversion spectroscopic
experiments reported in ref ( 33 ) .
the lifetime of the s2 state in solution was
treated as an adjustable parameter to achieve agreement between the
energy transfer efficiency of 56% computed from the dynamics data
and that measured by fluorescence excitation spectroscopy .
computed from eq 2 using the values of 1.2 ps ( 3 in table 4 ) and 4.1 ps measured here for the lifetime of the
s1 state of rhodopinal in the lh2 complex and as an average
value in solution ( 3 in table 3 ) , respectively .
computed
from eq 2 using the values of 10 and 100 fs
for the lifetime of the
s2 state in the lh2 complex and in solution , respectively .
in fact , the s2 lifetime obtained for rhodopinal glucoside
in solution was found here to be < 100 fs , which means that the k21 value of 10 ps based
on this lifetime is a lower limit to what the value could be .
the
lifetime of the s2 state in the complex was treated as
an adjustable parameter to achieve agreement between the energy transfer
efficiency of 97% computed from the dynamics data and that measured
by fluorescence excitation spectroscopy . the key result for rhodopin ( and rhodopin glucoside )
in the lh2
complex from rbl .
acidophilus 10050 is the extent
of partitioning between the s1 and s2 donor
states of the efficiency of energy transfer to bchl .
the data in table 5 show that et1 = 27% and et2 = 40% , which according to eq 1 leads
to an overall energy transfer efficiency of 56% .
however , it is important
to point out that , in the current analysis , the lifetime of the s2 state of rhodopin in solution was treated as an adjustable
parameter and set to 95 fs to achieve agreement between the energy
transfer efficiency of 56% computed from the dynamics data , and that
measured by fluorescence excitation spectroscopy ( figures 5 and 6 ) .
the value of 95
fs is in very good agreement with the s2 lifetime of 105
fs reported by macpherson et al . for
rhodopin glucoside in ethanol , and is consistent with the present
findings that the value is < 100 fs .
previous workers concur that
the s2 pathway is most important in the carotenoid - to - bchl
energy transfer mechanism in this lh2 complex ( table 5 ) .
when rhodopinal ( and rhodopinal
glucoside ) is present in the lh2
complex , as is the case for rbl . acidophilus 7050
ll and hl , the overall carotenoid - to - bchl energy transfer efficiency
becomes much greater .
as shown clearly from the analysis of the fluorescence
excitation spectra given in figure 6 , this
can be traced directly to the fact that rhodopinal is much more efficient
at transferring energy to bchl than rhodopin .
the data presented in
table 5 reveal that this enhancement is due
to a significant increase in both ket1 and ket2 for rhodopinal compared to
those obtained for rhodopin .
note from the data in table 5 that ket1 increases
from 0.089 ps to 0.59 ps and ket2 increases from 7.0 ps to 90 ps in going from rhodopin to rhodopinal
as the energy donor to bchl in the lh2 complex from 7050 ll .
these
combined increases result in a substantial enhancement in the carotenoid - to - bchl
eet efficiency from 56% for rhodopin to 97% for rhodopinal .
however ,
as was the case for the s2 lifetime of rhodopin discussed
above , owing to the fact that lifetimes of the s2 state
of rhodopinal in solution and in the lh2 complex are faster than the
time resolution of the laser spectrometer , these values were treated
as adjustable parameters to achieve agreement between the energy transfer
efficiency computed from the dynamics data and that measured by fluorescence
excitation spectroscopy .
a consideration of the fundamental
quantum mechanical expression
for the rate constant for eet4where t is the electronic
coupling term and j is the normalized spectral overlap5and fd( )
is the emission spectrum of the donor carotenoid and a( ) is the absorption spectrum of the bchl acceptor , leads
to the conclusion that the increase in the values of the rate constants
in going from rhodopin to rhodopinal ( table 5 ) can not be attributed solely to the different positions of the energy
levels of the carotenoids , and consequently neither to differences
in spectral overlap .
this is particularly evident for the route involving
the s2 ( 1bu ) state of
the carotenoid and the state corresponding to the qx band
of bchl . taking the s0 ( 1ag ) s2 ( 1bu )
absorption spectrum of rhodopin and rhodopinal reflected
about their spectral origins as approximations to their ( very weak )
emission profiles , and overlaying these lineshapes with the qx absorption band of bchl in the lh2 complex ( figure 16 ) , one obtains a rhodopinal - to - rhodopin spectral
overlap ratio of 1.2 .
this value is significantly different from the
rhodopinal - to - rhodopin ratio of ket2 rate
constants given in table 5 which indicate a
value of ( 90/7.0 ) = 13 .
although it is impossible to compute the spectral
overlap integrals associated with the s1 ( 2ag ) state of the carotenoid due to
the lack of either detectable s0 ( 1ag ) s1 ( 2ag ) absorption or fluorescence from the s1 ( 2ag ) state , the small
difference in the s1 lifetimes of rhodopin and rhodopinal
in solution ( table 3 ) suggests very similar
s1 excited state energies for the two molecules .
therefore ,
a minimal effect of the position of the energy levels , and consequently
of spectral overlap , is expected for the ket1 rate constant in the lh2 complexes .
however , a significant change
in ket1 is evidenced by comparing the
s1 lifetime of rhodopin in the lh2 complex from rbl .
acidophilus 10050 ( 3.0 ps ) with that of rhodopinal
( selectively excited at 570 nm ) in the lh2 complex from rbl .
acidophilus 7050 ll ( 1.2 ps ) . using eq 2 and an average lifetime of 4.1 ps for the carotenoids in the four
different solvents ,
the value of ket1 is
shown in table 5 to increase by a factor of
( 0.59/0.089 ) = 6.6 in going from rhodopin to rhodopinal . because only
small differences in excited state energies and spectral overlap with
the bchl qx and qy absorption bands are evident
for both the s1 and s2 states of these carotenoids ,
stronger electronic coupling between the donor and acceptor electronic
states induced by the presence of the aldehyde group on the -electron
polyene chain in rhodopinal must be a major factor determining why
rhodopinal is much more efficient at carotenoid - to - bchl eet than rhodopin
in the lh2 pigment protein complex .
spectral overlap of
the hypothetical fluorescence of the carotenoid
donor ( orange and purple traces ) and absorption of the bchl acceptor
( black trace ) .
the absorption spectra of the carotenoids were reflected
about their spectral origins to obtain approximations to the s2 ( 1bu ) s0 fluorescence
spectra .
although a rigorous analysis of
the coulomb coupling terms describing
eet from s1 and s2 is beyond the scope of this
paper , this issue is addressed for s1 by the level ordering
analysis shown in figure 13 .
the lowest excited
singlet state of both cis- and trans - configurations is calculated to be a forbidden
2ag state that gains oscillator
strength due to dipole - induced coupling with the higher energy , strongly
allowed 1bu state .
the above kinetics
analysis has revealed that energy is transferred from the s1 ( 2ag ) state to the bchl
acceptor 6.6 times faster than the corresponding state in rhodopin .
the computations predict that the enhanced energy transfer rate is
associated in part with a significant increase in the oscillator strength
of the s1 state in rhodopinal relative to rhodopin .
moreover ,
the increase in oscillator strength in trans - rhodopinal
is 35% larger than observed in 13-cis - rhodopinal
( figure 13 ) .
because the frster coupling
efficiency is proportional to the allowedness of the donor excited
state , the all - trans configuration of rhodopinal
provides added value to its light - harvesting function .
acidophilus 7050 is grown under ll conditions , two
major changes in the spectrum of the lh2 complex occur : a blue shift
of the bchl qy band from 850 nm to 820
nm , which is caused by a change in the orientation of the c3-acetyl
group of the b850 bchl ; and a red shift and broadening of the spectrum
of the carotenoid , which is a consequence of the enzymatic substitution
of an aldehyde group for a methyl group on rhodopin resulting in its
conversion to rhodopinal .
an important question is then , which of
these factors is most important for enhancing the rate and efficiency
of carotenoid - to - bchl eet ?
the answer is that the conversion of rhodopin
to rhodopinal is more important than the shift of the bchl qy band .
data suggestive of this conclusion was provided previously
by deinum et al . , who investigated the
b800 - 820 lh2 complex from rbl .
cells of this strain grown either at low temperatures or at ll do
not contain rhodopinal ( or rhodopinal glucoside ) , and
the carotenoid - to - bchl eet efficiency of its associated lh2 complex ,
which contains primarily rhodopin and rhodopin glucoside , was found
to be 45 5% .
if the change in the
position of the bchl qy band from 850 nm to 820
nm was important for enhancing the efficiency of eet from the carotenoid
to bchl , one would expect that the efficiency for rbl .
acidophilus strain 7750 would have a value above 45% or even higher than 56% ,
which is the value found for rhodopin in the b800 - 850 lh2 complex
from strain 10050 ( table 2 ) .
the fact that
it is not larger than 56% , and in fact is smaller than this value ,
i.e. , 45 5% , indicates that the change in the position of the
qy band of bchl is not a factor in determining the efficiency
of carotenoid - to - bchl eet . instead
, the conversion of rhodopin to
rhodopinal , which incidentally occurs prior to the conversion of b800 - 850
to b800 - 820 , is the primary controlling factor leading to more efficient
eet in the lh2 complex of rbl .
acidophilus 7050 grown under ll
conditions results
in a significant enhancement in the ability of the organism to transfer
absorbed light energy to the reaction center .
this is due almost entirely
to the fact that rhodopinal , and its corresponding glucoside , are
nearly 100% efficient at transferring excited state energy to bchl
in the lh2 complex .
also , the spectrum of rhodopinal occurs at a longer
wavelength compared to that of rhodopin , which allows the bacterium
containing this carotenoid to capture light energy in a spectral region
where its photosynthetic bacterial competitors do not absorb light .
this provides an important advantage for this organism as it competes
for life - sustaining photons in the water column .
moreover , based on
the ket1 and ket2 rate constants given in table 5 , the energy
absorbed by rhodopinal is transferred to bchl in the lh2 complex 6.6
times faster from the s1 state and > 10 times faster
from
the s2 state than energy absorbed by rhodopin .
more effective
electronic coupling between the electronic states of the carbonyl - containing
rhodopinal and bchl is the primary reason for the increase in these
rates . even though the blue shift of the bchl qy band
from
850 nm to 820 nm induced by growing rbl .
acidophilus 7050 under ll conditions does not have an effect on the rate and
efficiency of carotenoid - to - bchl eet , this spectral change has another
consequence .
singlet
annihilation measurements that , compared to the b800 - 850 complex ,
the b800 - 820 complex provides a more effective energy barrier for
preventing back transfer of excitation energy out of the reaction
center core complex and into the photosynthetic unit .
thus , the combined effects of alterations in carotenoid
and bchl structures and spectral properties resulting from the adaptation
of the bacterial photosynthetic organism to the challenging environmental
condition of limited light availability contribute in a very tangible
way to ensure survival of this species . | rhodopin , rhodopinal , and their glucoside
derivatives are carotenoids
that accumulate in different amounts in the photosynthetic bacterium , rhodoblastus ( rbl . ) acidophilus strain 7050 , depending on the intensity of the light under which
the organism is grown .
the different growth conditions also have a
profound effect on the spectra of the bacteriochlorophyll ( bchl ) pigments
that assemble in the major lh2 light - harvesting pigment
protein
complex . under high - light conditions
the well - characterized b800 - 850
lh2 complex is formed and accumulates rhodopin and rhodopin glucoside
as the primary carotenoids . under low - light conditions , a variant
lh2 , denoted b800 - 820 , is formed , and rhodopinal and rhodopinal glucoside
are the most abundant carotenoids .
the present investigation compares
and contrasts the spectral properties and dynamics of the excited
states of rhodopin and rhodopinal in solution .
in addition , the systematic
differences in pigment composition and structure of the chromophores
in the lh2 complexes provide an opportunity to explore the effect
of these factors on the rate and efficiency of carotenoid - to - bchl
energy transfer .
it is found that the enzymatic conversion of rhodopin
to rhodopinal by rbl .
acidophilus 7050 grown under
low - light conditions results in nearly 100% carotenoid - to - bchl energy
transfer efficiency in the lh2 complex .
this comparative analysis
provides insight into how photosynthetic systems are able to adapt
and survive under challenging environmental conditions . |
multiple sclerosis ( ms ) is a chronic inflammatory demyelinating disease of the central nervous system , characterized by various levels of physical , emotional , cognitive , and social difficulties .
thirty to fifty - five percent of ms patients consider fatigue as the most disabling symptom .
fatigue can occur in all ms forms at any stage and is reported to impact employment in 41% and social functioning in 25% of ms patients .
three dimensions of fatigue are described : cognitive , physical , and psychosocial , related to the impact of fatigue on cognitive , physical , and psychosocial functioning , respectively [ 4 , 5 ] .
the prevalence of depression in ms is reported to range from 16% to 46% and depression is considered to be more frequent in ms than in other chronic neurological diseases , with a higher risk of suicide [ 68 ] .
cognitive impairment is present in 40% to 65% of ms patients at any stage of the disease [ 9 , 10 ] and mainly affects long - term episodic and working memory , executive functions , information processing speed , and attention .
verbal episodic memory impairment and memory complaint are common in the early stage of the disease . among previous studies that examined the relationship between fatigue , cognition , and mood disorders ,
some found a link between depression and cognitive impairment whereas others did not [ 12 , 13 ] .
although a relationship has been demonstrated between fatigue and depression , the relationship between fatigue and cognitive impairment is still debated .
indeed , cognitive fatigue , defined as a decline of cognitive performance during a sustained cognitive task , seems to be correlated with cognitive impairment , whereas subjective fatigue is not associated with cognitive impairment .
cognitive complaint seems to be close to psychic factors , such as depression , fatigue , and anxiety , and is not correlated with an objective cognitive deficit evaluated by neuropsychological assessment .
such interactions between cognitive dysfunction , dysthymia , and fatigue make it unlikely that patients are able to perceive the real difficulty .
the primary objective of our study was to determine whether fatigue complaint in ms could reflect memory impairment .
the secondary objective was to investigate whether patients ' subjective fatigue is associated with memory complaint .
we consecutively included 50 ms patients who complained of fatigue between august 2011 and may 2012 .
fatigue was defined as a chronic fatigue that has occurred during at least six months and for more than half of that time .
exclusion criteria were ( i ) a history of any clinically relevant psychiatric disease or other neurological diseases , ( ii ) a history of drug or alcohol abuse , ( iii ) a relapse , steroid treatment , or recent onset of a new disease modifying drug during the month preceding the study , and ( iv ) difficulty in understanding french .
details of ms patients ' demographic and clinical characteristics and treatments were retrospectively extracted from medical files and according to a standardized questionnaire .
subjective fatigue was assessed using the fatigue impact scale ( fis , french validated version ) , a 40-item autoevaluation scale , each item being scored from 0 ( no problem ) to 4 ( extreme problem ) , providing a continuous scale from 0 to 160 .
it is made up of four subscales that describe how fatigue impacts on cognitive ( 10 items ) , physical ( 10 items ) , psychosocial ( 10 items ) , and social functioning ( 10 items ) .
the score is a reflection of functional limitations due to fatigue experienced within the previous month rather than a measure of the level of fatigue .
subjective memory complaint was evaluated by the 64 items of the questionnaire autovaluation de la mmoire ( qam ) , classified into 10 categories to assess memory lapses concerning conversations , films and books , people , and general knowledge .
each item is scored from 1 to 6 ( 1 : never , 2 : rarely , 3 : sometimes , 4 : often , 5 : very often , and 6 : always ) .
, patients completed the french validated version of the 21-item beck depression inventory ( bdi - ii ) .
each item is scored from 0 to 3 and the sum of all items gives the total bdi - ii score ranging from 0 to 63 .
scores between 0 and 13 define a minimal depressive state , between 14 and 19 a slight depressive state , between 20 and 28 a moderate depressive state , and between 29 and 63 a high depressive state .
every scale was performed by the patients themselves 2 weeks before the neuropsychological testing in order not to interfere with cognitive fatigue .
the battery , named bccogsep , is a modified version of the brief repeatable battery of neuropsychological tests for multiple sclerosis
( 1 ) the selective reminding test ( srt ) measures verbal episodic memory , with 3 scores considered : mean number of words in immediate recall , learning index , and delayed recall .
( 3 ) the symbol digit modality test ( sdmt ) measures information processing speed and selective attention . ( 4 ) the crossed tapping test evaluates mental flexibility . ( 5 ) the paced auditory serial addition test ( pasat ) evaluates sustained attention , information processing speed , and working memory . ( 6 ) memory span assesses immediate and working memory .
fourteen scores were obtained and a patient was considered impaired when a score was inferior to percentile 5 compared to the normal range .
a patient was considered to present global cognitive damage when at least 4 scores were lower than percentile 5 .
statistical analyses were performed using sas software , version 9.2 ( sas institute inc . ,
numerical variables are expressed as mean standard deviation ( sd ) and qualitative parameters as frequency and percentage .
for the primary objective , the outcome measurements were to assess the relationship between the 3 scores of the srt ( number of words , srt learning index , and delayed recall ) , the score of information processing speed and the global cognitive performance and each of the following 4 independent variables separately : total fis score , fis cognitive subscore , fis physical subscore , and the qam ( independent variables ) .
first , the influence of the clinical and therapeutic characteristics on the dependent variables ( the 3 scores of the srt and qam ) was studied by bivariate analyses .
the mann - whitney u test was used for qualitative binary values ( gender , occupational status , presence of autoimmune comorbidities , form of ms , and association with psychotropic and/or antiepileptic drugs ) and the kruskal - wallis test for evaluating qualitative values in more than 2 classes ( the treatment for ms ) . the correlation with quantitative variables ( disease duration , expanded disability status scale ( edss ) , age , and bdi - ii )
was assessed by a pearson correlation coefficient test . for each dependent variable , parameters with a p value less than 0.1
the correlation between each dependent variable and each of the 4 independent variables separately ( total fis score , fis cognitive subscore , fis physical subscore , and the qam ) was computed with a pearson correlation coefficient test . for each dependent variable ( e.g. ,
srt number of words ) and each independent variable ( e.g. , total fis score ) , a linear regression was performed in order to adjust for the confounding factors .
the same analysis was performed with qam as dependent variable and the 3 fis scores separately ( total fis score , the fis cognitive subscore , and the fis physical subscore ) as independent variables .
the sex ratio showed a predominance of women ( 66% ) in the patient group .
the mean education level was 12 2 years ; the median edss score was 3.0 [ 2.05.0 ] , and the mean disease duration was 13 7.9 years .
the mean scores for each fatigue dimension , that is , cognitive , physical , psychosocial , and social functioning scores , are reported in table 2 .
concerning memory complaint , the mean qam score was 2.75 0.84 . a memory complaint in at least one dimension of the qam
memory impairment was the most frequent ( 62% , n = 31 ) , followed by executive deficits ( 60% , n = 30 ) .
sixteen patients ( 32% ) presented a verbal episodic memory deficit on the three scores of the srt : number of words reminded , learning index , and delayed recall ( table 3 ) .
depression , edss status , concomitant treatment ( psychotropic and/or antiepileptic treatment ) , ms form ( progressive form ) , and ms treatments interfered with subjective fatigue and memory complaint scores and verbal episodic memory performance ( table 4 ) .
we verified that after adjustment for mood there was no link between any of the concomitant treatment classes ( such as benzodiazepines , antiepileptics , or antidepressants ) and fatigue and memory performances .
moreover , after adjustment for mood , the edss score was significantly associated with the physical subscore of fatigue ( p = 0.048 ) .
after adjustment for confounding factors ( table 4 ) , verbal episodic memory performance , assessed by the number of words recalled , learning , and delayed recall , was not correlated with either the total score ( number of words p = 0.1204 , learning p = 0.3216 , and delayed recall p = 0.6389 ) , the cognitive subscore ( number of words p = 0.1444 , learning p = 0.1249 , and delayed recall p = 0.5790 ) , and the physical subscore ( number of words p = 0.1511 , learning p = 0.4114 , and delayed recall p = 0.7430 ) of the fis or memory complaint ( number of words p = 0.4297 , learning p = 0.2569 , and delayed recall p = 0.3828 ) .
information processing speed was not correlated with either the total score ( p = 0.31 ) or the cognitive and physical subscore ( p = 0.65 and p = 0.14 , resp . ) .
global cognitive performance was not correlated with either the total score ( p = 0.93 ) or the cognitive and physical subscore ( p = 0.21 and p = 0.86 , resp . ) . both the total score ( p < 0.0001 ) and the cognitive subscore ( p
there was no significant link between physical subscore of subjective fatigue and memory complaint ( p = 0.053 ) .
our findings show that subjective fatigue complaint was associated with memory complaint but not with memory performances .
our study did not find any correlation between subjective fatigue in ms patients and verbal episodic memory performance evaluated by the srt test and global cognitive performance evaluated by the number of scores impaired on the bccogsep .
moreover , subjective fatigue was not correlated with information processing speed , a cognitive function frequently impaired in ms patients . in the literature ,
conflicting results have been reported concerning a correlation between fatigue complaint and episodic verbal memory performance and between fatigue complaint and information processing speed .
nocentini et al . reported that fatigue complaint on the fatigue severity scale ( fss ) was correlated with immediate and delayed recall scores on the 15-word rey 's test .
showed a correlation between the fis score and the delayed recall score on the srt .
however , in contrast to our study , these results were not adjusted for confounding factors . according to the literature
, the intensity of fatigue complaint in ms patients does not reflect their memory performance . in previous studies ,
the degree of fatigue , as defined by the fss or fis scales , was not significantly associated with paired associate learning test ( palt ) scores , evaluating episodic verbal memory . in the study by krupp and elkins , a decrease in memory performance on the srt test
was not correlated with an increase of subjective fatigue . more generally , no correlation was found between cognitive performance and subjective fatigue .
moreover , our results did not show a correlation between memory complaint and episodic verbal memory performance .
ms patients might have a poor ability for self - assessment of their memory difficulty .
however , this memory complaint in our results reflects subjective fatigue complaint and it therefore seems necessary to assess fatigue when memory complaint occurs .
in our study , a high percentage of the ms patients had a high score on the fis and the physical dimension of the fatigue was the most frequent complaint .
this physical dimension is more pronounced in ms patients with a higher edss score because edss assesses mainly the physical disability of ms . as in a study by pittion - vouyovitch
et al . , the physical dimension was more involved than the other dimensions of fatigue .
forty - four percent of ms patients in our study presented a cognitive impairment , a similar proportion to that found in other studies [ 10 , 28 ] .
there is a variability in the occurrence of memory performance disturbances reported in the literature , ranging from 29% to 69% of ms patients .
such variability is likely due to the different tools used to test verbal episodic memory . in our study , 32% of ms
patients presented verbal episodic memory impairment and , as expected , ms patients with a progressive form presented higher memory impairment than relapsing ms patients .
our study found that fatigue was not related to impairment of objective memory performance but could affect ms patients ' perception regarding their memory .
memory complaint of ms patients was associated not with their memory performance but with their fatigue . | background and purpose . fatigue and memory impairment are common symptoms in multiple sclerosis ( ms ) and both may interact with cognition . this can contribute to making a complaint misrepresentative of the objective disorder .
we sought to determine whether fatigue complaint in ms reflects memory impairment and investigated whether patients ' subjective fatigue is associated with memory complaint .
methods .
fifty ms patients complaining of fatigue underwent subjective assessment of fatigue and memory complaint measured using self - assessment scales .
cognitive functions were assessed using a battery of neuropsychological tests , including a test of verbal episodic memory , the selective reminding test ( srt ) .
correlations were studied between subjective fatigue , memory complaint , and performance in verbal episodic memory .
results .
depression score , psychotropic and/or antiepileptic drug use , expanded disability status scale ( edss ) score , and ms form were confounding factors . after adjusting for these confounding factors , neither fatigue complaint nor memory complaint
was correlated with srt performance .
subjective fatigue was significantly associated with memory complaint . conclusion .
although complaint of fatigue in ms was correlated with memory complaint , subjective fatigue was not the expression of memory impairment . |
placental abruption occurs when the placenta partially or completely detaches from the uterine wall during the pregnancy or during parturition before the delivery of the fetus ; it occurs in 0.5 - 1.5% of all pregnancies .
clinical presentation varies depending on the degree of placental detachment . however , blood flow through the placenta to the fetus can be partially or completely blocked , increasing fetal mortality .
morbidities affecting the mother also increase , because disseminated intravascular coagulation ( dic ) can cause severe blood loss .
placental abruption is most frequently observed during the 24 to 26 weeks of pregnancy , decreasing in frequency thereafter .
we report a case of placental abruption with severe blood loss at the 17 weeks of gestation .
she presented with sudden severe lower abdominal pain at 17 weeks gestation , accompanied by severe vaginal bleeding .
clinical findings : she was pale , blood pressure was 80/50 mmhg , and pulse rate was 120 per minute .
additionally , the placenta was attached to the anterior uterine wall , but a well - defined 53 cm high - intensity region and a 12-cm lowintensity region were noted within the placenta suggestive of intra - placental hemorrhage ( figure 1 ) .
initial laboratory findings : blood parameters were as follows : hemoglobin , 7.9 g / dl ; platelet count , 82,000/l ; prothrombin time , 320 seconds ( control , 11.4 seconds ) ; partial prothrombin time , > 400 seconds ( control , 11.4 seconds ) ; serum fibrinogen , 17 mg / dl ( reference range : 270 - 471 mg / dl ) .
a diagnosis of placental abruption at 17 weeks of gestation was made by clinical symptoms and ultrasonographic finding .
she was hospitalized to treat the dic , and hemorrhagic hypovolemic shock secondary to placental abruption .
a total of 16 units frozen plasma and 10 units packed red cells were transfused to treat the dic and hemorrhagic shock . however , hematological parameters 2 h post - transfusion worsened : hemoglobin , 5.0 g / dl ; platelet count , 42,000/l ; blood pressure , 70/40 mmhg ; pulse rate , 120 per minute .
however , since the dilation of the uterine cervix was only 2 cm , rapid delivery could not be expected .
therefore , we decided to perform a hysterotomy and terminate the pregnancy for life - threatening maternal hemorrhage .
blood infiltration was evident in the uterine muscle layer ( i.e. , couvelaire uterus ) ( figure 2 ) .
a hematoma covering 60% of the posterior wall of the placenta was observed when the uterus was excised and the fetus and placenta were removed ( figure 2 )
satisfactory uterine contraction and natural reduction in bleeding from the uterus were confirmed ; therefore , any other medication and gauze packing was unnecessary .
a total of 20 units frozen plasma and 12 units packed red cells were transfused during the operation .
uterine bleeding decreased , and the volume of blood loss was 380 ml 4 h after the operation .
twenty - four - hour postoperative hemoglobin was 9.9 g / dl , platelet count was 102,000/l , prothrombin time was 10.4 seconds ( control , 11.4 seconds ) , partial prothrombin time was 25.8 seconds ( control , 29.4 seconds ) , and serum fibrinogen was 296 mg / dl ( reference range : 270 - 471 mg / dl ) .
the dic improved , and vaginal bleeding became minimal ; therefore , treatment was halted , and the patient was discharged 6 days after the operation .
histopathological studies revealed a blood clot attached to the deciduous placenta and infiltration of blood into the surrounding intervillous space , confirming ischemic changes .
a total of 16 units frozen plasma and 10 units packed red cells were transfused to treat the dic and hemorrhagic shock . however , hematological parameters 2 h post - transfusion worsened : hemoglobin , 5.0 g / dl ; platelet count , 42,000/l ; blood pressure , 70/40 mmhg ; pulse rate , 120 per minute .
however , since the dilation of the uterine cervix was only 2 cm , rapid delivery could not be expected .
therefore , we decided to perform a hysterotomy and terminate the pregnancy for life - threatening maternal hemorrhage .
blood infiltration was evident in the uterine muscle layer ( i.e. , couvelaire uterus ) ( figure 2 ) .
a hematoma covering 60% of the posterior wall of the placenta was observed when the uterus was excised and the fetus and placenta were removed ( figure 2 )
satisfactory uterine contraction and natural reduction in bleeding from the uterus were confirmed ; therefore , any other medication and gauze packing was unnecessary .
a total of 20 units frozen plasma and 12 units packed red cells were transfused during the operation .
uterine bleeding decreased , and the volume of blood loss was 380 ml 4 h after the operation .
twenty - four - hour postoperative hemoglobin was 9.9 g / dl , platelet count was 102,000/l , prothrombin time was 10.4 seconds ( control , 11.4 seconds ) , partial prothrombin time was 25.8 seconds ( control , 29.4 seconds ) , and serum fibrinogen was 296 mg / dl ( reference range : 270 - 471 mg / dl ) .
the dic improved , and vaginal bleeding became minimal ; therefore , treatment was halted , and the patient was discharged 6 days after the operation .
histopathological studies revealed a blood clot attached to the deciduous placenta and infiltration of blood into the surrounding intervillous space , confirming ischemic changes .
placental abruption is one of the serious complications of pregnancy , because it leads to both poor maternal and fetal / neonatal outcome . in cases involving severe blood loss , incurring a risk of the maternal mortality , improvement from the life - threatening condition
however , since placental abruption with severe blood loss causing a life - threatening situation for the mother is rare for pregnancies before the 20 week of gestaion , the present case is an important case study .
the diagnosis of placental abruption usually depends on the clinical manifestations , and confirmed the placental detachment after delivery .
the usefulness of ultrasonography have been reported as the adjunctive diagnosis , and it is widely used for this purpose .
the patient presented with sudden and severe vaginal bleeding accompanied by lower abdominal pain . in addition ,
uterine stiffness was observed and intra - placental hematoma was confirmed by ultrasound . on the basis of these observations , we diagnosed the patient with placental abruption even though the pregnancy was only in the 17 weeks of gestation .
hypostasis of the uterus , i.e. , couvelaire uterus , was confirmed during the operation .
the histology of the placenta showed retroplacental blood clot as well as bleeding and ischemic changes in the intervillous space .
these findings demonstrate that placental abruption can occur and is diagnosable at the 17 weeks gestation .
placental abruption most frequently occurs during the 26 weeks gestation and rarely before the 20 weeks .
placental abruption at this stage of pregnancy is very uncommon , but the associated dic can be life threatening .
similar case reports and short communications were published in the literature in the late 1980s .
it is interesting to note that only 1 case was sufficiently severe to warrant dilation and evacuation , whereas the other obstetrics cases were followed with cautious conservative treatment as long as the fetus was alive , and the pregnancies were completed with fetal survival . in addition , although severe dic developed rapidly , the coagulopathy was completely resolved .
however , it is important to distinguish placental abruption as one of the causes of bleeding during the early phase of the second trimester .
next , the delivery method for fetuses before the 24 weeks gestation must be considered for severe placental abruption .
oyelese and ananth proposed an algorithm that calls for the delivery of premature fetuses before the 24 weeks regardless of the certainty of its survival , if there is danger to the mother ; however , they do not discuss the method of delivery . on the basis of the present patient s four prior normal pregnancies and stiff uterine contractions
however , dilation of the uterine cervix did not advance . on the other hand ,
bleeding continued , the mother s anemia worsened , blood pressure decreased , tachycardia was observed , and her general condition worsened .
termination led to rapid improvement of the mother s condition , suggesting that the surgical intervention was effective .
hematological findings at the time of hospitalization already revealed reduction of blood fibrinogen and platelets as well as continuous massive vaginal bleeding .
hence , we surmised that the placental abruption led to secondary dic , causing life - threatening blood loss for the mother .
therefore , we transfused fresh frozen plasma to correct the dic ; we simultaneously performed rapid transfusions to reduce circulating blood volumes .
dic is most common in patients with comparatively large ablated areas of the placenta or those with prior in utero fetal deaths .
however , in this case , the fetus was alive , and the mother still presented with dic , which differs from previously reported cases .
similar cases of dic with live fetuses accompanying placental abruption report that dic is more prevalent because there is more movement of tissue factors during the second trimester than the third . in such cases ,
dic can occur with placental abruption early in the pregnancy without adversely affecting the fetal heart rate ; however , it may be a distinct characteristic of placental abruption occurring early in pregnancy .
there is a report that pregnancy can be maintained even after anti - dic therapy for placental abruption of normally implanted placenta , which led to improvement .
seckin reported that progression from placental abruption to dic is rare during the second trimester ; if it does occur , the presence of factors for thromboembolic formation must be investigated . however , unfortunately , protein s , c for inherited thrombophilic conditions were untested in the presented case .
the risk factors leading to the onset of placental abruption include smoking ( odds ratio : 1.5 - 2.5 ) and age over 35 ( odds ratio : 1.3 - 2.6 ) .
furthermore , history of previous placental abruption has a high recurrence rate ( odds ratio : 3.2 - 25.8 ) .
this case illustrate that if there is a life - threatening obstetrical hemorrhage secondary to placental abruption , even early in the second trimester , pregnancy terminated promptly . | a 40-year old woman , gravida 4 , para 4 , presented with sudden lower abdominal pain and severe vaginal bleeding at 17 weeks of gestation . clinical symptoms and ultrasonographic finding revealed placental abruption .
the volume of bleeding was heavy and led to disseminated intravascular coagulation and hypovolemic shock .
we performed blood transfusion and therapy to treat the critical condition .
however , the mother s condition continued to worsen .
therefore , we performed a hysterotomy and aborted the pregnancy to save the mother .
since heavy bleeding caused by placental abruption leading to a life - threatening condition for a mother before the 20 weeks of gestation is very rare , the present case is an important case study . |
the majority of women with human immunodeficiency virus 1 ( hiv ) are of reproductive age and may use an efavirenz- ( efv- ) containing antiretroviral ( arv ) regimen [ 1 , 2 ] .
efv is a nonnucleoside reverse transcriptase inhibitor ( nnrti ) indicated in combination with other antiretroviral agents for the treatment of hiv .
efv is an fda pregnancy category d drug based on animal studies and human case reports of fetal neural tube defects [ 36 ] .
pregnancy rates for hiv - infected women range from 6.0 to 8.2 pregnancies per 100 person - years , and in 2001 , 49% of all pregnancies in the united states were unintended [ 79 ] .
women with hiv not desiring pregnancy are advised to use dual methods of contraception to prevent pregnancy and hiv transmission to their partners .
some women use emergency hormonal contraception to prevent pregnancy after unprotected sex or contraceptive failure ( condom breakage ) .
plan b is a levonorgestrel- ( lng- ) containing emergency contraceptive pill indicated for pregnancy prevention following unprotected intercourse or a known or suspected contraceptive failure .
it is taken as soon as possible within 72 hours after unprotected intercourse either as a single dose ( lng 1.5 mg ) or as two doses ( 0.75 mg ) taken twelve hours apart .
lng use for emergency hormonal contraception has been shown to reduce pregnancy rates by 85% .
few data are available on the pharmacokinetics ( pk ) of progesterone - based contraceptives with nnrtis .
a study of depomedroxyprogesterone acetate ( dmpa ) depot injections in hiv - infected women on antiretroviral therapy revealed no significant change in plasma levels of mpa or efv , nevirapine , or nelfinavir .
however , in a study of efv and the combination oral contraceptive pill orthotricyclen - lo and orthocyclen ( 25 mcg ethinyl estradiol plus 0.180.25 mg norgestimate ) , lng area under the concentration time curve from 0 to 12 hours ( auc12 ) , maximum concentration ( cmax ) , and minimum concentration ( cmin ) were decreased by 80% , 83% , and 86% , respectively . a case of contraceptive failure with ectopic pregnancy in an hiv - infected woman occurred with the etonogestrel contraceptive implant and efv .
subjects were hiv - seronegative women ages 1845 years with normal body mass index and no recent use of hormonal contraceptive agents ( oral or vaginal hormonal contraception use within 60 days or injectable hormonal contraception use within 180 days of study entry ; subjects with mirena iud were excluded ) or other medications / therapies known to interact with efv . subjects who had not undergone surgical sterilization used 2 nonhormonal types of contraception throughout the study period and for 2 weeks following study completion .
the protocol was approved by the institutional review board at participating sites , and informed consent was obtained from each woman before participation .
this was a prospective , open - label , single - arm , two - period , pk equivalence study .
the primary objective was to compare plan b lng auc12 prior to and during steady - state efv .
secondary objectives included ( 1 ) characterization of other lng plasma pk parameters , ( 2 ) assessment of the safety and tolerability of coadministration of plan b and efv , and ( 3 ) evaluation of potential effects of lng on efv auc24 with comparison to previous data in hiv+ women .
study participants received lng 0.75 mg at time 0 and 12 hours at baseline ( visit 1-day 0 ) and after steady state efv dosing ( visit 2-day 17 ) .
subjects were begun on efv 600 mg at bedtime on empty stomach 72 hours after visit 1 for a total duration of 14 days .
participants fasted at least 12 hours prior to the pk study visits and ate a standardized breakfast with lng dosing ( 600 kcal ; 15% protein , 30% fat , and 55% carbohydrates ) .
serial blood ( plasma ) sampling for lng pk analysis was performed after lng dosing at 0 ( predose ) , 2 , 3 , 4 , 6 , 8 , 10 , and 12 hours at visit 1 and 2 .
blood ( plasma ) sampling for efv pk analysis was performed prior to lng dose , 6 and 12 hours after lng dose at visit 2 only ( corresponding to approximately 10 , 16 , and 22 hours from efv dosing ) .
relevant clinical adverse events were assessed at study and 4 telephone visits ( study days 4 , 11 , 16 , and 2028 ) .
safety and laboratory profiles and pregnancy testing were performed at screening and visits 1 , 2 ( lft 's only ) , and 3 .
efv adherence was assessed by subject self - report at telephone visits approximately 7 and 17 days after efv was initiated .
lng plasma concentrations were determined with a liquid chromographic assay with ms / ms detection linear in the range of 5025000 pg / ml .
efv plasma concentrations were determined using a validated hplv / uc method linear in the range of 2020,000 ng / ml . accuracy and precision were within 15% with 0.2 ml plasma .
samples were frozen and shipped to ppd , inc . for lng analysis and university of colorado pharmacology lab for efv analysis .
sample size calculations assumed that expected lng auc12 was 123.1 nghr / ml with a standard deviation of 50.1 . assuming equal variances and a modest correlation of 0.5 , the standard deviation of the difference is also 50.1 .
18 subjects were required to detect a difference of 49.2 ( a 40% change ) in lng auc12 using a two - sided , paired t - test with a significance level of 0.05 and 97.5% power . to account for drop - outs
lng pk was determined by noncompartmental methods ( winnonlin v5.2.1 , pharsight corporation , mountain view , ca ) .
lng auc12 was calculated with the linear - log trapezoidal rule and lng cmin , cmax , and time to cmax ( tmax ) determined visually .
lng half - lives ( t1/2 ) were calculated as 0.693 divided by z , where z was the terminal elimination rate constant .
lng total apparent oral clearance ( cl / f ) was determined as dose divided by auc12 .
apparent volume of distribution ( v / f ) was determined by cl / f divided by z .
a post hoc bayesian approach ( nonmem vvi ) was used to estimate each subject 's efv auc24 based on the three measured efv levels .
the estimated auc24 was compared to data from a previous pk study of hiv+ women using a 2-sample t - test . for the primary hypothesis ,
equivalence was defined as a decrease of less than 40% lng auc12 after addition of efv based on previous studies utilizing a 40% difference in contraceptive steroid hormone auc as that which is clinically relevant .
the null hypothesis of equivalence was rejected if the corresponding 95% confidence interval included values 40% .
point estimates and 90% confidence intervals for geometric means of lng auc12 , cmax , cmin , v / f and cl / f , and t1/2 were determined for lng dosed alone and with efv .
geometric mean ratios ( gmr ) for lng auc12 , cmax , and cmin with versus without efv were calculated .
evaluable pk data was generated for 21 women who had a mean age of 31 years ( range 2145 ) and bmi of 27 ( range 2135 ) .
the majority of subjects were white ( 62% ) , and 24% were latina and 10% black .
contraception use included condoms , spermicide , diaphragm , abstinence , and intrauterine device .
the estimated percent decrease in lng auc12 with efv was 56% ( table 1 ) , and the corresponding 95% confidence interval ( 49% , 62% ) excluded a change of 40% ( p < 0.0001 ) , such that the equivalence hypothesis was rejected .
a decrease in lng auc12 of > 40% was observed in 90.5% ( 95% ci : 0.70% , 0.99% ) of women .
the geometric mean efv auc24 in combination with lng was 69597 nghr / ml .
this value was compared to a previous study of efv pk in hiv - infected females which demonstrated an efv geometric mean auc24 of 61361 nghr / ml ( 90% ci 19076 , 197379 nghr / ml ) ( p value = 0.35 ) .
study participants had a > 95% adherence with efv dosing , and all had detectable efv levels .
headache , abdominal pain , diarrhea , and menstrual cycle changes were the most common adverse events occurring in > 10% of subjects after plan b dosing alone .
the incidence of abdominal pain , diarrhea , and menstrual cycle changes was decreased , while incidence of fatigue was increased with efv .
the occurrence of rash , pruritus , abnormal dreams , and insomnia was similar to previous studies of efv .
adverse events were mild ( grade 1 ) to moderate ( grade 2 ) in majority and were resolved at follow - up visits .
one subject had a grade 2 rash likely related to study treatment ( efv ) and resolved at follow - up visits .
one subject had grade 3 syncope not related to study treatment and attributed to a vasovagal reaction with phlebotomy .
data are limited regarding the use of hormonal contraception in hiv - infected women on antiretroviral therapy .
interactions have been described between steroid hormones and both protease inhibitors and nnrti which could lead to decreased protection from pregnancy or increased contraceptive side effects .
previous studies have focused predominantly on combined oral contraceptive pills , injectable dmpa , and one small study evaluated pk with the transdermal contraceptive patch [ 12 , 13 , 18 ] .
women taking efv are specifically advised to avoid pregnancy due to this agents ' potential role in fetal neural tube defects [ 3 , 19 ] .
thus , emergency hormonal contraception , like plan b , may be even more important for these women .
we sought to evaluate the effect of efv on plasma concentration of lng in plan b in healthy hiv - negative women .
we found that pretreatment with efv for 14 days was associated with a 56% , 41% , and 67% decrease in lng auc12 , cmax , and cmin , respectively .
efv is an inducer of cyp3a and uridine - diphosphate glucuronosyl transferases ( ugts ) in vivo .
lng exposures are reduced approximately 40% with the anticonvulsants phenytoin and carbamazepine ( inducers of cyp3a ) and 19% with lamotrigine ( an inducer of glucuronidation enzymes ) [ 20 , 21 ] .
a study of rifampin and oral contraception demonstrated considerable reduction in contraceptive hormone levels ; however , ovulation suppression persisted .
these findings may have important ramifications with regard to the efficacy of plan b when taken with this arv .
however , the clinical relevance of this finding is unclear as the minimal effective lng plasma concentration is unknown .
it is possible the alternate plan b single dosing of lng 1.5 mg would mitigate this effect ; however , this is unlikely given the magnitude of our observed difference .
further clinical studies of plan b and efv are thus needed to inform providers of potential need for plan b dosing adjustments for these women .
hiv providers ' role in providing family planning services including contraception and preconception counseling is significant given the inherent complexities with hiv and antiretroviral therapy . | objectives . compare the plan b levonorgestrel ( lng ) area under the concentration- time curve ( auc12 ) prior to and with efavirenz ( efv ) . design .
prospective , open - label , single - arm , equivalence study . methods .
healthy hiv - negative subjects underwent 12 hr intensive pharmacokinetic ( pk ) sampling following single dose lng alone and after 14 days of efv .
geometric means , geometric mean ratios , and 90% confidence intervals ( ci ) are reported for pk parameters .
t - tests were utilized .
clinical parameters and liver function tests ( lfts ) were assessed .
results .
24 women enrolled and 21 completed the study .
with efv , lng auc12 was reduced 56% ( 95% ci : 49% , 62% ) from 42.9 to 17.8 nghr / ml , and maximum concentration ( cmax ) was reduced 41% ( 95% ci : 33% , 50% ) from 8.4 to 4.6 ng / ml .
lng was well tolerated with no grade 3 or 4 treatment - related toxicities . conclusions .
efv significantly reduced lng exposures .
higher lng doses may be required with efv .
these results reinforce the importance of effective contraception in women taking efv . |
artemisinin , derived from sweet wormwood , artemisia annua , is the spearhead of anti - malarial chemotherapy . in 2004 ,
the world health organization ( who ) recommendation that artemisinin - based combination therapy ( act ) should be the norm for the treatment of falciparum malaria in most endemic countries came into effect .
financial backing for this treatment from the global fund for aids , tuberculosis and malaria and other international sponsors has led to a massive increase in the demand for artemisinin , but with an estimated 1214 months lead - time from planting of a. annua to artemisinin extraction , stepping - up production ( or adjusting to fluctuations in demand ) from the plant is not simple , and alternative means of producing the raw material by complete chemical synthesis or recombinant technologies are being explored .
it would , however , be naive to believe that the cost of the raw material is the main determinant of the cost of a drug .
this article looks at issues surrounding the production of artemisinins , particularly in the light of a recent paper by jay keasling and colleagues ( ro et al . ) in bmc biotechnology , which describes a way of improving the yield of the precursor , artemisinic acid , in genetically engineered yeast .
artemisinin ( qinghaosu ) was discovered in 1971 by chinese scientists looking at the anti - malarial properties of herbs in the traditional chinese pharmacopeia : a. annua had been used as an antipyretic tea for more than 2,000 years . although no longer used as a drug itself , artemisinin is the starting compound for a class of highly active anti - malarials , which are produced by chemical modification of artemisinin .
these ' artemisinins ' include dihydroartemisinin , artemether and artesunate , the latter being a particularly interesting derivative because it is water - soluble and can be given intravenously .
artemisinin is a sesquiterpene lactone containing an endoperoxide bridge that is considered essential to its anti - malarial action : haem - catalyzed cleavage of the peroxide generates unstable free radicals to which malaria parasites are particularly sensitive .
although the peroxide activity is necessary , it may not be sufficient to explain the mechanism of action of artemisin , and alternative hypotheses might need to be considered .
specific interactions of artemisinin with parasite proteins , such as the translationally controlled tumor protein ( tctp ) and the parasite ortholog of sarcoplasmic / endoplasmic - reticulum ca - atpase ( pfatp6 ) , have been reported , but the interaction with haem within the parasite - containing vacuole inside the host cell is of special interest .
the conversion of toxic haem into non - toxic hemozoin , or malarial pigment , is crucial for parasite survival , and the inhibition of hemozoin formation is a major mechanism of action of many existing anti - malarials , including chloroquine .
kannan et al . have shown that there is a potent interaction between protein - bound haem in hemoglobin and artemisinin , with the formation of ' hemarts ' , which bind to pfhrp2 ( plasmodium falciparum histidine - rich protein-2 ) and inhibit hemozoin formation .
artemisinins have broad stage specificity in their anti - malarial action , killing all asexual stages , as well as gametocytes , but have no effect on the exo - erythrocytic stages .
where artemisinins differ from other anti - malarials is that they kill young trophozoites ( ' ring ' stage ) ; ( figure 1 ) . as a result , artemisins are rapidly effective and reduce the number of late trophozoites in the capillaries .
there is a rapid clinical improvement in patients and reduction of the risk of severe complications .
the effect on gametocytes means less efficient transmission of the parasite to the mosquito vector , which potentially contributes to an interruption of transmission to humans and also reduces the risk of dispersion of any drug - resistant mutants .
recent field studies have shown that artemisinin treatment significantly reduced gametocyte carriage compared with other drugs , an effect that was variable from one geographical area to another , depending on the level of malaria endemicity and access to treatment .
artemisinins kill parasites more effectively and at an earlier stage in the erythrocytic part of its life cycle than most of the other anti - malarial currently in use .
they do not work on the exo - erythrocytic forms , hence do not prevent relapses in p. vivax or p. ovale .
monotherapy with artemisinins has now mostly been abandoned . who finally agreed , after years of lobbying , to recommend the use of a three - day regimen of combination therapy that is , artemisinin together with a partner drug for the treatment of uncomplicated malaria .
because of their different kinetics of action , not only does one drug protect the other from provoking resistance , but as artemisinins effectively kill most of the parasites in the first few hours of treatment , the partner drug has a better chance of being effective against the remaining few .
two fixed - dose drug combinations are available , artemether - lumefantrine ( coartem or riamet ) and artesunate - amodiaquine ( coarsucam or asaq ) : these have broadly comparable efficacy and the fixed dose has many advantages over other presentations , such as blister - packs .
intravenous artesunate acts significatively faster than intravenous quinine , in terms of both parasite clearance and clinical improvement , and there is now a strong case for recommending parenteral artesunate instead of quinine for the treatment for severe malaria . as well as their anti - malarial activity ,
artemisinins are effective against a number of other parasitic diseases , particularly schistosomiasis , and there is now growing experimental evidence that they may potentially be useful against cancer , especially as their mechanism of action is so different from that of other anti - cancer drugs .
this question can be rephrased as ' how many cases of malaria need to be treated with act every year ? ' and is difficult to answer .
estimates of the true prevalence of malaria worldwide vary greatly because they are not based on accurate reporting at the country level . in most endemic countries
, treatment is given on a presumptive diagnosis , sometimes using a clinical algorithm with the presence of fever as a key factor but generally without laboratory confirmation .
at best this approach is only 50% reliable ( and less than 5% reliable in areas of low endemicity ) . in africa alone
, various estimates suggest that there are 215 to 375 million malaria episodes per year , and , on the basis of usage of the ' old anti - malarials ' , kindermans et al . calculated projected drug consumption to be between 113 and 314 million adult - equivalent treatment courses per year . in 2004 ,
the forecast was for 60 million treatment courses in 2005 and 130 million in 2006 .
although these figures already underestimated actual medical need , the amount of act delivered in africa in 2006 was even lower ( less than 100 million treatment courses ) .
underproduction leads to shortages and inflated prices , whereas overproduction leads to rapid expiry of the drugs and potential losses by suppliers , as outlined by cohen and colleagues , who looked at forecasting issues from a funding agency angle .
a. annua is an annual plant , growing to 150 cm tall and found wild in many countries ( figure 2 ) .
production of a. annua is dominated by china and vietnam , but it is also now grown in the highlands of tanzania , south africa , india and madagascar . the active pharmaceutical ingredient ( api ) or ' raw artemisinin '
is extracted from the leaves , just before the plant flowers , and from planting to extraction takes more than 12 months .
soil , climate , altitude and grower 's know - how can affect the content of artemisinin substantially . early problems with scaling - up production have , to a large extent , been solved by selection of plants for high artemisinin content and adaptation to the environment in which they are to be grown .
further improvement in yield and quality may be possible by further selection , like that being undertaken by artemisinin enterprise at the university of york , in collaboration with the swiss not - for - profit organization mdiplant , using fast - track genetic breeding technologies .
hydroponics and in vitro plant culture are other interesting options , which have not yet confirmed their potential .
there are claims that the plant simply infused in water could provide a ' home - grown ' malaria treatment in rural areas in the tropics . although trials of such a tea yielded higher than expected amounts of artemisinin , and the plasma concentration achieved by consumption of the tea was compatible with an effective anti - malarial activity ( reviewed in ) , the same trials showed considerable variation in artemisinin content from one batch of plants to another and a high rate of malaria recrudescence .
it would be wrong to promote the use of wormwood tea as a ' cheap ' alternative to drug treatment .
this use would certainly increase the risk of resistance emerging and a. annua does not grow in the tropical environment where malaria is endemic and can not , therefore , be ' home - grown ' where it would be most needed .
raw artemisinin is extracted with solvents , such as hexane , followed by chromatography to improve purity .
the extracts usually contain both artemisinin and its precursor , artemisinic acid , which can later be transformed into artemisinin . on average ,
40 hectares ( ha ) of land produce a tonne of dried leaves , which can yield 6 kg of artemisinin .
the production of 100 million adult treatment courses would require about 10,000 ha of a. annua plantations . despite the relatively low yield ,
the exploitation of a. annua is commercially viable as long as the price of api is sufficiently stable . in reality , there have been massive fluctuations since 2004 , with prices varying between us$1,600/kg , when there was a world shortage following the who recommendation , to as low as $ 150 at the end of 2007 , when there was a glut .
this see - saw effect , which is a direct result of the problems in forecasting demand , is not sustainable in the long term and efforts are being made to stabilize the market .
even more than cost , the difficulty in coping with fluctuations in the supply of raw artemisinin of plant origin is the major pressure for a switch to alternative sources .
it is considerably easier to cope with ups and downs in demand by switching on a chemical synthesis or a fermentation process than by dealing with the long lead - time of plantations .
the drugs currently used as components of act are no longer ' natural ' artemisinins , as extraction from the plant is followed by a semi - synthetic process to transform the molecule first into dihydroartemisinin , then into artemether or artesunate .
another alternative , which is , surprisingly , not used industrially outside china , is to focus the extraction process entirely on artemisinic acid , which being 810 times more abundant in the plant gives much better yields , and then to use one of the conversion processes described to transform it into artemisinin .
the full chemical synthesis of artemisinin was achieved in 1983 , but is too complex and expensive for commercialization .
these molecules have similarities with artemisinin , including the key endoperoxide bridge , but their mode of action is different in many ways . of
the many peroxides tested , one trioxolane showed promise ( oz277/rbx11160 or arterolane ) : it was highly potent against p. falciparum in vitro and its synthesis was sufficiently simple for industrial scale - up .
the drug proved too unstable in vivo , however , and clinical trials were stopped before phase iii .
more stable molecules are in development and a hybrid trioxane - aminoquinoline ( trioxaquine ) may achieve the looked - for objective of a fully synthetic anti - malarial peroxide .
artemisinic acid can be produced in escherichia coli and , even more effectively , in saccharomyces cerevisiae , following pioneering work by keasling and colleagues , who transferred 10 a. annua genes into e. coli to create an almost ' plant - like ' environment in the bacteria .
their approach circumvents some of the previous problems of engineering the precursors ( amorphadiene or artemisinic acid ) or enzymes ( amorphadiene oxidase ) involved in the artemisinin biosynthetic pathway .
expression levels in yeast are still insufficient for industrialized scale - up , but the new publication by the group describes a further improvement in production in yeast ( with a 10 times higher yield ) by using a high - copy plasmid system .
this opens the way for a production process that would completely bypass the need for plantations of a. annua , and in which the yeast - produced precursor would be chemically converted to artemisinin as indicated above .
there are still a few hurdles to clear , including confirmation of structural and functional identity between the natural and engineered molecules .
we know from past experience that , once emerged , resistance of the malaria parasite to a drug spreads fast : chloroquine resistance emerged in the late 1950s in south - east asia , was reported for the first time in east africa in the 1970s , and has since invaded the entire african continent .
it is probably inevitable that mutants resistant to artemisinins will eventually emerge and , if they do , this will probably happen in south - east asia , where artemisinins have been used extensively as monotherapy for nearly 20 years .
studies from the thai - cambodian border , and more recently from south cambodia , have already shown reduced susceptibility , as well as treatment failure rates with act exceeding 10% , which is higher than anywhere else .
this area is now carefully observed by the scientific community , but monitoring is difficult in the absence of reliable molecular markers for artemisinin resistance , and relies on in vivo and in vitro sensitivity testing , which is cumbersome . as long as there is no resistance to the partner drug , it is usually assumed that the risk of resistance spreading will remain low .
it is important that malaria treatment be most carefully controlled in south - east asia , banning monotherapy ( which is still frequent ) , ensuring access to prompt act treatment with good - quality drugs and , whenever possible , treating asymptomatic carriers , who contribute to the spread of resistance . in conclusion , even when synthetic or recombinant artemisinin - like molecules have gone through preliminary tests to confirm their worth , there will still be a long way to go at least five to six years and millions of dollars before they will be available as drugs .
it is , therefore , likely that a. annua will continue to be the main source of artemisinin for the foreseable future .
it is true that the effectiveness of increasing worldwide utilization of act relies on a sustainable supply of artemisinin , preferably at an affordable price .
it is also true that limited access to act , as is still the case in many african countries , is not necessarily due to a shortage of raw material , or insufficient production , or even the high cost of the drug .
major funding organizations , including the global fund , have begun to understand that , in order to be effective , they not only have to deal with the cost of the drug , but must also strengthen health - system infrastructure and improve provision for diagnosis and treatment in the field .
keeping the cost of the drug down means not only ensuring a sustainable supply of the raw material , but also putting pressure on manufacturers to keep the price of the final drug reasonable ; both of which could be achieved by using subsidies to pre - order drugs years in advance .
this , however , assumes that forecasting need becomes much better than is currently feasible .
the recent endorsement by the 2008 roll back malaria action plan of the affordable medicines facility for malaria ( amfm ) initiative , a proposed $ 1.9 billion financial resource , is likely to have a major role in improving access to the drug at all levels . | artemisinins are the most important anti - malarial drugs in use today , but are more costly than previous anti - malarials and production and price tend to fluctuate .
alternative ways of producing artemisinins are discussed here in the light of a recent paper in bmc biotechnology on improving the yield of the precursor , artemisinic acid , in genetically engineered yeast . |
the battle with diseases started since the advent of man on earth and it will continue in an endless warfare .
the discovery of antibiotics in the 1950s has turned the result of this war in favor of man , but few years later microbes returned with mutant strains , resistant to almost all inventive antibiotics .
stupendous plants were the main renewable source for medications since times immemorial . yet , many drugs from the modern medicine are originally derived from ancient herbal medicine .
currently , the dramatic increase in resistance of pathogens to current antibiotics leads to the requisite need for new antimicrobial agents .
plants , particularly those prescribed against microbial infections since a long time in traditional and folk medicine from different societies could be promising sources for new antimicrobials . in sudan , the majority of sudanese people , like many african countries , are still relying on traditional or folk medicine in treatment of diseases which are an integral part of an informal healthcare system , although this popular folk medicine has roots from islamic and west african medicine .
sabdariffa l ) belongs to malvaceae family , it is an annual tropical small shrub native to africa and also distributed in southeast asia and central america , it is known locally as karkadeh .
the macerate of the red calyces of this plant [ figure 1 ] is one of the most famous public sudanese beverages all over this country and all sudanese people know and use , which are consumed as hot or cold drinks for the treatment of respiratory tract infections , hypertension , colds , and fever .
the dried calyces of hibiscus sabdariffa internationally , h. sabdariffa is well known , many parts of hibiscus sabdariffa is employed and prescribed in traditional medicine in many countries such as african countries , india , mexico , brazil , china , and iran ; leaves which eaten as vegetables are diuretic , antiseptic , digestive , purgative , sedative , demulcent , and astringent ; calyces are used for treating of hypertension , liver disorders , diuretic , digestive , and sedative ; seeds are rarely mentioned in traditional medicine compared to the other parts of hibiscus , but seeds are roasted and consumed as food , also used traditionally as debility , diuretic , laxative , and tonic . although sudan is the biggest african countries cultivated h. sabdariffa but scientific studies on this product is not adequate .
the red hibiscus calyces were found to have a pigment called anthocyanins ; it is also rich in polyphenolic compounds such as flavonoids and phenolic acids such as gallic and protocatechuic acid .
the objective of this study was to evaluate the antibacterial potential of the calyces of the sudanese roselle ( h. sabdariffa ) which intensively used in sudanese folk medicine .
calyces of h. sabdariffa were purchased from local herbal markets in khartoum , sudan [ figure 1 ] ; dried calyces were crushed into fine powders , kept dry in a clean , well - tightened glass container until used .
a total of 100 g of the dried powder of h. sabdariffa calyces was weighted and put in a sterile glass container , 500 ml of 80% methanol ( to serve as hydroalcoholic solvent ) was added gradually and soaked , and then the container was well tightened and kept in the refrigerator to avoid any microbial contamination or fungal growth .
the closed container was subjected to frequent shaking ( 2 - 3 times a day ) and macerated for up to 3 days .
then , the macerate was filtered using whatman filter papers no.1 ; the filtrate was put in the incubator and allowed to evaporate at 45c for up to 10 days to get a semi - solid extract . for antibacterial testing , the extract was reconstituted in absolute methanol to get a working concentration ( 500 mg / ml ) and kept in a closed dark container until used .
eight referenced bacterial strains were used in testing the antibacterial activity of the methanol extract of h. sabdariffa calyces .
these bacteria are representing five gram - negatives ( escherichia coli atcc 25922 , salmonella enteric atcc 5174 , klebsiella pneumonia atcc 27736 , proteus vulgaris atcc 49132 , and pseudomonas aeruginosa atcc 27853 ) and three gram - positives ( staphylococcus aureus atcc 25923 , staphylococcus epidermidis atcc 49461 , and bacillus cereus atcc 10876 ) .
bacterial cultures were identified microscopically and biochemically , then sub - cultured in sterile bottles containing nutrient broth ( scharlab , s.l . , and spain ) and incubated overnight at 37c .
prior of the antibacterial experiment , the working bacterial samples were adjusted to 0.5 mcfarland to be equivalent to about 1 - 2 10 cfu / ml .
the antibacterial activity of methanol extract of h. sabdariffa calyces was evaluated by disc diffusion method as mentioned in abdallah and al - harbi ; 100 l from each working bacterial cultures were mixed with 20 ml warm autoclaved mueller - hinton agar ( watin - biolife , ksa ) in glass bottles size 50 ml , tighten and mixed well and then poured directly into 90 mm sterile plastic disposable plates ( jalil medicals ) and left to solidify at room temperature .
6 mm discs were previously prepared using whatman no.1 filter paper , sterilized and saturated with 300 mg / ml of the methanol extract of h. sabdariffa calyces , the pre - experimental test showed that the blank disk ( 6 mm ) could carry 20 l of the extract at a concentration of 500 mg / ml , which equivalent to 10 mg / disc .
then , the wet , saturated disks were directly loaded on the cultured mueller - hinton agar plate in aseptic conditions . on the same plate , 6 mm antibiotic discs ; penicillin g 10 units and gentamicin 10 g ( oxoid )
cultured plates with extract and antibiotic discs were incubated for about 24 h at 37c .
the mean zone of inhibition of two replicated disks on the same plate was taken in millimeter ( mm ) using a ruler , 6 mm zone diameter considered as no inhibition .
the phytochemical constituents of the same methanol extract which used in antibacterial testing were evaluated qualitatively as described elsewhere , for alkaloids ( mayer s test ) , saponins ( foam test ) , tannins ( ferric chloride test ) , anthraquinones ( borntrger test ) , phenolic compounds , and flavonoids were also investigated .
all the statistical analyzes were performed with the spss 17.0 ( spss inc . , chicago , usa ) statistical package , variables considered significant at p < 0.05 .
calyces of h. sabdariffa were purchased from local herbal markets in khartoum , sudan [ figure 1 ] ; dried calyces were crushed into fine powders , kept dry in a clean , well - tightened glass container until used .
a total of 100 g of the dried powder of h. sabdariffa calyces was weighted and put in a sterile glass container , 500 ml of 80% methanol ( to serve as hydroalcoholic solvent ) was added gradually and soaked , and then the container was well tightened and kept in the refrigerator to avoid any microbial contamination or fungal growth .
the closed container was subjected to frequent shaking ( 2 - 3 times a day ) and macerated for up to 3 days .
then , the macerate was filtered using whatman filter papers no.1 ; the filtrate was put in the incubator and allowed to evaporate at 45c for up to 10 days to get a semi - solid extract . for antibacterial testing , the extract was reconstituted in absolute methanol to get a working concentration ( 500 mg / ml ) and kept in a closed dark container until used .
eight referenced bacterial strains were used in testing the antibacterial activity of the methanol extract of h. sabdariffa calyces .
these bacteria are representing five gram - negatives ( escherichia coli atcc 25922 , salmonella enteric atcc 5174 , klebsiella pneumonia atcc 27736 , proteus vulgaris atcc 49132 , and pseudomonas aeruginosa atcc 27853 ) and three gram - positives ( staphylococcus aureus atcc 25923 , staphylococcus epidermidis atcc 49461 , and bacillus cereus atcc 10876 ) .
bacterial cultures were identified microscopically and biochemically , then sub - cultured in sterile bottles containing nutrient broth ( scharlab , s.l . , and spain ) and incubated overnight at 37c .
prior of the antibacterial experiment , the working bacterial samples were adjusted to 0.5 mcfarland to be equivalent to about 1 - 2 10 cfu / ml .
the antibacterial activity of methanol extract of h. sabdariffa calyces was evaluated by disc diffusion method as mentioned in abdallah and al - harbi ; 100 l from each working bacterial cultures were mixed with 20 ml warm autoclaved mueller - hinton agar ( watin - biolife , ksa ) in glass bottles size 50 ml , tighten and mixed well and then poured directly into 90 mm sterile plastic disposable plates ( jalil medicals ) and left to solidify at room temperature .
6 mm discs were previously prepared using whatman no.1 filter paper , sterilized and saturated with 300 mg / ml of the methanol extract of h. sabdariffa calyces , the pre - experimental test showed that the blank disk ( 6 mm ) could carry 20 l of the extract at a concentration of 500 mg / ml , which equivalent to 10 mg / disc .
then , the wet , saturated disks were directly loaded on the cultured mueller - hinton agar plate in aseptic conditions . on the same plate , 6 mm antibiotic discs ; penicillin g 10 units and gentamicin 10 g ( oxoid )
cultured plates with extract and antibiotic discs were incubated for about 24 h at 37c .
the mean zone of inhibition of two replicated disks on the same plate was taken in millimeter ( mm ) using a ruler , 6 mm zone diameter considered as no inhibition .
the phytochemical constituents of the same methanol extract which used in antibacterial testing were evaluated qualitatively as described elsewhere , for alkaloids ( mayer s test ) , saponins ( foam test ) , tannins ( ferric chloride test ) , anthraquinones ( borntrger test ) , phenolic compounds , and flavonoids were also investigated .
all the statistical analyzes were performed with the spss 17.0 ( spss inc . , chicago , usa ) statistical package , variables considered significant at p < 0.05 .
the current study revealed the considerable antibacterial activity of methanol extract of h. sabdariffa calyces against all tested bacteria , particularly the gram - positives , as shown in tables 1 and 2 , respectively .
disc inhibition zones above 10 mm for the tested crude extract considered a good antibacterial activity ( blank disc diameter 6 mm ) .
interestingly , the investigated extract was found to inhibit the growth of all tested gram - positives and gram - negatives , giving clear , obvious zones by disc diffusion method compared with the antibiotics [ figure 2 ] , being statistically significant ( p < 0.05 ) .
the highest antibacterial activity of h. sabdariffa calyces was recorded by s. aureus ( 18.5 0.5 mm ) , followed by s. epidermidis ( 17.5 1.5 mm ) , s. enteric ( 17.5 1.5 mm ) , k. pneumonia ( 17.5 0.5 mm ) , p. aeruginosa ( 15.5 0.5 mm ) , e. coli ( 14.50.5 mm ) , p. vulgaris ( 14.50.5 mm ) , and b. cereus ( 13.5 1.5 mm ) , respectively .
similar several previous studies were reported on the calyces of h. sabdariffa using different solvents , concentrations , and bacterial strains ( clinical and referenced strains ) ; borrs - linares et al . published that the ethanol extract from 25 varieties of mexican h. sabdariffa calyces was effective against all gram - negatives ( e. coli and s. enteritidis ) and gram - positives ( s. aureus and micrococcus luteus ) , the greater effect was against the gram - positive bacteria .
aqueous , ethanol , and methanol extracts h. sabdariffa calyxes revealed good antibacterial activity against salmonella cultures .
its 80% methanol extract was also found active against escherichia coli o157:h7 , a major foodborne pathogen and suggested as potential antibacterial in foods .
the current investigation and the majority of previous published studies unanimously agreed that h. sabdariffa calyces which collected from different localities around the world have effective antibacterial properties ; this feature makes it a unique and promising antibacterial plant .
it is known that the bioactivity of the extracts from the same plant species may vary according to the plant extraction process which affect greatly by many factors such as type of solvents , method of extraction , temperature ( hot or cold liquids ) , age of the plant and season of harvesting , this stability of h. sabdariffa calyces as distinguished antibacterial agent is very interesting . moreover , as represented in figure 3 , the antibacterial activity of the crude methanol extract of h. sabdariffa calyces ( 10 mg / disc ) was significantly higher than penicillin g ( 10 units ) , and non - significant statistically when compared with the gentamicin ( 10 g / disc ) , meaning that this plant extract is competitor to gentamicin .
this is interesting since antibiotics are suffering from deterioration in its effectiveness around the globe , s. aureus is one of the most prevalent antibiotics resistant pathogens worldwide , particularly the methicillin - resistant s. aureus .
more antibacterial studies on the effects of the calyces of h. sabdariffa on this bacterium in particular are recommended .
these considerable antibacterial properties of the methanol extract of h. sabdariffa calyces ( 80% v / v ) are attributed to some bioactive phytochemical constituents present in this extract .
table 3 shows that the methanolic extract is rich in phytochemicals such as alkaloids , phenolic compounds , flavonoids , and saponins while tannins and anthraquinones did not detect .
this is in agreement with djeussi et al . , who found many phytochemicals in the methanol extract of h. sabdariffa calyces such as alkaloids , flavonoids , phenols , polyphenols , saponins , triterpenes , and sterols but no anthraquinones , tannins , or anthocyanins .
figure out many bioactive phytochemicals in the aqueous extract of h. sabdariffa calyces such as saponins , phlobatannins , terpenoids , anthraquinones , tannins , steroids , and phenolic compounds .
however , the antibacterial agent(s ) in the calyces are not determined yet , it was believed that its antibacterial activity could be attributed to anthocyanins , but it was found that this antibacterial activity was high with h. sabdariffa calyces lacking totally these anthocyanins . accordingly
, more future studies should be conducted to find out and isolate a single compound or group of compounds which could serve as an antibacterial drug . certainly , additional pharmacological , microbiological , and toxicological studies are recommended .
the antibacterial activity of methanol extract of h. sabdariffa calyces by disc diffusion method ( 10 mg / disc ) against gram - positive bacteria the antibacterial activity of methanol extract of h. sabdariffa calyces by disc diffusion method ( 10 mg / disc ) against gram - negative bacteria representative photos showing the antibacterial activity of the methanol extract of hibiscus sabdariffa calyces at concentration 500 mg / ml ( 10 mg / disc ) .
p : penicillin g 10 units / disc , g : gentamicin 10 g / disc , m : 80% methanol extract of hibiscus sabdariffa calyces 10 g / disc , p.a : pseudomonas aeruginosa atcc 27853 , s.e : staphylococcus epidermidis atcc 49461 , e.c : escherichia coli atcc 25922 , b.c : bacillus cereus atcc 10876 comparison between the antibacterial effects of methanol extract hibiscus sabdariffa and antibiotics the phytochemical analysis of 80% methanol extract ( v / v ) of h. sabdariffa calyces
the results of the current study support the widespread use of this popular plant in sudanese folk medicine , particularly against some illnesses related to microbial infections .
the output of this investigation also introduces the calyces of h. sabdariffa as one of the most promising sources for new natural and effective antibacterial drugs competitor to antibiotics . | background : hibiscus sabdariffa l. is a plant native to tropical africa and intensively cultivated in sudan . its calyces are widely consumed with many uses in sudanese folk medicine.materials and methods : the dried calyces of h. sabdariffa were subjected to soak in 80% v / v methanol to get the methanolic extract , which was tested against five gram - negative and three gram - positive referenced bacterial strains using disc diffusion method .
selected bioactive phytochemical compounds were also investigated using qualitative methods.results:the results of the antibacterial test indicate that the methanol extract of h. sabdariffa calyces contained effective antibacterial agent(s ) , revealed a considerable zone of inhibition against all tested gram - negative and gram - positive bacteria , and it was a competitor to gentamicin and greatly higher than penicillin which showed weak or no effect.conclusion:the results of current investigation support the folk medicine application of this plant against different microbial ailments and suggest it as a promising source for new antibacterial agents . |
laparoscopic cholecystectomy is one of the most commonly practiced surgeries for gall bladder diseases in the present era . due to its well - known advantages like less post - operative pain , shorter hospitalization and faster functional recovery , laparoscopic cholecystectomy is also termed as patient friendly surgery .
however , like any other surgery , laparoscopic cholecystectomy is also associated with stress response induced by surgery and anaesthesia .
moreover , the pneumoperitoneum and carbon dioxide insufflations , required in laparoscopic surgeries , lead to increase in plasma nor - epinephrine , epinephrine levels and plasma renin activity .
all these changes lead to increase in heart rate , blood pressure , systemic and pulmonary vascular resistance , and reduced cardiac output .
the reverse trendelenburg position required for surgery leads to diminished venous return and thereby , further reduction in cardiac output .
the haemodynamic changes predispose the myocardium to ischemia that may be life threatening in a vulnerable patient .
modern anaesthesia practices , therefore , plan to prevent sympathetic discharge and provide haemodynamic stability perioperatively .
various agents in the form of opioid analgesics , benzodiazepines , beta blockers , calcium channel blockers and vasodilators have been used to achieve this objective with variable success . in last few years
, a great enthusiasm has been shown toward the use of 2 agonists in anaesthesia practice because of their anxiolytic , sedative , sympatholytic and analgesic sparing properties .
dexmedetomidine , introduced in 1999 for human use , is a selective 2 agonist with 8 times more affinity for 2 adrenergic receptors compared to clonidine and possesses all the properties of 2 agonist without respiratory depression .
intravenous use of dexmedetomidine in the perioperative period had been found to decrease serum catecholamine levels by 90% , to blunt the haemodynamic response to laryngoscopy , tracheal intubation , pneumoperitoneum and extubation , to provide sedation without respiratory depression and to decrease post - operative analgesic requirements .
the primary aim of this study was therefore , to evaluate the effects of low dose dexmedetomidine infusion on haemodynamic response to critical incidences like laryngoscopy , endotracheal intubation , creation of pneumoperitoneum and extubation in patients undergoing laparoscopic cholecystectomy .
the secondary aims were to observe the effects on extubation time , sedation levels , post - operative analgesia requirements , and occurrence of adverse effects .
the present study was carried out from august 2010 to july 2012 , after taking the permission and approval from the departmental ethical committee and the written informed consent from the patients .
sixty asa physical grades i and ii patients between 18 and 65 years , of either sex and posted for laparoscopic cholecystectomy under general anaesthesia were included in the study .
patients with decreased autonomic control such as the elderly , diabetic patients , patients with chronic hypertension or severe cardiac disease , patients on drugs like blockers or calcium channel blockers , pregnant or lactating women , patients with history of allergy to egg proteins and drugs particularly 2 agonists were not considered for the study .
the patients were randomly allocated by envelope method into three groups of 20 patients each , group ns ( patients receiving normal saline 0.9% infusion ) , group dex 0.2 ( patients receiving dexmeditomidine infusion 0.2 mcg / kg / h ) and group dex 0.4 ( patients receiving dexmedetomidine infusion 0.4 mcg / kg / h ) . infusion was prepared according to the group allotted in a separate o.t . to prepare the infusion , dexmedetomidine 0.5 ml containing 50 g of the drug
syringe and was diluted up to 12.5 ml with normal saline resulting in the final concentration of 4 mcg / ml .
depending on the weight of the patient , the pump was set so as to deliver the targeted infusion rate .
after the setting the infusion pump , it was covered with blue cloth so that the assessor did not come to know about the grouping of the patient .
thus , the syringe was same , volume of prepared solution was same , only the rate of injection was different according to the weight and group of patient .
decoding of blinding to the assessor was done only at the time of tabulation and result analysis . after taking the patient on the operation table ,
a multipara monitor was attached and the baseline pulse rate ( pr ) , mean arterial pressure ( map ) and oxygen saturation were noted down .
a wide bore intravenous cannula was inserted for giving the intravenous fluids , and another line was taken up for the infusion pump .
premedication was administered 45 min before induction of anaesthesia to all the patients in the form of injection glycopyrrolate 5 mcg / kg i m and injection tramadol 1.0 mg / kg i m as per the institutional protocol . at the time of induction ,
all the patients received injection ranitidine 50 mg iv and injection ondansetron 4 mg iv .
fifteen minutes after starting the drug infusion , pre - oxygenation was performed for 3 min .
patients were induced with injection propofol 2 mg / kg intravenously followed by injection succinyl choline 1.5 mg / kg intravenously .
anaesthesia was maintained with o2:n2o ( 50:50 ) , isoflurane and injection vecuronium bromide as a muscle relaxant on fabius plus work station .
intra - abdominal pressure was maintained between 12 and 14 mmhg throughout the laparoscopic procedure .
the patients were mechanically ventilated using circle system to keep the etco2 between 35 and 45 mm hg .
all the patients were observed for vital parameters like pr , map and spo2 at regular intervals including before starting the infusion , 15 min after starting the infusion , after induction , after intubation , after creation and release of pneumoperitoneum and after extubation .
patients were also observed for time to extubate trachea , post - operative sedation level , time to first rescue analgesic requirement ( time from completion of injection of drug to the time post- operatively , when pain reported by patient was 4 on visual analogue scale [ vas ] , total amount of analgesic drug required during the first 24 h post - operatively and the adverse effects .
injection diclofenac sodium 1.5 mg / kg i m was used as rescue analgesic and thereafter whenever the vas score became 4 .
extubation time was counted from stoppage of anaesthetic agent to time , when the extubation was done .
sedation was assessed at 1 , 15 , 30 , 60 to 120 min post - operatively using ramsay sedation score ( rss ) . throughout the study , patients were observed for any adverse effects like bradycardia , tachycardia ( pr less than or more than 20% of pre - operative level respectively on two consecutive readings ) , hypo and hypertension ( map less than or more than 20% of pre - operative level respectively on two consecutive readings ) , sedation score more than rss 4 , respiratory depression ( sao2 < 90% ) and dryness of mouth and they were managed conventionally .
( medcalc software bvba , acacialaan 22 , 8400 ostend , belgium ) based on minimum mean difference of 25% in parameters with = 0.01 and = 0.20 , sample size for each group was estimated as 18 . rounding up this figure
the results were tabulated and statistically analysed using spss ( statistical package for social sciences ) software version 15.0 , chi - square test was used for qualitative data ( sex , asa grade ) , pr , blood pressure , oxygen saturation , end tidal carbon dioxide etc . , were compared within the group against baseline values using paired t - test .
anova test was used for three group comparisons of continuous variables ; if anova was found significant , tuckey post - hoc test was used for comparing two groups and the results were expressed as mean standard deviation .
p > 0.05 was considered insignificant , < 0.05 as significant and highly significant if < 0.001 .
all the three groups under study were comparable to each other with respect to age , sex , weight , asa grading , duration of surgery and anaesthesia [ table 1 ] .
there was no significant difference among the three groups in reference to the baseline pr and the map .
demographic characteristics and duration of surgery and anaesthesia in group ns , after starting the infusion there was no significant change in pr and map but these increased highly significantly above pre - infusion level after intubation and extubation ( p < 0.01 ) and significantly after pneumoperitoneum [ p < 0.05 , tables 2 and 3 ] . changes in pr ( beats per minute ) ( meansd ) changes in map ( mm of hg ) ( meansd ) in both the dexmedetomidine groups , after starting the infusion , the pr decreased highly significantly below the pre - infusion level .
the map decreased significantly in dex 0.2 group and highly significantly in dex 0.4 group .
after intubation and extubation , the pr and map increased significantly above the pre - infusion level in dex 0.2 group , though , this increase was less compared to increase in group ns ( p < 0.05 ) . unlike these changes in dex 0.2 group , pr and map in dex 0.4 group remained below pre - infusion level after intubation and extubation ( p < 0.05 when compared with dex 0.02 ) .
the mean sedation scores [ table 4 ] were more in dexmedetomidine groups compared to normal saline group patients .
none of the patients in dexmedetomidine groups developed siginificant sedation levels and the patients were cooperative , oriented and tranquil all the time .
in group ns sedation score , which was less initially , improved subsequently due to early requirement of analgesia in this group .
changes in mean sedation score there was no significant difference among the three groups with reference to extubation time ( 7.15 1.20 , 6.55 1.27 and 6.40 1.04 min in group ns , dex 0.2 and dex 0.4 group respectively . the rescue analgesia was required early ( 55.5 min . ) in group ns compared to dexmedetomidine groups ( 173 min in dex 0.2 and 249 min in dex 0.4 group ) .
all the patients in group ns , 15 patients in group dex 0.2 and 10 patients in group dex 0.4 group required rescue analgesia .
total analgesic requirement of first 24 h post - operative period also decreased by 36.11% in group dex 0.2 and by 63.88% in group dex 0.4 [ table 5 ] .
post - operative analgesic requirements tachycardia and hypertension were seen in 7 and 6 patients of group ns compared to 1 and 2 patients of group dex 0.2 .
hypotension was noted in 1 patient of group dex 0.2 and bradycardia was seen in 1 patient of dex 0.4 group .
it acts through three types of 2 receptors-2 a , 2 b and 2 c situated in brain and spinal cord .
the resultant action is sedation , anxiolysis , analgesia and sympatholysis , the latter leading to hypotension and bradycardia .
activation of 2 a receptors in brain stem vasomotor centre results in suppression of norepinephrine release , hypotension and bradycardia .
stimulation of 2 a and 2 c in locus ceruleus causes sedation . in the spinal cord ,
activation of both 2 a and 2 c receptors directly reduce pain transmission by reducing release of substance p. looking at these pharmacological properties , it has been evaluated in the past to assess its effect on haemodynamic responses in patients undergoing laparoscopic surgeries .
infusion rates varying from 0.1 to 10 mcg / kg / h have been studied . however , with higher dose infusion of dexmedetomidine , high incidence of adverse cardiac effects have been observed .
stimulation of 2 b receptors in vascular smooth muscles is said to be responsible for this .
low dose infusion of 0.250.5 mcg / kg / h results in a monophasic response of 1015% fall in mean arterial blood pressure and pr .
furthermore , in low dose , dexmedetomidine exhibits linear kinetics , meaning that a constant amount of drug is eliminated per hour rather than a constant fraction of drug .
hence , in a pilot study , we used low dose infusions of dexmedetomidine without any bolus .
initially , we used dexmedetomidine 0.2 mcg / kg / h infusion . though it controlled the rise in pr and map at pneumoperitoneum ,
the control was not very effective at the time of tracheal intubation and extubation , and the pr and map both increased above pre - infusion levels .
hence , we increased the dose to 0.4 mcg / kg / h infusion in our next two patients .
we also studied few cases with dex 0.6 mcg / kg / h dose but the hypotension was seen in greater number of patients , and the sedation was more ( rss 45 ) .
hence , we decided to have three groups in our study , which were group ns , group dex 0.2 and group dex 0.4 .
our study confirms the fact that critical incidences like laryngoscopy and intubation , pneumoperitoneum and extubation do significantly increase the map and pr in patients undergoing laparoscopic cholecystectomy as seen in group ns .
the effective attenuation dose with minimum side effects noted in our study was 0.4 mcg / kg / h infusion .
apart from providing stress response attenuation , the added effects of dexmedetomidine are sedation and analgesia .
sedation produced by 2 agonists is unique in the sense that the patients can be easily aroused to co - operate during procedures and also respond to the verbal commands and then can return to sleep like state when not stimulated .
we observed patients for 120 min as elimination half - life of dexmedetomidine is 2 h. initially , the sedation score was more in group dex 0.4 compared to group ns or group dex 0.2 . over a period of time mean sedation score settled around
2 in group dex 0.2 and group dex 0.4 , while in group ns , score improved from 1 to 2 because of early administration of analgesia 3040 min post - operatively .
it may be hypothesised that due to its sedative effect , response to verbal command and extubation time may be delayed with dexmedetomidine .
however , in our study the mean extubation time did not show any significant difference between three groups .
bhattacharjee et al . also observed no significant effect of dexmedetomidine on response to verbal command and extubation time .
dexmedetomidine has been found to reduce the intra and post - operative requirement of opioids .
we also observed an increase in the time to receive first rescue analgesia and a decrease in total analgesic requirements in first 24 h post - operatively in both dexmedetomidine groups .
low dose infusion of dexmedetomidine at the rate of 0.4 mcg / kg / h without any bolus dose serves as a very useful anaesthesia adjuvant to control haemodynamic stress response to intubation , pneumoperitoneum and extubation in patients undergoing laparoscopic cholecystectomy .
it also provides lighter sedation and reduces the post - operative analgesic requirements without any significant adverse effects . | background and aim : dexmedetomidine is a 2 agonist with sedative , sympatholytic and analgesic properties and hence , it can be a very useful adjuvant in anaesthesia as stress response buster , sedative and analgesic .
we aimed primarily to evaluate the effects of low dose dexmedetomidine infusion on haemodynamic response to critical incidences such as laryngoscopy , endotracheal intubation , creation of pneumoperitoneum and extubation in patients undergoing laparoscopic cholecystectomy .
the secondary aims were to observe the effects on extubation time , sedation levels , post - operative analgesia requirements and occurrence of adverse effects.methods:sixty patients of american society of anaesthesiologists(asa ) physical grades i and ii undergoing laparoscopic cholecystectomy were randomly allocated into three groups of 20 patients each .
group ns patients received normal saline , group dex 0.2 and group dex 0.4 patients received dexmedetomidine infusion at 0.2 mcg / kg / h and 0.4 mcg / kg / h respectively , starting 15 min before induction and continued till end of surgery .
parameters noted were pulse rate , mean arterial pressure , oxygen saturation , post - operative sedation and analgesia requirements .
spss 15.0 version software was used for statistical analysis .
anova test for continuous variables , post - hoc test for intergroup comparison , and chi - square test for discrete values were applied.results:in group ns significant haemodynamic stress response was seen following laryngoscopy , tracheal intubation , creation of pneumoperitoneum and extubation . in dexmedetomidine groups ,
the haemodynamic response was significantly attenuated .
the results , however , were statistically better in dex 0.4 group compared with dex 0.2 group .
post - operative 24 hour analgesic requirements were much less in dexmedetomidine groups .
no significant side effects were noted.conclusion:low dose dexmedetomidine infusion in the dose of 0.4 mcg / kg / h effectively attenuates haemodynamic stress response during laparoscopic surgery with reduction in post - operative analgesic requirements . |
a 58-year - old man was admitted to our opthalmology department with the complaint of decreased visual acuity .
best - corrected visual acuity ( bcva ) was 0.2 on snellen 's acuity chart in the right eye , 1.0 in the left eye and bilateral intraocular pressure meaurements were in normal limits .
fundus fluorescein angiography revealed non - ischemic brvo and macular edema in the right eye .
optical coherance tomography revealed cystoid macular edema with a central retinal thickness of 430 m .
the patient underwent intravitreal ranibizumab injection afterwards ; however there was not any improvement in macular edema .
as a result , we decided to implant intravitreal sustained - release dexamethasone ( ozurdex ) to the patient with cystoid macular edema because of crvo .
the procedure was performed in the operating room adhering strictly to the rules of sterilization .
we used a single - use sterile injection set and the instruments were prepared on a sterile tray .
a sterile anesthetic drop was placed in the appropriate eye . a periocular scrub using 10% povidone iodine ( pvi )
was performed , and 5% pvi drop was placed on the ocular surface before the injection . a fenestrated self - adhesive surgical drape that covered the patient 's nose and mouth , and a sterile lid speculum
two days after the procedure , the patient presented with ocular pain , blurred vision and photophobia .
significant anterior chamber reaction with hypopyon , moderate corneal edema and severe vitritis were detected on examination ( fig .
b - scan ultrasonography showed vitreous opacification , but no retinal detachment . according to the results described above , a diagnosis of endophthalmitis was made .
we subsequently performed emergent 23-gauge pars plana vitrectomy ( ppv ) and the implant was removed from the vitreous cavity using a retinal forceps ( fig .
we diagnosed cataract formation one week after the procedure and the blurring in the vitreus was still present .
five days after the last procedure , the signs and symptoms of endophthalmitis were resolved with total remission of pain and hypopyon .
endophthalmitis is one of the most feared complications of ophthalmic invasive procedures , and appears to be a very rare complication of sustained - release dexamethasone intravitreal implant .
ozurdex is a type of intravitreal therapy which is designed to provide sustained delivery of 700 g of preservative - free dexamethasone to the vitreous cavity and retina .
ozurdex has been recently approved by the united states food and drug administration for the treatment of macular edema associated with crvo and brvo .
several clinical studies revealed favorable results regarding the beneficial effects of ozurdex in the treatment of brvo .
in addition , researchers reported limited side effects without any infection or endophthalmitis , but pain , ocular hypertension , anterior chamber cells , cataracts , vitreous hemorrhage , and retinal detachment did occur .
despite the attempts to provide aseptic conditions before injection in order to avoid microbial contamination causing acute endophthalmitis , the patient developed complications involving endophthalmitis 2 days after ozurdex implantation .
therefore , because of the loss in vision to hand motion , we immediately performed ppv and removed the implant on the same day as the intravitreal antibiotic injection . unlike endophthalmitis after cataract extraction for which specific guidelines were developed by the endophthalmitis vitrectomy study , there is no established treatment protocol for endophthalmitis after intravitreal implantation of ozurdex .
although our patient was treated with intravitreal antibiotics and ppv on the same day of presentation , we did not follow a strict treatment protocol for endophthalmitis regarding indications for vitrectomy surgery .
similarly , previous reports recommended immediate vitrectomy to reduce the bacterial overload and improve the outcome before the loss of vision according to the prespecified criteria defined by the endophthalmitis vitrectomy study .
we think that surgical removal of the implant and immediate ppv is an important step in the management of endophthalmitis . given the increasing number of injections in clinical practice , the overall number of endophthalmitis cases is expected to increase as well .
therefore , there seems to be an emerging need to make a consensus statement for endophthalmitis regarding the intravitreal implant procedure . despite the unique characteristics seen in this case report , we are aware of some limitations associated with this case including the culture negativity and the nonmethodical treatment algorithm that we followed .
the culture was negative in our case ; therefore we can not exclude the possibilty that noninfectious injection - related inflammation might be mistakenly attributed to infectious endophthalmitis . in cases of identified bacterial strains ,
however , the issue of empirical treatment with antibiotics covering potential microorganisms should be kept in mind , even if the culture is negative . in conclusion
surgical removal of the implant and immediate vitrectomy seems to be a useful treatment option in these cases .
further studies are needed to establish a treatment algorithm for the treatment of endophthalmitis after sustained - release dexamethasone intravitreal implant . | a 58-year - old man admitted to our opthalmology department with the complaint of branch retinal vein occlusion .
he was treated with intravitreal ozurdex in the right eye .
two days after the injection , the patient presented with ocular pain and the visual acuity was hand movement .
a diagnosis of endophthalmitis was made .
we performed emergent pars plana vitrectomy ( ppv ) and the implant was removed from the vitreous cavity using a retinal forceps . a combination of vancomycin 1.0 mg and amikacin 0.4 mg was injected intravitreally
.
however , because of the blurring in the vitreus one week after the procedure , phacoemulsification and a repeat ppv was performed .
five days after the last procedure the signs and symptoms of endophthalmitis were resolved .
our case demonstrated that endophthalmitis could develop after intravitreal implantation of ozurdex .
surgical removal of the implant and immediate vitrectomy seems to be a useful treatment option in these cases . |
retained foreign body granulomas in the intervertebral disc space due to penetrant traumas are very rare like other paraspinal ones2,3,57,10 ) . to our knowledge ,
all of the reported retained foreign bodies in the intervertebral disc space were from missile injuries and there was not any report involving a retained foreign body granuloma in this region from wooden penetrant trauma2,3,5,6,10 ) .
a 30-year - old man sought medical care with a history of low back pain ( lbp ) that radiated to lower extremities .
he reported that he had fallen from a tree and a tree branch had been penetrated into him left lower back before 17 years . that tree branch had been taken out manually and he had recovered without any complaint . in the past two months
, the patient has presented a lbp radiating to his lower limbs . physical examination revealed a bad - healed scar at about level of the third lumbar vertebra on left paraspinal region ( fig .
the straight - leg - raising sign was negative and there was not any neurologic deficit on lower limbs .
computerized tomography ( ct ) demonstrated a hypodenselytic lesion in the l4 vertebral body surrounding a linear hyperdense structure ( fig .
2 ) . anterior part of the disc space between fourth and fifth lumbar vertebrae had disappeared with fusion of these bodies in magnetic resonance ( mr ) images .
mr images also revealed a lesion that lodged in posterior part of this disc space and expanding into the corpuses of these vertebrae ( especially into l4 vertebral corpus ) .
it was seen a linear horizontal area of decreased signal intensity representing the intranuclear cleft in the center of posterior part of disc space between fourth and fifth lumbar vertebrae in sagittal mri image of the stir sequence .
a firm mass was found lodged in posterior part of the intervertebral disc space between the fourth and fifth lumbar vertebrae .
excision of the mass revealed a tough granulation tissue surrounding a tiny piece of wood in a little cavity filled with a clear yellowish fluid ( fig .
there was a fusion between anterior parts of third and fourth lumbar vertebrae and a degeneration in the discal material in the rest of intervertebral disc space .
postoperatively , the pain resolved gradually and the patient was discharged without any problem 10 days after operation .
he had a total symptomatic cure after two months from excision of the foreign body .
biological responses of tissues to foreign bodies are influenced by the location and the nature of them17,10 ) . in a soft tissue ,
a wooden foreign body can cause either a mild inflammatory tissue reaction resulting in a localized granuloma or a severe reaction resulting in a significant infectious lesion with or without osseous reaction in adjacent bone1,4,6,8 ) .
paraspinal disorders like ones in the intervertebral disc space may cause important complications because they may affect vital structures in and near the spinal canal . in a similar way , the majority of reported cases with foreign bodies that embedded and retained near the spinal canal presented with delayed symptoms or findings resulting from compression of neural elements due to developed granuloma or abscess as well as in creased bony growths or migration of foreign body in the spinal canal2,3,57,10,11 ) . in the literature , there are only four case reports presented with the retained foreign body granulomas in the intervertebral disc space from penetrant traumas2,5,6,8,10 ) .
two of these cases presented with cauda equina syndrome resulted from compression of neural structures due to granuloma and/or bony growths while two of them presented with symptoms and findings other than neurological deficits like our case2,5,6,8,10 ) .
we thought that our patient s complaints probably were due to expanding of granuloma into the corpora of adjacent vertebrae .
his complaints totally disappeared after removing the granuloma that in a way supported our thought .
the detection of wooden foreign bodies with plain radiographies and ct has limited value because they are radiolucent .
therefore , ultrasound and mr imaging are recommended in the detection of these foreign bodies like of other nonmetallic ones9 ) .
but , the identification of wooden foreign bodies may also be difficult on mr imaging when they are small and on ultrasound when they are in the deeper paraspinal tissues .
thus , a wooden foreign body in paraspinal tissues like intervertebral disc space may be easily missed especially in acute cases .
more over , it is very difficult to predict the natural history of a retained foreign body in the intervertebral disc space , because , a sufficient number of cases is unfortunately not exist in the literature .
if a retained wooden body in intervertebral disc space is missed and it is not removed , it may be potentially hazardous and result in significant morbidity because of the nature of wood and the paraspinal location of intervertebral disc space .
moreover , a missed foreign body in a paraspinal tissues may cause further damages for patients and malpractice claims for physicians .
therefore , physicians and radiologist need to keep their diagnostic suspicions height in terms of retained foreign bodies during examination or follow up a patient with paraspinal wooden penetrant traumas . | we report a patient with a wooden foreign body granuloma in the intervertebral disc space being symptomatic 17 years after a paraspinal penetrant trauma . according to the our result of the search for wooden foreign body granulomas ,
this is the first case suffered from a wooden foreign body granuloma in the intervertebral disc space that reported in the literature . in this report
, we emphasized the importance of rigorous examination and follow up in paraspinal wooden penetrant traumas . |
due to the increased incidence of cancer , the production and use of antitumor drugs is on the rise . majority of antitumor drugs , cytotoxic , which may cause cancer , mutations , and other effects .
several studies revealed side - effects of these drugs on health workers , for instance , the increasing chance of chromosomal damages , the decrease in the immune system , increasing possibility of infertility and abortion , premature labor , low birth weight , irritation of the eyes , skin and mucosa and allergic reactions due to skin contacts , vomiting , headache and dizziness , hair loss , and liver damages are the mentioned side - effects . as a result , increased concern of handling these drugs by the pharmacy team , nurses , and physicians . till date , the challenge of protecting workers health is persisting and expanding , with an increasing number of publications demonstrating that contamination of cytotoxic drugs is still present on work surfaces after cleaning procedures are concluded . the traditional approach to workers health protection from exposure to cytotoxic drugs
was pioneered in the 1970s and 1980s , when special guidelines and protective measures were introduced ; e.g. the canadian society of hospital pharmacists issued the very first guideline for the handling of cytotoxic drugs in 1981 . in the following decades
, we investigated the occupational protection status of shiraz university of medical sciences hospitals nursing staff when handling cytotoxic drugs and range of skin and mucosal contamination with cytotoxic drugs , expecting to provide some clues for improving occupational protection ability and reducing the related injury in nursing staff .
the research population was comprised all nurses who work in oncology units of all three hospitals and four clinics in fars province that is the most important centers for chemotherapy , in the south of iran .
all oncology wards had a population of 130 nurses including 90 qualified nurses ( participating in all preparation , administration , and disposal of cytotoxic drugs process ) for participation .
this questionnaire contains 19 items including four closed ended questions about demographics , 12 closed - ended , and three open - ended questions to determine working conditions of nurses . before using the questionnaire in this research ,
it was shown to five specialists for their opinions about its validity and ability to obtain information to answer the research questions .
after recommended changes had been made , it was given to 15 oncology nurses in one hospital as a pilot test .
based on the oncology nurses recommendations , it was further revised before it was used in this research . to study the nurses practice , the checklist was used .
, only the protective aspects that should be practiced by staff during medication preparation , administration , and disposal were evaluated .
therefore , five experts in this field revised it and some protective remarks were added to the checklist and the none - protective remarks were eliminated .
the checklist included fifty practical cases in three fields of preparing ( 25 cases ) , administrating ( 12 cases ) , and disposing of cytotoxic drugs ( 13 cases ) , all having the same value as 1 .
for reliability of the checklist , interobserver reliability test was used ; the obtained correlation coefficient was 0.94 . for ethical considerations , they told the nurses this is an approved project .
the aim of this study and the procedures were explained to them to obtain their cooperation .
written informed consent was obtained from them to ensure willingness to engage in the study .
nurses were allowed to choose whether to participate or not , and they had the right to withdraw from the study at any time without penalty . the questionnaire was completed by all nurses ( n = 86 ) , they filled the question forms more or less 30 min during their free times and returned them to the researcher .
the high response rate may be a result of the researchers careful follow - up as well as the nurses feeling encouraged to have their voices heard about the problems they face . to evaluate the performance of nurses , researcher went to their work place without prior notice and observed ( with the checklist ) their function during the preparation , administration and disposal of cytotoxic drugs .
for an exact observation , the personnel 's practice in two opposite shifts was observed and mean of the two observations was considered as the performance for each individual .
to statistically analysis of the data , spss software ( version 18 , spss inc . , chicago , il , usa ) was applied .
percentage calculations and the independent samples t - test were used to see the general distribution and analysis of data .
percentage calculations and the independent samples t - test were used to see the general distribution and analysis of data .
in the examination of the participated oncology nurses individual characteristics , it was determined that their mean age was 30.52 6.50 years , most of them were female ( 98.8% ) , all of them had a bachelor 's degree in nursing , more than half of them ( 53.5% ) had work experience in oncology units between 1 and 5 years .
there were no clear guidelines for safe handling of cytotoxic drugs during pregnancy and lactation . just in pregnancy , they did not participate in preparation of cytotoxic drugs but they administered and disposed these drugs .
about the percentage of skin or mucosal exposures to cytotoxic drugs , majority of nurses ( 65.1% ) have been exposed .
the maximum exposure range ( 30.2% ) was between 1 and 4 times . in this study ,
66.27% of the nurses worked on inpatient oncology wards and 33.73% in outpatient treatment units . in the participating hospitals , all of the nurses prepared cytotoxic drugs by themselves .
almost all of the nurses ( 91.7% ) stated that before working in an oncology unit , they did not receive any education about methods of protection ; however , eight nurses ( 9.3% ) stated that they had taken a short course about of it .
they stated these teachings are not enough and all nurses desired to learn about safety standards with cytotoxic agents .
favorite nurse 's training methods were workshop ( n = 59 , 68.6% ) , lecture ( n = 9 , 10.5% ) , electronic learning ( n = 8 , 9.3% ) , module ( n = 7 , 8.1% ) , and informal learning in workplace ( 3 , 3.5% ) , respectively . although the majority ( 69.23% ) of the nurses who worked in oncology units and who administered chemotherapy did not receive , higher salary than nurses did working in other units did .
all of them stated that they were not given any extra specific payment for their unit . when nurses were accidentally contaminated with cytotoxic drugs during preparation they frequently did nothing other than normal cleaning , took care to wash off the drug from the area contaminated .
in addition , when the environment where cytotoxic drugs were prepared was contaminated , they used similar procedures as stated above of doing nothing different and normal cleaning and there was no emergency spills kits to use .
the independent samples t - test showed the outpatient nurses were weaker in practice ( 17.2 2.52 ) in comparison with university hospitals ( 23.35 3.02 , p < 0.001 ) .
the findings showed that for preparation of cytotoxic drugs biologic safety cabinet class i was used in all hospitals and clinics .
fifteen nurses ( 17.44% ) prepared drug in open space . in 52.9% of canters ,
biologic safety cabinet was in separate room , in other canters was found in the patient rooms .
the mean of two observations of nurse 's practice in two opposite shifts about the use of personal protective equipment such as gown , mask , goggles , and gloves in preparation , administration and disposal of cytotoxic drugs were as follows [ table 1 ] .
the status of protective behaviors in preparing , administration and disposal of cytotoxic drugs ( n=86 )
in this research , the occupational protection status of clinical nursing staff exposed to cytotoxic drugs in chemotherapy center of shiraz university of medical sciences was examined .
generally , it was determined that the working conditions of oncology nurses are more negative than nurses working in other areas .
when the length of employment of nurses working in oncology units was examined , it was determined that in general , nurses started to work in oncology units after they had 3 and 4 years of experience ( 53.5% ) .
almost 20.9% ( n = 18 ) of the oncology nurses began their work in oncology ward without having work experience in other wards .
in which the mean job experience of the nurses was 8.6 years and experience in oncology units was 4.9 years .
the research was conducted by the european oncology nurses society ( eons ) to determine the status of oncology nurses in europe , 20 out of 22 countries responded .
they reported that the nurses mean age was 43 , they had 21 years of professional experience , the level of education in 13 countries was diploma and academic education in seven countries .
the reason for this may be that these hospitals do not have a clinical pharmacist on staff .
in addition according to national legislation , drugs are prepared by nurses and because there is no specific legislation about cytotoxic drugs , nurses are under obligation to prepare them . in research conducted by baykal et al . with 171 oncology nurses throughout turkey similar results
were found as the majority of the nurses ( 78.9% ) prepared cytotoxic drugs by themselves and only 13.5% stated that the drugs were prepared in the pharmacy .
the research was conducted by the eons to determine the status of oncology nurses in europe determined they took 26 weeks of annual leave . in this research ,
similar to results from the studies by baykal et al . and yanqin et al .
, lack of continuing education was a problem . in this study , the nurses who prepared and administered cytotoxic drugs had not been given education on safety standards with cytotoxic drugs ( 90.7% ) .
however , it is an international labor organization recommendation that employees need to have knowledge and abilities appropriate for their working area and it is clearly necessary for nurses to be better prepared through the education . in study of yanqin et al . in china , with the aim of protecting nurses ( n = 168 ) during the exposure to cytotoxic drugs showed that of the 108 ( 64.8% ) of nurses had received training about job security .
only 50 ( 30.3% ) of them had received special training in preparation of these drugs . in terms of performance ,
almost half of the nurses during medication preparation used basic protective equipment including gowns , masks , goggles , and gloves .
use of this equipment during the administration and disposal of medicines was insufficient . in the research study conducted by yanqin et al . revealed all of nurses used mask 168 ( 100% ) , 81 ( 48.8% ) wore double gloves and 6 ( 3.6% ) used gowns and goggles , when prepared these drugs . in the other research conducted by baykal et al .
, the nurses were asked what kind of protective measures they used , the majority of them ( 94.7% ) stated that they wore gloves , followed by masks ( 89.5% ) , gowns ( 52.0% ) , and goggles ( 18.7% ) .
cytotoxic drugs were prepared by 40.4% of the nurses under a hood ( biological safety cabin ) , by 37.4% in the nurses office , and by 15.8% in the room which is also used for tea breaks , meals , and for other purposes . in this study , it was found that the majority of oncology nurses ( 65.1% ) had skin or mucous membrane exposure while handling cytotoxic drugs .
the absorption of these drugs is likely to be higher than this value . because inhalation is one of the important absorbable forms of these drugs , was not considered , because nurses did not feel it .
on the other hand , studies from several countries have demonstrated surface contamination of biological safety cabinets , countertops , cabinets , and floors within the drug preparation area .
however , this study was determined when accidental spills occur during the preparation of cytotoxic drugs and the environment or the person becomes contaminated that no special cleaning was done by the majority of the nurses , they just rinsed off as they would for normal cleaning . in the studies by valanis et al . and
they did not use appropriate methods of cleaning areas that were contaminated by cytotoxic drugs and did not adequately protect themselves against contamination .
however , various studies , the positive role of protective equipment ( gloves , gowns , masks and eye protection ) have been shown to reduce occupational exposure to these drugs .
in summary , cytotoxic drugs have been widely used clinically , the rules of occupational protection are still under improvement in iran . in addition , nursing staff exposed to anti - tumor drugs do not generally attach enough importance to self - protection . to raise the self - protection consciousness and ability of nursing staff exposed to anti - tumor drugs , hospitals should build a perfect monitoring system , set standard training programs , and introduce protective facilities , so that the hazards could be limited to the least .
| context : the use of antineoplastic agents for the treatment of cancer is an increasingly common practice in hospitals . as a result , workers involved with handling antineoplastic drugs may be accidentally exposed to these agents , placing them at potential risk for long - term adverse effects .
this study aimed to determine the occupational protection status of clinical nursing staff exposed to cytotoxic drugs.subjects and methods : the study was designed as an analytic descriptive survey . the research settings took place in six centers of chemotherapy in shiraz , iran .
the participants were 86 nurses who worked in oncology units and administered cytotoxic drugs .
data were collected using a questionnaire and a checklist which was developed by the investigators to determine occupational protection status of clinical nursing staff exposed to cytotoxic drugs .
percentage calculations and the independent samples t - test were used to see the general distribution and analysis of data . to statistically analyze of the data , spss software ( version 16 ) was applied.results:the mean age of participants was 30.52 6.50 years and
66.27% of the nurses worked on inpatient oncology wards .
the mean practice score was 21.1 3.76 that ranged from 12.5 to 31 .
the independent samples t - test showed the outpatient nurses were weaker in practice ( 17.2 2.52 ) in comparison with university hospitals ( 23.35 3.02 , p < 0.001 ) . occupational protection status of clinical nursing staff exposed to cytotoxic drugs especially during administration and disposal of medicines was poor and rarely trained with this subject and
was observed under the standard conditions.conclusions:there is deficiency in the understanding and related protection practices of clinical nursing staff vocationally exposed to cytotoxic drugs .
it is recommended that all clinical nursing staff should receive full occupational protection training about these matters and the authorities provide standard conditions of oncology wards . |
this randomized , double blind , placebo - controlled , 2-period , 2-treatment crossover trial evaluated the safety and efficacy of 21 days of twice daily ( bid ) treatment with tv-45070 ointment in phn patients .
the study was conducted between august , 2010 and march , 2011 at 24 sites in the united states .
the study included 2 , 1-week patient - blind placebo run - in periods ( before each treatment period ) , and 2 , 3-week double - blind treatment periods ( fig .
1 ) . a 1-week washout separated treatment period 1 ( tp1 ) and the second placebo run - in period , effectively separating each randomized treatment period by 2 weeks .
the first single - blind , placebo run - in period was 1 week in duration ( day-7 to day-1 ) . at randomization ( visit 3 ) , patients were randomized in a crossover design to 1 of 2 treatment sequences : tv-45070/placebo or placebo / tv-45070 .
the 1-week between - treatment washout period ( between visits 6 and 7 ) , study medication was not applied , but patients continued to record pain scores using the interactive voice response system .
the second single - blind , placebo run - in period was 1 week in duration ( day-29 to day-35 ) . in tp2 , patients received the medication that was not received during tp1 .
one week after the end of tp2 , patients returned for a follow - up visit ( visit 12 ) .
patients with mean daily pain scores of 4 ( using an 11-point numerical rating scale ) for at least 4 days during the initial placebo 7 day run - in period were randomized in a 1:1 ratio through an interactive voice response system to 1 of 2 treatment sequences ( tv-45070/placebo or placebo / tv-45070 ) . throughout the study ( including washout ) , patients recorded pain scores 4 times each day ( upon waking , 12 noon1 h , 4 pm1 h , and 8 pm1 h ) using an 11-point numerical rating scale , and reported them through telephone to the interactive voice response system .
after treatment period 1 ( tp1 ) and the 1-week washout , patients entered a second patient - blinded , placebo run - in period . unlike tp1 , patients were not required to report a minimum mean daily pain score before entering treatment period 2 .
all patients applied 7.5 l of ointment per cm of affected skin , covering the entire painful area ( up to a maximum of 400 cm ) .
the actual dose applied varied according to the size of the treatment area ( determined by the investigator ) .
patients were given 1 of 4 area - specific dosing cards , indicating the amount of ointment to be applied ( 60 , 120 , 180 , or 240 mg of tv-45070 bid , table 1 ) .
patients were allowed to rescue with acetaminophen ( up to 4000 mg / d ) and recorded any use in a diary .
doses administered by size of treatment area a central independent review board approved the study protocol and amendments . all participants provided written informed consent .
the study was performed in accordance with the declaration of helsinki and international conference on harmonization guideline on good clinical practice and is registered with clinicaltrials.gov ( nct01195636 ) . males and females of nonchildbearing potential , aged 18 to 80 years , with a clinical diagnosis of phn , persistent pain for > 6 months from the appearance of herpes zoster rash and intact skin over the treatment area were eligible for inclusion .
exclusion criteria were patients with systemic disease that would have put them at an additional risk or limited their ability to participate , phn on the face or in proximity to mucous membranes , creatinine clearance < 30 ml / min , history of serious mental or psychiatric illness , known sensitivity to topical products , active herpes zoster lesions or dermatitis , other severe or chronic pain conditions , or patients who had participated in > 1 topical pain study and/or > 3 phn studies . patients were also excluded if they had received a local anesthetic in 2 weeks before randomization , nerve blocks within 30 days of randomization , qutenza or other capsaicin preparations in the 90 days before screening , or if they used opioids , local prescription or nonprescription therapy ( lidocaine patch , transcutaneous electrical nerve stimulation , etc . ) and were unable to washout for the study .
patients were permitted to remain on stable doses of up to 2 of the following : gabapentin , pregabalin , duloxetine , or amitriptyline .
stable doses were defined as remaining unchanged for the 4 weeks before the start of the first placebo run - in period , with no planned doses changes during the study .
safety assessments included adverse event reporting , laboratory measurements ( serum chemistry , hematology , and urinalysis ) , 12-lead electrocardiograms , vital signs , and physical examinations ( including assessments for local irritation and numbness were performed at regular clinic visits .
pharmacokinetic plasma samples were collected at each weekly visit to determine the extent of systemic exposure of tv-45070 using a validated bioanalytical method .
efficacy assessments included patient reported pain intensity ( 4 times per day ) and daily sleep interference scores ( dsis , an 11-point numerical rating scale where 0=pain does not interfere with sleep and 10=completely interferes ) .
the 4 daily pain intensity scores were combined to generate a mean daily pain score . the neuropathic pain symptom inventory ( npsi , a 12-point questionnaire to evaluate different neuropathic pain symptoms ) and patient global impression of change ( pgic ) were also assessed at clinic visits . as an exploratory endpoint , for patients who consented to genetic testing , a dna sample was collected and genotyped for nav1.7 r1150w carrier status .
the sample size was estimated to provide 80% power to detect a 1.0-point difference in mean pain intensity .
the calculation assumed a significance level of 0.05 and a within - patient sd of 2.25 . the treatment effect and sd were selected based on review of other similar phn studies.1214 an interim analysis
was performed after 21 patients completed the study to determine if the estimated sd was appropriate and if the sample size required adjustment . as a result
, the significance level for the final analysis of the primary variable was adjusted to =0.049 .
safety variables including pharmacokinetic exposure measures were summarized using descriptive statistics for continuous variables and frequency and percentage for categorical variables .
the primary efficacy endpoint was the difference in mean change from baseline in mean daily pain score between the last week ( week 3 ) of tv-45070 treatment and the last week of placebo treatment .
baseline for each treatment period was the average of the 7 mean daily pain measurements in the preceding patient - blind placebo run - in period .
the mean daily pain scores for weeks 1 , 2 , and 3 of each treatment period were similarly defined .
if the third week in a treatment period had < 4 days of recordings , the last observation carried forward ( locf ) algorithm was used ( ie , the average of the last 4 d with at least 1 pain score ) .
the hypothesis that the mean change in mean daily pain scores from baseline would differ between tv-45070 and placebo was tested using a mixed effects analysis of covariance model , with period and sequence as fixed effects , patient within treatment sequence as a random effect , and the baseline pain score as a covariate .
secondary efficacy analyses included : number of patients with 30% and 50% improvement in mean pain score from baseline .
changes in mean daily pain scores from baseline to each week of treatment and over the entire treatment period .
number of patients with a mean improvement from baseline of 1 , 2 , and 3 or more points for each week of treatment and overall .
changes in dsis , npsi , and pgic scores from baseline to the end of treatment .
for categorical variables the numbers and percentages of patients in each treatment group in each category were summarized .
exploratory analyses included examining response to treatment in the subgroup of patients who were carriers of the nav1.7 r1150w polymorphism .
this randomized , double blind , placebo - controlled , 2-period , 2-treatment crossover trial evaluated the safety and efficacy of 21 days of twice daily ( bid ) treatment with tv-45070 ointment in phn patients .
the study was conducted between august , 2010 and march , 2011 at 24 sites in the united states .
the study included 2 , 1-week patient - blind placebo run - in periods ( before each treatment period ) , and 2 , 3-week double - blind treatment periods ( fig .
1 ) . a 1-week washout separated treatment period 1 ( tp1 ) and the second placebo run - in period , effectively separating each randomized treatment period by 2 weeks .
the first single - blind , placebo run - in period was 1 week in duration ( day-7 to day-1 ) . at randomization ( visit 3 ) , patients were randomized in a crossover design to 1 of 2 treatment sequences : tv-45070/placebo or placebo / tv-45070 .
the 1-week between - treatment washout period ( between visits 6 and 7 ) , study medication was not applied , but patients continued to record pain scores using the interactive voice response system .
the second single - blind , placebo run - in period was 1 week in duration ( day-29 to day-35 ) . in tp2 , patients received the medication that was not received during tp1 .
one week after the end of tp2 , patients returned for a follow - up visit ( visit 12 ) .
patients with mean daily pain scores of 4 ( using an 11-point numerical rating scale ) for at least 4 days during the initial placebo 7 day run - in period were randomized in a 1:1 ratio through an interactive voice response system to 1 of 2 treatment sequences ( tv-45070/placebo or placebo / tv-45070 ) . throughout the study ( including washout ) , patients recorded pain scores 4 times each day ( upon waking , 12 noon1 h , 4 pm1 h , and 8 pm1 h ) using an 11-point numerical rating scale , and reported them through telephone to the interactive voice response system .
after treatment period 1 ( tp1 ) and the 1-week washout , patients entered a second patient - blinded , placebo run - in period . unlike tp1 , patients were not required to report a minimum mean daily pain score before entering treatment period 2 .
all patients applied 7.5 l of ointment per cm of affected skin , covering the entire painful area ( up to a maximum of 400 cm ) .
the actual dose applied varied according to the size of the treatment area ( determined by the investigator ) .
patients were given 1 of 4 area - specific dosing cards , indicating the amount of ointment to be applied ( 60 , 120 , 180 , or 240 mg of tv-45070 bid , table 1 ) .
patients were allowed to rescue with acetaminophen ( up to 4000 mg / d ) and recorded any use in a diary .
a central independent review board approved the study protocol and amendments . all participants provided written informed consent
the study was performed in accordance with the declaration of helsinki and international conference on harmonization guideline on good clinical practice and is registered with clinicaltrials.gov ( nct01195636 ) .
males and females of nonchildbearing potential , aged 18 to 80 years , with a clinical diagnosis of phn , persistent pain for > 6 months from the appearance of herpes zoster rash and intact skin over the treatment area were eligible for inclusion .
exclusion criteria were patients with systemic disease that would have put them at an additional risk or limited their ability to participate , phn on the face or in proximity to mucous membranes , creatinine clearance < 30 ml / min , history of serious mental or psychiatric illness , known sensitivity to topical products , active herpes zoster lesions or dermatitis , other severe or chronic pain conditions , or patients who had participated in > 1 topical pain study and/or > 3 phn studies .
patients were also excluded if they had received a local anesthetic in 2 weeks before randomization , nerve blocks within 30 days of randomization , qutenza or other capsaicin preparations in the 90 days before screening , or if they used opioids , local prescription or nonprescription therapy ( lidocaine patch , transcutaneous electrical nerve stimulation , etc . ) and were unable to washout for the study .
patients were permitted to remain on stable doses of up to 2 of the following : gabapentin , pregabalin , duloxetine , or amitriptyline .
stable doses were defined as remaining unchanged for the 4 weeks before the start of the first placebo run - in period , with no planned doses changes during the study .
safety assessments included adverse event reporting , laboratory measurements ( serum chemistry , hematology , and urinalysis ) , 12-lead electrocardiograms , vital signs , and physical examinations ( including assessments for local irritation and numbness were performed at regular clinic visits .
pharmacokinetic plasma samples were collected at each weekly visit to determine the extent of systemic exposure of tv-45070 using a validated bioanalytical method .
efficacy assessments included patient reported pain intensity ( 4 times per day ) and daily sleep interference scores ( dsis , an 11-point numerical rating scale where 0=pain does not interfere with sleep and 10=completely interferes ) .
the 4 daily pain intensity scores were combined to generate a mean daily pain score .
the neuropathic pain symptom inventory ( npsi , a 12-point questionnaire to evaluate different neuropathic pain symptoms ) and patient global impression of change ( pgic ) were also assessed at clinic visits . as an exploratory endpoint , for patients who consented to genetic testing ,
the sample size was estimated to provide 80% power to detect a 1.0-point difference in mean pain intensity .
the calculation assumed a significance level of 0.05 and a within - patient sd of 2.25 . the treatment effect and sd were selected based on review of other similar phn studies.1214 an interim analysis
was performed after 21 patients completed the study to determine if the estimated sd was appropriate and if the sample size required adjustment . as a result
, the significance level for the final analysis of the primary variable was adjusted to =0.049 .
safety variables including pharmacokinetic exposure measures were summarized using descriptive statistics for continuous variables and frequency and percentage for categorical variables .
the primary efficacy endpoint was the difference in mean change from baseline in mean daily pain score between the last week ( week 3 ) of tv-45070 treatment and the last week of placebo treatment .
baseline for each treatment period was the average of the 7 mean daily pain measurements in the preceding patient - blind placebo run - in period .
the mean daily pain scores for weeks 1 , 2 , and 3 of each treatment period were similarly defined .
if the third week in a treatment period had < 4 days of recordings , the last observation carried forward ( locf ) algorithm was used ( ie , the average of the last 4 d with at least 1 pain score ) . the hypothesis that the mean change in mean daily pain scores from baseline would differ between tv-45070 and placebo
was tested using a mixed effects analysis of covariance model , with period and sequence as fixed effects , patient within treatment sequence as a random effect , and the baseline pain score as a covariate .
secondary efficacy analyses included : number of patients with 30% and 50% improvement in mean pain score from baseline .
changes in mean daily pain scores from baseline to each week of treatment and over the entire treatment period .
number of patients with a mean improvement from baseline of 1 , 2 , and 3 or more points for each week of treatment and overall .
changes in dsis , npsi , and pgic scores from baseline to the end of treatment .
continuous variables were summarized using similar methods as for the primary efficacy variable . for categorical variables
the numbers and percentages of patients in each treatment group in each category were summarized .
exploratory analyses included examining response to treatment in the subgroup of patients who were carriers of the nav1.7 r1150w polymorphism .
in total , 129 patients were screened and 109 ( 84.5% ) entered the first placebo run - in period , 70 patients were randomized : 35 ( 50.0% ) to each treatment sequence .
fifty - four ( 77.1% ) patients completed the study and 16 ( 22.9% ) discontinued ( fig .
reasons for discontinuation included adverse event ( 8 [ 11.4% ] patients ) , withdrawal of consent ( 6 [ 8.6% ] patients ) , protocol violation ( 1 [ 1.4% ] patient ) , and lost to follow - up ( 1 [ 1.4% ] patient ) .
the tv-45070/placebo sequence had a slightly lower mean age than the placebo / tv-45070 sequence ( 58.3 vs. 63.1 y ) .
overall , the mean number of months since herpes zoster rash healing was 77.1 months ( range , 3.6 to 558.1 mo ) .
patients were relatively evenly distributed between the 4 categories of treatment area ; 61.4% of patients were allocated to the 2 smaller areas of application ( ie , < 201 cm ) .
seventeen ( 24.3% ) patients remained on one or more of the permitted phn medications during the study , ( 15 [ 21.4% ] patients remained on gabapentin and 2 [ 2.9% ] on pregabalin ) .
patient demographics and disease characteristics by treatment sequence for all randomized patients the safety population ( n=68 ) comprised patients who received at least 1 application of study medication .
there were 2 serious adverse events ( saes ) , neither considered related to study medication : an sae of tooth abscess occurred during placebo treatment , and an sae of worsening of coronary artery disease ( cad ) occurred on the first day of tv-45070 treatment .
this cardiac event occurred in a patient with well - documented cad ( including angina and a prior myocardial infarction ) .
angiography performed at the time of the sae revealed coronary artery occlusion that was managed with angioplasty and stent placement to the left anterior descending coronary artery .
there were no clinically meaningful changes in blood chemistry , hemodynamic parameters , electrocardiograms , vital signs , or in physical examination findings .
the incidence of teaes was similar between tv-45070 and placebo ( 53.2% and 50.8% , respectively ) . however , study medication - related teaes were more frequent on placebo than tv-45070 ( 30.2% vs. 17.7% ) . generally , application site related teaes were more common on placebo treatment , for example , application site pain and pruritus ( table 3 ) .
in addition , a very low incidence of dizziness was reported and no somnolence was observed ( table 3 ) . summary and incidence of the most frequently reported treatment emergent adverse events safety population eight patients discontinued because of a teae . for 7 of these 8 patients ,
the events leading to discontinuation were local skin reactions ( 5 during placebo and 2 during tv-45070 ; table 4 ) and the other discontinuation was the patient with the worsening cad .
adverse events leading to discontinuation overall , systemic exposure was low , ranging from < 0.1 ng / ml ( lower limit of quantitation ) to 13.7 ng / ml .
the mean maximum plasma concentration ( cmax ) was comparable between treatment sequences : the mean cmax for the tv-45070/placebo sequence was 2.1 ng / ml and the mean cmax for the placebo / tv-45070 treatment sequence was 2.2 ng / ml
. similar findings of low plasma levels have been observed with application of topical tv-45070 in a prior human phase 1 study ( unpublished ) which also showed high skin concentration ( mean concentration 12,000 ng / g ) .
the efficacy evaluable population ( n=57 ) comprised all patients with baseline and postbaseline efficacy data in both treatment periods .
the least squares ( ls ) mean change in mean daily pain score from baseline to week 3 with locf was 0.97 with placebo and 0.94 with tv-45070 ( difference in change from baseline ls means between treatments=0.03 , 95% confidence interval , 0.38 , 0.43 ; p=0.8885 ) .
analysis of the change in mean daily pain scores by each week of treatment did not result in significant differences between tv-45070 and placebo and there were no significant differences in the numbers or percentages of patients with a mean improvement from baseline of 1 , 2 , and 3 points for each week of treatment .
more patients achieved 50% improvement with tv-45070 ( n=15 , 26.8% ) than with placebo ( n=6 , 10.7% ) at week 3 with locf ( p=0.0039 ) .
a similar trend was observed in the proportion of patients achieving 30% improvement at week 3 with locf , with 22 ( 39.3% ) patients achieving 30% improvement on tv-45070 versus 13 ( 23.2% ) patients on placebo ( p=0.0784 ) . over the entire 3-week treatment period ,
the difference between treatments in the proportion of patients achieving 30% improvement was statistically significant ( p=0.0213 ) , with 19 ( 33.9% ) patients achieving 30% improvement on tv-45070 versus 9 ( 16.1% ) patients on placebo ( table 5 ) .
the separation between treatments increased over time a week - to - week improvement in the percentage of responders on tv-45070 versus placebo was observed ( table 5 ) .
proportion of patients achieving at least 30% or 50% pain improvements efficacy evaluable population cumulative percentage of patients versus percent change in mean daily pain score at week 3/locf .
the figure displays the cumulative percentage of patients versus percent change in mean daily pain score at week 3 with locf for the efficacy evaluable population .
there is a noticeable separation between the 2 curves , indicating that more patients experienced a 10% to 80% reduction in mean daily pain scores at the end of tv-45070 treatment compared with placebo treatment .
for example , approximately 40% of patients reported a 30% or greater reduction in pain when on tv-45070 treatment , compared with just 24% of patients during placebo treatment .
overall , the mean total number of rescue medication pills taken was slightly lower with tv-45070 treatment compared with placebo treatment ( 24.8 vs. 29.2 pills , not statistically significant ) .
analyses of dsis , npsi , and pgic did not demonstrate any significant differences between treatments .
the ls mean change in dsis score from baseline to week 3 with locf was 0.84 on placebo and 0.81 on tv-45070 .
however , a statistically significant increase , favoring tv-45070 , in the proportion of patients with 30% and 50% improvement in sleep interference scores in week 3 was observed ( p<0.05 ) .
the mean change in npsi score from baseline was 6.1 on placebo and 6.7 on tv-45070 .
for pgic , patients reported their overall status during each treatment period as follows : very much improved
( 5.4% tv-45070 : 1.8% placebo ) , or very much worse ( 0% tv-45070 : 0% placebo ) .
one randomized patient discontinued due to lack of efficacy ( in treatment period 2 while on placebo treatment due to worsening of pain at the site of phn ) .
forty - five of these patients were included in the efficacy evaluable population and contributed data to the exploratory r1150w subgroup analyses .
thirty - seven patients did not have the r1140w genotype , whereas 8 were heterozygous carriers of the nav1.7 r1150w polymorphism .
given the small number of patients contributing data , no inferential analyses were performed and results were summarized .
sixty - three percent of r1150w carriers achieved 30% reduction in pain scores on tv-45070 treatment compared with 35% of wild - type patients , and 38% of r1150w carriers achieved 50% reduction in pain scores compared with 24% of wild - type patients ( table 6 ) . in addition , the r1150w carriers showed a greater reduction in mean pain after 3 weeks of tv-45070 treatment compared with their response to placebo treatment ( mean treatment difference=0.78 points ) .
in total , 129 patients were screened and 109 ( 84.5% ) entered the first placebo run - in period , 70 patients were randomized : 35 ( 50.0% ) to each treatment sequence .
fifty - four ( 77.1% ) patients completed the study and 16 ( 22.9% ) discontinued ( fig .
reasons for discontinuation included adverse event ( 8 [ 11.4% ] patients ) , withdrawal of consent ( 6 [ 8.6% ] patients ) , protocol violation ( 1 [ 1.4% ] patient ) , and lost to follow - up ( 1 [ 1.4% ] patient ) .
the tv-45070/placebo sequence had a slightly lower mean age than the placebo / tv-45070 sequence ( 58.3 vs. 63.1 y ) .
overall , the mean number of months since herpes zoster rash healing was 77.1 months ( range , 3.6 to 558.1 mo ) .
patients were relatively evenly distributed between the 4 categories of treatment area ; 61.4% of patients were allocated to the 2 smaller areas of application ( ie , < 201 cm ) .
seventeen ( 24.3% ) patients remained on one or more of the permitted phn medications during the study , ( 15 [ 21.4% ] patients remained on gabapentin and 2 [ 2.9% ] on pregabalin ) .
the safety population ( n=68 ) comprised patients who received at least 1 application of study medication .
there were 2 serious adverse events ( saes ) , neither considered related to study medication : an sae of tooth abscess occurred during placebo treatment , and an sae of worsening of coronary artery disease ( cad ) occurred on the first day of tv-45070 treatment .
this cardiac event occurred in a patient with well - documented cad ( including angina and a prior myocardial infarction ) .
angiography performed at the time of the sae revealed coronary artery occlusion that was managed with angioplasty and stent placement to the left anterior descending coronary artery .
there were no clinically meaningful changes in blood chemistry , hemodynamic parameters , electrocardiograms , vital signs , or in physical examination findings .
the incidence of teaes was similar between tv-45070 and placebo ( 53.2% and 50.8% , respectively ) . however , study medication - related teaes were more frequent on placebo than tv-45070 ( 30.2% vs. 17.7% ) . generally , application site related teaes were more common on placebo treatment , for example , application site pain and pruritus ( table 3 ) .
in addition , a very low incidence of dizziness was reported and no somnolence was observed ( table 3 ) .
summary and incidence of the most frequently reported treatment emergent adverse events safety population eight patients discontinued because of a teae . for 7 of these 8 patients ,
the events leading to discontinuation were local skin reactions ( 5 during placebo and 2 during tv-45070 ; table 4 ) and the other discontinuation was the patient with the worsening cad .
overall , systemic exposure was low , ranging from < 0.1 ng / ml ( lower limit of quantitation ) to 13.7 ng / ml .
the mean maximum plasma concentration ( cmax ) was comparable between treatment sequences : the mean cmax for the tv-45070/placebo sequence was 2.1 ng / ml and the mean cmax for the placebo / tv-45070 treatment sequence was 2.2 ng / ml
. similar findings of low plasma levels have been observed with application of topical tv-45070 in a prior human phase 1 study ( unpublished ) which also showed high skin concentration ( mean concentration 12,000 ng / g ) .
the efficacy evaluable population ( n=57 ) comprised all patients with baseline and postbaseline efficacy data in both treatment periods .
the least squares ( ls ) mean change in mean daily pain score from baseline to week 3 with locf was 0.97 with placebo and 0.94 with tv-45070 ( difference in change from baseline ls means between treatments=0.03 , 95% confidence interval , 0.38 , 0.43 ; p=0.8885 ) .
analysis of the change in mean daily pain scores by each week of treatment did not result in significant differences between tv-45070 and placebo and there were no significant differences in the numbers or percentages of patients with a mean improvement from baseline of 1 , 2 , and 3 points for each week of treatment .
more patients achieved 50% improvement with tv-45070 ( n=15 , 26.8% ) than with placebo ( n=6 , 10.7% ) at week 3 with locf ( p=0.0039 ) .
a similar trend was observed in the proportion of patients achieving 30% improvement at week 3 with locf , with 22 ( 39.3% ) patients achieving 30% improvement on tv-45070 versus 13 ( 23.2% ) patients on placebo ( p=0.0784 ) . over the entire 3-week treatment period ,
the difference between treatments in the proportion of patients achieving 30% improvement was statistically significant ( p=0.0213 ) , with 19 ( 33.9% ) patients achieving 30% improvement on tv-45070 versus 9 ( 16.1% ) patients on placebo ( table 5 ) .
the separation between treatments increased over time a week - to - week improvement in the percentage of responders on tv-45070 versus placebo was observed ( table 5 ) .
proportion of patients achieving at least 30% or 50% pain improvements efficacy evaluable population cumulative percentage of patients versus percent change in mean daily pain score at week 3/locf .
the figure displays the cumulative percentage of patients versus percent change in mean daily pain score at week 3 with locf for the efficacy evaluable population .
there is a noticeable separation between the 2 curves , indicating that more patients experienced a 10% to 80% reduction in mean daily pain scores at the end of tv-45070 treatment compared with placebo treatment .
for example , approximately 40% of patients reported a 30% or greater reduction in pain when on tv-45070 treatment , compared with just 24% of patients during placebo treatment .
overall , the mean total number of rescue medication pills taken was slightly lower with tv-45070 treatment compared with placebo treatment ( 24.8 vs. 29.2 pills , not statistically significant ) .
analyses of dsis , npsi , and pgic did not demonstrate any significant differences between treatments .
the ls mean change in dsis score from baseline to week 3 with locf was 0.84 on placebo and 0.81 on tv-45070 .
however , a statistically significant increase , favoring tv-45070 , in the proportion of patients with 30% and 50% improvement in sleep interference scores in week 3 was observed ( p<0.05 ) .
the mean change in npsi score from baseline was 6.1 on placebo and 6.7 on tv-45070 .
for pgic , patients reported their overall status during each treatment period as follows : very much improved
( 28.6% tv-45070 : 28.6% placebo ) , minimally improved ( 17.9% tv-45070 : 33.9% placebo ) , no change ( 25.0% tv-45070 : 21.4% placebo ) , minimally worse
( 5.4% tv-45070 : 1.8% placebo ) , or very much worse ( 0% tv-45070 : 0% placebo ) .
one randomized patient discontinued due to lack of efficacy ( in treatment period 2 while on placebo treatment due to worsening of pain at the site of phn ) .
forty - five of these patients were included in the efficacy evaluable population and contributed data to the exploratory r1150w subgroup analyses .
thirty - seven patients did not have the r1140w genotype , whereas 8 were heterozygous carriers of the nav1.7 r1150w polymorphism .
given the small number of patients contributing data , no inferential analyses were performed and results were summarized .
sixty - three percent of r1150w carriers achieved 30% reduction in pain scores on tv-45070 treatment compared with 35% of wild - type patients , and 38% of r1150w carriers achieved 50% reduction in pain scores compared with 24% of wild - type patients ( table 6 ) . in addition , the r1150w carriers showed a greater reduction in mean pain after 3 weeks of tv-45070 treatment compared with their response to placebo treatment ( mean treatment difference=0.78 points ) .
we conducted a proof - of - concept trial in 70 patients with phn using tv-45070 , a topical novel potent inhibitor of nav1.7 and additional navs.10 overall , tv-45070 was safe and well tolerated and unlike many commonly used orally active medications used to treat neuropathic pain , topically applied tv-45070 demonstrated an absence of drug - related central nervous system ( cns ) teaes .
this was an expected property of the product due to the low plasma concentrations ( mean cmax was 2.1 ng / ml with a maximal individual cmax of 14 ng / ml ) relative to plasma levels previously shown to mediate cns effects ( data not shown ) .
the most common adverse events were local skin reactions where application site pain and pruritus were observed more frequently on placebo than tv-45070 treatment , suggesting that the tv-45070 api itself does not primarily cause dermal irritation .
the local skin reactions were mostly mild ( 66% ) , with occasional moderate events ( 28% ) . these resolved spontaneously without intervention .
eight patients terminated because of an adverse event ( 5 during placebo treatment and 3 during tv-45070 treatment ) .
neither of the 2 saes reported in this trial were considered related to study treatment .
the phn population studied was consistent with the typical demographic profile for other reported phn trials with one major exception ; the mean duration since diagnosis was substantially higher ( 76.6 mo ) than reported in other phn studies ( 30 mo).12,13,15 although some studies have been able to demonstrate efficacy in phn patients who have failed prior therapies , in this study the longer duration of disease suggests a more refractory population that may have compromised the ability to demonstrate an overall treatment difference in this study . nevertheless , despite a potentially more refractory study population , while the primary efficacy analysis did not show differences between the treatment arms , statistical significance was observed for responder rate analyses .
a statistically higher percentage of patients had a 50% improvement in pain during tv-45070 treatment compared with placebo and a trend to 30% improvement in pain during tv-45070 treatment compared with placebo was observed .
this positive responder rate observation suggests that a subpopulation of phn patients exists that is more likely to have an analgesic effect and , further suggests the possibility exists that this response to topical tv-45070 could be mechanism based .
although the sample size is very small and definitive conclusions can not be drawn , phn patients who were carriers of the common gain - of - function r1150w polymorphism appeared more likely to experience a more robust response to tv-45070 .
an increase in the proportion of responders for both 30% and 50% improvement in pain was observed for heterozygous carriers compared with wild type .
interestingly , the placebo response was reduced in the r1150w carriers , further widening the effect size seen in this carrier subpopulation .
although the nav1.7 r1150w polymorphism has been associated with an increase in pain perception for a variety of painful disorders including both nociceptive and neuropathic pain as well as experimentally induced heat pain ( mean increase in visual analog scale of 2.0 for carriers vs. wild type),9 these data have not yet been replicated.16 using in vitro electrophysiological testing in transfected dorsal root ganglion cells , the r1150w variant channel has previously been shown to demonstrate gain - of - function and hyperactivity.17 we postulate that enhanced pain perception mediated through an increased activity of the r1150w polymorphism may render patients more susceptible to pharmacological intervention by nav1.7 inhibitors such as tv-45070 compared with their wild - type counterparts .
these preliminary findings reported herein , when combined with the previously reported efficacy of the active ingredient of tv-45070 in iem patients where the mutations in the scn9a gene are known to cause a gain - of - function of nav1.7 channel , support the continued evaluation of the r1150w polymorphism in larger clinical studies in common disorders of pain .
human genetics has uncovered the vital importance of nav1.7 in pain perception illuminating a critical mechanism for analgesic development.3,4,18 here , we show that topically applied tv-45070 , which has potent nav1.7 inhibition and is currently in clinical development , was safe , well tolerated , and devoid of drug - related cns or cardiac toxicity .
although a mean difference between treatment groups was not detected , significant improvements were demonstrated with tv-45070 in a subset of phn patients .
furthermore , based on a small analysis subpopulation , heterozygous carriers of the r1150w polymorphism appeared more likely to report a clinically meaningful response than their wild - type counterparts .
these results support the potential clinical utility of pharmacological inhibition of nav1.7 for the treatment of peripheral neuropathic pain . given the favorable safety profile coupled with demonstrated efficacy in this phn study , these data warrant further development of topical tv-45070 in larger , longer , parallel - design studies of patients with phn or other peripheral neuropathic pain conditions . | objective : the objective was to evaluate the safety and efficacy of tv-45070 ointment , as a treatment for postherpetic neuralgia , and to explore the response in patients with the nav1.7 r1150w gain - of - function polymorphism.materials and methods : this was a randomized , placebo - controlled , 2-period , 2-treatment crossover trial .
patients with postherpetic neuralgia with moderate or greater pain received tv-45070 and placebo ointments , each applied twice daily for 3 weeks .
the primary efficacy measure was the difference in change in mean daily pain score from baseline compared with the last week of placebo and active treatment .
secondary endpoints included responder rate analyses and a further exploratory analysis of response in carriers of the nav1.7 r1150w polymorphism was conducted.results:seventy patients were enrolled and 54 completed the study .
tv-45070 was safe and well tolerated .
no statistical difference was observed between treatments for the primary endpoint .
however , the proportion of patients with 50% reduction in mean pain scores at week 3 was greater on tv-45070 than on placebo ( 26.8% vs. 10.7% , p=0.0039 ) .
similarly , a greater proportion of patients on tv-45070 had a 30% reduction in mean pain scores at week 3 ( 39.3% on tv-45070 vs. 23.2% on placebo , p=0.0784 ) .
of note , 63% of patients with the r1150w polymorphism versus 35% of wild - type carriers had a 30% reduction in mean pain score on tv-45070 at week 3 ( no inferential analysis performed).conclusions : the 50% responder analysis suggests a subpopulation may exist with a more marked analgesic response to tv-45070.the trend toward a larger proportion of responders within nav1.7 r1150w carriers warrants further investigation . |
pyrexia ( fever ) is an important clinical sign / symptom that occurs either as an isolated event or as part of a constellation of signs and symptoms of some hazards of blood transfusion .
transfusion pyrexia ( tp ) is the elevation of temperature 1c from baseline or temperature > 38c , with or without chills or rigors occurring in a recipient of a unit of blood or blood component with no other explanation other than the transfused unit .
the correct evaluation of fever in a blood transfusion recipient is important as this sign / symptom is manifested by several distinct clinical entities varying from simple febrile non - haemolytic transfusions ( fnhtr ) to life threatening complications as transfusion related acute lung injury ( trali ) and acute haemolytic transfusion reactions .
in addition , transfusion pyrexia is an independent factor that predicts platelet recovery , increment , or survival in transfusion recipients [ 3 , 4 ] .
the decision to stop the administration of blood in a case of transfusion pyrexia is often a difficult one .
many , but not all cases , can be tolerated by the transfusion recipient with supportive care and analgesics [ 5 , 6 ]
the onset of pyrexia in a transfusion recipient correlates with the pathophysiology of the specific etiology ; thus , while it could be a few minutes as a result of the presence of accumulated cytokines in the transfused unit ( in the case of fnhtr ) , its onset could be delayed up to two weeks as a result of transfusion transmitted malaria and even up to 4 weeks for transfusion associated graft versus host disease ( tagvhd ) in which engraftment of viable t cell is central to the pathogenesis of the entity .
the appropriate evaluation of pyrexia after a reasonable interval from the transfusion event in particular requires a high index of suspicion by the clinician as the transfusion may inadvertently be completely overlooked in relation to the febrile episode .
an important fundamental in the approach to the differential diagnoses and management in all cases is the early detection of fever arising from transfusion .
an optimal approach to management should incorporate quarter hourly vital signs monitoring from the onset of transfusion commencement for the first 30 minutes and half hourly monitoring thereafter till transfusion is ended .
monitoring of vital signs chart after transfusion is equally important in the days immediately following the transfusion .
transfusion related acute lung injury ( trali ) . transfusion associated graft versus host disease ( tagvhd ) .
transfusion transmitted malaria . transfusion related acute lung injury ( trali ) . transfusion associated graft versus host disease ( tagvhd ) .
the occurrence of a febrile nonhaemolytic reaction is an important complication of a blood component transfusion because of its possible confusion with other more dangerous transfusion reactions , such as acute haemolysis , sepsis , and transfusion - related acute lung injury ( trali ) , with which it shares common features .
febrile non - haemolytic reactions were thought to be mainly due to antileucocyte antibodies , with antibodies directed against hla antigens , or against granulocyte - specific antigens [ 79 ] . as a result
, universal leucoreduction of blood components has been advocated by some to reduce the incidence of febrile nonhaemolytic transfusion reaction .
some other workers found no significant impact of leucodepletion of red cells on the incidence of fnhtr [ 10 , 11 ] .
recent studies , however , have shown that the dominant factor determining the risk of a febrile reaction was not white cell contamination , but the age of the component which predisposes to accumulation of cytokines in the transfused unit [ 1214 ] .
another frequent cause of a non - haemolytic febrile reaction is sensitization to white cell or platelet antigens .
a rise in temperature may be the sole symptom , but the recipient may suffer chills , rigors , or headache .
febrile responses have also been reported as being more common in patients receiving platelet transfusions than red cell transfusion .
this has been attributed to raised levels of cd-154 , a potent inducer of cyclooxygenase 2 ( cox-2 ) enzyme and thus pge-2 , an important fever inducer [ 16 , 17 ] .
furthermore , the incidence of fnhtr with single - donor platelet ( sdp ) is much less as compared with random donor platelet ( rdp ) , and transfusion of platelet concentrate as soon as possible after collection minimized the risk of accumulation of cytokines .
those who advocate halting the transfusion while screening tests are undertaken to exclude acute haemolysis , sepsis , and trali , with resumption of the transfusion of the same unit of blood product , risk not completing the transfusion , while those who advocate the routine permanent disconnection of the unit from the administration set , returning it to the blood bank and substituting a different unit to complete the patient 's transfusion , risk exposing the patient to multiple donors thus increasing the recipient 's risk of alloimmunization and transfusion transmitted disease acquisition , as well as potentially compromising the inventory of the blood bank . both of these strategies , apart from the risks they pose , imply more discomfort for the patient and more cost for the patient and the health care provider . in a study of transfusion reactions at a tertiary hospital in nigeria ,
70% of discontinued transfusions were as a result of fnhtr ; out of these , 58% of the discontinued transfusion episodes were successfully completed with tepid sponge and antipyretic cover following review by a haematologist .
the association of allergic reaction with a febrile episode is not uncommon [ 5 , 22 ] . in such instances ,
the addition of antihistamine and or hydrocortisone is beneficial to the management of the patient . in some cases
, the symptoms of an fnhtr may be sufficiently severe that the patient becomes apprehensive and reluctant to have further transfusions ; therefore , elimination of fnhtrs will be beneficial to these patients .
acetaminophen , a common nonprescription nonsteroidal anti - inflammatory drug ( nsaid ) is sometimes given as a premedication and has been reported to lead to a significant reduction in the incidence of fnhtr .
the issue of premedication with antipyretics for fnhtr has been a subject of debate amongst transfusionists .
while some have posited that premedication can mask fever and thus make it difficult to quickly identify some more dangerous conditions such as trali , acute haemolysis , and sepsis , some other workers have found no evidence to corroborate such fears .
furthermore , even if the thermal response to these reactions can be suppressed by antipyretics , other manifestations of these reactions remain , as hypotension , haemolysis , rigors , nausea , vomiting , and tachycardia are not suppressed by antipyretics .
still , others advocate the use of pretransfusion medication , but only in those patients who have had prior febrile episodes .
to do so though means denying those patients a useful prophylaxis during their original transfusion .
. the lower rate of reaction makes feasible a policy of the return of implicated units to the blood bank for laboratory evaluation .
secondly , the use of antipyretic medications reduces the chance of symptoms of an fnhtr that may obscure the clinical findings of a patient 's underlying illness and place additional burden on the hospital 's resources , as well as the resources of medical , nursing , and laboratory personnel .
furthermore , termination of a prescribed necessary transfusion , with resultant wasting of the products , will also be avoided .
finally , the use of intravenous pethidine could be indicated in some cases of troublesome febrile non - haemolytic reactions with severe rigors especially associated with platelet transfusions .
this has been found particularly useful in cancer patients who require large volumes of platelet concentrate transfusions while on myeloablative therapy or recovering from the transplant .
however , a haemolytic transfusion event as well as bacterial contamination must be excluded before the use of pethidine for the management of febrile non - haemolytic transfusion reactions .
haemolytic reactions could be immediate or delayed , depending on whether signs and symptoms occur within or after 24 hrs
it is believed to be the most dangerous type of transfusion reaction and highly avoidable .
there is pain at the site of the intravenous access as well as severe constricting chest and loin pains , tachycardia , hypotension , and haemoglobinemia with subsequent haemoglobinuria and hyperbilirubinemia .
uncontrollable bleeding due to disseminated intravascular coagulation may occur and may actually be the only sign of a haemolytic transfusion reaction in an unconscious or anesthetized patient .
the severity of the reaction is dependent on the site of red cell destruction , which is dependent on antibody characteristics .
intravascular red cell destruction associated with the activation of full - complement cascade is the most dangerous type of hemolytic reaction ; however , weak antibodies that do not seem to be clinically significant in vitro have been reported to cause severe acute hemolytic transfusion reactions .
delayed hemolytic transfusion reactions ( dhtrs ) are characterized by a triad of pyrexia , anaemia , and hyperbilirubinemia and are well - recognized hazards of blood transfusion that may occur as a result of an anamnestic immune response [ 2729 ] .
dhtrs are seen more frequently in patients with sickle cell disorders ( scd ) and haemoglobinopathies than in other groups of patients .
such reactions are neither predictable nor preventable ; usually an individual has been previously sensitized to one or more red cell antigens by transfusion or pregnancy .
antibody is not detectable in routine pre - transfusion screening , but the transfusion of blood , containing antigens to which the recipient has previously been sensitized provokes a brisk anamnestic response .
however , patten et al . reported a case of dhtr resulting from a primary immune response .
awareness of dhtr in particular for the patient at risk can limit wastage of scarce resources by the patient and the medical , nursing , and laboratory personnel in septic workup .
management of dhtr is mainly supportive , and no definitive treatment may be necessary .
a transfusion reaction form should be completed , and notification of the blood bank at the time the reaction is suspected is mandatory to allow prompt investigation .
adequate attention must be given to the urinary output of the patient with strict input - output monitoring .
where the facility is available , haemodialysis is helpful as circulating immune complexes which are generated as a result of the haemolytic reaction are removed in the process thus attenuating the inflammatory response .
immediate haemolytic transfusion reactions could be prevented through the avoidance of clerical errors by the laboratory staff as well as the clinical staff before the administration of blood .
transfusion pyrexia could be a sign or symptom of the systemic inflammatory response syndrome ( sirs ) complicating the transfusion episode as a result of bacterial contamination of blood for transfusion ( septicaemia ) [ 32 , 33 ] .
transfusion of heavily contaminated blood will usually lead to high fever , collapse , shock , and hemorrhagic phenomena due to disseminated intravascular coagulation ( dic ) .
a number of gram - negative , psychrophilic , and endotoxin - producing contaminants found readily in dirt and soil ( pseudomonades , coliforms ) may very rarely enter a unit and grow readily under the storage conditions of blood and even more rapidly at room temperature .
severe fulminant toxic symptoms can be seen after transfusion of blood contaminated by staphylococcus or yersinia .
yersinia enterocolitica grows well in red cell components due to its dependence on citrate and iron .
bacterial contamination is commoner with platelet transfusion apparently due to the storage temperature for platelet concentrates ( 22c24c ) , a temperature conducive for rapid proliferation of most bacteria contaminants often arising from inadequate cleaning of the phlebotomy site on the donor .
the use of single donor platelets as against the preparation of platelet concentrates from pooled donor ( random donor platelets ) has been shown to reduce the risk of bacterial contamination from donor skin flora or asymptomatic bacteraemia .
careful examination of the blood bag before transfusion could lead to identification of a contaminated blood bag as a result of colour change of the donor unit , and such units should not be transfused .
inactivation of pathogen in platelet concentrate using photochemical techniques is targeted not only to bacteria but also to a wide spectrum of viruses , spirochetes , parasites , and leukocytes .
pathogen inactivation is a proactive method which anticipates the contamination of the blood pool by emerging pathogens . in cases where
transfusion of a contaminated blood component has been inadvertently carried out , stopping the transfusion immediately reduces the bacteria load of the patient , and the hospital blood bank should be immediately notified .
after initial supportive care , blood cultures should be taken and broad - spectrum antimicrobials commenced .
diagnosis is established by gram stain and blood culture of both the blood component and the recipient .
transmission of this parasite through blood is important as only a small number of infected cells from the donor can lead to malaria in the recipient of the unit .
transfusion - acquired plasmodium falciparum - induced malaria fevers predispose to significant morbidity , not only after whole blood transfusion , but also after infusion of components , such as platelet cryoprecipitate and leucocytes , with the average incubation period being 710 days [ 38 , 39 ] . this could , however , be up to three weeks in some cases .
the risk of acquiring malaria via the transfusion of blood components is extremely low in nonendemic countries such as canada and the united states .
this is largely due to the strict donor deferral criteria . a transfusion malaria risk of 0.25 cases / million donor units has been estimated in the united states , with a fairly steady incidence of one to three cases per year reported by the united states centers for disease control and prevention ( cdc ) .
in contrast , the risk in endemic regions which include central and south america , hispaniola sub - saharan africa , the indian subcontinent , the middle east , southeast asia , and oceania may be more than 50 cases / million donor units .
however , the malaria endemic status of nigeria makes the issue of donor deferral on account of malaria status unrealistic as exclusion would include nearly all eligible donors .
deferral policies for malaria are not practical for endemic areas [ 37 , 41 ] .
the symptoms developed by the recipient include fever , chills , headache , muscle aches , and malaise .
if the film is positive for malaria parasite , appropriate antimalaria therapy should be immediately instituted in accordance with the current treatment guidelines for the region .
in recent years , transfusion - related acute lung injury ( trali ) has developed from an almost unknown transfusion reaction to one of the most common cause of transfusion - related major morbidities and fatalities [ 42 , 43 ] .
trali , a condition also known as noncardiogenic pulmonary edema presents with fever , cough , tachypnea , tachycardia , wheeze , cyanosis , hypotension and evidence of pulmonary infiltrate on chest x - ray .
a clinical definition of trali was established in 2004 , based on acute respiratory distress , non - cardiogenic pulmonary edema temporal association with transfusion and hypoxaemia .
it could be confused as a case of severe anaphylaxis , and a high index of suspicion is needed to make the diagnosis .
the onset typically occurs within 6 hours of transfusion , but most cases present within 1 to 2 hours .
the incidence of trali has been reported as 0.02% of all units or 0.16 of all patients , although it is believed to be underdiagnosed [ 4446 ] .
clinical predisposing factors may be associated with the development of trali , as it has been observed more frequently in patients with sepsis , cancer , or patients who had received multiple transfusions .
yang et al . reported two cases of trali resulting from designated blood transfusion between mother and child and suggests that designated blood transfusion between multiparous mothers and children might add an additional transfusion - related risk owing to the higher likelihood of the hla antibody - antigen specificity between mother and child .
the pathophysiology is unclear but has been attributed to hla antibodies , granulocyte antibodies , and more recently to biologically active mediators in stored blood components .
immune complexes are formed , and entering the pulmonary vascular bed stimulates the release of vasoactive substances that cause the leakage of fluid into alveolar spaces , activation of complement , leukostasis , and activation of polymorphonuclear neutrophils [ 42 , 43 ] .
ventilatory and hemodynamic assistance are utilized as required , and with good ventilatory support , most of the symptoms resolve within 96 hrs of instituting such assistance .
although steroids are often given as part of treatment in trali , there are no clear indications for the use of corticosteroids , and their use remains controversial in this setting .
there is , however , enough evidence to warrant permanent deferral of a donor whose donated unit is frequently implicated in the etiology of trali .
ta - gvhd occurs when donor lymphocytes in cellular blood products engraft in a susceptible transfusion recipient .
thus , an index of suspicion is necessary when blood components are given to at risk categories of patients ( see table 1 ) .
the clinical syndrome comprises fever , skin rash , pancytopenia , abnormal liver function , and diarrhoea .
transfusion - associated gvhd occurs when viable t lymphocytes in blood components are transfused , and they engraft and react against the recipient 's tissues causing damage to target organs especially bone marrow , skin , liver , and gastrointestinal tract , and the recipient is unable to reject the donor lymphocytes .
normally , recipient lymphocytes are capable of recognising foreign hla and prevent the development of a donor antihost immune response .
firstly , sharing of hla - haplotypes between donor and recipient which occurs when hla - selected components are transfused or when donations are obtained from relatives .
this is particularly true when hla homozygous blood components are transfused [ 47 , 48 ] .
the second factor is defective recipient cell - mediated immunity which may be inherited , for example , severe combined immune deficiency - scid or acquired , for example , hodgkin 's disease .
other factors which may be relevant are the age of the component as the number of viable lymphocytes diminishes with storage .
lymphocyte dose is important , but leucodepletion does not prevent ta - gvhd . not all cases of acquired immune suppression states are , however , at risk for tagvhd , thus there is no need for irradiation of components for transfusion in such cases ( table 2 ) . generally , however , the most commonly reported setting for ta - gvhd is in immunocompetent recipients of blood from biologically related ( directed ) or hla identical donors .
the most frequent reports of ta - gvhd in immunocompetent individuals are from japan , where there is a greater hla homogeneity in the general population .
transfusion - associated gvhd carries a very poor prognosis ; it is fatal in over 90% of cases .
gamma irradiation of cellular blood components is the recommended method of preventing this complication .
the blood bank must be appraised of the immune status , or diagnosis , of the patient so that cellular components intended for transfusion of immunocompromised patients and blood components from designated donors will be irradiated .
the dose of gamma irradiation should be a minimum of 25 gy to any part of the blood component container .
irradiation of blood red cell containing components , however , decreases the red cell survival and increases the potassium of the component .
fresh frozen plasma [ ffp ] and cryoprecipitated ahg ( cryo ) need not be irradiated , because these components do not contain enough viable lymphocytes to cause gvhd . in conclusion , transfusion pyrexia is an important sign and or symptom of blood transfusion that should be properly evaluated by the transfusionist .
a good understanding of the pathophysiology of the differential diagnoses is indispensable to the correct evaluation of fever in blood transfusion .
the algorithm developed in ( figure 1 ) , may be used in patient evaluation in order to institute appropriate management . | background / purpose . transfusion pyrexia ( fever ) is an important clinical sign / symptom occurring either as an isolated event or as part of a constellation of signs and symptoms in relation to blood transfusion .
it is an important cause of morbidity and may be an important sign of life - threatening complications of blood transfusion .
pyrexia is often a reason for the discontinuation of a blood transfusion episode , and adequate evaluation remains a challenge for clinicians .
the decision to stop a blood transfusion episode on account of fever is often a difficult one .
this paper reviews the differential diagnosis of transfusion pyrexia ( tp ) , the pathogenesis as well as current management measures .
study selection and data source .
literature sources include medical texts , journals , dissertations , and internet - based electronic materials
results and conclusion .
adequate evaluation of pyrexia accompanying blood transfusion remains a challenge for clinicians .
an algorithm to assist the clinician in the evaluation of fever occurring in a blood transfusion recipient is developed and presented .
continuous medical education is necessary for clinicians towards improved patient care in transfusion medicine . |
thyroid - stimulating hormone ( also known as thyrotropin , tsh ) stimulates the thyroid gland to produce thyroxine ( t4 ) and triiodothyronine ( t3 ) , which is a metabolism - stimulating hormone .
tsh is synthesized and secreted by thyrotrope cells in the anterior pituitary gland and regulates the endocrine function of the thyroid gland .
production and secretion of tsh is stimulated by the hypothalamus , which produces thyrotropin - releasing hormone ( trh ) .
production of tsh is inhibited by somatostatin , which is also produced by the hypothalamus , and via a negative feedback loop by t3 and t4 .
they have high prevalence , chronic course , significant mental and somatic comorbidity and very high personal and societal costs ( lost productivity and increased medical expenses ) .
also , many patients respond poorly to medications and have frequent and disrupting relapses . finally , although several biological markers have been identified , such as cortisol or brain derived neurotrophic factor ( bdnf ) , none of them is conclusive , partly because they are shared by different disorders .
tsh also can not be used as a single biomarker of any mental disorder , but could serve as an additional biomarker for improving diagnostic or therapeutic procedures . the relation between thyroid function and mental disorders
thyroid disorders , including both hypothyroidism and hyperthyroidism , may be accompanied by various neuropsychiatric manifestations , ranging from depression and anxiety to psychosis .
hypothyroidism clinical symptoms may mimic melancholic depression and dementia , while in elderly patients hyperthyroidism may mimic depression and pseudodementia . also , tsh levels are correlated with depression severity and hyperthyroidism may increase the risk of developing bipolar disorders .
have found higher rates of panic disorder , simple phobia , obsessive compulsive disorder , major depressive disorder , bipolar disorder and cyclothymia in thyroid patients than in the general population .
these findings would suggest that the co - occurrence of psychiatric and thyroid diseases may be the result of common biochemical abnormalities .
reported that depression was observed more frequently among individuals with subclinical ( 49.7 % ) hypothyroidism than among individuals with overt hypothyroidism ( 16.8 % ) ( p < 0.001 ) and subclinical hypothyroidism increased the risk for a patient to present depression more than four times ( or = 4.9 ) . in schizophrenia results of thyroid function studies
rao et al . concluded that increased dopaminergic activity affects pituitary secretory functions and may lead to reduced tsh levels .
subclinical hypothyroidism may be present in treatment - naive patients with schizophrenia and treatment with antipsychotics may increase basal tsh levels , while rao et al .
higher basal tsh levels may be associated with a poorer treatment response in schizophrenia , while t4 levels showed a positive correlation with the severity of illness and the degree of clinical response to neuroleptic treatment .
the above findings would suggest that the co - occurrence of psychiatric and thyroid diseases may be the result of common biochemical abnormalities .
therefore , we have carried out this study in order to investigate differences in tsh level in patients with schizophrenia , unipolar depression , bipolar depression and bipolar mania .
our psychiatry clinical hospital database was screened for serum thyroid - stimulating hormone ( tsh ) level . only the first entry for each patient from inpatient care units
thus , we have assumed that most patients that we included in the study were in acute phase of their disorder .
we focused on patients with schizophrenia ( all subtypes ) , bipolar disorder and unipolar depression .
patients were grouped under diagnostic criteria as schizophrenia ( f20 according to icd-10 , 295 according to dsm - iv ) , unipolar depression ( f31 and f32 according to icd-10 , 296.2 and 296.3 according to dsm - iv ) , bipolar disorder ( f30 and f31 according to icd-10 , 296 . according to dsm - iv ) ,
bipolar depression ( f31.3f31.5 according to icd-10 , 296.6 according to dsm - iv ) and bipolar mania ( f30 and f31.0f31.2 according to icd-10 , 296.0 and 296.4 according to dsm - iv ) . in our unit diagnosis
is based on the icd-10 criteria , dsm - iv codes were given as reference .
blood samples were drawn for all patients between 8 and 9 a.m. after 12 h overnight fast . immediately after collecting blood samples
tsh serum level was determined using automatic analyzer dirui cs-400 ( dirui , china ) . for the normal
range three different criteria were used : ( 1 ) the currently accepted 0.45.0 iu / ml range ; ( 2 ) the 0.33.0 iu / ml range proposed by the american association of clinical endocrinology ; and ( 3 ) the 0.42.5 iu / ml range recommended by the national academy of clinical biochemistry ( nacb ) .
simple descriptive statistics ( means and standard deviations ) were generated for continuous variables . for discrete variables number of patients and percentages are given .
wallis test ( for tsh level ) or one - way anova ( for age ) , intra - group differences were analyzed using wilcoxon rank - sum test .
ine the study group of 1,685 patients included in the analysis there were 63.1 % women ( n = 1,064 ) . the proportion of women in the schizophrenia group ( n = 769 ) was 50.5 % ( n = 388 ) , in the unipolar depression group ( n = 652 ) was 75.8 % ( n = 494 ) , in the bipolar disorder group ( n = 264 ) was 68.9 % ( n = 182 ) , in the bipolar depression group ( n = 203 ) was 71.9 % ( n = 146 ) and in the bipolar mania ( n = 61 ) was 59.0 % ( n = 36 ) .
the difference between the groups in the proportion of women was significant ( = 105.01 , df = 3 , p < 0.001 ) , with the lowest proportion of women in the schizophrenia group . in the schizophrenia group
( n = 769 ) there were three patients with hebephrenic subtype , 20 patients with paranoid schizophrenia and catatonic symptoms , four with residual schizophrenia and two with simple - type schizophrenia .
these subgroups were to small to include them into separate analysis and therefore we decided to combine all patients with schizophrenia into one group .
figure 1 shows the total number of patients and stratification of patients into diagnostic groups.fig .
1total number of patients and stratification of patients into diagnostic groups total number of patients and stratification of patients into diagnostic groups the age ( mean standard deviation ) of the study group was 46.4 19.8 years .
mean age in the subgroups was : schizophrenia 40.0 16.2 , unipolar depression 52.1 21.7 , bipolar depression 52.8 18.4 , bipolar mania 45.3 19.8 years .
one - way anova showed that there were significant age differences between the groups ( f = 56.72 , df = 3 , p < 0.001 ) .
post hoc comparisons using the bonferroni test showed that patients with unipolar- and bi - polar depression groups were significantly older ( p < 0.001 for both groups ) .
age distribution in the study sample is shown in table 1.table 1age distribution in the study samplediagnosisage categorytotal [ n ( % ) ] < 2020404060>60schizophrenia men262487631381 ( 49.5 ) women3117211570388 ( 50.5 ) total57420191101769unipolar depression men24205757158 ( 24.2 ) women9334165202494 ( 75.8 ) total11754222259652bipolar disorder men1124212682 ( 31.1 ) women8436170182 ( 68.9 ) total19678296264bipolar depression men516122457 ( 28.1 ) women4355057146 ( 71.9 ) total9516281203bipolar mania men689225 ( 41.0 ) women48111336 ( 59.0 ) total1016201561 age distribution in the study sample we have found several significant correlations between age and tsh level . in the whole study group tsh level was negatively correlated with age ( r = 0.23 , p < 0.001 ) , but not in the bipolar mania ( p = 0.07 ) group .
weak correlations with age have been found in the schizophrenia ( r = 0.21 , p < 0.001 ) , unipolar depression ( r = 0.23 , p < 0.001 ) , bipolar depression ( r = 0.25 , p = 0.002 ) and bipolar disorder ( r = 0.21 , p = 0.005 ) groups .
for the whole study group there was no difference between men and women for tsh levels ( 1.58 1.39 vs. 1.77 2.82 iu / ml , z = 1.13 , p = 0.25 ) .
mean serum tsh level in the study groups was : schizophrenia 1.71 1.49 iu / ml , unipolar depression 1.63 1.95 iu / ml , bipolar disorder group 1.86 4.58 iu / ml , bipolar depression 2.00 5.18 iu / ml , bipolar mania 1.38 1.17 iu / ml , see fig .
2 . there was a significant difference between the serum level of tsh between patients with schizophrenia , unipolar depression , bipolar depression and bipolar mania ( h = 11.58 , df = 3 , p = 0.009 ) .
the highest level of tsh was found in the bipolar depression group ( 2.00 5.18 iu / ml ) and the lowest in patients with bipolar mania ( 1.38 1.17 iu / ml ) .
the difference was also significant for the subgroup of men ( h = 9.89 , df = 3 , p = 0.019 ) , but not for women ( h = 4.93 , df = 3 , p = 0.17 ) . when patients with bipolar mania or depression were combined into one group ( bipolar disorder ) ,
there were also significant inter - group differences between patients with schizophrenia , unipolar depression and bipolar disorder ( h = 8.62 , df = 2 , p = 0.013 ) .
the lowest level was in patients with unipolar depression ( 1.63 1.95 iu / ml ) and the highest level was in patients with bipolar disorder ( 1.86 4.58 iu / ml ) , see fig .
the difference was also significant for the subgroup of men ( h = 7.49 , df = 2 , p = 0.023 , but not for women ( h = 4.17 , df = 2 , p = 0.12).fig .
2mean tsh levels [ iu / ml ] with standard error in subjects with schizophrenia , unipolar depression , bipolar depression and bipolar maniafig .
3mean tsh levels [ iu / ml ] with standard error in subjects with schizophrenia , unipolar depression and bipolar disorder mean tsh levels [ iu / ml ] with standard error in subjects with schizophrenia , unipolar depression , bipolar depression and bipolar mania mean tsh levels [ iu / ml ] with standard error in subjects with schizophrenia , unipolar depression and bipolar disorder table 2 shows sex distribution in diagnostic groups for normal ranges of tsh .
evaluation of the 0.45.0 iu / ml normal range revealed significant differences between diagnostic groups with regard to serum tsh categories ( = 16.29 , df = 6 , p = 0.012 ) .
when patients with bipolar depression and bipolar mania were combined into one group ( bipolar disorder ) the difference was also significant ( = 14.78 , df = 4 , p = 0.005 ) .
the overall rate of being above or below the normal range was 7.9 % for patients with schizophrenia , 13.9 % for patients with unipolar depression , 10.8 % for patients with bipolar disorder , 12.2 % for patients with bipolar depression , and 11.4 % for patients with bipolar mania .
the rate of patients being below the normal range was the highest in patients with unipolar depression ( 11.0 % ) , while the rate of patients being above the normal range was the highest in patients with bipolar depression ( 4.4 % ) .with the exception of bipolar mania , the rate of patients being below or above the normal range was higher in women ( where it was higher only in patients with tsh level above the normal range ) .
there was a significant difference between age of patients with tsh level below or above normal range ( below 56.3 17.6 years , normal range 45.5 19.8 years , above 48.2 19.8 years , f = 19.26 , df = 2 , p < 0.001 ) . post hoc analysis showed that patients with tsh level below the normal range were significantly older comparing to patients with tsh level above ( p = 0.042 ) or within the normal range ( p < 0.001).table 2sex distribution in diagnostic groups for normal ranges of tshdiagnosistsh category [ ( iu / ml ) n ( % ) ] < 0.40.45.0>5.0<0.30.33.0>3.0<0.40.42.5>2.5schizophrenia44 ( 5.7)706 ( 91.8)19 ( 2.5)24 ( 3.1)659 ( 85.7)86 ( 11.2)44 ( 5.7)597 ( 77.6)128 ( 16.6 ) men20 ( 5.2)354 ( 92.9)7 ( 1.8)8 ( 2.1)331 ( 86.9)42 ( 11.0)20 ( 5.2)300 ( 78.7)61 ( 16.0 ) women24 ( 6.2)352 ( 90.7)12 ( 3.1)16 ( 4.1)328 ( 84.5)44 ( 11.3)24 ( 6.2)297 ( 76.5)67 ( 17.3)unipolar depression72 ( 11.0)561 ( 86.0)19 ( 2.9)47 ( 7.2)541 ( 83.0)64 ( 9.8)72 ( 11.0)482 ( 73.9)98 ( 15.0 ) men15 ( 9.5)141 ( 89.2)2 ( 1.3)10 ( 6.3)136 ( 86.1)12 ( 7.6)15 ( 9.5)123 ( 77.8)20 ( 12.7 ) women57 ( 11.5)420 ( 85.0)17 ( 3.4)37 ( 7.5)405 ( 82.0)52 ( 10.5)57 ( 11.5)359 ( 72.7)78 ( 15.8)bipolar disorder22 ( 8.3)232 ( 87.9)19 ( 2.5)15 ( 5.7)214 ( 81.1)35 ( 13.3)22 ( 8.3)191 ( 72.3)51 ( 19.3 ) men6 ( 7.3)74 ( 90.2)7 ( 1.8)3 ( 3.7)72 ( 87.8)7 ( 8.5)6 ( 7.3)64 ( 78.0)12 ( 14.6 ) women16 ( 8.8)158 ( 86.8)12 ( 3.1)12 ( 6.6)142 ( 78.0)28 ( 15.4)16 ( 8.8)127 ( 69.8)39 ( 21.4)bipolar depression16 ( 7.8)178 ( 87.7)9 ( 4.4)12 ( 5.9)162 ( 79.8)29 ( 14.3)16 ( 7.8)145 ( 71.4)42 ( 20.7 ) men2 ( 3.51)53 ( 93.0)2 ( 3.5)1 ( 1.7)50 ( 87.7)6 ( 10.5)2 ( 3.51)45 ( 78.9)10 ( 17.5 ) women14 ( 9.59)125 ( 85.6)7 ( 4.8)11 ( 7.5)112 ( 76.7)23 ( 15.7)14 ( 9.59)100 ( 68.5)32 ( 21.9)bipolar mania6 ( 9.8)54 ( 88.5)1 ( 1.6)3 ( 4.9)52 ( 85.2)6 ( 9.8)6 ( 9.8)46 ( 75.4)9 ( 14.7 ) men4 ( 16.0)21 ( 84.0)02 ( 8.0)22 ( 88.0)1 ( 4.0)4 ( 16.0)19 ( 76.0)2 ( 8.0 ) women2 ( 5.6)33 ( 91.7)1 ( 2.8)1 ( 2.8)30 ( 83.3)5 ( 13.9)2 ( 5.6)27 ( 75.0)7 ( 19.4 ) sex distribution in diagnostic groups for normal ranges of tsh evaluation of the 0.33.0 iu / ml normal range revealed significant differences between diagnostic groups with regard to serum tsh categories ( = 15.55 , df = 6 , p = 0.016 ) .
when patients with bipolar depression and bipolar mania were combined into one group ( bipolar disorder ) the difference was also significant ( = 14.45 , df = 4 , p = 0.006 ) .
the overall rate of being above or below the normal range was 14.3 % for patients with schizophrenia , 17.0 % for patients with unipolar depression , 19.0 % for patients with bipolar disorder , 20.2 % for patients with bipolar depression , and 14.7 % for patients with bipolar mania .
the rate of patients being below the normal range was the highest in patients with unipolar depression ( 7.2 % ) , while the rate of patients being above the normal range was the highest in patients with bipolar depression ( 14.3 % ) .with the exception of bipolar mania , the rate of patients being below or above the normal range was higher in women ( where it was higher only in patients with tsh level above the normal range ) .
there was a significant difference between age of patients with tsh level below or above normal range ( below 57.9 17.1 years , normal range 46.1 19.8 years , above 43.7 19.3 years , f = 16.50 , df = 2 , p < 0.001 ) .
post hoc analysis showed that patients with tsh level below the normal range were significantly older comparing to patients with tsh level above ( p < 0.001 ) or within the normal range ( p < 0.001 ) .
evaluation of the 0.42.5 iu / ml normal range revealed significant differences between diagnostic groups with regard to serum tsh categories ( = 16.69 , df = 6 , p = 0.010 ) . when patients with bipolar depression and bipolar mania were combined into one group ( bipolar disorder ) the difference was also significant ( = 15.36 , df = 4 , p = 0.004 ) .
the overall rate of being above or below the normal range was 22.3 % for patients with schizophrenia , 26.0 % for patients with unipolar depression , 27.6 % for patients with bipolar disorder , 28.5 % for patients with bipolar depression , and 24.5 % for patients with bipolar mania .
the rate of patients being below the normal range was the highest in patients with unipolar depression ( 11.0 % ) , while the rate of patients being above the normal range was the highest in patients with bipolar depression ( 20.7 % ) .with the exception of bipolar mania , the rate of patients being below or above the normal range was higher in women ( where it was higher only in patients with tsh level above the normal range ) .
there was a significant difference between age of patients with tsh level below or above normal range ( below 56.3 17.6 years , normal range 46.4 19.7 years , above 42.0 20.2 years , f = 24.58 , df = 2 , p < 0.001 ) .
post hoc analysis showed that patients with tsh level below the normal range were significantly older comparing to patients with tsh level above ( p < 0.001 ) or within the normal range ( p < 0.001 ) , while patients with tsh level above the normal range were younger than patients with tsh level within the normal range ( p = 0.002 ) .
patients were divided into four group : 20 , > 20 years and 40 , > 40 years and 60 years and > 60 years .
age distributions of tsh level are shown in table 3 for patients with schizophrenia , in table 4 for patients with unipolar depression , in table 5 for patients with bipolar disorder , in table 6 for patients with bipolar depression and in table 7 for patients with bipolar mania .
there were significant differences between tsh level in age groups of schizophrenia patients ( p < 0.001 ) , in men ( p < 0.001 ) but not in women ( p = 0.077 ) . comparing men with women , tsh level was higher in women only in the subgroup of patients aged 4060 ( p = 0.008 ) , while other differences were not significant . in patients with unipolar depression
there were significant differences between tsh level in age groups ( p < 0.001 ) , both in men ( p < 0.001 ) and in women ( p < 0.001 ) . comparing men with women , tsh level was higher in women only in the subgroup of patients aged > 60 ( p = 0.002 ) , while other differences were not significant . in patients with bipolar disorder
there were significant differences between tsh level in age groups ( p = 0.026 ) , in women ( p = 0.031 ) but not in men ( p = 0.23 ) .
there were no differences in age groups between men and women with bipolar disorder . in patients with bipolar depression
there were significant differences between tsh level in age groups ( p = 0.014 ) but not in men ( p = 0.06 ) and in women ( p = 0.19 ) .
there were no differences in age groups between men and women with bipolar depression . in patients with bipolar mania
there were no significant differences between tsh level in age groups ( p = 0.64 ) , also in men ( p = 0.11 ) and in women ( p = 0.07 ) . comparing men with women , tsh level was higher in women only in the subgroup of patients aged 2040 ( p = 0.012 ) , while other differences were not significant ( table 7 ) .
table 3mean tsh level ( in iu / ml ) in patients with schizophreniatotalage category
p
< 2020 - 4040 - 60>60men1.68 1.571.93 0.901.75 1.321.42 2.301.64 1.72h = 29.62
p < 0.001women1.73 1.401.84 1.031.81 1.361.62 1.561.63 1.38h = 6.84
p = 0.077total1.71 1.491.88 0.961.77 1.331.54 1.891.63 1.48h = 27.56
p < 0.001
p
z = 0.87
p = 0.38z = 0.68
p = 0.49z = 0.57
p = 0.56z = 2.66
p = 0.008z = 0.42
p = 0.66
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum testtable 4mean tsh level ( in iu / ml ) in patients with unipolar depressiontotalage category
p
< 2020404060>60men1.39 0.982.20 1.191.72 1.041.37 0.910.96 0.63h = 31.18
p < 0.001women1.71 2.171.98 1.101.59 1.221.86 3.341.47 1.22h = 26.72
p < 0.001total1.63 1.952.03 1.121.64 1.151.74 2.921.36 1.14h = 48.46
p <
0.001
p
z = 1.49
p = 0.13z = 1.03
p = 0.30z = 0.66
p = 0.51z = 0.04
p = 0.96z = 3.04
p = 0.002
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum testtable 5mean tsh level ( in iu / ml ) in patients with bipolar disordertotalage category
p
< 2020 - 4040 - 60>60men1.43 1.121.74 1.021.53 1.361.59 1.281.09 0.70h = 4.21
p = 0.23women2.05 5.462.73 2.281.94 1.221.49 1.372.51 8.63h = 8.87
p = 0.031total1.86 4.582.16 1.691.80 1.271.52
7.39h = 9.28
p = 0.026
p
z = 0.89
p = 0.37z = 0.57
p = 0.56z = 1.73
p = 0.08z = 0.48
p = 0.62z = 0.86
p = 0.38
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum testtable 6mean tsh level ( in iu / ml ) in patients with bipolar depressiontotalage category
p
< 2020404060>60men1.56 1.201.90 0.871.84 1.471.84 1.581.15 0.69h = 4.71
p = 0.19women2.17 6.053.54 2.591.93 1.271.63 1.472.71 9.54h = 7.11
p = 0.06total2.00 5.182.63 1.911.90 1.321.67 1.482.25 8.02h = 10.63
p = 0.014
p
z = 0.28
p = 0.77z = 0.98
p = 0.32z = 0.44
p = 0.65z = 0.39
p = 0.69z = 0.45
p = 0.64
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum testtable 7mean tsh level ( in iu / ml ) in patients with bipolar maniatotalage category
p
< 20204040 - 60>60men1.16 0.891.61 1.190.91 0.861.26 0.630.33 0.35h = 5.94
p = 0.11women1.54 1.311.92 1.922.01 1.040.90 0.421.66 1.66h = 6.77
p = 0.07total1.38 1.171.74 1.431.46 1.081.06 0.541.48 1.61h = 1.66
p = 0.64
p
z = 1.07
p = 0.28z = 0.00
p = 1.00z = 2.52
p = 0.012z = 1.17
p = 0.23z = 1.69
p = 0.09
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum test mean tsh level ( in iu / ml ) in patients with schizophrenia
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum test mean tsh level ( in iu / ml ) in patients with unipolar depression
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum test mean tsh level ( in iu / ml ) in patients with bipolar disorder
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum test mean tsh level ( in iu / ml ) in patients with bipolar depression
inter age - subgroups , kruskal
wallis test
men versus women , wilcoxon rank - sum test mean tsh level ( in iu / ml ) in patients with bipolar mania
inter age - subgroups , kruskal
the aim of this retrospective , cross - sectional , naturalistic study was to investigate differences in tsh level in acute phase of schizophrenia , unipolar depression , bipolar depression and bipolar mania .
we have analyzed tsh level in age subgroups of diagnostic groups , as well as distribution of results within and outside three different normal ranges .
we have found that patients with bipolar disorder have the highest level of tsh , while the lowest level was seen in patients with unipolar depression . when patients with bipolar mania and bipolar depression were analyzed as separate groups , we have found that patients with bipolar depression had the highest level of tsh , while the lowest level was found in patients with bipolar mania .
this difference can not be explained only by differences in age between study groups since patients with unipolar- and bi - polar depression were significantly older comparing to other study groups .
the direction of tsh abnormalities ( to hypothyroidism or hyperthyroidism ) depended on the normal range used for analysis .
when the widest normal range was used ( 0.45.0 iu / ml ) the abnormality was mainly in the direction of hyperthyroidism , while for other two ranges the abnormality was mainly in the direction of hypothyroidism .
these observations were independent on diagnostic group and sex , with the exception of patients with bipolar mania , but this group was much smaller and thus under representative comparing to other diagnostic groups .
patients with bipolar depression had the highest rate of being above or below normal range of tsh level . with the exception of bipolar mania ,
in all diagnostic groups there were differences in tsh level between predefined age groups . in patients with schizophrenia and unipolar depression older patients
had the lowest level of tsh . in patients with bipolar disorder and bipolar depression lowest tsh levels were in patients aged 4060 ( there were no significant differences between tsh level in age groups of patients with bipolar mania ) .
this is contradictory to the results of the population - based national health and nutrition examination survey ( nhanes ) , where positive correlation between age and tsh level was found in a large ( n = 17,353 ) sample .
this observation might be explained by the fact that our study sample has various potential risk factors of thyroid dysfunction , associated with clinical diagnosis and resulting from treatment . on the other hand ,
our results are consistent with results of the german study , which show that age was the only independent factor and was significantly inversely associated with tsh .
meta - analysis of thyroid dysfunctions in europe showed that the prevalence of undiagnosed hypothyroidism was 4.94 % and the prevalence of undiagnosed hyperthyroidism was 1.72 % .
the prevalence of both previously diagnosed and undiagnosed hypothyroidism and hyperthyroidism was 3.05 and 0.75 % , respectively . for depression , 14 % of patients are found to have overt hypothyroidism while subclinical hypothyroidism occurs in 440 % of these patients . in 868 elderly psychiatric long - term care patients
10.8 % had elevated tsh , 8 % in those with a prior diagnosis of hypothyroidism , while low tsh levels were found in 0.07 % patients .
according to our data the prevalence of clinical and subclinical hypothyroidism in the study sample may be as high as 4.420.7 % ( in patients with bipolar depression , for normal range 0.45.0 and 0.42.5 iu / ml , respectively ) . as reported by cole et al .
patients with bipolar depression and with optimal thyroid profile experienced remission 4 months faster than the remainder of the study group .
this study provides further evidence that patients with bipolar disorder are particularly sensitive to variations in thyroid function within the normal range and nearly three - quarters of patients with bipolar disorder have a thyroid profile that may be suboptimal for antidepressant response .
regarding hyperthyroidism , the prevalence of clinical and subclinical hyperthyroidism in the study sample may be as high as 7.211.0 % ( in patients with unipolar depression , for normal range 0.33.0 and 0.42.5 or 0.45.0 iu / ml , respectively ) . the rate of low tsh in patients with bipolar disorder ( tsh < 0.4 iu / ml , 8.3 % ) in this study was higher than previously reported ( 6.2 % ) . as noted by these authors ,
since we have no data on free t4 level , our interpretation of tsh level might be inaccurate and can be used only as rough estimations .
moreover , it should be noted that a state of brain hypothyroidism was reported in patients with depression .
this state of brain hypothyroidism occurs in systemic euthyroidism and may result from a defect of thyroid hormone receptor or impaired thyroid hormone transporter . in the large nhanes study
mean serum tsh was 1.50 iu / ml and was higher in females than males .
comparing to this result , in our study sample mean serum tsh levels were higher in patients with schizophrenia ( 1.71 iu / ml ) , unipolar depression ( 1.63 iu / ml ) , bipolar disorder ( 1.86 iu / ml ) , bipolar depression ( 2.00 iu / ml ) and lower in patients with bipolar mania ( 1.38 iu / ml ) .
as above , this may also be explained by the fact that our study sample was not population - based and it reflects associations between mental disorders and thyroid dysfunctions . in conclusion ,
our results confirm that there may be a higher prevalence of thyroid dysfunctions in patients with mood disorders ( both unipolar and bipolar ) and that these two diagnostic groups differ in terms of direction and frequency of thyroid dysfunctions .
this also confirms the need to monitor tsh level regularly in patients with all types of mood disorders .
when low normal tsh ranges are used ( 0.3 - 3.0 or 0.4 - 2.5 iu / ml ) , we could detect subclinical thyroid dysfunctions in higher number of highly susceptible patients ( lowering the normal tsh range from 0.4 - 5.0 to 0.4 - 2.5 caused a five to eight - fold increase in the number of patients who had tsh levels outside the normal range ) .
this is particularly important in the light of recommendations of the american association of clinical endocrinologists , which state that the diagnosis of subclinical hypothyroidism must be considered in every patient with depression .
our study has some limitations , which results mainly from its retrospective and naturalistic design .
we have no clinical data , data for subtypes of bipolar disorder type i and ii and rapid cycling .
diagnostic groups were based on diagnosis at discharge from the hospital , so it might be inaccurate in some cases , where strict diagnostic procedure using clinical criteria was not done .
we only have data representing current medical condition and have no information regarding past thyroid disorders and their treatment .
we also have no date on detailed thyroid assessment ( t3 , t4 , anti - thyroid autoantibodies , results of ultrasonograph examinations ) .
diagnostic groups of our study were not homogenous , there were less women in the schizophrenia group comparing to other groups , while patients with unipolar and bipolar depression were significantly older comparing to other groups . also , the bipolar disorder group was smaller comparing to schizophrenia and unipolar disorder ( this is particularly true for bipolar mania group ) .
these factors may affect results of inter- and intra - group comparisons . in case of patients with bipolar disorder
lithium may cause hypothyroidism , it was also reported that anti - depressants as well as haloperidol may affect thyroid functions and we have no detailed data on treatment that could be included in the analysis . on the other hand , the large sample size and ability to compare three major clinical groups ( schizophrenia , unipolar depression and bipolar disorder ) are strengths of the study . | the aim of this study is to investigate differences in thyroid - stimulating hormone ( tsh ) level in patients with acute schizophrenia , unipolar depression , bipolar depression and bipolar mania .
serum level of tsh was measured in 1,685 caucasian patients ( 1,064 women , 63.1 % ; mean age 46.4 ) .
mean serum tsh concentration was : schizophrenia ( n = 769 ) 1.71 iu / ml , unipolar depression ( n = 651 ) 1.63 iu / ml , bipolar disorder ( n = 264 ) 1.86 iu / ml , bipolar depression ( n = 203 ) 2.00 iu / ml , bipolar mania ( n = 61 ) 1.38 iu / ml ( h = 11.58 , p = 0.009 ) . depending on the normal range used , the overall rate of being above or below the normal range was 7.922.3 % for schizophrenia , 13.926.0 % for unipolar depression , 10.827.6 % for bipolar disorder , 12.228.5 % for bipolar depression , and 11.424.5 % for bipolar mania .
we have also found differences in tsh levels between the age groups ( 20 , > 20 years and 40 , > 40 years and 60 years and > 60 years ) .
tsh level was negatively correlated with age ( r = 0.23 , p < 0.001 ) . weak correlations with age have been found in the schizophrenia ( r = 0.21 , p < 0.001 ) , unipolar depression ( r = 0.23 , p < 0.001 ) , bipolar depression ( r = 0.25 , p = 0.002 ) and bipolar disorder ( r = 0.21 , p = 0.005 ) groups .
our results confirm that there may be a higher prevalence of thyroid dysfunctions in patients with mood disorders ( both unipolar and bipolar ) and that these two diagnostic groups differ in terms of direction and frequency of thyroid dysfunctions . |
trauma history refers to an individual 's total range of experiences of possibly distressing events during a defined period of time , either a lifetime or a shorter period . in accordance with the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) ptsd a1a2 criteria ( american psychiatric association , 2000 )
, events are classified into two categories : potentially traumatic events ( ptes ) and traumatic events ( tes ) ( corcoran , green , goodman & krinsley , 2000 ; norris & hamblen , 2004 ; weathers & keane , 2008 ) .
the focus in the pte category is on whether a person has been exposed to an event of a possibly distressing nature , corresponding to criterion a1 ( such as natural disasters , motor vehicle accidents , war , etc . ) .
the te category ( criterion a2 ) indicates whether an individual felt severe distress ( fear , helplessness , or horror ) because of the exposure .
ptsd is a severe anxiety disorder caused by experiencing or witnessing a subjectively distressing event ( american psychiatric association , 2000 ) .
the disorder is diagnosed on the basis of symptoms such as reexperiencing the trauma , hyperarousal , and avoidance .
as pointed out by vogt , king d. w. , and king l. a. ( 2007 ) , weathers and keane ( 2007 ) , and other researchers , only a minority of those exposed to a distressing event will develop full - blown ptsd .
comorbidity studies of trauma - related distress symptoms have shown that depression is the most frequently coexisting disorder with ptsd ( e.g. , breslau , 2009 ; breslau , davis , peterson & schultz , 2000 ; keane , brief , pratt & miller , 2007 ; kessler , 1997 ; ragheb & zimmerman , 2008 ) .
however , the comorbidity issue in relation to psychological trauma is complex as the inclusion criteria and the chronology of disorders need to be taken into account . for instance , while , by definition , ptsd is caused by a distressing event , depression does not have a similar external criterion ( american psychiatric association , 2000 ) .
in addition , symptoms of depression may have been present before the distressing event occurred , and this may have enhanced the risk for developing ptsd . in trauma studies ,
the chronology of included disorders has mainly been studied as relationships between main effects measured at several time points after the pte had occurred ( breslau , 2009 ; breslau et al . , 2000 ; erickson , wolfe , king d. w. , king l. a. & sharkansky , 2001 ; ginzburg , ein - dor & solomon , 2010 ) .
following the aforementioned logic of possible patterns of comorbidity and chronology , studies investigating the mediating role of trauma - related disorders have been designed in various ways depending on the study theme .
for instance , the level of ptsd symptoms has been analyzed as a predictor in relation to several combinations of mediators and outcome variables , sometimes including depression as a subsequent disorder ( subica , claypoole & wylie , 2011 ) .
depression has been studied in parallel with ptsd as a central post - trauma intervening factor , mediating a relationship between a trauma history factor and another identified mental illness or trauma - related behavior ( see for example dedert et al . , 2010 , or nelson et al . , 2011 )
ptsd has also been studied as an outcome factor in relation to previous trauma history and a chosen intervening variable .
to the authors knowledge , however , depression as an intervening variable between trauma history and ptsd has received no scholarly attention .
scholars providing overviews of studies on trauma - related symptoms in news journalists point out that only a limited number of studies have been published on the topic . among these studies , sample sizes and the inclusion criteria have varied , and in some cases , they have been insufficiently reported , which in turn affects the comparability of results ( newman , shapiro & nelson , 2009 ; simpson & cot , 2006 ; smith & newman , 2009 ) .
traumatic reactions caused by one identified assignment , as well as current trauma symptoms in relation to trauma history , have been investigated . in regard to the latter , trauma history has been defined either as work - related trauma exposure , as private life experiences , or , in some cases , combining both trauma contexts ( backholm & bjrkqvist , 2010 ; pyevich , newman & daleiden , 2003 ; simpson & boggs , 1999 ; weidmann & papsdorf , 2010 ) .
current studies indicate that 86100% of daily newsgatherers have been exposed to events at work that should be regarded as possibly traumatic ( smith , 2008 ; smith & newman , 2009 ) .
studies on the relationship between work trauma history and psychological distress have shown varying results .
some ( newman et al . , 2003 ; pyevich et al . , 2003 ;
simpson & boggs , 1999 ) have reported that the number of experienced work - related crises were connected to ptsd symptoms , whereas others ( backholm & bjrkqvist , 2010 ; dworznik , 2008 ; smith , 2008 ) have not found connections .
the latter studies have reported that the characteristics of each specific work - related crisis is of importance rather than the number of such crises experienced .
some studies have shown that having worked with an event that included a large range of distressing characteristics was related to severe current distress symptoms .
distressing characteristics can consist of witnessing someone being killed , knowing the victims , and/or oneself being directly attacked while working on the assignment ( pyevich , 2001 ) .
studies of journalists , including private life trauma history as well , have usually found a relationship between exposure to a larger range of personal traumas and elevated levels of symptoms ( backholm & bjrkqvist , 2010 ; newman et al . , 2003 ; pyevich et al . , 2003 ) .
the vast majority of studies of news workers trauma - related experiences include measures of ptsd ( smith & newman , 2009 ) .
depending on the type of journalism , sample size , and situational factors in different studies , the proportion of cases with ptsd have varied . in their overview of studies , smith and newman ( 2009 ) estimated that possible ptsd in journalists varied from 4 to 29% .
for instance , dworznik ( 2008 ) reported that in a group of american domestic news journalists , 7% of respondents fulfilled the diagnosis criteria , whereas hatanaka et al .
( 2010 ) noted that 6% of japanese news reporters with similar work descriptions had full - blown ptsd at the time of the survey .
war correspondents have been shown to be at a higher risk ; feinstein , owen , and blair ( 2002 ) reported that 29% suffered from ptsd at some point in their career .
trauma - induced depression also has been studied in only a few journalist samples ( backholm & bjrkqvist , 2010 ; feinstein et al . , 2002 ; mcmahon , 2001 ; weidmann , fehm & fydrich , 2008 ) . among these , the study design has varied .
some scholars have investigated the link between trauma - related assignment / trauma history and depression as well as the ptsd - depression comorbidity , while others have focused on one topic only .
backholm and bjrkqvist ( 2010 ) , in a general sample of finnish domestic news journalists , and weidmann et al .
( 2008 ) , studying european reporters working with the 2004 asian tsunami disaster , found few or no direct relationships between the range of work - related traumatic exposure and changes in depressive symptoms .
mcmahon ( 2001 ) reported that among australian journalists with a work history that included at least one crisis assignment , 43% experienced significant , long - term depression , which they attributed to potentially traumatic exposure . in regard to comorbidity between ptsd and depression
, results have usually shown that more severe ptsd symptoms are correlated with more symptoms of depression ( backholm & bjrkqvist , 2010 ; feinstein et al . , 2002 ; weidmann et al . ,
however , to the best of the authors knowledge , studies with journalist samples have not investigated the underlying relationships between ptsd and depression in detail .
in this study , the complex relationship between trauma history and trauma - related symptoms in news journalists is investigated .
the aim of the study is to investigate whether possible direct relationships between work - related or personal trauma history and posttraumatic symptoms may be mediated by symptoms of depression .
in total , 407 finnish news journalists who had experienced one or several crisis - related work assignments during their career ( including work on the location of the crisis , in the newsroom , or regional work in another , non - affected , part of the country ) participated in the study .
the sample was extracted from a larger sample of journalists . the choice to exclude journalists with no experience of crisis assignments was made to ensure a strictly defined sample of participants with a relevant work description .
journalists in the sample had a mean age of 43 years ( sd = 11.1 ) .
participant data was collected via a web - based survey on the topic of trauma history and current psychological distress within the profession and sent out to finnish news journalists in the spring of 2008 ( backholm & bjrkqvist , 2010 ) . the total sample of the present study consisted of 178 males ( 44% ) and 229 females ( 56% ) . the main media affiliation of the journalists was as follows : newspapers , n=232 ( 57% of the sample ) , television , n=74 ( 18% ) , radio , n=73 ( 18% ) , and other types of media , n=28 ( 7% ) .
work - related exposure to potentially traumatic assignments ( ptas ) was measured with the journalist trauma exposure scale ( jtes ) ( newman et al . , 2003 ; pyevich , 2001 ) . in the jtes ,
14 items measure the range of ptas during the past 12 months , while a further nine items focus on the intensity of possible work exposure . in the collection of data , the authors wanted to distinguish between exposure to ptas at the scene of the crisis and work tasks carried out in the newsroom or somewhere else .
accordingly , participants were asked to indicate whether they had experienced any of the above mentioned types of exposure on the location , indirectly , or both . for this study , only at - the - scene data for the 14-item pta subscale was used in the analyses , and a summed score of provided yes / no - answers was derived . since scales measuring trauma history ( e.g. , jtes ) reflect exposure to various types of events rather than a unified underlying construct , reliability assessment for the trauma history scales is not meaningful and therefore is not presented . to measure trauma history in personal life , the traumatic life events questionnaire ( tleq ) ( kubany et al . , 2000 )
the tleq consists of 23 items measuring the range of lifetime trauma history , corresponding to the dsm - iv ptsd a1 ( ptes ) and a2 ( tes ) criteria ( american psychiatric association , 2000 ) . in this study ,
a total of 18 trauma history items were used , 17 from the original tleq and one item added reflecting exposure to bullying .
the item was added because of previous research showing that exposure to bullying is related to symptoms of ptsd ( bjrkqvist , 2001 ) .
the wording of items was modified somewhat to reduce length , and only data from the pte subscale was used in this study .
answers for each item were provided as yes / no alternatives , and as in the case of the jtes , a summed score was derived . for measuring symptoms of ptsd , the ptsd checklist , civilian version ( pcl - c ) ( national center for ptsd , 2010 ; weathers , litz , herman , huska & keane , 1993 ) was used .
the pcl - c consists of 17 items with response choices arranged on a five - point likert scale , ranging from not at all to extremely .
the wording of the pcl - c in the study was modified to refer to past traumatic exposure in general . moreover ,
a total summed score of items was used in the statistical analyses . in the study ,
the pcl - c had excellent internal consistency , with a reliability of =.91 . for symptoms of depression ,
the short version of the beck depression inventory ( bdi-13 ) ( beck a. t. & beck r. w. , 1972 ) was used . in the bdi-13 , participants respond by choosing one out of four given alternatives per item .
a summed score for the 13 items was used in the analysis . in this study ,
in total , 407 finnish news journalists who had experienced one or several crisis - related work assignments during their career ( including work on the location of the crisis , in the newsroom , or regional work in another , non - affected , part of the country ) participated in the study .
the sample was extracted from a larger sample of journalists . the choice to exclude journalists with no experience of crisis assignments was made to ensure a strictly defined sample of participants with a relevant work description .
journalists in the sample had a mean age of 43 years ( sd = 11.1 ) .
participant data was collected via a web - based survey on the topic of trauma history and current psychological distress within the profession and sent out to finnish news journalists in the spring of 2008 ( backholm & bjrkqvist , 2010 ) . the total sample of the present study consisted of 178 males ( 44% ) and 229 females ( 56% ) . the main media affiliation of the journalists was as follows : newspapers , n=232 ( 57% of the sample ) , television , n=74 ( 18% ) , radio , n=73 ( 18% ) , and other types of media , n=28 ( 7% ) .
work - related exposure to potentially traumatic assignments ( ptas ) was measured with the journalist trauma exposure scale ( jtes ) ( newman et al . , 2003 ; pyevich , 2001 ) . in the jtes ,
14 items measure the range of ptas during the past 12 months , while a further nine items focus on the intensity of possible work exposure . in the collection of data , the authors wanted to distinguish between exposure to ptas at the scene of the crisis and work tasks carried out in the newsroom or somewhere else .
accordingly , participants were asked to indicate whether they had experienced any of the above mentioned types of exposure on the location , indirectly , or both . for this study , only at - the - scene data for the 14-item pta subscale was used in the analyses , and a summed score of provided yes / no - answers was derived . since scales measuring trauma history ( e.g. , jtes ) reflect exposure to various types of events rather than a unified underlying construct , reliability assessment for the trauma history scales is not meaningful and therefore is not presented . to measure trauma history in personal life , the traumatic life events questionnaire ( tleq ) ( kubany et al . , 2000 )
the tleq consists of 23 items measuring the range of lifetime trauma history , corresponding to the dsm - iv ptsd a1 ( ptes ) and a2 ( tes ) criteria ( american psychiatric association , 2000 ) . in this study ,
a total of 18 trauma history items were used , 17 from the original tleq and one item added reflecting exposure to bullying .
the item was added because of previous research showing that exposure to bullying is related to symptoms of ptsd ( bjrkqvist , 2001 ) .
the wording of items was modified somewhat to reduce length , and only data from the pte subscale was used in this study .
answers for each item were provided as yes / no alternatives , and as in the case of the jtes , a summed score was derived . for measuring symptoms of ptsd , the ptsd checklist , civilian version ( pcl - c ) ( national center for ptsd , 2010 ; weathers , litz , herman , huska & keane , 1993 ) was used .
the pcl - c consists of 17 items with response choices arranged on a five - point likert scale , ranging from not at all to extremely .
the wording of the pcl - c in the study was modified to refer to past traumatic exposure in general . moreover ,
a total summed score of items was used in the statistical analyses . in the study ,
the pcl - c had excellent internal consistency , with a reliability of =.91 . for symptoms of depression ,
the short version of the beck depression inventory ( bdi-13 ) ( beck a. t. & beck r. w. , 1972 ) was used . in the bdi-13 , participants respond by choosing one out of four given alternatives per item .
a summed score for the 13 items was used in the analysis . in this study ,
differences between males and females were found on most factors ( table 1 ) . males reported a history of significantly more pta as well as pte exposure , whereas females had higher ptsd scores .
summary of sex differences in ptas , ptes , ptsd , and depression journalists could indicate a maximum range of 14 pta types exposed to at the location of the event . among participants ( n=357 ) , 196 ( 55% ) had worked with at least one such event during the past year . as a group ,
the journalists reported having worked with a mean of 1.71 different types of events ( sd = 2.41 , range 012 ) during the past year .
most frequent types of ptas in the past year participants ( n=407 ) indicated the types of ptes they have experienced from a list of 18 types of ptes .
results showed that 395 ( 97% ) journalists had experienced at least one of the listed ptes in their life .
a mean of 3.92 types of exposure ( sd = 2.41 , range = 013 ) was found for the group , with a sudden death of a family member or friend as the most frequent type of exposure , experienced by 284 journalists ( 70% ) .
the occurrence of an indirect , or mediating , effect is supported if a significant relationship between a predictor variable and an outcome variable diminishes after introducing a third variable , the mediator ( baron & kenny , 1986 ) .
a mediation effect can be either complete , when the significant relationship between predictor and outcome no longer prevails after adding the mediator , or partial , when the relationship is reduced in size , but still exists ( kenny , 2012 ) . a theoretical model of the mediation analysis
is provided by kenny ( 2012 ) and presented in a slightly modified format in fig .
a is the predictive value between predictor and mediator , and path b is the regression coefficient between mediator and outcome , while controlling for the effect of the original predictor .
path c represents the regression coefficient between the predictor and the outcome variable before introducing the mediator .
c stands for the new predictive value between the original predictor and outcome , with the mediator included , and thus should be smaller than c if an indirect effect is present .
this can be represented by the following equation : c = c'+aba series of regression analyses ( table 3 ) were conducted to investigate whether exposure to ptas had a direct predictive effect on ptsd symptoms in the sample , and if so , whether symptoms of depression had an indirect effect on the relationship . in all analyses , sex was controlled for .
first , a regression analysis was conducted to test for a predictive effect of pta exposure on higher level of ptsd symptoms .
this model is a modified version of the model by kenny ( 2012 ) ( reproduced with permission from david a. kenny ) .
summary of an analysis of the mediating effect of depressive symptoms on the relationship between exposure to ptas and ptsd symptoms .
c represents the predictive value of ptas on ptsd before including depression into the equation ; c the value after including depression .
summary of an analysis of the mediating effect of depression between exposure to ptas and ptsd , controlling for sex ( n=352 ) regression analysis of ptas as predictor of symptoms of ptsd , controlling for sex .
regression analysis of ptas and symptoms of depression as predictors of symptoms of ptsd , controlling for sex .
since a significant effect between ptas and ptsd symptoms was found , the relationship between the predictor and the mediator , or path a , was investigated .
the regression analysis showed that pta exposure was a significant predictor for the proposed mediator , depression ( table 3 and fig .
2 , path a ) . in the final step of the mediation analysis , paths b and c were investigated by conducting a regression analysis that focused on the predictive value of the mediator ( depression ) on the outcome variable ( ptsd ) , including the original predictor ( ptas ) in the analysis .
when introducing depression as a mediator , the predictive effect of ptas on ptsd symptoms was no longer significant ( see paths b and c in fig . 2 , and table 3 )
furthermore , a sobel test ( mackinnon , lockwood , hoffman , west & sheets , 2002 ; sobel , 1982 ) showed that the complete indirect effect of pta range via depression on ptsd was significant ( z=2.53 , p<.05 ) .
a similar mediation model was used for investigating whether a possible predictive effect of ptes in personal life on ptsd symptoms would be indirectly affected by the level of depressive symptoms . in table 4 and
the predictive value of pte exposure on ptsd symptoms diminished after introducing the mediator , but they still remained significant .
a sobel test showed that the partial indirect effect of ptes experienced in personal life via symptoms of depression on symptoms of ptsd was significant ( z=3.97 , p<.001 ) .
summary of an analysis of the mediating effect of depressive symptoms on the relationship between exposure to ptes in personal life and ptsd symptoms .
c represents the predictive value of ptes on ptsd before including depression into the equation ; c the value after including depression .
summary of an analysis of the mediating effect of depression between exposure to ptes in personal life and ptsd , controlling for sex ( n=352 ) regression analysis of ptes as predictor of symptoms of ptsd , controlling for sex .
regression analysis of ptes and symptoms of depression as predictors of symptoms of ptsd , controlling for sex .
differences between males and females were found on most factors ( table 1 ) . males reported a history of significantly more pta as well as pte exposure , whereas females had higher ptsd scores .
journalists could indicate a maximum range of 14 pta types exposed to at the location of the event
. among participants ( n=357 ) , 196 ( 55% ) had worked with at least one such event during the past year . as a group ,
the journalists reported having worked with a mean of 1.71 different types of events ( sd = 2.41 , range 012 ) during the past year .
most frequent types of ptas in the past year participants ( n=407 ) indicated the types of ptes they have experienced from a list of 18 types of ptes .
results showed that 395 ( 97% ) journalists had experienced at least one of the listed ptes in their life .
a mean of 3.92 types of exposure ( sd = 2.41 , range = 013 ) was found for the group , with a sudden death of a family member or friend as the most frequent type of exposure , experienced by 284 journalists ( 70% ) .
the occurrence of an indirect , or mediating , effect is supported if a significant relationship between a predictor variable and an outcome variable diminishes after introducing a third variable , the mediator ( baron & kenny , 1986 ) .
a mediation effect can be either complete , when the significant relationship between predictor and outcome no longer prevails after adding the mediator , or partial , when the relationship is reduced in size , but still exists ( kenny , 2012 ) . a theoretical model of the mediation analysis
is provided by kenny ( 2012 ) and presented in a slightly modified format in fig .
a is the predictive value between predictor and mediator , and path b is the regression coefficient between mediator and outcome , while controlling for the effect of the original predictor .
path c represents the regression coefficient between the predictor and the outcome variable before introducing the mediator .
c stands for the new predictive value between the original predictor and outcome , with the mediator included , and thus should be smaller than c if an indirect effect is present .
this can be represented by the following equation : c = c'+aba series of regression analyses ( table 3 ) were conducted to investigate whether exposure to ptas had a direct predictive effect on ptsd symptoms in the sample , and if so , whether symptoms of depression had an indirect effect on the relationship . in all analyses , sex was controlled for .
first , a regression analysis was conducted to test for a predictive effect of pta exposure on higher level of ptsd symptoms .
this model is a modified version of the model by kenny ( 2012 ) ( reproduced with permission from david a. kenny ) .
summary of an analysis of the mediating effect of depressive symptoms on the relationship between exposure to ptas and ptsd symptoms .
c represents the predictive value of ptas on ptsd before including depression into the equation ; c the value after including depression .
summary of an analysis of the mediating effect of depression between exposure to ptas and ptsd , controlling for sex ( n=352 ) regression analysis of ptas as predictor of symptoms of ptsd , controlling for sex .
regression analysis of ptas and symptoms of depression as predictors of symptoms of ptsd , controlling for sex .
since a significant effect between ptas and ptsd symptoms was found , the relationship between the predictor and the mediator , or path a , was investigated .
the regression analysis showed that pta exposure was a significant predictor for the proposed mediator , depression ( table 3 and fig .
2 , path a ) . in the final step of the mediation analysis , paths b and c were investigated by conducting a regression analysis that focused on the predictive value of the mediator ( depression ) on the outcome variable ( ptsd ) , including the original predictor ( ptas ) in the analysis . when introducing depression as a mediator , the predictive effect of ptas on ptsd symptoms was no longer significant ( see paths b and c in fig . 2 , and table 3 )
furthermore , a sobel test ( mackinnon , lockwood , hoffman , west & sheets , 2002 ; sobel , 1982 ) showed that the complete indirect effect of pta range via depression on ptsd was significant ( z=2.53 , p<.05 ) .
a similar mediation model was used for investigating whether a possible predictive effect of ptes in personal life on ptsd symptoms would be indirectly affected by the level of depressive symptoms . in table 4 and
the predictive value of pte exposure on ptsd symptoms diminished after introducing the mediator , but they still remained significant .
a sobel test showed that the partial indirect effect of ptes experienced in personal life via symptoms of depression on symptoms of ptsd was significant ( z=3.97 , p<.001 ) .
summary of an analysis of the mediating effect of depressive symptoms on the relationship between exposure to ptes in personal life and ptsd symptoms .
c represents the predictive value of ptes on ptsd before including depression into the equation ; c the value after including depression .
summary of an analysis of the mediating effect of depression between exposure to ptes in personal life and ptsd , controlling for sex ( n=352 ) regression analysis of ptes as predictor of symptoms of ptsd , controlling for sex .
regression analysis of ptes and symptoms of depression as predictors of symptoms of ptsd , controlling for sex .
the aim of this study was to investigate whether the amount of trauma history at work and in personal life was related to levels of ptsd symptoms in journalists , and if so , whether this relationship was mediated by the severity of depressive symptoms .
the work - related pta results showed that among the participants , 55% had been deployed to the location of a crisis in the past 12 months .
this number is not directly comparable with smith and newman 's ( 2009 ) overview in which 86100% of news journalists had had assignments including potentially traumatic exposure at some point .
it is worth noting that work - related trauma history questionnaires have varied between studies . in this study ,
only exposure to ptas among journalists directly witnessing a crisis was investigated , not exposure among journalists working with ptas , for example , in the newsroom .
also , the sample was more strictly defined than that in some previous studies , including only journalists who had worked with at least one crisis assignment during their career .
finally , the data was collected via a web - based survey ; therefore , the included sample may not be representative for finnish news journalists in general . still , the results from this study support the overall picture that exposure to ptas is common in journalistic work .
these findings are roughly similar to those from other studies ; see , for example , dworznik ( 2008 ) , pyevich et al .
the nature of the most frequent pta reflect an important aspect sometimes neglected within journalism and trauma research or teaching : crisis - related work relatively infrequently involves being sent out to large - scale disasters or war zones , but rather usually consists of small local incidents with a limited number of victims affected ( norwood , walsh & owen , 2003 ; simpson & cot , 2006 ) . in the sample ,
the numbers are in line with the conclusion drawn by norris and slone ( 2007 ) : by the age of 45 , most people will have been exposed to at least one pte .
an indirect , or mediating , effect is defined by kenny ( 2012 ) as the possible effect of a third variable , intervening in an existing relationship between a predictor and an outcome . in this study ,
the range of depressive symptoms had a complete indirect effect on the relationship between work - related ptas during the past 12 months and current ptsd . also , depression had a partial indirect effect on the significant relationship between the range of lifetime personal ptes and ptsd symptoms .
some conclusions can be made regarding the relationship between the two types ( work - related and private life ) of measured trauma history and ptsd . after including depression as an intervening variable ,
only private life trauma history had a significant predictive value on ptsd . on a broad level , these results support the epidemiological literature on risk factors for ptsd , where trauma history has been indicated as a central factor for heightened risk ( brewin , andrews & valentine , 2000 ; ozer , best , lipsey & weiss , 2003 ; vogt et al . , 2007 ) . in these studies , overall trauma history has been highlighted as a risk factor , and no differentiation between personal life and work - related exposure has been made .
the connection found between personal ptes and current ptsd symptoms reinforces previously reported results for other journalist samples ( newman et al .
some have not found any direct link between work exposure and trauma - induced distress ( dworznik , 2008 ; smith , 2008 ) , whereas the results of others suggest that such an impact exists ( newman et al . , 2003 ; pyevich et al . , 2003 ; simpson & boggs , 1999 ) .
in relation to these results , the reader is reminded of the nature of the measurement of trauma history in this study .
only potentially traumatic exposure was studied , and no data on the range of events provoking outspoken subjective severe distress was analyzed .
furthermore , the exposure time period varied between instruments ( ptas during the past 12 months , personal ptes during a lifetime ) . as posited by dworznik ( 2008 ) ,
there is a need to refine the instruments used for measuring exposure to ptas in newsgatherers .
for example , an added subscale probing for assignments experienced as traumatic ( dsm - iv ptsd criterion a2 ; american psychiatric association , 2000 ) would be useful in future research . as pointed out elsewhere by the authors ( backholm & bjrkqvist , 2010 ) , more research on the possible impact of ptas on journalists is clearly needed . in this study ,
depression had a strong indirect effect on the relationship between predictors and outcome , in the case of ptas to the point that the direct effect on ptsd disappeared .
these findings indicate that previous symptoms of depression have a strong influence on the development of ptsd in journalists exposed to a potentially traumatic event during work or in personal life , supporting the notion of prior psychiatric history being a risk factor for ptsd ( brewin et al .
, 2000 ; ozer et al . , 2003 ) . furthermore , this study design underlines the importance of taking the analysis a step further than merely treating both ptsd and depression as outcomes of traumatic exposure .
although , as discussed in the introduction , a few studies in the broader area of trauma research have looked at the complex relationships among trauma history , ptsd , and depression in a more general sense , this study is of relevance since , to the authors knowledge , no studies with journalist samples have investigated depression as an intervening variable .
the results in regard to the intervening effect of depression should , however , be interpreted with caution .
the main limitation is related to the cross - sectional study design . although trauma history by definition consists of experiences that have occurred in the past , the assumptions behind included factors in the mediation models are theoretical only .
more definitive results could be obtained by using a longitudinal design ( breslau , 2009 ; vogt et al . , 2007 ) .
there is no conflict of interest in the present study for any of the authors . | backgroundnews journalists are an occupational group with a unique task at the scene of an unfolding crisis to collect information and inform the public about the event . by being on location , journalists put themselves at risk for being exposed to the potentially traumatic event.objectiveto compare potentially traumatic exposure during work assignments at a crisis scene and in personal life as predictors of the development of post - traumatic stress disorder ( ptsd ) in news journalists .
further , to investigate the mediating effect of depression between the predictor and predicted variables.methodwith a web - based questionnaire , information from a sample of finnish news journalists ( n=407 ) was collected .
the data collected included details on the range of potentially traumatic assignments ( ptas ) at the crisis scene during the past 12 months , lifetime potentially traumatic events ( ptes ) in personal life , ptsd symptoms , and level of depression.resultsapproximately 50% of the participants had worked with a pta during the past 12 months .
depression had a significant indirect effect on the relationship between ptas at the scene and symptoms of ptsd .
a similar result was found regarding the relationship between personal life ptes and ptsd .
depression had a complete indirect effect in the case of ptas and a partial indirect effect in regard to pte exposure in personal life.conclusionsexposure to ptas is common within journalistic work .
the results reflect the importance of understanding the underlying mechanisms of the measured symptoms ( ptsd , depression ) in relation to trauma history . the main limitations of the study include the cross - sectional design and the nature of the instruments used for the collection of work - related trauma history . |
uterine artery anatomic variants have been the subject of detailed study , since the beginning of last century , given fibromyoma role in the pathogenesis of arterial vasculature .
according to various studies , there are certain classifications of uterine artery anatomical variants and paper proposes such a classification , the observations selected aspects of examinations performed angiographic fibromyoma embolization .
110 examinations were conducted in women aged between 20 and 47 years , with symptoms and clinical diagnosis of uterine fibromyoma .
embolization procedure was performed by unilateral brachial vascular access , using seldinger technique with a sheath pressure mounting and placing a 4f or 5f cobra catheter tip , running through the axillary artery , subclavian artery , ( brachiocephalic arterial trunk ) descending aorta , iliac artery common internal iliac artery , uterine artery tracing is the purpose of selective catheterization of it .
retrospective evaluation of imaging uterine artery arteriography allowed classification into four groups , depending on the origin of the uterine artery .
220 were observed in the uterine artery catheterization and were classified number 200 , which represents 90.1% of the total , the remaining 20 were not conclusive arteriogram
.
analysis arteriogram interpretation allowed a classification into 4 types , given the classifications listed in the literature as follows : - type i - the origin of the uterine artery inferior gluteal artery ram ( fig .
1 ) - type ii - the origin of the uterine artery bifurcation ram inferior gluteal artery ( fig .
2 ) - type iii - the origin of the uterine artery to the internal iliac artery trifurcation ram , with lower and upper gluteal artery ( fig .
3 ) - type iv - uterine artery proximal to the origin of the origin of the gluteal arteries top and bottom ( fig .
4 ) the origin of the uterine artery inferior gluteal artery ram the origin of the uterine artery bifurcation ram inferior gluteal artery the origin of the uterine artery to the internal iliac artery trifurcation ram , with lower and upper gluteal artery uterine artery proximal to the origin of the origin of the gluteal arteries top and bottom
anatomy descriptive notes uterine artery origin from the internal iliac artery anterior trunk , with arteries bladder , rectal average obturatory artery , internal pudendal artery , sciatica , however note the variant of origin of uterine artery from a common trunk artery umbilical [ 5 - 8 ] .
in fact , the origin of the uterine artery is variable , according to recent studies , which are based on angiographic examinations .
currently there is a classification of the uterine artery origin : type i ( the origin of the uterine artery inferior gluteal artery ram ) in 45% , type ii ( the origin of the uterine artery bifurcation ram inferior gluteal artery ) 6% , type iii ( origin uterine artery to the internal iliac artery of the ram trifurcation , with the upper and lower gluteal artery ) 43% and 6% type iv ( proximal to the origin of the uterine artery to the origin of the upper and lower gluteal arteries ) .
recent studies , which are based on angiographic examination , note the presence of two trunks of bifurcation of the internal iliac artery , anterior and posterior in 70% of cases , and uterine artery is inferior gluteal artery origin or branch of the internal iliac artery trifurcation .
the present study shows different values of such a percentage , such type i is 24% of the total arteriograms classifiable and type ii is in a proportion of 10% . for type iii percentage
different values are observed to specialized studies , with a much increased type iv also shows that the type iii and type i are halfway to the classification established values .
this again demonstrates the variability of uterine artery origin in different percentages , but major practical importance .
in addition to the above aspects of the present and the emergence of uterine artery dissection of the cadavers in department of anatomy , university of medicine craiova . this notice these issues . in fig.5 the origin of the uterine artery
uterine artery dissection origin proximal to the superior and inferior gluteal arteries emerging ( arrow ) in fig.6 uterine artery arises from the internal iliac artery , making common core with superior gluteal artery and inferior gluteal .
knowledge of emerging variants uterine artery has an important role in the success of fibromyoma embolization , thus avoiding damage to other vascular structures , also correct assessment of uterine artery origin is reflected in the reduction of radiation dose and shortening the time of intervention . | uterine artery embolization as a therapeutic method in fibromyoma requires a good knowledge of the origin of the uterine artery to the success of this procedure involving selective catheterization .
this study presents a classification of anatomical variants of uterine artery as a retrospective review of consecutive arteriogram , complete with various aspects of the origin of the uterine artery in cadaver dissection , in the department of anatomy . |
the
major reactive aldehyde produced during peroxidation of polyunsaturated
fatty acids , malondialdehyde ( mda ) , reacts with nucleophilic sites
of dna and protein to form oxopropenyl adducts .
reaction of mda with
dna produces primarily 3-(2-deoxy--d - erythro - pentofuranosyl)pyrimido[1,2-]purine-10(3h)-one ( m1dg ) and lesser amounts of 2-oxopropenyl - deoxyadenosine
( figure 1 ) .
m1dg is mutagenic in vitro and in vivo , and 2-oxopropenyl - deoxyadenosine alters dna polymerase
activity .
both species
can
react with da or dg , resulting in oxypropenylation of the base .
the reaction of mda with protein
occurs predominantly at lysine
residues under physiological conditions .
protein adduct formation is rudimentary , as the exact
targets and sites of adduction are largely unknown .
however , a particularly
interesting effect of mda exposure in human cells is the inhibition
of nucleotide excision repair ( ner ) , which is associated with increased
sensitivity to the mutagenicity of ultraviolet light and benzo[a]pyrene dihydrodiolepoxide .
ner is the primary process for repair of bulky dna adducts , and
in humans , ner deficiencies result in xeroderma pigmentosum , a spectrum
of disorders characterized by hypersensitivity to sunlight , neurological
degeneration , and a greatly increased incidence of cancer .
although not verified experimentally , inhibition
of ner by mda was proposed to arise from loss of function of mda - modified
ner proteins .
a potential mda target
is the ner scaffold protein , xeroderma pigmentosum complementation
group a ( xpa ) , which binds to the damaged dna substrate via a c - terminal
basic cleft comprising multiple lysine residues .
xpa coordinates recruitment and assembly of the enzymes that repair
the damaged dna , and mutations in its dna - binding domain are associated
with the most severe clinical xp phenotypes .
oxopropenyl adducts to dna and protein can also be formed
through
reaction with base propenal , a reactive aldehyde produced by oxidant - mediated
hydrogen abstraction from the 4-position of dna bases ( figure 1 ) .
base propenals are approximately 100-fold more reactive to dna than
mda , and experiments in which escherichia coli strains of varying membrane composition were exposed to oxidants
demonstrated that base propenals are more important than mda as a
source of m1dg .
currently ,
very little is known about potential protein targets of base propenal
adduction ; however , as a highly reactive form of dna damage , base
propenals are ideally situated to oxopropenylate dna - binding proteins
such as xpa .
this has led us to hypothesize that base propenal - mediated
protein damage contributes to toxicity and mutagenicity associated
with oxidative or electrophile stress . to test this hypothesis , a
comprehensive evaluation of base propenal s ability to directly
damage proteins
adenine propenal is known to react with
glutathione via michael addition to form glutathionylpropenal and
a glutathione adenine propenal cross - link , but , to date , no detailed investigation of the reaction
of any base propenal with amino acids or proteins has been reported .
one approach to evaluate the protein adduction potential of an
electrophile is to identify all sites of modification and the structures
of those modifications using a model protein .
a frequently used target
for this purpose is human serum albumin ( hsa ) .
albumin is highly abundant and has affinity for a broad range of
ligands , a relatively long half - life , and the potential for noninvasive
sampling .
thus , it is an ideal candidate for the global qualitative
and quantitative evaluation of exposure to electrophiles of endogenous
or exogenous origin ( the exposome ) .
in fact , in a large multilaboratory
comparison of biomarkers of in vivo oxidant generation ,
the level of carbonylated plasma protein ( i.e. , albumin ) was one of
only two markers that correlated with oxidant exposure and tissue
pathology , and it was the only marker that was effective longer than
6 h after exposure . to exploit albumin s
potential as a biomarker , however , requires a more complete understanding
of its major sites of adduction and determinants of their reactivity
to a range of electrophiles .
for example , whereas much attention has
been directed to hsa s highly reactive cys-34 as a sensor of
oxidant damage , the major site of oxopropenylation
by mda is lys-525 . here , we report
the application of a recently described mass spectrometric
approach to identify adenine propenal
modifications of hsa and xpa .
our data demonstrate that , like mda ,
adenine propenal primarily reacts at protein lysine residues ; however ,
the major site of attack on hsa is distinct from that of mda .
we also
report that adenine propenal modifies lysine residues of xpa , including
residues within its dna - binding domain and residues reported to be
sites of regulation via sirt1-mediated deacetylation .
biophysical analysis confirms
that modification of xpa by adenine propenal decreases its affinity
for damaged dna .
n--acetyllysine , trichloroacetic acid ( tca ) , dithiothreitol ,
iodoacetate ( sigma ultra ) , and nacnbh3 were obtained from
sigma - aldrich ( st .
nmr
experiments were
acquired using a 14.0 t bruker magnet equipped with a bruker av - iii
console operating at 600.13 mhz . all spectra were acquired in 3 mm
nmr tubes using a bruker 5 mm tci cryogenically cooled nmr probe .
chemical shifts were referenced internally to d2o ( 4.70
ppm ) , which also served as the h lock solvent . for 1d h nmr , experiments were acquired with presaturation in order
to suppress the residual h2o signal , and typical experimental
conditions included 32k data points , 13 ppm sweep width , a recycle
delay of 1.5 s , and 32 scans . for 2d h
h cosy , presaturation was also performed , and experimental conditions
included a 2048 1024 data matrix , 13 ppm sweep width , recycle
delay of 1.5 s , and 8 scans per increment .
the data were processed
using squared sinebell window function , symmetrized , and displayed
in magnitude mode .
c hsqc experiments were acquired using a 1024 128 data matrix ,
a j(c h ) value of 145 hz , which resulted in
a multiplicity selection delay of 34 ms , a recycle delay of 1.5 s ,
and 80 scans per increment along with garp decoupling on c during the acquisition time ( 150 ms ) .
the data were processed using
a p/2 shifted squared sine window function and displayed with ch / ch3 signals phased positive and ch2 signals phased
negative .
c hmbc experiments were acquired using
a 2048 128 data matrix , a j(c h ) value
of 9 hz for detection of long - range couplings resulting in an evolution
delay of 55 ms , j1(c h ) filter
delay of 145 hz ( 34 ms ) for the suppression of one - bond couplings ,
a recycle delay of 1.5 s , and 144 scans per increment .
the hmbc data
were processed using a p/2 shifted squared sine window function and
displayed in magnitude mode .
compounds were greater than 90% pure ,
and mass spectral analysis was accomplished on an applied biosystems
3200 q trap mass spectrometer ( mds sciex ) .
the mass spectrometer was
equipped with an electrospray source and operated in positive ion
mode .
h nmr ( dmso - d6 ) : ( ppm ) 7.17 ( dd , j1 = 7.88 hz , j2 = 14.4 hz ,
1h ) , 7.55 ( br s , 2h ) , 8.27 ( s , 1h ) , 8.35 ( d , j =
14.4 hz , 1h ) , 8.63 ( s , 1h ) , 9.67 ( d , j = 7.88 hz ,
1h ) .
c nmr ( dmso - d6 ) :
( ppm ) 119.2 , 120.5 , 136.4 , 142.8 , 147.6 , 151.4 , 158.2 , 190.8 .
h nmr
( d2o ) : ( ppm ) 9.15 ( d , j = 10.0
hz , 2h ) , 5.73 ( t , j = 10.0 hz , 1h ) .
the sodium salt of
malondialdehyde ( mda ) ( 100 mg , 1.06 mmol ) was added to a solution
of n--acetyllysine ( 200 mg , 1.06 mmol ) in
5 ml of ammonium acetate buffer ( ph 4.5 ) .
the reaction was then stirred
at room temperature for 15 h. the reaction mixture was lyophilized ,
and the residue was purified by column chromatography ( etoac / meoh ,
5:1 ) on silica gel ( 60 porosity , 4063 m particle
size ) .
h nmr ( d2o ) : ( ppm ) 1.33 ( m ,
2h ) , 1.561.61 ( m , 3h ) , 1.71 ( m , 1h ) , 1.93 ( s , 3h ) , 2.91 ( t , j = 7.3 hz , 2h ) , 4.05 ( m , 1h ) , 5.31 ( m , 1h ) , 7.44 ( d , j = 13.0 hz , 1h ) , 8.59 ( d , j = 8.5 hz ,
1h ) .
c nmr ( d2o ) : ( ppm ) 22.0 , 23.4 ,
27.2 , 32.0 , 39.3 , 55.3 , 86.6 , 163.7 , 173.7 , 179.1 , 191.2 .
the sodium salt of mda ( 60 mg , 0.319 mmol ) was added to
a solution of n--acetyllysine ( 15 mg , 0.159
mmol ) in 2 ml of anhydrous methanol .
the mixture was heated at reflux ,
and the progress of the reaction was monitored by uv vis spectroscopy .
the product was then purified by column chromatography ( etoac / meoh ,
5:1 ) on silica gel ( 60 porosity , 4063 m particle
size ) and hplc .
hplc chromatography was carried out on a luna c18
column ( 250 4.60 mm , 5 m ) at a flow rate of 1.25 ml / min .
the eluting solvents included solvent a ( 10 mm ammonium acetate ) and
solvent b ( acetonitrile ) .
the following solvent gradient was used :
010.5 min linear gradient from 5% b to 20% b , followed by
a 2 min linear gradient to 80% b , hold for 6.50 min at 80% b , followed
by a 1 min linear gradient to the initial conditions of 5% b. the
product was observed with a retention time of 6.1 min .
h nmr ( d2o ) : ( ppm ) 1.30 ( m , 4h ) , 1.591.61
( m , 6h ) , 1.73 ( m , 2h ) , 1.88 ( s , 6h ) , 3.22 ( m , 2h ) , 3.30 ( t , j = 7.3 hz , 2h ) , 4.08 ( m , 2h ) , 5.45 ( m , 1h ) , 7.49 ( d , j = 11.5 hz , 1h ) , 7.56 ( d , j = 11.5 hz ,
1h ) .
c nmr ( d2o ) : ( ppm ) 21.3 , 21.8 ,
25.7 , 30.2 , 42.0 , 53.7 , 88.5 , 158.9 , 160.5 , 177.5 , 178.9 .
n--acetyllysine
( various concentrations ) was mixed with 10 mm adenine propenal in
50 l reaction mixtures containing 10 mm nacl and 10 mm 4-morpholinepropanesulfonic
acid ( mops ) ( ph 6.5 ) . at 1 h intervals , aliquots of each reaction
mixture
were injected onto a waters hplc system equipped with a 717
plus autosampler , 1525 binary hplc pump , and 2996 photodiode array
detector .
analytes were separated using a phenomenex polar - rp synergi
hplc column , 75 cm 2 mm with 4 m particle size , at a
flow rate of 0.6 ml / min .
the mobile phase solvents were h2o + 0.05% acetic acid ( v / v , solvent a ) and 3:1 meoh / acn + 0.05% acetic
acid ( v / v , solvent b ) delivered in a 19 min gradient consisting of
the following : 010.5 min , 520% b ; 10.512.5
min , 2080% b ; 12.516 min , 80% b ; 1619 min ,
5% b. data were analyzed using empower software , and peak areas were
manually integrated .
reactions
were performed
as described above and analyzed using a thermo accela hplc pump ( thermo
fisher scientific ) and htc pal autosampler ( leap technologies ) in - line
with a thermo quantum triple quadrupole mass spectrometer ( thermo
fisher scientific ) .
the quantum was equipped with an electrospray
source operated in q1 full scan , positive ion mode .
q1 was scanned
from 100600 amu in 0.45 s. analytes were chromatographically
separated on a synergi polar - rp hplc column ( phenomenex , 7.5
0.2 cm , 4 m particle size ) using the following gradient : 010.5
min , 520% b ; 10.512.5 min , 2080% b ; 12.516
min , 80% b ; 1617 min , 5% b. the mobile phase solvents were
h2o + 0.05% acetic acid ( v / v , solvent a ) and acetonitrile
+ 0.05% acetic acid ( v / v , solvent b ) , and they were delivered at a
flow rate of 0.6 ml / min .
all data were acquired and analyzed using
thermo xcalibur software . solutions containing
known concentrations of synthetic 3 and 4
were used to measure extinction coefficients of each compound . the
value obtained for both compounds (
33 600 200 mcm ) was the same within experimental
error ; however , the wavelengths of maximal absorbance were different
( 280 nm for 3 and 300 nm for 4 ) ( figure s1 ) .
adenine propenal ( 10 mm ) was
incubated with increasing concentrations of n--acetyllysine
( 1 , 2 , 5 , 10 , 50 , and 100 mm ) in reaction mixtures containing 10 mm
nacl and 10 mm mops ( ph 6.5 ) .
the mixtures were incubated for 2 h
at room temperature and then diluted 1:1000 in h2o .
the
absorbance at 300 nm of each dilution was then obtained using a du800
spectrophotometer ( beckman coulter ) , and the experimental extinction
coefficient of 4 was used to estimate its concentration
in the reaction mixture .
note that , for reaction mixtures containing
> 10 mm n--acetyllysine , complete consumption
of adenine propenal eliminates concerns of its contribution to the
absorbance of the final reaction mixture .
the adenine product does
not absorb significantly at 300 nm ( figure s1 ) . on the basis of its full uv spectrum ( figure
s1 ) ,
the extinction coefficient of 3 at 300 nm
is estimated to be 7.8 10 m cm .
the presence of this product at a concentration
equal to approximately 10% of the concentration of 4 ,
as found in the reaction mixtures , indicates that its contribution
to the absorbance at 300 nm will be no more than 3% .
consequently ,
the absorbance at 300 nm can be used to estimate the concentration
of 4 with reasonable accuracy .
n--acetyllysine
( 10 mm ) was incubated with adenine propenal ( 10 mm ) in 10 mm mops
( ph 6.5 ) containing 10 mm nacl in a 200 l reaction mixture
for 30 min .
after incubation , 50 mm nacnbh3 ( in h2o ) ( sigma - aldrich ) was added for an additional 30 min , and samples
were then analyzed using lc - ms as described below .
lc - ms analysis
was performed on the thermo quantum triple quadrupole
mass spectrometer . here
, the instrument was operated in q1 full - scan
negative ion mode using electrospray ionization .
q1 was scanned over
the range 100400 amu over 0.5 s. chromatography was carried
out on a synergi max - rp column ( phenomenex , 7.5 0.2 cm , 5 m
particle size ) using the following gradient : 05 min linear
gradient from 2% b to 50% b , 57 min at 50% b , 710
min 50% b to 2% b. the mobile phase solvents were h2o +
0.05% formic acid ( v / v , solvent a ) and 3:1 ch3cn / meoh +
0.05% formic acid ( v / v , solvent b ) , and they were delivered at a flow
rate of 400 l / min .
all data were acquired and analyzed using
thermo xcalibur software . using this chromatographic system , the reduced
intermediate eluted at approximately 2.9 min .
n - acetylcysteine ( 200
m ) was combined with 50 m adenine propenal in 50 mm
sodium phosphate buffer , ph 7.4 , and incubated for 6 h at 37 c .
nabh4 ( 50 mm )
( sigma - aldrich ) was used to reduce samples as indicated .
analytes
were separated on a supelco ascentis c18 column ( 50 2.1 mm ,
3 m ) with buffers a and b comprising 0.1% formic acid in water
and 0.1% formic acid in acetonitrile , respectively .
the gradient was
as follows : 13 min , 1% b ; 37 min , 98% b ; 711
min , 98% b ; 1117 min , 1% b. samples were analyzed on a thermofinnigan
tsq quantum mass spectrometer with esi source interfaced to a thermofinnigan
ms pump plus and autosampler plus ( thermo , san jose , ca ) .
n - acetylcysteine - adducted species were analyzed with data - dependent
scanning enabled in negative ion mode , scanning from m / z 150650 over 0.5 s. the top two m / z peaks from each ms scan were fragmented
with 10 ev collision energy for 0.5 s. hsa ( 15 m ) ( abcam ,
cambridge , ma ) was incubated with increasing
amounts of adenine propenal ( 0.15 , 0.38 , 1.5 , 3 , and 7 mm ) , representing
a molar excess of adenine propenal to hsa of 10 , 25 , 100 , 250 , and
500 , respectively .
reactions were incubated for 6 h at room temperature
in a buffer containing 10 mm nacl and 10 mm mops ( ph 6.5 ) . following
incubation ,
samples were precipitated with 25% trichloroacetic acid
( sigma - aldrich ) on ice for 1 h , washed with cold acetone , dried , and
reconstituted in 50 mm tris ( ph 8.0 ) containing 50% 2,2,2-trifluoroethanol
( acros organics , pittsburgh , pa ) .
samples were reduced with dtt ( sigma - aldrich ) ,
carbamidomethylated with iodoacetamide ( sigma - alrich ) , and diluted
5-fold with 100 mm tris ( to obtain a final solution containing 10%
2,2,2-trifluoroethanol ) . following dilution , samples were digested
with sequencing - grade trypsin ( promega , madison , wi ) overnight , acidified ,
and diluted in 0.1% formic acid .
the resulting solutions of trypsin - generated
peptides were loaded onto a capillary reverse - phase analytical column
( 360 m o.d .
100 m i.d . ) using an eksigent nanolc
ultra hplc and autosampler .
the 20 cm analytical column was directly
packed into a laser - pulled emitter tip using jupiter c18 reverse - phase
medium ( 3 m beads , 300 pore size , phenomenex ) .
mobile
phase solvents consisting of 0.1% formic acid in water ( solvent a )
and 0.1% formic acid in acetonitrile ( solvent b ) at a flow rate of
500 nl / min were used to elute the peptides .
the 90 min gradient consisted
of the following : 010 min , 2% b ; 1050 min , 235%
b ; 5060 min , 3595% b ; 6065 min , 95% b ; 6570
min 952% b ; 7090 min , 2% b. mass analysis of eluting
peptides was performed on an ltq orbitrap xl mass spectrometer ( thermo
scientific ) , equipped with a nanoelectrospray ionization source with
detection in positive ion mode . the ltq orbitrap was operated using
a data - dependent method , enabling dynamic exclusion .
full scan ( m / z 4002000 ) spectra were acquired
with the orbitrap ( resolution 60 000 ) , and the five most abundant
ions in each ms scan were selected for fragmentation in the ltq .
an
isolation width of 2 m / z , 30 ms
activation time , and collision energy normalized to 35% were used
to generate ms spectra .
dynamic exclusion settings allowed
for a repeat count of 2 within a 10 s duration , with exclusion duration
time set to 15 s. for identification of modified peptides , tandem
mass spectra were searched with sequest ( thermo fisher scientific )
against a human subset database created from the uniprotkb protein
database ( www.uniprot.org ) . variable modifications of + 57.0214
on cys ( carbamidomethylation ) , + 15.9949 on met ( oxidation ) , and + 54.0105
on lys or cys ( oxypropenylation ) were included for database searching .
search results were assembled with scaffold 3.0 ( proteome software )
using threshold filtering criteria consisting of 95% peptide probability
to achieve a peptide false discovery rate estimate of 0.2% .
sites of modification were validated by manual interrogation of tandem
mass spectra using xcalibur 2.1 qual browser software ( thermo scientific ) .
calculations of mass errors ( in ppm ) of precursor ions mass analyzed
in the orbitrap were performed using the following equation : ( ( theoretical
mass observed mass)/theoretical mass ) 10 . to obtain theoretical masses ,
analysis of cross - links was performed
in a similar manner with inclusion of + 36 on lys for database searching .
purified hsa ( 0.75 m ) was incubated with increasing concentrations
of adenine propenal in reaction mixtures containing 10 mm nacl and
10 mm mops ( ph 6.5 ) for 6 h at room temperature . following incubation ,
the protein was heated at 95 c for 10 min , cooled to room temperature ,
and then treated with 1 mg of pronase ( calbiochem , gibbstown , nj )
per mg of protein at 37 c for 24 h. pronase was inactivated
by heating at 95 c for 10 min , followed by cooling to room temperature ,
and treating with 1 l of aminopeptidase m ( calbiochem , gibbstown ,
nj ) at 37 c for 24 h. a final incubation at 95 c for 10
min inactivated the aminopeptidase m , and the samples were then cooled
to room temperature in preparation for adduct purification . for use as an internal standard ,
[ h]-[c]-4 was prepared by reaction
of mda with [ h]-lysine containing [ c]-lysine
as a tracer and quantified using a method similar to that described
for the [ h]-[c]-lysine - lactam internal standard
as described .
an oasis hlb cartridge
( 1 cm ) ( waters , milford , ma ) was equilibrated with 2 ml
of methanol followed by 2 ml of h2o .
following addition
of [ h]-[c]-4 , the sample was
loaded onto the cartridge using gravity flow .
the cartridge was washed
with 1 ml of h2o , and 4 was eluted with 3
ml 1:1 methanol / ethyl acetate and dried to 100 l under n2 gas .
the sample was then diluted in 0.1% formic acid solution
to a final volume of 1 ml and filtered using a 0.22 m nylon
spin - x centrifuge tube spun at 6000 rpm for 5 min .
samples were
purified using a thermo scientific aquasil c18 5 m reverse - phase
column using liquid scintillation counting to monitor elution of the
radioactive [ h]-[c]-lysine lysine
cross - link standard .
the internal standard [ h]-[c]-lysine lysine cross - link was prepared by reaction of mda
with [ h]-lysine containing [ c]-lysine as
a tracer and quantified using a method similar to that described for
the [ h]-[c]-lysine - lactam internal standard . for quantitation , the transition between m / z 341 and m / z 306 was monitored ; it corresponds to the loss of one molecule
of water and one molecule of ammonia .
portions of samples containing
the standard were then concentrated using 1 cc oasis hlb columns and
dried under nitrogen .
the purified samples were analyzed using a thermo
scientific tsq vantage quadrupole mass spectrometer equipped with
xcalibur software for data collection and manipulation .
all measured
mda - adduct signal in each sample was divided by the signal for the
[ h]-[c]-lysine lysine cross - link standard
of known concentration to quantify experimental results .
xpa was expressed and
purified as described . to modify xpa ,
3.2 m of purified protein was incubated
with 500 ( 1.6 mm ) of adenine
propenal for 6 h at room temperature
in a buffer containing 10 mm nacl and 10 mm mops ( ph 6.5 ) .
a high - throughput
fluorescence anisotropy assay was used to measure the dna - binding
activity of xpa .
the substrate for this assay
was a fluorescein - labeled y - shaped ssdna
dsdna junction substrate
containing 8 base pairs of duplex and 12 nucleotide noncomplementary
ssdna overhangs at both the 3 and 5 ends .
xpa was
modified with nhs biotin as described previously . modified or mock - treated xpa protein and the
dna substrate were diluted in binding buffer ( 20 mm hepes , ph 7.9 ,
75 m kcl , 5 mm mgcl2 , 5% glycerol , 1 mm dtt ) .
fluorescence anisotropy
was measured using a synergy h1 plate reader ( ex = 485 nm , em = 528 nm ) .
apparent dissociation constants ( kd ) were
determined for each individual titration by plotting fluorescence
anisotropy against protein concentration and fitting to a simple two - state
binding model with kaleidagraph ( v4.03 ) software .
adenine propenal was reacted with n--acetyllysine , n--acetylhistidine , n--acetylarginine , or n--acetylcysteine
in 10 mm nacl buffered with 10 mm mops ( ph 6.5 ) .
reaction progress
was monitored hourly by hplc using a c18 reverse - phase column for
product separation .
adenine propenal eluted at 8 min and displayed
a maximum absorbance of 260 nm ( figures 2 and s1 ) .
reaction with n--acetyllysine
resulted in the appearance of three new chromatographic peaks at 1.0 ,
4.8 , and 5.3 min , which were analyzed by uv / vis , nmr , and ms ( figures 2 and s1 ) .
the peak eluting
at 1 min displayed a uv absorbance maximum at 260 nm and an m / z of 136 , corresponding to protonated
adenine ( [ m + h ] ) ( 1 ) .
on the basis of nmr analysis , this product
was identified as 3 , and lc - ms analysis gave an m / z of 243 , which corresponds to the protonated
species ( [ m + h ] ) .
the 5.3 min peak , with a maximum absorbance
at 300 nm and an m / z of 413 , which
corresponds to the protonated species ( [ m + h ] ) , was identified
as 4 by nmr analysis .
the total amount of product formed
was unaffected by variations in ph over a range of 6.5 to 9.5 .
n--acetyllysine
( 50 mm ) was incubated with adenine propenal ( 10 mm ) in a 50 l
reaction mixture for 2 h at room temperature .
the column
eluate was monitored at 260 nm ( a ) or 300 nm ( b ) .
analytes present
in each peak were subjected to ms , uv , and nmr spectrometry .
chemical synthesis of 3 and 4 yielded
standards that verified the product structure assignments .
the molar
extinction coefficients of the pure compounds ( = 33.6
10 m cm at 280
nm for 3 and = 33.6 10 m cm at 300 nm for 4 ) were similar to that of adenine propenal ( = 32.5
10 m cm at 260
nm ) .
hplc analysis of reaction mixtures of n--acetyllysine
and adenine propenal using diode - array detection enabled direct comparison
of peak areas for each compound measured at its optimal wavelength
( figure 3 ) .
the essentially identical absorbance
coefficients of 3 and 4 allowed peak areas
to be used to estimate relative product quantity .
these comparisons
indicated that the major product formed from the reaction was 4 , with much lower quantities of 3 .
hplc analysis
of product formation in the reaction of adenine propenal
and n--acetyllysine .
the indicated amounts
of n--acetyllysine ( nal ) were incubated with
10 mm adenine propenal , and reactions were monitored hourly for 910
h. peaks corresponding to 3 and 4 were monitored
at 280 and 300 nm , respectively , by diode - array detection .
variation of the amount of n--acetyllysine
( from 2 to 100 mm ) in reaction mixtures while holding the amount of
adenine propenal ( 10 mm ) fixed demonstrated that the amount of 3 formed increased with n--acetyllysine
concentrations from 2 to 10 mm and then decreased somewhat at the
higher concentrations .
in contrast , the amount of 4 produced
increased with n--acetyllysine concentrations
up to 50 mm . on the basis of the relative peak area , the ratio of 4 to 3 in most reaction mixtures was approximately
10:1 .
estimation of the concentration of 4 using absorbance
at 300 nm indicated a maximum of 9 mm formed in reaction mixtures
containing 50100 mm n--acetyllysine
( figure s2 ) .
our initial hypothesis for the production of 4 was
that it arose by reaction of n--acetyllysine
with 3 , but this reaction did not occur under conditions
comparable to those used for the reaction of adenine propenal with n--acetyllysine . an alternative reaction scheme ,
in which an enaminoimine cross - link between adenine propenal and n--acetyllysine is first formed and then reacts with
h2o to produce 3 or another equivalent of n--acetyllysine to form 4 , is shown
in figure 4 .
if this scheme is correct , then
our data indicate that , at the concentrations of n--acetyllysine and adenine propenal used here , reaction of
the proposed intermediate with another molecule of n--acetyllysine is more favorable than its reaction with water .
hplc analysis with uv or in - line nmr was unable to detect
the putative
intermediate 5 ( figure 4 ) . therefore ,
we attempted to trap it by reduction with nacnbh3 .
adenine
propenal was incubated with n--acetyllysine
in 10 mm nacl/10 mm mops ( ph 6.5 ) in the presence of nacnbh3 .
we utilized lc - ms analysis of the reaction mixture to detect ions
with m / z 360 , which corresponds
to the partially reduced intermediate ( 6 ) .
a peak corresponding
to this mass eluted at 2.9 min , consistent with the transient formation
of the adenine propenal adduct to n--acetyllysine
( figure 5 ) .
( a ) representative lc - ms chromatogram
of 6 . in this
example , 5 mm n--acetyllysine was reacted
with 10 mm adenine propenal for 30 min followed by reduction with
50 mm nacnbh3 .
( b ) mass spectrum of the parent ion of the
reduced intermediate displaying m / z 360 with detection in negative ion mode .
reactions of adenine propenal with n--acetylhistidine
and n--acetylarginine did not result in the
formation of new chromatographic peaks .
reaction with n--acetylcysteine resulted in the formation of an oxopropenylated
product , which displayed m / z 216 ,
corresponding to the deprotonated molecular ion , and its major fragmentation
product , m / z 86.9 .
treatment of
the reaction mixture with nabh4 yielded the partially reduced
product ( m / z 218 ) and its major
fragmentation product ( m / z 89 ) ( figure s3 ) . to ascertain adenine propenal s ability to
react with protein , we incubated increasing amounts of the electrophile
with a fixed amount of purified hsa for 6 h , and the samples were
prepared for lc - ms / ms by trichloroacetic acid precipitation , resolubilization
of the precipitated protein pellet , reduction and carbamidomethylation
of cysteine residues , and proteolytic digestion with sequencing - grade
trypsin .
the resultant proteolytic peptides were analyzed via lc - ms / ms
using an ltq - orbitrap velos mass spectrometer with collision - induced
dissociation .
sequence coverage for unmodified hsa was found to be
90% , but exposure to increasing concentrations of adenine propenal
reduced coverage to a minimum value of 62% ( table
s1 ) .
hsa peptides were interrogated for the presence of a + 54.0105
mass increase , as would be expected from the addition of an oxopropenyl
group . to verify positively identified residues ,
extracted ion chromatograms were
generated from precursor peptide masses , and theoretical masses calculated
from protein prospector were compared to observed masses to calculate
ppm mass error .
for modified precursor peptides displaying a ppm mass
error less than 10 , ms / ms spectra were interrogated with xcalibur
software .
at least 40% of each theoretical b- and y - product ion series was present in the observed product
ion spectra of positively identified peptides , and 50% sequence coverage
was obtained for each peptide validated .
to validate the site of adduction ,
at least two ions within a product ion series with a + 54.0105 adduction
mass shift were required .
multiple lysine residues were found to be
modified in adenine propenal - exposed hsa , including lys26 , lys36 ,
lys161 , lys183 , lys214 , lys219 , lys236 , lys249 , lys257 , lys347 , lys375 ,
lys402 , lys413 , lys438 , lys549 , lys569 , and lys598 .
residues verified
to have a + 54.0105 adduction mass were mapped to the crystal structure
of hsa ( pdb i d : 1ao6 ) and were found to be largely confined to the protein surface ( figure 6a ) .
the 17 modified lysine residues represented
28% of the total lysines within the protein ( table 1 ) .
although we determined that adenine propenal can also form
oxopropenylated cysteine , searching for adducts on cysteine residues
within hsa did not result in the identification of any modified sites .
s - oxopropenal - n - acetylcysteine was stable
to treatment with tca as well a dtt , which are reagents used for proteomic
analysis , so any oxopropenylated cysteine residues on hsa - derived
peptides should have survived the workup prior to mass spectrometry .
therefore , adenine propenal is acting as a lysine - specific
modifier of hsa .
( a ) modified
lysine residues identified in table 1 were
mapped to the crystal structure of hsa ( 1ao6 ) .
( b ) the most highly modified
residues by individual electrophiles are highlighted with the modifying
electrophile noted .
hsa was treated at the indicated
molar ratios of adenine propenal and analyzed for lysine residues
modified with + 54.0105 .
the presence of each modified lysine in a
sample is indicated with . the modified lysine in each peptide
sequence is indicated with an asterisk .
equivalent lysine residues
within the crystal structure of hsa are provided in parentheses . the use of hsa as a biomarker
has primarily been described in the
context of electrophilic modifications at cys34 .
adenine propenal did not modify cys34 , so we sought to
identify the most reactive lysine residue(s ) that might be used as
a biomarker for modification by adenine propenal .
we reduced the levels
of adenine propenal relative to protein to a ratio of 10:1 , which
was the lowest ratio at which modification was observed . using the
interrogation methods described above to verify adduction sites , two
lysine residues , k190 and k351 , displayed the highest reactivity ( table 1 ) .
mapping of those residues to the crystal structure
of hsa revealed that both are located within domain ii , a recognized
binding region for many compounds ( figure 6a ) .
decreased hsa sequence coverage observed
with increasing concentrations of adenine propenal suggested that
adducts impede protein digestion and/or fragmentation required for
peptide identification .
lysine cross - links are formed
from the reaction of adenine propenal with n--acetyllysine ,
we hypothesized that at high concentrations of adenine propenal , lysine
lysine
cross - links may be induced in hsa . to investigate the presence of
cross - links , we interrogated previously acquired proteomics data for
a + 36 adduct mass , corresponding to an unreduced 3-carbon linkage
between lysine residues .
lysine cross - links ,
which was not surprising considering the decreased sequence coverage
and potential hindrance of peptide fragmentation by cross - links .
although
cross - linked sites were not identified with these algorithm - based
data searching methods , we manually interrogated the data to look
for the presence of precursor peptides containing cross - linked residues .
to predict lysine residues likely to participate in cross - link formation ,
we performed in silico analysis of the hsa crystal
structure ( 1ao6 ) .
carbon bond is approximately 1.5
in length , we hypothesized that the 3-carbon cross - link would be 57
long .
flexible , dynamic protein movements in solution may result
in cross - link formation between lysines at a distance greater than
7 .
we therefore examined the crystal structure to identify
lysine residue pairs separated by a distance of less than or equal
to 20 as potential sites of cross - link formation . following
identification of these residues ,
tryptic peptide sequences were determined
from the primary sequence , and theoretical peptide masses containing
the cross - link mass of + 36 were calculated using protein prospector .
we then thoroughly interrogated the data manually for the presence
of ions of the theoretical precursor peptide masses .
no evidence for
the presence of cross - link - containing peptides was obtained . as proteomics - based mass spectrometric analyses were unsuccessful ,
we employed a different analytical approach to search for lysine lysine
cross - links , in which modified hsa was degraded to the level of amino
acids , and the resultant hydrolysate was analyzed by ms .
this methodology
employed selected reaction monitoring to identify analytes exhibiting
a mass transition from m / z 329 to m / z 294 , as exhibited by analysis of the
lysine lysine cross - link standard .
addition of a [ h]-[c]-4 internal standard ( m / z 341 to m / z 306
transition ) enabled identification of analyte - containing fractions
during purification by scintillation counting and quantification by
stable isotope dilution .
application of this approach to hsa that
had been incubated with adenine propenal resulted in the identification
of 30 12 ng cross - link / mg protein in vehicle - treated hsa and
57 17 ng cross - link / mg protein in adenine propenal - treated
hsa .
these differences were not statistically significant , supporting
the conclusion that adenine propenal did not induce intramolecular
cross - links in hsa .
having confirmed
adenine propenal s capacity to modify lysine residues in hsa ,
we investigated its reaction with xpa .
adenine propenal was incubated
with xpa for 6 h at room temperature , and the samples were prepared
for and analyzed by lc - ms / ms using the same methods as described for
hsa . as for hsa
, peptides were interrogated for the presence of a
+ 54.0105 mass addition , and rigorous criteria were applied to peptide
and site of adduction identification .
of note , three
of these , k145 , k183 , and k204 , are located in the proposed dna - binding
domain of xpa ( figure 7a ) , and two , k63 and k67 , are known sites of acetylation that reduces
xpa s interaction with replication protein a. adenine propenal modifies xpa and reduces its dna - binding activity .
(
a ) lysine residues modified with an adduct mass of + 54.0105 were
mapped to the xpa nmr solution structure ( 1d4u ) .
( b ) plots of the change in fluorescence
anisotropy for a y - shaped 8/12 ssdna dsdna junction substrate
( 50 nm ) versus added protein treated with dmso , adenine propenal ,
or nhs - biotin .
purified xpa was incubated with
a 500-fold molar excess of adenine propenal and analyzed for lysine
residues modified with + 54.0105 .
modification of residues within the xpa dna - binding
domain has
the potential to affect dna - binding activity . using a plate - based
fluoresence anisotropy assay ,
the effect of adenine propenal - mediated
oxopropenylation on xpa s affinity for a y - shaped ssdna
fitting
of the data for binding of unmodified xpa to the fluorescein - labeled
20-nucleotide substrate provided a dissociation constant ( kd ) of 300 30 nm ( figure s4 ) .
binding affinities for the 20-nucleotide substrate
were measured for purified xpa incubated with vehicle ( dmso ) , adenine
propenal , or nhs biotin ( biotin n - hydroxysuccinimide
ester ) ( table 3 ) . as expected , incubation of xpa with dmso had no effect on its dna - binding
activity .
in contrast , exposure to nhs biotin , which efficiently
modifies lysine residues and disrupts the dna - binding activity of
many proteins , resulted in a nearly complete loss of xpa s dna - binding affinity .
xpa modified with adenine propenal bound to the substrate , but with
a greater than 3-fold reduction in affinity as compared to that of
the unmodified protein ( figure 7b ) .
in fact ,
oxopropenylation of the protein reduced the affinity to such an extent
that it could not be accurately measured ; thus , the kd value reported in table 3 ( 1
0.1 m ) is an estimate .
dissociation constants ( kd ) were determined by fitting fluorescence anisotropy
data to a simple two - state binding model .
the biotin - modification of xpa causes such a large loss of dna - binding
affinity that there is no significant change in fluorescence anisotropy ;
hence , a kd value could not be determined .
covalent
modifications of dna and proteins by electrophiles produced
during oxidative stress have multiple consequences on cellular function ,
including mutagenesis , stress responses , cellular dysfunction , and
ultimately cell death .
all of these may contribute to pathological
processes such as carcinogenesis , neurodegeneration , atherosclerosis ,
and chronic inflammation .
however , elucidation of the exact mechanisms
by which specific macromolecular damage leads to pathology requires
a better understanding of the sites and nature of that damage .
the most frequently used biomarker of electrophile damage to protein
during oxidative stress is the change in global protein carbonylation . although useful as a general indicator that
stress has occurred
, this approach does not identify the exact type
of modification or the modifying electrophile .
recent developments
in highly sensitive ms - based proteomics techniques have allowed for
expansion of this approach to the investigation of the specific types
of modifications induced and the impact of these modifications on
protein activity .
the resultant increased ability to identify specific
protein modifications induced by a single electrophile enables the
search for biomarkers that distinguish between different mechanisms
of oxidant damage .
the generation of adenine propenal occurs
predominantly in intact
dna , suggesting that most of its targets will likely be dna - associated
proteins in the nucleus or mitochondria .
nevertheless , we initiated
our investigation of adenine propenal s protein - modifying profile ,
using hsa as the model protein .
hsa s reactivity with electrophilic
agents has been studied extensively in the past , allowing us to place adenine propenal - dependent modifications in
the context of those existing data .
for example , teucrin a was found
to preferentially modify lys351 and lys545 , while 4-hydroxynonenal
modifies lys195 , lys199 , lys233 , and lys525 , and polycyclic aromatic
hydrocarbon epoxides modify lys137 .
mda has been shown to
modify multiple lysine residues including lys136 , lys174 , lys240 ,
lys281 , lys525 , and lys541 . as mda and
adenine propenal both produce oxopropenyl adducts on lysine residues ,
it is interesting to note that the major sites of adduction for the
two electrophiles are different : lys190 and lys351 for adenine propenal
versus lys525 for mda .
these findings suggest that distinct hsa modification
patterns may be used as markers for different electrophiles ( figure 6b ) .
clearly , the use of hsa as a biomarker of electrophile
damage is attractive , given its high concentration in the blood and
ease of sampling .
however , as noted above , adenine propenal generation
is largely confined to intact dna , so it remains to be seen if enough
of the electrophile reaches the circulation to allow the modification
of hsa to serve as a biomarker of exposure .
the impact of electrophiles
has long been studied in the context
of covalent modification of dna and its potential to lead directly
to mutations .
however , adenine propenal produced in the nucleus also
has the potential to modify dna - associated proteins , including those
involved in dna repair .
we report here that adenine propenal covalently
modifies the ner protein xpa in vitro and that this
modification leads to a reduction in xpa s dna - binding activity .
oxopropenylation of residues known to be acetylated further suggests
that adenine propenal - dependent modification could result in failure
of regulation of xpa s activity through sirt-1-dependent deacetylation .
xpa is a critical component of the ner machinery ,
without which repair activity ceases .
mutations in xpa , particularly in the dna - binding domain , are associated
with severe xp clinical phenotypes .
thus ,
it is possible that decreased xpa - binding activity resulting from
adenine propenal - dependent modification could result in reduced ner
activity in cells and increased potential for mutagenesis and carcinogenesis .
it should be noted that , in this model , the modification of repair
proteins may have a synergistic effect on mutagenesis by preventing
the repair of the original dna lesion . in this case , even moderate
reductions in the activity of the repair proteins have the potential
to result in significant increases in genomic instability .
further
analysis will be required to determine whether reactive base propenal
species modify dna repair proteins in cells under oxidative stress
and to characterize the effects of modification on dna repair activity in vivo . | base propenals are products of the
reaction of dna with oxidants
such as peroxynitrite and bleomycin .
the most reactive base propenal ,
adenine propenal , is mutagenic in escherichia coli and reacts with dna to form covalent adducts ; however , the reaction
of adenine propenal with protein has not yet been investigated .
a
survey of the reaction of adenine propenal with amino acids revealed
that lysine and cysteine form adducts , whereas histidine and arginine
do not .
n-oxopropenyllysine , a
lysine lysine cross - link , and s - oxopropenyl
cysteine are the major products .
comprehensive profiling of the reaction
of adenine propenal with human serum albumin and the dna repair protein ,
xpa , revealed that the only stable adduct is n-oxopropenyllysine .
the most reactive sites for modification
in human albumin are k190 and k351 .
three sites of modification of
xpa are in the dna - binding domain , and two sites are subject to regulatory
acetylation . modification by adenine propenal dramatically reduces
xpa s ability to bind to a dna substrate . |
cyclic episodes result in the momentary closure , partial or complete , of the upper airway at the level of the pharynx .
the repeated pauses in breathing can lead to intermittent hypoxia ( ih ) and increased reactive oxygen species ( ros ) .
the increase of ros in osa is likely due to the repeated oxygen depletion followed by the hyperoxia that develops to restore oxygen pressure ( po2 ) .
a similar phenomenon is observed in ischemia followed by reperfusion [ 25 ] . in ischemia
/ reperfusion , xanthine oxidase generates free radicals in the presence of oxygen , contributing to oxidative stress [ 68 ] .
osa is associated with chronic liver diseases , such as nonalcoholic steatohepatitis ( nash ) [ 916 ] .
second hit to liver disease by amplifying the tissue damage induced by a high dose of paracetamol [ 17 , 18 ] .
the injury mechanism , triggered by osa , appears to be related to the formation of peroxynitrite , depletion of glutathione , and apoptosis of hepatocytes . in osa ,
inflammatory factors , such as nuclear factor kappa b ( nf-b ) , are activated at a systemic level [ 17 , 19 ] .
nf-b is a master regulator of the inflammatory process , by inhibiting its inhibitor ikk - b , and its activation leads to the increased expression of tumor necrosis factor ( tnf- ) , interleukins 1 and 6 , and inducible nitric oxide synthase ( inos ) .
alternatively , these factors can be activated by hypoxia inducible factor ( hif-1 ) [ 2023 ] , which results in apoptosis .
several studies have shown that osa is associated with inflammation , nash , oxidative stress , and apoptosis .
this is the first experimental study that evaluated the inflammatory process in the lung and liver with intermittent hypoxia , suggesting that there is a recruitment of inflammatory mediators recognized during ischemia and reperfusion . here
, we investigate the molecular mechanism involved in the lung and liver injury in an animal model of osa .
the experiments were approved and completed according to the research and ethics committee of the research and postgraduation at the hospital de clnicas de porto alegre , brazil .
a total of 12 c57bl/6 mice ( 811 weeks old ) were housed in plastic boxes ( 30 19 13 cm ) at the animal experimentation unit of the hospital de clnicas de porto alegre .
the mice were kept on a 12-hour light / dark cycle ( lights on from 7 am to 7 pm ) at 22 4c and given free access to food ( purina - nutripal , porto alegre , rs , brazil ) and water .
the mice were randomly divided into two experimental groups ( n = 6 per group ) .
the groups consisted of mice exposed to intermittent hypoxia for 35 days ( ih group ) and mice that underwent a simulation of the ih procedure ( sih group ) .
the mice were placed in intermittent hypoxia chambers 8 hours a day ( 9 am to 5 pm ) for 5 weeks ( figure 1 ) .
the animals were exposed to a gas mixture consisting of 90% nitrogen and 10% carbon dioxide for 30 seconds .
the gas mixture reduces the oxygen fraction in the chambers by 6 1% . in sequence , the gas release is then blocked and fans are triggered to restore ambient air for the remaining 30 seconds .
the sih group was housed in a cage and subjected to the same adjacent fan activity as the ih group but no gas was introduced into the cage .
after 35 days , the animals were deeply anesthetized with an intraperitoneal injection of ketamine hydrochloride ( 100 mg / kg ) and xylazine hydrochloride ( 50 mg / kg ) and the liver and lungs were removed .
the organs were immediately frozen in liquid nitrogen and kept at 80c for subsequent analysis .
the organs were cut and divided for biochemical and protein analyses . for analysis of oxidative stress , 100 mg of tissue
was added to 0.9 ml of buffer ( 140 mm kcl , 20 mm phosphate , ph 7.4 ) and homogenized with a micropestle in microtubes .
after centrifugation at 2150.4 g for 10 minutes in a refrigerated centrifuge ( 4c ) , the supernatant was discarded and the pellet was stored at 80c for further analysis . for western blotting , a nuclear extraction protocol was used .
briefly , 100 mg of tissue was added to 0.6 ml of lysis buffer ( 25 mm hepes , 1% triton x-100 , 2 mm edta , 0.1 ml nacl , 25 mm naf , 1 mm sodium orthovanadate , and a protease inhibitor cocktail ) and homogenized with a micropestle in microtubes .
after centrifugation at 15,000 g for 10 minutes at 4c , the supernatant was discarded and the pellet was stored at 80c for further analysis .
the protein concentration in the homogenate was measured spectrophotometrically at 595 nm using the bradford method .
the values are expressed in mg / ml and were used in the calculations for the tbars and antioxidant enzymes .
the tbars technique consists of heating the homogenate with thiobarbituric acid to produce a colored product that is subsequently measured at 535 nm using a spectrophotometer .
the change in color is due to the presence of malondialdehyde and other substances produced from lipid peroxidation in the biological material .
briefly , 0.25 ml of 10% trichloroacetic acid ( tca ) , 0.10 ml of homogenate , 0.067 ml of 0.67% thiobarbituric acid ( tba ) , and 0.033 ml of distilled water were added to a tube , stirred , and then heated at 100c .
after the tubes cooled , 0.20 ml of n - butyl alcohol was added to extract the pigment .
the tubes were then stirred and centrifuged for 10 minutes at 1110 g. a 0.20 ml aliquot of the supernatant was added to a 96-well plate .
the technique used to measure sod was based on the level of inhibition caused by the reaction of the enzyme with o. we used adrenaline in an alkaline medium to produce adrenochrome and o . in a 96-well plate , we measured sod activity in the reaction medium ( 50 mm glycine - naoh , ph 10 ) and three samples containing different concentrations of homogenate .
after addition of 10.5 l epinephrine ( 60 mm , ph 2.0 ) , the reaction was monitored for 2 min at 480 nm .
the rate of decomposition of hydrogen peroxide is directly proportional to enzyme activity and follows pseudo - first - order kinetics with respect to hydrogen peroxide .
the decrease in absorption at 240 nm was determined after adding 7 l of 300 mm h2o2 to the reaction medium ( 50 mm phosphate regulator ) .
a total of 50 mg of protein was added to a buffer ( 60% glycerol , 2 m tris , sds , and 10% pyrroline 0.5% ) and incubated for four minutes at 100c .
after electrophoresis was performed on a 912% polyacrylamide gel , the protein was transferred to a polyvinylidene difluoride ( pvdf ) membrane .
the membrane was washed with pbs contained 0.5% tween 20 and then incubated in a blocking solution ( 5% skim milk powder and 0.5% tween 20 in cold pbs ) for 30 minutes .
next , the membrane was washed and incubated in the secondary antibody ( hrp ) for two hours at room temperature .
after another wash , the protein was visualized using chemiluminescent detection ( chemiluminescent hrp substrate ) , film , and a transiluminator ( l - pix chemi molecular imaging
the results were quantified using labimage 1d ( loccus biotechnology ) and are expressed as arbitrary units . for analyzing the result
, the student 's t - test was performed using spss version 18.0 ( statistical package for social science ) .
lipid peroxidation , a marker of oxidative damage , was significantly increased in the lung ( 14% ) and liver ( 29% ) of the ih group when compared with the sih group ( table 1 ) .
the activity of endogenous sod was significantly lower in lung tissue ( 56% ) and higher in liver tissue ( 87% ) from ih animals when compared with the control group ( table 1 ) .
the activity of cat was significantly higher in both organs ( 32% in the lung and 184% in the liver ) from the ih group when compared with the sih group ( table 1 ) .
the activated ( phosphorylated ) p65 subunit of nf-b was increased by 30% in the lung and 39% in the liver of ih mice when compared with sih mice .
the expression of hif-1 and tnf- was significantly increased in the ih group when compared with the sih group ( figures 2 and 3 ) . in the lung tissue , hif-1 increased by 96% and tnf- increased by 38% . in the hepatic tissue , hif-1 was increased by 19% and tnf- was increased by 48% .
the expression of inos and vegf was significantly higher in the ih group when compared with controls ( figures 4 and 5 ) .
there was a 35% increase in inos and a 22% increase in vegf in the lung tissue .
the liver showed a 79% increase in inos levels and a 71% increase in vegf .
cleaved caspase 3 was increased by 237% in the lung and 182% in the liver of ih animals when compared to the sih group ( figure 6 ) .
animal models that use intermittent hypoxia can help elucidate the mechanism of damage to various systems caused by sleep apnea .
independent of body mass index , the respiratory disturbance index is directly related to the degree of liver damage and is recognized as a risk factor for nonalcoholic fatty liver disease ( nafld ) [ 10 , 34 ] .
it has been proposed that the development of nash is produced in two phases consisting first of the accumulation of triglyceride , which is attributed to insulin resistance and obesity , and then the presence of inflammation and fibrosis , which is correlated with oxidative stress and hepatic lipid peroxidation [ 36 , 37 ] .
our research group has described oxidative damage to membrane lipids measured by tbars and changes in endogenous antioxidant enzymes in the liver tissue that indicate the role of oxidative stress in our model system .
these data are in agreement with the results described in other model systems [ 3841 ] .
oxidative stress occurs through xanthine oxidase by producing the superoxide anion radical ( o2 ) and hydrogen peroxide [ 42 , 43 ] ; it is suggested that the o2 and h2o2 , formed by the activity of xanthine oxidase , act independently on the activity of sod and cat .
nitrosative stress includes the formation of nitric oxide ( no ) that binds o2 to form the radical peroxynitrite [ 38 , 42 , 45 , 46 ] .
hif-1 regulates the concentration of oxygen , and it can be the initiator of inflammation in intermittent hypoxia [ 47 , 48 ] or stimulated by oxidative stress . this protein is correlated with chronic alcohol use and the presence of nafld .
hif-1 also stimulates macrophages , increases the production of vegf and inos [ 51 , 52 ] , reduces apoptosis [ 24 , 53 ] , and stimulates cell proliferation .
it is suggested that inflammatory activity is dependent on nf-b , indicating that nf-b can regulate hif-1 transcription .
although indirect , inhibition of ikk experimentally prevents the activation of nf-b and was found to prevent the development of steatosis and nash . in the present study
, we observed an increase in the expression of hif-1 in the liver and the lung of mice exposed to hypoxia .
the stimulation of tnf- leads to phosphorylation of ib , which results in activation of nf-b .
activation of nf-b causes it to translocate to the nucleus and promote the transcription of numerous proinflammatory genes . here
, we showed that tnf- and nf-b were increased in animals exposed to intermittent hypoxia .
vegf is essential for the initiation of angiogenesis , and it has a strong effect on vascular elements in response to hypoxia [ 59 , 60 ] . in this study , we found increased expression of vegf in both organs when mice were subjected to intermittent hypoxia . in our previous work
, we found that there is an increase in nitric oxide metabolites ( no ) after exposure to intermittent hypoxia . in the present study
, we evaluated an enzyme responsible for no production , inos , and found that the levels of this enzyme were increased in the lung and liver of animals exposed to hypoxia .
apoptosis in all cells is regulated by caspases . after cleavage , caspases become active and initiate pathways that lead to apoptosis .
we found that cleaved caspase 3 expression is increased in the liver and lung of the ih group , demonstrating that there was activation of this apoptotic cascade .
thus , the data suggest that intermittent hypoxia leads to liver and lung damage that can result from a cascade of signals initiated by oxidative stress , inflammation , and apoptosis .
in mice , the cyclic oxygen deprivation observed in sleep apnea induces oxidative stress and activation of hif-1 , which stimulates a cascade of inflammatory signaling , nitric oxide generation , angiogenesis , and apoptosis in the lung and liver . | sleep apnea is a breathing disorder that results from momentary and cyclic collapse of the upper airway , leading to intermittent hypoxia ( ih ) .
ih can lead to the formation of free radicals that increase oxidative stress , and this mechanism may explain the association between central sleep apnea and nonalcoholic steatohepatitis .
we assessed the level of inflammation in the lung and liver tissue from animals subjected to intermittent hypoxia and simulated sleep apnea .
a total of 12 c57bl/6 mice were divided into two groups and then exposed to ih ( n = 6 ) or a simulated ih ( sih ) ( n = 6 ) for 35 days .
we observed an increase in oxidative damage and other changes to endogenous antioxidant enzymes in mice exposed to ih .
specifically , the expression of multiple transcription factors , including hypoxia inducible factor ( hif-1 ) , nuclear factor kappa b ( nf-b ) , and tumor necrosis factor ( tnf- ) , inducible no synthase ( inos ) , vascular endothelial growth factor ( vegf ) , and cleaved caspase 3 were shown to be increased in the ih group .
overall , we found that exposure to intermittent hypoxia for 35 days by simulating sleep apnea leads to oxidative stress , inflammation , and increased activity of caspase 3 in the liver and lung . |
spermatocytic seminoma ( ss ) is an uncommon testicular germ cell neoplasm with distinct histopathological features .
it usually occurs in older men with mean age of 53.6 years ( range 19 - 72 years ) .
it bears an excellent prognosis but tends to behave aggressively and renders a poor prognosis if associated with sarcomatous transformation .
patients with sarcomatous transformation at the time of presentation already have metastatic disease and this is almost fatal
. the sarcoma component may present as rhabdomyosarcoma , angiosarcoma , leiomyosarcoma , myxoid liposarcoma or undifferentiated sarcoma .
we describe a case of a young man with ss developing rhabdomyosarcomatous transformation . a 38-year - old man presented with long standing history of swelling in right hemiscrotum .
he underwent right testicular biopsy in a local medical college , which was diagnosed as seminoma .
there was a right testicular mass measuring 10 10 cm , with scrotal violation and left testis was normal .
the serum tumor markers i.e. , alfa - fetoprotein ( afp ) and beta hcg were within normal .
retroperitoneal lymph node dissection ( rplnd ) was not performed as radiologically there was no lymphadenopathy .
in addition to local and abdominal radiotherapy , chemotherapy ( with vincristine , actinomycin d and cyclophosphamide ) was given .
two months postoperatively , the patient is on follow - up and doing well . the right high inguinal orchidectomy specimen measured 7 6 4 cm with attached vas deferens and scrotal skin .
most of the nodules were gelatinous with some appearing darker in color and firm in consistency than the rest [ figure 1a ] .
microscopically , the spermatocytic component consisted of polymorphic cell population with large cells having characteristic spermine chromatin ; intermediate - sized cells and small lymphocyte - like cells [ figure 1b ] .
the cells were immunopositive with c - kit ( cd117 ) [ figure 1d ] but were negative with plap ( placental - like alkaline phosphatase).the other component of the tumor consisted of fascicles of spindle shaped cells with fusiform nuclei [ figures 2a and b ] .
admixed with both the components were many multinucleated and bizarre tumor giant cells with hyperchromatic nuclei .
few strap cells and globoid cells were identified within the fascicles of spindle cells [ figure 2c ] .
no areas of intratubular germ cell neoplasia ( itgcn ) or other associated germ cell components were identified .
immunohistochemically , the spermatcocytic component was negative for placental alkaline phosphatase ( plap ) , cd30 , smooth muscle - actin , and the spindle cells were strongly positive for desmin [ figure 2d ] and weakly for myoglobin . ( a ) lobulated , soft and homegenous cut surface of the tumor with few nodules appearing form and dark in color ( arrow ) .
( b ) spermatocytic seminoma with three types of cells ( small , intermediate and large ) with fine fibrovascular septa traversing between them ( h&e 400 ) .
( d ) spermatocytic seminoma cells show cytoplasmic positivity with c - kit ( immunoperoxidase 400 ) ( a ) long fascicles of spindle cells ( h&e 200 ) .
( b ) sarcomatous component in close apposition with the seminomatous component ( hxe 200 ) .
a 38-year - old man presented with long standing history of swelling in right hemiscrotum .
he underwent right testicular biopsy in a local medical college , which was diagnosed as seminoma .
there was a right testicular mass measuring 10 10 cm , with scrotal violation and left testis was normal .
the serum tumor markers i.e. , alfa - fetoprotein ( afp ) and beta hcg were within normal .
retroperitoneal lymph node dissection ( rplnd ) was not performed as radiologically there was no lymphadenopathy .
in addition to local and abdominal radiotherapy , chemotherapy ( with vincristine , actinomycin d and cyclophosphamide ) was given .
two months postoperatively , the patient is on follow - up and doing well . the right high inguinal orchidectomy specimen measured 7 6 4 cm with attached vas deferens and scrotal skin
most of the nodules were gelatinous with some appearing darker in color and firm in consistency than the rest [ figure 1a ] .
microscopically , the spermatocytic component consisted of polymorphic cell population with large cells having characteristic spermine chromatin ; intermediate - sized cells and small lymphocyte - like cells [ figure 1b ] .
the cells were immunopositive with c - kit ( cd117 ) [ figure 1d ] but were negative with plap ( placental - like alkaline phosphatase).the other component of the tumor consisted of fascicles of spindle shaped cells with fusiform nuclei [ figures 2a and b ] .
admixed with both the components were many multinucleated and bizarre tumor giant cells with hyperchromatic nuclei .
few strap cells and globoid cells were identified within the fascicles of spindle cells [ figure 2c ] .
no areas of intratubular germ cell neoplasia ( itgcn ) or other associated germ cell components were identified .
immunohistochemically , the spermatcocytic component was negative for placental alkaline phosphatase ( plap ) , cd30 , smooth muscle - actin , and the spindle cells were strongly positive for desmin [ figure 2d ] and weakly for myoglobin .
( a ) lobulated , soft and homegenous cut surface of the tumor with few nodules appearing form and dark in color ( arrow ) .
( b ) spermatocytic seminoma with three types of cells ( small , intermediate and large ) with fine fibrovascular septa traversing between them ( h&e 400 ) .
( d ) spermatocytic seminoma cells show cytoplasmic positivity with c - kit ( immunoperoxidase 400 ) ( a ) long fascicles of spindle cells ( h&e 200 ) .
( b ) sarcomatous component in close apposition with the seminomatous component ( hxe 200 ) .
a 38-year - old man presented with long standing history of swelling in right hemiscrotum .
he underwent right testicular biopsy in a local medical college , which was diagnosed as seminoma .
there was a right testicular mass measuring 10 10 cm , with scrotal violation and left testis was normal .
the serum tumor markers i.e. , alfa - fetoprotein ( afp ) and beta hcg were within normal .
retroperitoneal lymph node dissection ( rplnd ) was not performed as radiologically there was no lymphadenopathy .
in addition to local and abdominal radiotherapy , chemotherapy ( with vincristine , actinomycin d and cyclophosphamide ) was given .
the right high inguinal orchidectomy specimen measured 7 6 4 cm with attached vas deferens and scrotal skin . the testicular shape was maintained . on cut surface ,
most of the nodules were gelatinous with some appearing darker in color and firm in consistency than the rest [ figure 1a ] .
microscopically , the spermatocytic component consisted of polymorphic cell population with large cells having characteristic spermine chromatin ; intermediate - sized cells and small lymphocyte - like cells [ figure 1b ] .
the cells were immunopositive with c - kit ( cd117 ) [ figure 1d ] but were negative with plap ( placental - like alkaline phosphatase).the other component of the tumor consisted of fascicles of spindle shaped cells with fusiform nuclei [ figures 2a and b ] .
admixed with both the components were many multinucleated and bizarre tumor giant cells with hyperchromatic nuclei .
few strap cells and globoid cells were identified within the fascicles of spindle cells [ figure 2c ] .
no areas of intratubular germ cell neoplasia ( itgcn ) or other associated germ cell components were identified .
immunohistochemically , the spermatcocytic component was negative for placental alkaline phosphatase ( plap ) , cd30 , smooth muscle - actin , and the spindle cells were strongly positive for desmin [ figure 2d ] and weakly for myoglobin .
( a ) lobulated , soft and homegenous cut surface of the tumor with few nodules appearing form and dark in color ( arrow ) .
( b ) spermatocytic seminoma with three types of cells ( small , intermediate and large ) with fine fibrovascular septa traversing between them ( h&e 400 ) .
( d ) spermatocytic seminoma cells show cytoplasmic positivity with c - kit ( immunoperoxidase 400 ) ( a ) long fascicles of spindle cells ( h&e 200 ) .
( b ) sarcomatous component in close apposition with the seminomatous component ( hxe 200 ) .
ss is a relatively rare , well - defined pathological entity first described by masson comprising 3 - 7% of all seminomas .
it is an indolent neoplasm with long duration of symptoms , early stage at presentation , absence of metastasis and bears an excellent prognosis .
however , dedifferentiation or transformation to a sarcoma which occurs in approximately 6% of cases renders a poor prognosis with a metastatic potential .
literature review cites small series and a few case reports[810 ] of occurrence of sarcomatous differentiation in ss .
the occurrence of rhabdomyosarcomatous differentiation is rare and has been described in the literature in eight previous case reports [ table 1 ] . in the present case ,
the sarcomatous transformation occurred at a much early age of 38 years compared to the previous cases . the sudden increase in size
however , it is important to be aware of misinterpreting the focal areas of atypical stromal overgrowth within conventional germ cell tumor .
true sarcomatous component can be distinguished by its expansile growth pattern , infiltration into the surrounding elements and characteristic morphology of the distinct sarcomatous components .
review of reported cases of spermatocytic seminoma with rhabdomyosarcomatous differentiation the seminomatous component was well circumscribed but the rhabdomyosarcomatous component was seen infiltrating and destroying the rete testis and epididymis .
identification of epithelial structures of rete testis is important as these may be mistaken for epithelial component of a mixed nonseminomatous germ cell tumor .
the origin of the sarcomatous elements of these tumors and the relation to the spermatocytic component is uncertain .
few have implied this sarcoma component to be a teratomatous element of a mixed germ cell tumor as the occasional occurrence of sarcomatous elements ( chiefly rhabdomyosarcoma , angiosarcoma , and leiomyosarcoma ) in gonadal and extragonadal germ cell tumors is well known .
the serum tumor markers were not elevated in the present case . however , other authors view the sarcomatous component as an
the development of this tumor , first with slow enlargement of the testis followed by rapid increase in size tends to favor the explanation of sarcomatous differentiation occurring in ss .
the treatment of choice in ss is orchidectomy , followed by surveillance to detect tumor in the contralateral testis .
however , in the presence of sarcomatous differentiation , adjuvant chemotherapy or radiotherapy is beneficial . as the risk of metastases
if remarkably increased with this transformation , a close follow - up is essential ; however , prognosis is dismal despite aggressive multimodality approach . in conclusion
, this case describes the rare sarcomatous differentiation occurring in ss heralded by a rapid increase in size , which portends a poor prognosis to this indolent neoplasm . | the sarcomatous differentiation occurring in spermatocytic seminoma ( ss ) renders an aggressive behavior with metastatic potential to this relatively indolent neoplasm .
correct identification of this sarcomatous component is essential as further management differs .
herein , we report a case of young male with ss with rapid increase in size of the tumor .
histopathology revealed a rhabdomyosarcomatous component infiltrating the rete - testis and epididymis along with a well - circumscribed ss . |
in the previous issue of critical care , joulin and colleagues describe an animal model of lipopolysaccharide ( lps)-induced impaired myocardial systolic and diastolic function .
diastole is composed of two physiological phenomena : myocardial stiffness , which is energy independent , and active relaxation , which is an atp - requiring process .
current echocardiographic techniques can help distinguish which of the two is the predominant pathway , although these techniques may not always be practical in critically ill patients . in some patients with sepsis ,
a reversible component to impaired ventricular relaxation has been demonstrated , which would imply that the energy - requiring component of diastolic function is more perturbed in certain patients .
one might wonder why this is an important distinction to make , but considering that increased mortality in sepsis may be correlated with increasing fluid administration , it would seem that being able to distinguish between isolated systolic , diastolic , or combined dysfunction may prove to be life saving .
this was achieved by serial transesophageal echocardiographic measurement of standard indices of systolic function , as well as analyzing diastolic mitral inflow and annular tissue doppler patterns , the current standard in echocardiographic grading of diastolic function .
this study highlighted the fact that we should be cognizant that not all patients suffering from sepsis and shock should be treated uniformly , and that choices of intravenous resuscitation and vasopressor therapies need careful consideration .
the study further elucidated the reversible nature of the impaired ventricular relaxation in humans , suggesting that a metabolic or molecular process may be responsible .
an attractive mechanism currently thought to play a major role is that of calcium flux .
systole is initiated by rapid elevation of myocyte intracellular calcium , both through influx ( through l - type calcium channels ) and calcium - mediated calcium release from the sarcoplasmic reticulum ( through the ryanodine receptor ) .
re - uptake of calcium into the sarcoplasmic reticulum by sarcoplasmic reticulum ca - atpase ( serca ) allows for cardiac relaxation .
the ability of serca to pump calcium back into the sarcoplasmic reticulum is governed by phospholamban , a sarcoplasmic reticulum membrane - bound modulatory protein .
the rapidity at which calcium is returned to the sarcoplasmic reticulum is directly related to the rapidity of cardiac relaxation . in the unphosphorylated state
signaling by protein kinase leads to phosphorylation of phospholamban , which , in turn , diminishes its inhibitory activity on serca , promoting cardiac relaxation . in a knockout mouse model lacking phospholamban ,
while other pathways , such as sodium - calcium exchange , are important in myocardial calcium trafficking , those are currently beyond the scope of this comment , and will not be described .
joulin and colleagues , in their experiments , were able to demonstrate a serca - dependent aberration in diastolic cardiomyocyte behavior .
in particular , in their experiments , they were able to demonstrate a serca - dependent aberration in diastolic cardiomyocyte behavior .
the force - frequency relationship ( that is , heart - rate - dependent increase in cardiomyocyte shortening ) and frequency - dependent acceleration of relaxation are physiological phenomena that ensure maintenance of cardiac output with the decreased ejection and filling times that are consequences of higher heart rates .
the group hypothesized that lps would disrupt this delicate balance by exerting an effect on the molecular workings within the cardiomyocyte principally through the inhibition of the phosphorylation of phospholamban . by utilizing echocardiographic measurements of diastole , and through western blot analyses , they were able to demonstrate a correlation between lps - induced myocardial relaxation dysfunction ( frequency - dependent acceleration of relaxation depression ) and serca function .
it is important to note that this work was done in a murine model of lps infusion , a model with potentially important differences from septic patients being treated in the intensive care unit .
anesthetic agents were used to regulate heart rates , and real - time alterations in physiological functioning may have been missed .
the group studied only the sarcoplasmic reticulum when calcium trafficking is also linked to mitochondrial function and integrity , nitric oxide production , the beta - adrenergic response , and potential protective effects on the myocardium during prolonged sepsis .
would specific targeting of intracellular calcium , or serca - related protein kinase and phosphatase result in better hemodynamics in septic shock ?
there is already a body of evidence to suggest that calcium - sensitizing agents , such as levosimendan , may improve hemodynamics in sepsis . in two animal models ,
left ventricular relaxation was improved after treatment with levosimendan in contrast to inotropes , such as milrinone or dobutamine , and in a clinical trial , levosimendan proved useful in improving global hemodynamic measurements .
the study by joulin and colleagues is certainly thought provoking , and will hopefully lead us closer to developing better strategies for dealing with sepsis - induced myocardial dysfunction both its systolic and diastolic components .
| sepsis - induced myocardial dysfunction has traditionally been thought of as principally affecting systolic heart function .
one of the primary reasons for this concept is that systolic dysfunction is relatively easy to conceptualize , visualize , and measure . with the advent of preload - independent measurements for diastolic function ,
both measurement and conceptual difficulties are being resolved , and a new realm of evidence is beginning to emerge regarding the aberrations that are found during cardiac relaxation in sepsis .
a recent article in critical care brings this issue into sharper focus . |
hepatitis a virus ( hav ) infection is considered as an important public health concern .
it is estimated that about 1.4 million cases of hav infection occur every year worldwide ( 1 , 2 ) .
healthcare workers ( hcws ) can always be exposed to hav infected patients and if they were non - immune , they will also be infected .
hence hospital personnel can be proposed as a high risk group for hav infection and evaluation of a suitable preventive method for them seems to be reasonable . on the other hand , investigation of hav seropositivity among this group
some countries have evaluated this rate and some of them proposed hav vaccination as preventive strategy for their hcws ( 2 - 5 ) . in this review article , we highlighted some points about virology , epidemiology , preventive strategies and importance of hav infection and then focused on available data regarding hav seroprevalence among iranian healthcare personnel to evaluate the need for using vaccination as a preventive method .
for this purpose , scopus , pubmed and google scholar were comprehensively searched using all appropriate combinations of following keywords ; healthcare provider , healthcare worker , healthcare personnel , nurse , medical students ,
furthermore , to find related persian evidences we searched google scholar and scientific information database ( sid ) .
it is a 27 nanometer rna virus , containing 7474 nucleotides , positive stranded , non - enveloped and icosahedral virus .
four genotypes have been identified for hav in humans that are a single serotype without biological differences .
the spread route for hav is fecal - oral and risk factors are travel to endemic areas , near contact with an infected person , homosexual activity , waterborne , being in a daycare center and injection drug use .
the infection , as an important public health problem , occurs worldwide with more prevalence in low socioeconomic areas ( 1 , 2 , 9 ) . in a population study in tehran province of iran ,
the overall rate of anti - hav seroprevalence has been reported as 90% ( 10 ) .
also a gradual shift for age of hav infection from childhood to adulthood has been reported .
control of some of the aforementioned risk factors can be the reason of this gradual shift ( 9 ) .
hav causes an acute illness that is self - limited , but it can cause fulminant hepatitis rarely in some patients .
manifestation of hepatitis a infection could be silent or with non - specific symptoms in children , but it can cause a mild flu - like illness to hepatic failure in adults ( 11 ) . considering different causes of acute hepatitis , diagnosis of hav infection based on clinical manifestations
detecting serum hav - rna using pcr amplification is an expensive method and usually used for research purposes .
however , assessment of immunoglobulin ( ig ) antibody to hav has been suggested for diagnosis of hav infection .
igm anti - hav anti - body emerges one to two weeks after exposure to virus and disappears after 3 to 6 months , so diagnose of acute phase of hav infection can be possible .
positive results for testing igg hav anti - body also shows previous history of hav infection ( 8 , 13 ) .
fulminant hepatic failure is an ominous complication of hepatitis a and in this case , liver transplantation is a suggested option .
approximately 30% of symptomatic patients admitted to hospital for management of complications including dehydration , severe prostration , coagulopathy , encephalopathy and other evidences of hepatic decompensation ( 14 ) . as we explained before
intramuscular administration of hav immune globulin provides passive immunization , which is short - term and temporary and usually takes from three to five months .
passive immunization has its special indications like persons travelling to or working in countries and communities with intermediate and high rate of hav infection and etc .
however , it is not related to the topic of this review and more information can be obtained from related review articles ( 8 , 15 , 16 ) .
also there is another vaccine , a combined vaccine namely twinrix , which contains both hav and hepatitis b antigens ( 17 ) .
the two mentioned types of vaccines has been approved and used in the united states . both of them are administered intramuscularly and as two separated injections with a six - month interval .
it is said that after one month of receiving two doses of these vaccines , about 100% of immunocompetent vaccinated subjects can earn a probable lifelong immunity against hav infection ( 8 , 17 ) .
nowadays , we know that vaccination is an important and effective preventive method for decreasing fulminant hepatitis . in a study in the united states , vaccination rate for hav among healthcare personnel was higher than general population , however overall protection has remained suboptimal ( 18 ) .
though , selecting an appropriate preventive strategy in each area depends on evaluation of some issues related to that area and they are annual incidence rate of fulminant hepatitis due to hav infection , hav seropositivity in different age and occupation groups , costs for hospitalization and treatment of patients with hav infection .
these are on one side of the equation and the costs for providing hav vaccines and running vaccination program are on the other side .
furthermore , gross national income of each country is the major factor that influences this equation ( 19 - 22 ) .
therefore , each country or territory needs to run some cost - effectiveness studies for making a decision about running hav vaccination program ( 23 ) .
here is the other epidemiologic factor that should be considered in vaccination program of each country , especially developing countries like iran .
controlling risk factors for hav infection with improvement in hygiene of food and water has led to a gradual shift of occurrence of hav infection from childhood to adulthood . in the first view it is good , but
with this condition children remain without immunity to hav and we know that hav infection in adulthood is more severe than childhood ( 20 , 23 , 24 )
. combination of hav vaccine with hepatitis b virus vaccine , as a mandatory vaccine in some healthcare systems ( 25 ) and applying hav vaccination in non - immune healthcare personnel ( based on the previous screening ) are some of the cost - effective methods for hav prevention ( 26 ) .
it is a 27 nanometer rna virus , containing 7474 nucleotides , positive stranded , non - enveloped and icosahedral virus .
four genotypes have been identified for hav in humans that are a single serotype without biological differences .
the spread route for hav is fecal - oral and risk factors are travel to endemic areas , near contact with an infected person , homosexual activity , waterborne , being in a daycare center and injection drug use .
the infection , as an important public health problem , occurs worldwide with more prevalence in low socioeconomic areas ( 1 , 2 , 9 ) . in a population study in tehran province of iran ,
the overall rate of anti - hav seroprevalence has been reported as 90% ( 10 ) .
also a gradual shift for age of hav infection from childhood to adulthood has been reported .
control of some of the aforementioned risk factors can be the reason of this gradual shift ( 9 ) .
hav causes an acute illness that is self - limited , but it can cause fulminant hepatitis rarely in some patients .
manifestation of hepatitis a infection could be silent or with non - specific symptoms in children , but it can cause a mild flu - like illness to hepatic failure in adults ( 11 ) .
considering different causes of acute hepatitis , diagnosis of hav infection based on clinical manifestations is not possible ( 12 ) .
detecting serum hav - rna using pcr amplification is an expensive method and usually used for research purposes .
however , assessment of immunoglobulin ( ig ) antibody to hav has been suggested for diagnosis of hav infection .
igm anti - hav anti - body emerges one to two weeks after exposure to virus and disappears after 3 to 6 months , so diagnose of acute phase of hav infection can be possible .
positive results for testing igg hav anti - body also shows previous history of hav infection ( 8 , 13 ) .
fulminant hepatic failure is an ominous complication of hepatitis a and in this case , liver transplantation is a suggested option .
approximately 30% of symptomatic patients admitted to hospital for management of complications including dehydration , severe prostration , coagulopathy , encephalopathy and other evidences of hepatic decompensation ( 14 ) .
intramuscular administration of hav immune globulin provides passive immunization , which is short - term and temporary and usually takes from three to five months .
passive immunization has its special indications like persons travelling to or working in countries and communities with intermediate and high rate of hav infection and etc .
however , it is not related to the topic of this review and more information can be obtained from related review articles ( 8 , 15 , 16 ) .
also there is another vaccine , a combined vaccine namely twinrix , which contains both hav and hepatitis b antigens ( 17 ) .
the two mentioned types of vaccines has been approved and used in the united states . both of them are administered intramuscularly and as two separated injections with a six - month interval .
it is said that after one month of receiving two doses of these vaccines , about 100% of immunocompetent vaccinated subjects can earn a probable lifelong immunity against hav infection ( 8 , 17 ) .
nowadays , we know that vaccination is an important and effective preventive method for decreasing fulminant hepatitis . in a study in the united states , vaccination rate for hav among healthcare personnel was higher than general population , however overall protection has remained suboptimal ( 18 ) .
though , selecting an appropriate preventive strategy in each area depends on evaluation of some issues related to that area and they are annual incidence rate of fulminant hepatitis due to hav infection , hav seropositivity in different age and occupation groups , costs for hospitalization and treatment of patients with hav infection .
these are on one side of the equation and the costs for providing hav vaccines and running vaccination program are on the other side .
furthermore , gross national income of each country is the major factor that influences this equation ( 19 - 22 ) .
therefore , each country or territory needs to run some cost - effectiveness studies for making a decision about running hav vaccination program ( 23 ) . here is the other epidemiologic factor that should be considered in vaccination program of each country , especially developing countries like iran .
controlling risk factors for hav infection with improvement in hygiene of food and water has led to a gradual shift of occurrence of hav infection from childhood to adulthood . in the first view it is good , but with this condition children remain without immunity to hav and we know that hav infection in adulthood is more severe than childhood ( 20 , 23 , 24 ) .
combination of hav vaccine with hepatitis b virus vaccine , as a mandatory vaccine in some healthcare systems ( 25 ) and applying hav vaccination in non - immune healthcare personnel ( based on the previous screening ) are some of the cost - effective methods for hav prevention ( 26 ) .
data about hav prevalence among iranian hcws are very limited . in a cross - sectional study in a hospital from babol ( a city of mazanadaran province of iran ) between 2011 and 2012
, 466 hcws ( paramedical technicians , nurses and nurses aid ) were tested for anti - hav antibody ( igg ) .
it was reported that 330 ( 71% ) of participants had positive results for the mentioned test . on the other hand
however , about 30% of healthcare personnel are not immuned against hav and in exposure with hav infected patients and need confident preventive strategy . as conclusion of this project , role of vaccination program in protecting susceptible hcws
, 150 personnel of a military hospital in tehran were investigated regarding previous immunity to hav infection .
this is a very considerable result in four years ago and suggests a wisdom preventive method for healthcare personnel against hav infection as authors of this project recommended hav vaccination as their solution ( 28 ) .
comparison of hav seropositivity among hcws in different times can help making a better decision about the best hav preventive method .
however , the literature has not enough data about this issue , especially in the past . in a cross - sectional project
performed about 12 years ago in a hospital of sari ( another city of mazanadaran province ) , hav seropositivity rate was reported as 90.36% for hcws ( 29 ) .
this is the maximum hav seropositivity rate for hcws among included studies in this project .
perhaps this high rate of hav immunity in comparison with recent studies emphasizes on improvement in health status , controlling hav risk factors and finally gradual shift of hav infection from childhood toward adulthood .
such important points can only be proved by cohort studies or only comparison of available cross - sectional studies with similar ones in the past .
a study in one of the medical universities of tehran province in 2015 revealed that just 34% of evaluated patients (
n = 270 ) had positive results for anti - hav anti - body test ( 30 ) .
another study in medical university of babol in 2014 showed that more than 30% of medical students were not immune against hav infection ( 31 ) .
medical students in some other provinces of iran like isfahan , kermanshah and hamedan had been evaluated for hav prevalence .
it has been reported that more than one third of these students in each university are hav seronegative ( 32 ) . as a result of occupational exposure , medical students , as future hcws , are at risk of hav infection and therefore all of these studies suggested vaccination as solution .
regardless of this factor , there are some high risk groups like travelers to endemic countries , patients with chronic liver diseases , food handler , sewage workers and etc . that are suggested for hav vaccination ( 33 , 34 ) ;
data about hav prevalence among iranian hcws are very limited . in a cross - sectional study in a hospital from babol ( a city of mazanadaran province of iran ) between 2011 and 2012
, 466 hcws ( paramedical technicians , nurses and nurses aid ) were tested for anti - hav antibody ( igg ) .
it was reported that 330 ( 71% ) of participants had positive results for the mentioned test . on the other hand
however , about 30% of healthcare personnel are not immuned against hav and in exposure with hav infected patients and need confident preventive strategy . as conclusion of this project , role of vaccination program in protecting susceptible hcws
, 150 personnel of a military hospital in tehran were investigated regarding previous immunity to hav infection .
this is a very considerable result in four years ago and suggests a wisdom preventive method for healthcare personnel against hav infection as authors of this project recommended hav vaccination as their solution ( 28 ) .
comparison of hav seropositivity among hcws in different times can help making a better decision about the best hav preventive method .
however , the literature has not enough data about this issue , especially in the past . in a cross - sectional project
performed about 12 years ago in a hospital of sari ( another city of mazanadaran province ) , hav seropositivity rate was reported as 90.36% for hcws ( 29 ) .
this is the maximum hav seropositivity rate for hcws among included studies in this project .
perhaps this high rate of hav immunity in comparison with recent studies emphasizes on improvement in health status , controlling hav risk factors and finally gradual shift of hav infection from childhood toward adulthood .
such important points can only be proved by cohort studies or only comparison of available cross - sectional studies with similar ones in the past .
a study in one of the medical universities of tehran province in 2015 revealed that just 34% of evaluated patients (
n = 270 ) had positive results for anti - hav anti - body test ( 30 ) .
another study in medical university of babol in 2014 showed that more than 30% of medical students were not immune against hav infection ( 31 ) .
medical students in some other provinces of iran like isfahan , kermanshah and hamedan had been evaluated for hav prevalence .
it has been reported that more than one third of these students in each university are hav seronegative ( 32 ) . as a result of occupational exposure , medical students , as future hcws , are at risk of hav infection and therefore all of these studies suggested vaccination as solution .
regardless of this factor , there are some high risk groups like travelers to endemic countries , patients with chronic liver diseases , food handler , sewage workers and etc . that are suggested for hav vaccination ( 33 , 34 ) ;
a gradual shift for occurrence of hav infection from childhood toward adulthood has been reported in iran . on the other hand , hav infection can be more severe when it occurs in adulthood .
healthcare personnel are at a more risk to be infected with hav due to occupational exposure ( 23 , 35 ) .
hav vaccination is an effective method for preventing morbidity and mortality due to fulminant hepatitis .
it also helps to decrease the spread of hav in the community and subsequently prevent from hav outbreaks ( 35 ) .
more original studies should be performed to investigate hav seroprevalence among hcws ( 15 ) .
limited available studies show that a considerable number of hcws are hav seronegative and therefore vaccination in this high risk special group should be considered as a wisdom method ( 24 , 35 ) . | context : hepatitis a virus ( hav ) infection is an important public health problem . it is estimated that about 1.4 million cases of hav infection occur every year worldwide .
non - immune healthcare workers ( hcws ) can be at higher risk of hav infection in comparison to general population and an appropriate preventive method should be considered for them.evidence acquisition : for finding related articles , a comprehensive search was performed in scopus , pubmed and google scholar and all appropriate combinations of following keywords were considered ; healthcare provider
, healthcare personnel , healthcare worker , nurse medical students ,
iran ,
hepatitis a and vaccination .
also we did a search in persian language in google scholar and scientific information database ( sid ) to find related persian literature.results:a gradual shift in age of hav infection has been seen from childhood toward adulthood .
data about hav seropositivity among iranian hcws are very limited .
however based on the recent studies , it seems that hav seropositivity has been reduced among hcws in comparison with the past .
all recent studies have suggested hav vaccination for hcws.conclusions:available limited studies show that iranian healthcare personnel need hav vaccination .
however , for selecting an appropriate preventive method for this high risk group , more original studies are still needed . |
dna - based therapeutics may become a new generation of drugs for the treatment of brain disorders provided that the problem of its delivery across the blood - brain barrier ( bbb ) and into brain cells is solved .
a global distribution of the transgene throughout the brain is needed for most of the enzyme replacement therapy protocols , and this could be possible by the transvascular route to brain via transport across the bbb . however , in the absence of either facilitated or receptor mediated transport systems , only lipophilic molecules of less than 400 da are able to cross the bbb by simple diffusion .
viruses have been used as brain dna delivery systems with disappointing results associated with preexisting immunity , immunological response induced by viral coat proteins , and inflammation that led to demyelination [ 515 ] .
cationic lipids are widely used for transfection of dna in in vitro tissue culture models .
however , cationic lipid - dna complexes in vivo are unstable or form large molecular weight aggregates that deposit in the pulmonary vascular bed [ 1618 ] , which decreases its bioavailability for delivery to the brain . an alternative approach for dna delivery to the central nervous system ( cns )
trojan horse liposome ( thl ) technology [ 3 , 4 , 1923 ] ( figure 1(a ) ) .
the encapsulation of the transgene in the interior of a liposome protects the coding dna against degradation by ubiquitous nucleases .
any dna not fully encapsulated in the interior of the thl is removed by treatment of the thl with a mixture of exo / endonucleases .
the thl is constructed with polyethylene glycol- ( peg- ) conjugated lipids , and the peg strands on the surface of the thl stabilizes the liposome in vivo and increases the plasma residence time [ 24 , 25 ] .
a small fraction of the peg molecules , that is , 1 - 2% , carry a terminal maleimide functional group to allow for conjugation of the liposome surface with thiolated targeting ligands .
the targeting ligand acts as a molecular trojan horse ( mth ) and is directed at an endogenous bbb receptor / transporter , such as the insulin receptor ( ir ) or transferrin receptor ( tfr ) receptor ( table 1 ) [ 3 , 4 , 1923 ] .
the extension of the peg - conjugated mab from the surface of the thl is illustrated by electron microscopy ( figure 1(b ) ) .
the ir or tfr are also expressed on the plasma membrane of brain cells , which enables the thl to traverse the brain cell membrane following delivery across the bbb ( figure 1(c ) ) .
mabs against the ir or tfr are almost always species specific , and a mab against the mouse tfr will not recognize the tfr on human cells .
therefore , in mixed animal models such as a brain tumor model produced by the intracranial growth of a human glioma in the mouse , a combination of targeting mabs is used , so that the thl is targeted across both the mouse bbb and the human tumor cell membrane .
for example , thls were constructed with a mab to the mouse tfr , to target the thl complex across the mouse bbb , and with a second mab against the human insulin receptor ( hir ) , to target the thl across an intracranial human u87 glioma , as illustrated in figure 1(a ) . with the development of genetically engineered forms of the hirmab
the engineering of plasmid dna encoding the therapeutic transgene under the influence of brain cell - specific promoters eliminates ectopic transgene expression and enables transgene expression in targeted regions of the cns [ 2 , 1923 , 27 , 28 ] .
thls are pegylated liposomes containing a supercoiled plasmid dna molecule in the interior of the liposome ( figure 1(a ) ) .
thls are engineered with a mixture of naturally occurring lipids that has been optimized for the encapsulation of plasmid dna [ 4 , 19 ] .
the liposomes are comprised of 93% 1-palmitoyl-2-oleoyl - sn - glycerol-3-phosphocholine ( popc ) , 3% didodecyldimethylammonium bromide ( ddab ) , 3% distearoylphosphatidylethanolamine ( dspe)-peg2000 , and 1% dspe - peg2000-maleimide .
the maleimide functional group allows for covalent conjugation of a thiolated mab via a stable thioether linkage ( figure 1(a ) and table 1 ) .
a panel of species - specific peptidomimetic mabs has been developed ( table 1 ) , and their efficacy in delivering thls to brain has been demonstrated in experimental animal models in vivo [ 1 , 3 , 4 , 1923 , 27 ] . the 83 - 14 murine mab to the hir and the ox26 murine mab to the rat tfr are used to target human and rat tissues , respectively ( table 1 ) .
the ox26 tfrmab is active only in rats , so the rat 8d3 mab against the mouse tfr is used in mice ( table 1 ) [ 20 , 21 , 2733 ] .
the 83 - 14 hirmab does not cross - react with the insulin receptor in rodents or even new world primates such as the squirrel monkey .
however , this hirmab does cross - react with the insulin receptor of old world primates such as the rhesus monkey .
since the plasmid dna must be delivered to the nuclear compartment of brain cells , the thl must traverse both the bbb and the brain cell plasma membrane ( bcm ) behind the bbb ( figure 1(c ) ) .
owing to high expression of the tfr or ir on both the bbb and bcm barriers , the targeting mab enables the sequential receptor - mediated transcytosis of the thl across the bbb followed by the receptor - mediated endocytosis of the thl into the brain cell ( figure 1(c ) ) .
thls have also been successfully constructed to target human tumor cells in a scid mouse model wherein dual targeting mabs were directed to the mouse tfr and hir ( table 1 and figure 1(a ) , i.e. , mab1 and mab2 ) .
in vivo applications of thls were initially investigated with luciferase and lacz reporter genes in vivo [ 3 , 4 , 20 , 21 , 34 ] .
thls were constructed with the expression plasmid of interest ( i.e. , luciferase reporter gene ) and engineered with either the tfrmab for rodents or the hirmab for rhesus monkeys , respectively .
the doses of dna encapsulated in thls and administered iv was 5 or 70 g per rat or primate , respectively , which are equivalent 20 and 12 g / kg body weight , respectively .
when the transgene is driven by the widely read sv40 promoter , the levels of luciferase were 10 pg luciferase / mg protein in the monkey brain .
high levels of expression were also seen in peripheral tissues that are rich in the target receptor , including liver , spleen , and lung [ 27 , 34 ] . a 50-fold increase in the tissue levels of luciferase
the high levels of expression in monkey tissues were associated with intrinsic properties of the hirmab that targets the nuclear compartment of the cell .
time course studies in both rodents and primates demonstrated that the peak of luciferase expression occurs 48 hs following injection of a single iv dose of thls .
there are 2 potential mechanisms for the decline in the expression of the transgene , that is , promoter inactivation and plasmid degradation .
the levels of both luciferase enzyme activity and plasmid dna decay in the primate brain and liver were measured , and both processes decayed with a t1/2 of approximately 2 days , which indicates that the transient duration of the luciferase gene expression is mainly due to plasmid degradation .
the organ distribution of the lacz transgene was also investigated at the cellular level with histochemistry following thl delivery of a reporter gene driven by the sv40 promoter and designated sv40-lacz [ 4 , 20 , 34 ] .
the latter was used to engineer thls with either the tfrmab or the hirmab ( table 1 ) .
the histochemical detection of the -galactosidase is shown in figure 2 for the mouse and the rhesus monkey .
the expression of the transgene was widely detected through the cortical and subcortical structures of mouse and monkey brain , with a greater gene expression in gray matter relative to white matter in both cerebrum and cerebellum ( figure 2 ) .
on the contrary , the -galactosidase histochemistry of control uninjected primate brain shows no -galactosidase activity ( figure 2(b ) ) .
light micrographs of the primate brain shows gene expression within the choroid plexus epithelium ( figure 2(d ) ) and the capillary endothelium within white matter ( figure 2(f ) ) .
the gene expression was also confirmed within the neurons of the occipital cortex showing the columnar organization of this region in primate brain ( figure 2(e ) ) .
the molecular and granular layers of the cerebellum and the purkinje cells were also positive for the transgene ( figure 2(f ) ) .
confocal microscopy studies with antibodies against either bacterial -galactoside or the neuronal neun marker colocalized transgene expression in the neuronal compartment of brain [ 27 , 34 ] .
the ectopic expression of the -galactosidase with the sv40-lacz vector was also observed in tissues expressing either the tfr or the ir , such as spleen , providing that the transgene was driven by the widely read sv40 promoter ( figure 2 , top left panel ) .
it is possible to produce thls that carry plasmid dna engineered with a tissue - specific promoter .
this is of particular relevance in gene therapy protocols wherein ectopic expression of the transgene is not desired when the lacz expression plasmid is driven by the brain - specific promoter derived from the 5-flanking sequence of the glial fibrillary acid protein ( gfap ) gene , the expression of -galactosidase in brain was widely detected ( figure 2 , top right panel ) , as previously seen with the sv40-lacz plasmid ( figure 2 , top left panel ) . on the contrary , there was no expression of the transgene in peripheral tissues ( figure 2 , bottom right panel ) when the transgene was under the influence of the brain - specific gfap promoter .
tissue - specific gene expression with the combined use of thls and the opsin promoter was demonstrated in vivo in the rhesus monkey .
the in vivo efficacy of thls was investigated in a model of type vii mucopolysaccharidosis ( mps ) , which is caused by mutations in the gene encoding the lysosomal enzyme -glucuronidase ( gusb ) .
mps is a lysosomal storage disorder , and the majority of lysosomal storage disorders adversely affect the central nervous system . therefore , therapeutic transgenes must be delivered to all parts of the brain , and this is only possible with a transvascular route to brain .
thls were prepared with a plasmid dna encoding for gusb , and with the tfrmab to target thls across both the bbb and the bcm in a transgenic mouse model of mps - vii .
the gusb expression plasmid , designated pcmv - gusb , is driven by the widely read cytomegalovirus ( cmv ) promoter .
the latter was preferred over a brain - specific promoter , as mps - vii affects both the cns and peripheral tissues .
the gusb enzyme activity was investigated in cultured fibroblasts obtained from gusb null mice [ gusb( ) ] , and > 50-fold increase in the gusb activity was observed following incubation with the thl carrying the pcmv - gusb as compared with control gusb( ) fibroblasts ( figure 3(a ) ) .
the gusb enzyme activity persisted at high levels for over 2-week period . in vivo studies in gusb null mice
were also performed with a single dose of 10 ug / mouse of pcmv - gusb encapsulated in tfrmab - targeted thls .
the gusb enzyme activity in brain and peripheral organs was determined 48 hours after iv injection .
gusb enzyme activity was increased > 10-fold in brain , liver , spleen , lung , and kidney , but not in heart ( figure 3(b ) ) .
the expression pattern of gusb gene among mouse organs in vivo is consistent with the local expression of the tfr in the vascular barriers of these tissues .
the liver and spleen are perfused with fenestrated capillaries that are highly porous , so the 100 nm thls can freely cross their vascular barrier .
thus , the observation that gusb enzyme activity is increased in lung and kidney with tfrmab - targeted thls in vivo provides additional evidence for the expression of the tfr on the vascular barrier in these organs in the mouse .
lack of expression in heart supports prior work with reporter genes showing that tfrmab - targeted thls are not delivered across the vascular barrier in heart [ 20 , 21 , 27 ] .
the brain gusb enzyme activity observed at 48 h after a single thl administration approximated 2 u / mg protein ( figure 3(b ) ) , which represents 55% of the brain level in heterozygotes .
since the replacement of just 15% of lysosomal enzyme activity in an organ may be sufficient to cause therapeutic effects and a reversal of lysosomal storage disease , the levels of gusb enzyme activity generated in the brain of null mice with a single iv injection of thls is within the therapeutic range .
the plasmid dna is expressed episomally in brain cells without integration into the host genome .
therefore , long - term treatment of lysosomal storage disorders with intravenous administration of thls will require repeat administration of the gene medicine at intervals that are determined by both the persistence of transgene expression and the turnover of the expressed protein in brain and peripheral organs .
the therapeutic efficacy of thls was demonstrated in vivo in a model of parkinson 's disease ( pd ) , wherein the therapeutic gene encoded for tyrosine hydroxylase ( th ) .
pd is associated with a loss of dopaminergic neurons in the substantia nigra , which terminate in the striatum [ 40 , 41 ] .
the rate limiting enzyme in the synthesis of dopamine is th , and a potential treatment for pd is th gene replacement therapy .
studies were performed in the rat 6-hydroxydopamine ( 6-ohda ) model , and with thl packaged with a th expression plasmid driven by the gfap brain - specific promoter , designated clone 951 .
gfap - th - thls were constructed with the ox26 mab to target the rat tfr ( table 1 ) .
the intracerebral injection of 6-ohda produced a 98% reduction in the levels of th in the ipsilateral striatum as compared with the contralateral or nonlesioned control animals ( table 2 ) .
animals with positive lesion were identified by apomorphine - induced contralateral rotation and administered 10 g clone 951 dna encapsulated in either ox26-thl or in thls targeted with a non - specific isotype control igg1a mouse igg , as a negative control .
the apomorphine - induced contralateral rotation test was used to demonstrate therapeutic efficacy . in the negative control group administered with the 951-thls targeted with the non - specific igg2a , the drug - induced rotation increased in all animals . on the contrary ,
in the rats injected with the 951-thls targeted with the tfrmab , there was an 82% reduction in the apomorphine - induced contralateral rotations .
the therapeutic effect of the th gene replacement was correlated with the levels of th determined by enzyme activity ( table 2 ) or immunocytochemistry ( figure 4 ) .
the latter was performed in coronal sections of brain and showed complete normalization of the immunoreactive th in the striatum of 6-ohda lesioned rats 3 days after a single injection of the gene therapy ( figures 4(a)4(c ) ) .
in contrast , lesioned control animals treated with the thls targeted with the non - specific igg2a isotype control antibody show a marked reduction in striatal immunoreactive th ( figures 4(d)4(f ) ) .
the levels of the th enzyme activity were also normalized in the ipsilateral striatum ( table 2 ) .
additional studies were performed in the 6-ohda pd rat model with thls carrying the th gene under the widely read sv40 promoter , that is , clone 877 ( table 2 ) .
similar data were obtained in both the restoration of the th expression pattern in brain and in the reduction of the apomorphine - induced contralateral rotation . the only difference between the studies performed with the th expression plasmid driven by the sv40 promoter , or the gfap promoter , was a 10-fold increase in the levels of th activity in liver of animals injected with the sv40-th construct , which is not seen with the gfap - th plasmid ( table 2 and figure 2 ) .
the stability of the th is associated with the availability of the biopterin cofactor , and the expression of the th enzyme is found in regions of the brain that express gtp cyclohydrolase 1 ( gtpch ) [ 4244 ] .
the gtpch is also expressed in peripheral tissues , like liver , which supports the increased expression in liver th activity when the th transgene is driven by the sv40 promoter ( table 2 ) .
the gene therapy in this pd model with either sv40- or gfap - th plasmids produced normalization of the expression pattern of th and without expression of supranormal levels of th activity ( table 2 ) [ 22 , 30 ] .
this observation parallels findings observed in th transgenic mice , which showed only a minor increase in either immunoreactive th or th activity in striatum despite a 50-fold increase in the level of th mrna in the substantia nigra .
the latter , in conjunction with the th gene therapy with thls ( table 2 and figure 4 ) , suggests that the expression of the th gene is regulated at the posttranscriptional level in brain , so that the striatal th activity is maintained within a narrow range and by neurons expressing the gtpch cofactor gene .
as discussed above for the gusb gene therapy , the plasmid dna in thl is not integrated into the host genome .
therefore , long - term treatments with repetitive intravenous administration of thls are needed to produce a long - term therapeutic effect .
the engineering of plasmid dna vectors that incorporate intronic or chromosomal - derived gene elements may produce more sustained expression of the transgene following thl delivery .
a series of 4 rat th expression plasmids , designated clone 877 , prgth2 , prgth3 , and prgth4 , were derived from the rat th gene or cdna , as outlined in figure 5(a ) .
clone prgth2 encodes a 12 kb th genomic expression cassette , which includes a 3.0 kb th 5-flanking sequence ( fs ) , the 7.3 kb rat th coding region , and a 1.9 kb 3-fs .
the 3 kb rat th 5-fs in prgth2 was expanded to 8.4 kb with the engineering of clone prgth3 .
the introns and 3-fs were eliminated by engineering clone prgth4 ( figure 5(a ) ) .
the cdna form of th gene therapy , clone 877 , caused a 26-fold increase in striatal th enzyme activity at 3 days after the iv injection , but this declined over 12-fold by 10 days ( table 3 ) .
there was a significant 86% improvement in motor function at 3 days after the injection of clone 877 , but this improvement was not significant at 6 and 10 days after the single iv injection ( table 3 ) .
the genomic th expression plasmids produced in general a lower peak of striatal th enzyme activity in vivo , but a more lasting therapeutic effect .
striatal th enzyme activity at 3 days after iv injection of prgth2 , or prgth4 , was less than that observed with clone 877 , but the striatal th enzyme activity at 10 days after injection with prgth2 was significantly higher than with clone 877 ( table 3 ) .
the iv administration of prgth3 resulted in no significant increase in striatal th enzyme activity at 3 or 6 days after administration , relative to clone 877 or prgth2 , but yielded the highest striatal th enzyme activity , and the lowest drug - induced rotation , of any single therapy at 10 days after administration ( table 3 ) .
therefore , the clone 877 cdna form of th gene therapy is fast acting with short duration , whereas the genomic form of th gene therapy with prgth3 is slow acting with long duration ( table 3 ) .
the combination cdna / genomic th gene therapy was further investigated with thls carrying both 877 and prgth3 plasmids ( figures 5(b ) and 5(c ) ) .
the striatal th enzyme activity was significantly higher with the combination gene therapy as compared to clone 877 alone at 10 days after injection , and it was significantly higher as compared to prgth3 alone at 3 and 6 days after injection ( figure 5(b ) ) .
the combination therapy also produced a parallel reduction in apomorphine rotation behavior ( figure 5(c ) ) .
the rotation behavior was significantly reduced with combination gene therapy as compared to clone 877 alone at 10 days after injection , and it was significantly reduced as compared to prgth3 alone at 3 days after injection . in summary , combination gene therapy is superior to single cdna gene therapy .
the combination gene therapy using both short - acting cdna - derived th transgene and long - acting genomic - derived th transgene provides a more sustained therapeutic duration in experimental pd as compared to single gene therapy using either cdna - derived or genomic - derived transgene .
plasmid dna - based gene therapy with thl technology involves episomal gene expression and must be given on a chronic basis , which raises concerns about potential toxic side effects from chronic repeat thl dosing .
a 6-week toxicological study was conducted with repeated weekly intravenous administration of thls carrying a 7 kb expression plasmid encoding for rat th and targeted with either the ox26 mab to the rat tfr or with the mouse igg2a isotype control antibody .
animals were divided into 3 treatment groups : ( a ) saline , ( b ) 5 ug dna / week of the thls targeted with the tfrmab , and ( c ) 5 ug dna / week of the thls targeted with the nonspecific isotype control igg2a antibody . at the end of 6 weeks of chronic weekly treatment
, there was no measurable differences in the 3 groups with respect to body weights , 14 serum chemistries ( table 4 ) , or organ histology of brain , liver , spleen , kidney , heart , or lung .
the immunocytochemistry showed no evidence of inflammation in brain using antibodies that react with multiple components of the immune system .
these results demonstrate the lack of toxicity of chronic dosing of mab - targeted thls carrying plasmid dna .
the efficiency of gene delivery to the brain and gene expression in target cells with thls may be potentially enhanced by optimizing the formulation of thls .
the use of ethanol - mediated dna condensation was recently shown to increase the efficiency of dna encapsulation in thls , and the polymer polyethylenimine ( pei ) allowed for encapsulation of pei / oligodeoxynucleotide polyplexes in thls .
mabs directed to the mouse or rat tfr and the human hir are the most potent bbb trojan horses developed to date for drug delivery across the mouse , rat , or primate bbb , respectively [ 26 , 5355 ] , and the thl technology has been validated in numerous animals models ( see above ) .
as new targeting molecules with increased brain uptake , as compared to tfr- and hir - mab , become available , it may also be possible to engineer thls with improved brain uptake and therapeutic efficacy .
other ligands have been tested in the construction of dna liposomes , but demonstrated limitations in terms of specificity and/or global distribution of the transgene in the brain .
tat - peptide - modified liposomes were able to target human brain tumors in mice , but not the normal brain adjacent to the tumor .
immunoliposomes labeled with anti - gfap mab targeted gliomas that had disruption of the bbb , but they were unable to penetrate unimpaired bbb .
glycosylation of dna lipoplexes and liposomes have been proposed to increase biodistribution most likely via absorptive endocytosis [ 58 , 59 ] ; however , the application of these constructs to gene delivery to the brain remains to be demonstrated .
the thl plasmid dna gene transfer technology has been validated in multiple animal models in mice , rats , and rhesus monkeys , and this work shows that it is possible to deliver transgenes to brain following the noninvasive intravenous administration of nonviral formulations . the ectopic expression of the transgene is shown to be eliminated by the combined use of thls and plasmid dna engineered with tissue - specific gene promoters .
transgene expression following thl delivery is reversible secondary to degradation of the plasmid dna , which is not integrated into the host genome .
this nonintegrating property of plasmid dna is considered advantageous , since the integration of viral genomes into the host dna can lead to insertional mutagenesis .
increase in the duration of plasmid dna expression is possible with the engineering of plasmid dna that incorporates chromosomal elements .
thls can be administered chronically without toxicity or immune reactions the thl technology can be translated to humans with the use of human - specific antibodies that are genetically engineered to reduce immunogenicity .
the murine hirmab , which is active at the human bbb , has been genetically engineered , and a humanized hirmab has been produced .
therefore , it is possible to produce thls with the humanized hirmab for gene transfer to the human brain ( table 1 ) . | the application of blood - borne gene therapy protocols to the brain is limited by the presence of the blood - brain barrier ( bbb ) .
viruses have been extensively used as gene delivery systems . however , their efficacy in brain is limited by the lack of transport across the bbb following intravenous ( iv ) administration .
recent progress in the trojan horse liposome
( thl ) technology applied to transvascular non - viral gene therapy of the brain presents a promising solution to the trans - vascular brain gene delivery problem .
thls are comprised of immunoliposomes carrying nonviral gene expression plasmids .
the tissue target specificity of the thl is provided by peptidomimetic monoclonal antibody ( mab ) component of the thl , which binds to specific endogenous receptors located on both the bbb and on brain cellular membranes , for example , insulin receptor and transferrin receptor .
these mabs mediate ( a ) receptor - mediated transcytosis of the thl complex through the bbb , ( b ) endocytosis into brain cells and ( c ) transport to the brain cell nuclear compartment .
the expression of the transgene in brain may be restricted using tissue / cell specific gene promoters .
this manuscript presents an overview on the thl transport technology applied to brain disorders , including lysosomal storage disorders and parkinson 's disease . |
adjuvant radiotherapy ( rt ) after conservative surgery for early stage breast cancer is a standard of care that is well tolerated by the patients in terms of compliance and risk of adverse effects . in most cases ,
breast cancer patients have a long life expectancy with a risk of late normal tissue complications that may continue to accumulate years after treatment concludes .
for this reason , late effects remain important health concerns for long - term survivors .
common late toxicity manifestations in the breast include fibrosis , cutaneous atrophy and skin telangiectasia . in particular , fibrosis is the development of excess fibrous connective tissue due to fibroblasts proliferation because of tissue injury .
there is considerable interindividual variability in the development of late toxicity , which depends on rt parameters such as total dose , dose per fraction , irradiated volume , and dose inhomogeneity , as well as on patient related factors such as age and life style factors . in the last decade , several studies have suggested that development of late normal tissue complications in the breast after rt is at least in part genetically determined . currently , clinical radiosensitivity is nowadays regarded as a complex polygenic trait resulting from the combined effects of multiple factors , each with relatively modest effects . therefore , an approach based on the combination of multiple genetic factors into a genetic risk score ( grs ) may be an attractive strategy for prediction of adverse rt effects .
dna methylation is an epigenetic mechanism established and maintained by a family of dna methyltransferase ( dnmt ) enzymes that catalyze the addition of a methyl group to cytosine residues , using s - adenosylmethionine as the methyl group donor .
dna methylation generally occurs on cytosine residues located in cpg dinucleotides within the promoter region , resulting in chromatin compaction and gene silencing .
three active dnmts have been described in mammals : dnmt1 is required for maintenance of methylation through generations , while dnmt3a and dnmt3b are mainly involved in establishment of de novo dna methylation patterns . in vitro and in vivo experimental evidence
suggests that enzymes of the methylation machinery play a role in fibrogenesis and radiation response .
for instance , upregulation of dnmt1 has been detected in the fibrotic tissue of the skin , kidneys , lungs , and liver , whereas activation of myofibroblasts or hepatic stellate cells can be reversed via inhibition of dnmt1 by dna - demethylating drugs or by specific sirna shutdown .
in addition , reduction of global methylation levels has been reported after irradiation , probably due to decreased expression of dnmt1 , dnmt3a , and dnmt3b . despite evidence of involvement of dna methylating enzymes in fibrogenesis and radiation response ,
no information is currently available regarding whether common genetic variants of dnmts genes contribute to the development of radiation - induced fibrosis in cancer patients .
however , recent in vitro studies suggest that mitochondria are the primary loci of rt effects , and that mitochondrial dna haplogroups differently affect mrna expression of dnmt1 , dnmt3a , and dnmt3b , as well as global dna methylation levels . in the present study , we assessed the role of four single nucleotide polymorphisms ( snps ) of dnmt genes ( dnmt1 rs2228611 , dnmt3a rs1550117 , dnmt3a rs7581217 , and dnmt3b rs2424908 ) as risk factors for subcutaneous fibrosis in a cohort of italian breast cancer patients who received rt after breast conserving surgery .
in addition to dnmt snps , we evaluated the predictive role of xrcc1 rs2682585 , which was previously reported to be associated with the standardized total average toxicity ( stat ) score , an index of overall toxicity combining skin toxicities and fibrosis of the breast .
we also assessed the ability of the aforementioned snps to improve prediction accuracy when combined with mitochondrial haplogroup h , which we recently found to be independently associated with a lower hazard of radiationinduced fibrosis in breast cancer patients .
this study included 286 caucasian patients affected by histologically confirmed breast cancer who underwent conservative surgery and adjuvant rt from 1989 to 2010 at our department of radiotherapy .
briefly , rt consisted of two opposite tangential wedged beams , followed by a boost on the tumor bed .
patients underwent whole breast rt with conventional fractionation to a total dose of 50 gy followed by boost dose on the tumor bed in cases of invasive tumors . at the time of patient recruitment , a peripheral blood sample
was taken and stored at 4c until analysis . during annual follow - up visits ( last update on january 2015 ) , radiation oncologists evaluated the appearance of subcutaneous and cutaneous late toxicities , with particular attention to the onset of fibrosis
toxicity was scored according to the late effects of normal tissue - subjective objective management analytical ( lent - soma ) scale .
patients with moderate to severe fibrosis ( grade 2 ) were referred to as the " radiosensitive group " and compared to patients with no or minimal fibrotic reactions ( grade 0 - 1 , control group ) .
this study was approved by the local ethics committees of our university hospital and met the requirements of the declaration of helsinki .
determination of snps was conducted on genomic dna by real - time polymerase chain reaction ( pcr ) using the following taqman pre - designed snp genotyping assays ( applied biosystems , milan , italy ) : c_27838930_10 ( dnmt1 rs2228611 ) ; c_8722920_10 ( dnmt3a rs1550117 ) ; c_7863728_10 ( dnmt3a rs7581217 ) ; c_16013055_10 ( dnmt3b rs2424908 ) ; and c_16269889_10 ( xrcc1 rs2682585 ) . real - time pcr amplification and detection was performed in 96-well pcr plates using a cfx connect real - time pcr detection system ( bio - rad , milan , italy ) .
each polymorphism was tested for deviation from the hardy - weinberg equilibrium ( hwe ) by use of pearson s chisquared test as implemented in finetti s program ( http://ihg.gsf.de/cgi-bin/hw/hwa1.pl ) .
for the selected polymorphisms , we considered the co - dominant , dominant , and recessive modes of inheritance . the time to event end - point ( grade 2 fibrosis )
was calculated from the first session of rt , and patients not experiencing the end - point were censored at the last follow - up performed .
the cumulative incidence of grade 2 fibrosis was calculated by the kaplan - meier method and comparisons between genotype groups were performed using the log - rank test .
univariate cox regression analyses were performed to calculate the hazard ratio ( hr ) and the 95% confidence interval ( ci ) to evaluate the influence of genotypes on grade 2 fibrosis risk .
multivariate adjustments were made for the mitochondrial haplogroup ( h vs. non - h ) and clinical variables having a cut - off p - value of < 0.20 in univariate cox analysis ( body mass index , breast diameter , adjuvant treatment , dose per fraction , radiation quality , acute skin toxicity , and postsurgical complications ) . to avoid the risk of over - fitting biases , only significant genetic factors were considered for construction of a grs . to accomplish this ,
the cox proportional hazard regression coefficient of each genetic factor was converted into an integer risk score by rounding the quotient and dividing the regression coefficient by a single constant .
discrimination capabilities of genetic models were assessed using the area under the receiver operating characteristic curve ( roc ) .
the area under the roc curve and comparisons between roc curves were calculated using the method described by delong et al . .
because of the exploratory nature of this study , we reported nominal statistical associations ( p < 0.05 ) .
adjusted p - values based on the bonferroni correction were also considered to avoid chance findings due to multiple testing of five snps , and the significance was lowered to p < 0.01 .
this study included 286 caucasian patients affected by histologically confirmed breast cancer who underwent conservative surgery and adjuvant rt from 1989 to 2010 at our department of radiotherapy .
briefly , rt consisted of two opposite tangential wedged beams , followed by a boost on the tumor bed .
patients underwent whole breast rt with conventional fractionation to a total dose of 50 gy followed by boost dose on the tumor bed in cases of invasive tumors . at the time of patient recruitment , a peripheral blood sample
was taken and stored at 4c until analysis . during annual follow - up visits ( last update on january 2015 ) , radiation oncologists evaluated the appearance of subcutaneous and cutaneous late toxicities , with particular attention to the onset of fibrosis
toxicity was scored according to the late effects of normal tissue - subjective objective management analytical ( lent - soma ) scale .
patients with moderate to severe fibrosis ( grade 2 ) were referred to as the " radiosensitive group " and compared to patients with no or minimal fibrotic reactions ( grade 0 - 1 , control group ) .
this study was approved by the local ethics committees of our university hospital and met the requirements of the declaration of helsinki .
determination of snps was conducted on genomic dna by real - time polymerase chain reaction ( pcr ) using the following taqman pre - designed snp genotyping assays ( applied biosystems , milan , italy ) : c_27838930_10 ( dnmt1 rs2228611 ) ; c_8722920_10 ( dnmt3a rs1550117 ) ; c_7863728_10 ( dnmt3a rs7581217 ) ; c_16013055_10 ( dnmt3b rs2424908 ) ; and c_16269889_10 ( xrcc1 rs2682585 ) .
real - time pcr amplification and detection was performed in 96-well pcr plates using a cfx connect real - time pcr detection system ( bio - rad , milan , italy ) .
each polymorphism was tested for deviation from the hardy - weinberg equilibrium ( hwe ) by use of pearson s chisquared test as implemented in finetti s program ( http://ihg.gsf.de/cgi-bin/hw/hwa1.pl ) . for the selected polymorphisms , we considered the co - dominant , dominant , and recessive modes of inheritance . the time to event end - point ( grade 2 fibrosis )
was calculated from the first session of rt , and patients not experiencing the end - point were censored at the last follow - up performed .
the cumulative incidence of grade 2 fibrosis was calculated by the kaplan - meier method and comparisons between genotype groups were performed using the log - rank test .
univariate cox regression analyses were performed to calculate the hazard ratio ( hr ) and the 95% confidence interval ( ci ) to evaluate the influence of genotypes on grade 2 fibrosis risk .
multivariate adjustments were made for the mitochondrial haplogroup ( h vs. non - h ) and clinical variables having a cut - off p - value of < 0.20 in univariate cox analysis ( body mass index , breast diameter , adjuvant treatment , dose per fraction , radiation quality , acute skin toxicity , and postsurgical complications ) . to avoid the risk of over - fitting biases , only significant genetic factors were considered for construction of a grs . to accomplish this ,
the cox proportional hazard regression coefficient of each genetic factor was converted into an integer risk score by rounding the quotient and dividing the regression coefficient by a single constant .
discrimination capabilities of genetic models were assessed using the area under the receiver operating characteristic curve ( roc ) .
the area under the roc curve and comparisons between roc curves were calculated using the method described by delong et al . .
13.3.3 ( medcalc software , mariakerke , belgium ) software . because of the exploratory nature of this study , we reported nominal statistical associations ( p < 0.05 ) .
adjusted p - values based on the bonferroni correction were also considered to avoid chance findings due to multiple testing of five snps , and the significance was lowered to p < 0.01 .
overall , 51 of the 286 participants ( 17.8% ) experienced moderate to severe fibrosis ( lent - soma grade 2 ) , while 235 patients ( 82.2% ) had no or minimal fibrosis ( lentsoma grade 0 - 1 ) .
detailed demographic and clinical data in the whole cohort of breast cancer patients and following stratification according to their radiosensitive status have been reported elsewhere .
the distributions of genotype frequencies for the polymorphisms analyzed were in hwe ( all p > 0.05 ) .
for the entire set of breast cancer patients , frequencies of minor variant alleles were 0.48 ( dnmt1 rs2228611 g ) , 0.09 ( dnmt3a rs1550117a ) , 0.35 ( dnmt3a rs7581217 t ) , 0.14 ( dnmt3b rs2424908 t ) , and 0.22 ( xrcc1 rs2682585 ) .
kaplan - meier curves for dnmt1 rs2228611 showed differences among genotypes in the cumulative incidence of grade 2 subcutaneous fibrosis ( log - rank test p - value=0.018 ) ( fig .
univariate cox regression analysis revealed that dnmt1 rs2228611 was associated with a lower risk of grade 2 fibrosis under either the codominant ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.70 ; p=0.007 ) or the recessive contrast ( gg vs. aa+ag : hr , 0.31 ; 95% ci , 0.12 to 0.78 ; p=0.013 ) ( table 1 ) . multivariate cox regression analysis adjusted for mitochondrial haplogroup ( h vs. non - h ) and clinical confounding factors revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.71 ; p=0.009 ; gg vs. aa+ag : hr , 0.29 ; 95% ci , 0.12 to 0.75 ; p=0.011 ) ( table 1 ) .
conversely , none of the other dnmt snps ( table 1 ) , nor xrcc1 rs2682585 ( table 2 ) were associated with radiation - induced fibrosis of the breast upon both univariate or multivariate cox regression analysis .
it is worth noting that mitochondrial haplogroup h emerged as a significant protective factor in these multivariate models when dnmt1 rs2228611 was considered under the codominant ( hr , 0.48 ; 95% ci , 0.26 to 0.88 ; p=0.018 ) or the recessive mode of inheritance ( hr , 0.48 ; 95% ci , 0.26 to 0.89 ; p=0.019 ) . to understand the joint effects of genetic markers on individual risk of grade
2 fibrosis , we built a grs for grade 2 fibrosis based on different combinations of dnmt1 rs2228611 and/or mitochondrial haplogroups according to five cox regression models , as shown in table 3 .
model 1 included mitochondrial haplogroups only ( h vs. non - h ) , model 2 and model 3 comprised the codominant or the recessive contrast of rs2228611 , respectively , model 4 combined mitochondrial haplogroup h with the codominant contrast of dnmt1 rs2228611 and model 5 incorporated mitochondrial haplogroup h with the recessive contrast of dnmt1 rs2228611 .
we then performed roc curve analyses to determine the performance of the five models at discriminating patients with or without grade 2 fibrosis ( fig .
the lowest discrimination accuracy , quantified by area under the receiver operating characteristic curves ( auc ) , was obtained for models 2 and 3 , which were based on the two contrasts of rs2228611 ( both auc , 0.587 ; 95% ci , 0.28 to 0.645 ) , and the highest was observed for model 5 , which included the combination of mitochondrial haplogroup h with the recessive contrast of rs2228611 ( auc , 0.655 ; 95% ci , 0.597 to 0.710 ) .
2 , adding the recessive contrast of dnmt1 rs2228611 to haplogroup h determined an increase of the discrimination accuracy from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710 ) .
when applying the score developed in model 5 , the proportion of patients with grade 2 radiation - induced fibrosis for each score group showed an increasing trend from lower to higher sum risk scores : 3.4% ( score 0 ) , 10.0% ( score 1 ) , 14.0% ( score 2 ) , and 27.3% ( score 2 ) .
the kaplan - meier analysis of model 5 revealed that patients with an increasing sum risk score showed a trend towards higher frequencies of grade 2 fibrosis ( p trend=0.0005 ) ( fig .
overall , 51 of the 286 participants ( 17.8% ) experienced moderate to severe fibrosis ( lent - soma grade 2 ) , while 235 patients ( 82.2% ) had no or minimal fibrosis ( lentsoma grade 0 - 1 ) .
detailed demographic and clinical data in the whole cohort of breast cancer patients and following stratification according to their radiosensitive status have been reported elsewhere .
the distributions of genotype frequencies for the polymorphisms analyzed were in hwe ( all p > 0.05 ) .
for the entire set of breast cancer patients , frequencies of minor variant alleles were 0.48 ( dnmt1 rs2228611 g ) , 0.09 ( dnmt3a rs1550117a ) , 0.35 ( dnmt3a rs7581217 t ) , 0.14 ( dnmt3b rs2424908 t ) , and 0.22 ( xrcc1 rs2682585 ) .
kaplan - meier curves for dnmt1 rs2228611 showed differences among genotypes in the cumulative incidence of grade 2 subcutaneous fibrosis ( log - rank test p - value=0.018 ) ( fig .
univariate cox regression analysis revealed that dnmt1 rs2228611 was associated with a lower risk of grade 2 fibrosis under either the codominant ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.70 ; p=0.007 ) or the recessive contrast ( gg vs. aa+ag : hr , 0.31 ; 95% ci , 0.12 to 0.78 ; p=0.013 ) ( table 1 ) . multivariate cox regression analysis adjusted for mitochondrial haplogroup ( h vs. non - h ) and clinical confounding factors revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.71 ; p=0.009 ; gg vs. aa+ag : hr , 0.29 ; 95% ci , 0.12 to 0.75 ; p=0.011 ) ( table 1 ) .
conversely , none of the other dnmt snps ( table 1 ) , nor xrcc1 rs2682585 ( table 2 ) were associated with radiation - induced fibrosis of the breast upon both univariate or multivariate cox regression analysis .
it is worth noting that mitochondrial haplogroup h emerged as a significant protective factor in these multivariate models when dnmt1 rs2228611 was considered under the codominant ( hr , 0.48 ; 95% ci , 0.26 to 0.88 ; p=0.018 ) or the recessive mode of inheritance ( hr , 0.48 ; 95% ci , 0.26 to 0.89 ; p=0.019 ) .
to understand the joint effects of genetic markers on individual risk of grade 2 fibrosis , we built a grs for grade 2 fibrosis based on different combinations of dnmt1 rs2228611 and/or mitochondrial haplogroups according to five cox regression models , as shown in table 3 .
model 1 included mitochondrial haplogroups only ( h vs. non - h ) , model 2 and model 3 comprised the codominant or the recessive contrast of rs2228611 , respectively , model 4 combined mitochondrial haplogroup h with the codominant contrast of dnmt1 rs2228611 and model 5 incorporated mitochondrial haplogroup h with the recessive contrast of dnmt1 rs2228611 .
we then performed roc curve analyses to determine the performance of the five models at discriminating patients with or without grade 2 fibrosis ( fig .
the lowest discrimination accuracy , quantified by area under the receiver operating characteristic curves ( auc ) , was obtained for models 2 and 3 , which were based on the two contrasts of rs2228611 ( both auc , 0.587 ; 95% ci , 0.28 to 0.645 ) , and the highest was observed for model 5 , which included the combination of mitochondrial haplogroup h with the recessive contrast of rs2228611 ( auc , 0.655 ; 95% ci , 0.597 to 0.710 ) .
2 , adding the recessive contrast of dnmt1 rs2228611 to haplogroup h determined an increase of the discrimination accuracy from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710 ) .
when applying the score developed in model 5 , the proportion of patients with grade 2 radiation - induced fibrosis for each score group showed an increasing trend from lower to higher sum risk scores : 3.4% ( score 0 ) , 10.0% ( score 1 ) , 14.0% ( score 2 ) , and 27.3%
the kaplan - meier analysis of model 5 revealed that patients with an increasing sum risk score showed a trend towards higher frequencies of grade 2 fibrosis ( p trend=0.0005 ) ( fig .
in the present study , we tested whether the occurrence of radiation - induced fibrosis in breast cancer patients could be at least partially explained by genetic variability in dnmt genes , which encode for epigenetic enzymes known to be involved in fibrogenesis and radiation response . to accomplish this , we investigated the association between common polymorphisms in dnmt1 , dnmt3a , and dnmt3b genes and the risk of subcutaneous fibrosis in a cohort of breast cancer survivors who received rt after breast - conserving surgery .
multivariate cox analysis revealed dnmt1 rs2228611gg genotype as an independent predictor of lower risk for radiation - induced skin fibrosis . to the best of our knowledge ,
this is the first study to suggest that genetic variability at the dnmt1 locus contributes to the development of late normal tissue complications after radiation therapy , which further supports the importance of dnmt1 on fibrogenesis and wound healing after radiation exposure .
it is worth noting that xrcc1 rs2682585 , previously reported to be associated with an overall toxicity score combining skin toxicities and fibrosis of the breast , was not found to be correlated with fibrosis alone in the present study .
snps in dnmts genes have primarily been investigated as risk factors for cancer susceptibility given that aberrant dna methylation is one of the earliest molecular events during carcinogenesis .
in addition , since snps in dnmts genes influence dna methylation at global and gene - specific levels , they have also been also investigated as susceptibility factors for common complex diseases other than cancer . among the snps investigated in the present study , dnmt3a rs1550117 and dnmt3b rs2424908 have been associated with a lower cancer risk , as shown by recent meta - analyses . with regard to dnmt1 rs2228611 , the dnmt1 rs2228611gg genotype has been reported to be associated with a higher risk of ovarian cancer .
although dnmt1 rs2228611 is a synonymous snp located in exon 17 , it has a possible splice regulatory function with a > g resulting in gaining of three exonic splicing enhancer binding motifs .
in addition , dnmt1 rs2228611 has significant modifying effects on the inverse relationship between line-1 methylation , a marker of global methylation , and cadmium exposure in argentinean women , suggesting a possible role in regulating the methylation level in response to environmental cues .
reactive oxygen species ( ros ) caused by radiation could be a common trigger mechanism in both fibrosis and radiation response . in oxygenated tumor and normal cells ,
mitochondria play a major role in the generation of ros and appear to be the primary loci of rt effects .
conversely , mitochondrial polymorphisms defining major haplogroups have been suggested to alter oxidative phosphorylation coupling as well as ros production . in addition , cybrids containing mitochondrial haplogroup h have lower levels of dnmt1 , dnmt3a , and dnmt3b and global methylation than those with mitochondrial haplogroup j .
based on our previous findings highlighting a protective role of mitochondrial haplogroup h on radiation - induced fibrosis , we built a grs based on its combination with dnmt1 rs2228611 .
in line with the concern recently raised for high risk of over - fitting and false positive results of multifactorial genetic models , for the grs construction we only considered dnmt snps significantly associated with radiationinduced fibrosis , rather than using the total number of putative risk alleles .
our results provided evidence of an increased discrimination accuracy when dnmt1 rs2228611 was combined with mitochondrial haplogroup h , suggesting potential usefulness of rs2228611 in genetic - based models for prediction of radiation - induced skin fibrosis .
in addition , the method used for weighting predictors , which was based on cox hazard regression coefficients , can be easily applied for inclusion of clinical factors as well . unfortunately , none of the clinical or dosimetric variables analyzed emerged as independent predictors for grade 2 fibrosis , as we reported in our previous analysis .
first , the lack of a second cohort of breast cancer patients precluded the possibility of validating dnmt1 rs2228611 as a predictive factor for radiation - induced fibrosis .
thus , our results need to be interpreted cautiously because of the small sample size and risk of false positive findings given that very few radiogenomic studies with a candidate gene approach have been replicated in independent cohorts .
second , in the present study we did not evaluate gene polymorphisms involved in enzymatic dna demethylation , such as the ten - eleven translocation ( tet ) protein family , which are known to play a role in fibrogenesis .
therefore , a more comprehensive investigation of genes encoding epigenetic enzymes should be conducted to develop and validate a clinically useful geneticbased model for prediction of radiation - induced skin fibrosis .
third , the predictive performance of the genetic model , based solely on rs2228611 and haplogroup h , is rather modest .
however , this is expected for complex polygenic traits such as clinical radiosensitivity , which is probably the result of the combined effects of multiple polymorphic loci .
in addition , further investigation in cellular and in vivo animal models is still required to provide mechanistic insights into the protective role of rs2228611gg on radiation - induced fibrosis .
our findings support a protective effect of the dnmt1 rs2228611gg genotype on the risk of radiation - induced fibrosis in breast cancer patients . in addition , we provide evidence that inclusion of dnmt1 rs2228611 in a genetic - based model has the potential to increase prediction accuracy for radiation - induced fibrosis .
however , further investigation to provide mechanistic insights into the role of dnmt1 rs2228611 in fibrogenesis and radiation response is warranted .
in addition , a more comprehensive investigation of the role of germline polymorphisms should be conducted to develop and validate a clinically useful genetic - based model for prediction of radiation - induced fibrosis after treatment of breast cancer . | purposethis study was conducted to investigate the role of four polymorphic variants of dna methyltransferase genes as risk factors for radiation - induced fibrosis in breast cancer patients .
we also assessed their ability to improve prediction accuracy when combined with mitochondrial haplogroup h , which we previously found to be independently associated with a lower hazard of radiation - induced fibrosis.materials and methodsdnmt1 rs2228611,dnmt3a rs1550117,dnmt3a rs7581217 , and dnmt3b rs2424908 were genotyped by real - time polymerase chain reaction in 286 italian breast cancer patients who received radiotherapy after breast conserving surgery .
subcutaneous fibrosis was scored according to the late effects of normal tissue subjective objective management analytical ( lent - soma ) scale .
the discriminative accuracy of genetic models was assessed by the area under the receiver operating characteristic curves ( auc).resultskaplan - meier curves showed significant differences among dnmt1 rs2228611 genotypes in the cumulative incidence of grade 2 subcutaneous fibrosis ( log - rank test p - value= 0.018 ) .
multivariate cox regression analysis revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa ; hazard ratio , 0.26 ; 95% confidence interval [ ci ] , 0.10 to 0.71 ; p=0.009 ) .
adding dnmt1 rs2228611 to haplogroup h increased the discrimination accuracy ( auc ) of the model from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710).conclusiondnmt1 rs2228611 may represent a determinant of radiation - induced fibrosis in breast cancer patients with promise for clinical usefulness in genetic - based predictive models . |
g protein - coupled receptors ( gpcrs ) are regarded to take up more than one - fourth of marketed human medicines [ 1 , 2 ] .
drugs targeting gpcrs account for the majority of the best - selling drugs and about 40% of all prescription pharmaceuticals in the marketplace .
histamine receptors belong to one family of rhodopsin - like class a gpcrs , and four subtypes are named in chronological order as h1 , h2 , h3 , and h4 .
each type of histamine receptor reacts to subtype - specific ligands into an active or inactive form .
gpcrs are integral membrane proteins that consist of 7 transmembrane segments connected by 3 intracellular and 3 extracellular loops of variable length .
the crystal structures of gpcrs with their binding ligands have revealed the features of the ligand binding pockets and extracellular loops [ 4 - 6 ] .
a ligand of gpcr activates the receptor by changing the receptor structure to the active form .
1 ) is produced by decarboxylation of l - histidine and acts as a chemical mediator and neurotransmitter in central and peripheral tissues .
it acts as an important pharmacological modulator involved in the processes of allergy , inflammation , neurophysiology , and cancer [ 7 - 10 ] .
the molecular understanding of ligand - receptor interactions of gpcr remains unclear and is still the subject of investigations .
there are several theories explaining the ligand - receptor interaction mechanism , such as shape theory , binding theory , and vibration theory [ 11 , 12 ] .
the structures of histamine receptor ligands are so variable that we can not easily classify the pharmacological function of the ligand . to find any other characteristic in the molecular patterns between agonists and antagonists of histamine receptors , a computational approach to molecular vibration was carried out in an attempt to find a bit of the molecular interaction mechanisms .
the simplified molecular - input line - entry system ( smiles ) and 3-dimensional structure data format ( sdf ) files of the dataset were downloaded from the pubchem compound database in national center for biotechnology information ( ncbi ) and used in the further analyses .
all 47 ligand molecules in the dataset , comprising 9 histamine agonists and 38 histamine antagonists , are shown in table 1 .
the molecular similarities between histamine and other 46 chemicals were calculated from smiles of the chemicals .
the structural similarity was calculated and represented as the tanimoto distance of each molecule from histamine .
the tanimoto coefficient for pairwise comparison of molecules is the most widely used measure of molecular structural similarity .
this coefficient is defined as tc = nab/(na + nb - nab ) , with nab being the number of common bits , na the unique bits in molecule a , and nb the unique bits in molecule b , using a molecular fingerprint . in this study ,
the molecular similarity was calculated as the tanimoto coefficient using the 38-bit set . in order to calculate molecular vibrational frequency
since each provided theoretical 3-d conformer sdf is not at an energy minimum and may not represent the lowest energetic form in a vacuum , solvent , or a binding pocket , each sdf file of a ligand molecule underwent conversion to a single low - energy conformation using the general atomic and molecular electronic structure system ( gamess ) program package .
restricted hartree - fock ( rhf ) calculations using becke 's exchange and lee - yang - parr 's correlation functionals ( blyp ) density functional theory ( dft ) method with 6 - 31 g basis set were performed to optimize the geometries of the molecules .
each result was taken as the representative conformation of the molecule , although the calculation of molecular vibrational frequency has some dependence on conformation .
each geometry optimization result was subjected to the calculation step for the vibrational frequency with runtyp of hessian in the gamess program . for the simplified molecular comparison ,
the calculated vibrational frequencies of a molecule were then sorted in increasing order and taken into the corrals , the step size of which was 5 .
the intensities of each frequency in the same corral were summed up as the representative of the corral in the frequency range of 0 - 5,000 cm . as a final outcome
, this potential molecular descriptor of each molecule was displayed in a 1-dimensional vector containing 1,000 elements .
finally , the similarity matrix , comprising the descriptors of 47 ligands of histamine receptor , was then subjected to hierarchical clustering in the agglomerative manner . in this study ,
the calculations of cimvf were performed by in - house scripts , written in python .
the simplified molecular - input line - entry system ( smiles ) and 3-dimensional structure data format ( sdf ) files of the dataset were downloaded from the pubchem compound database in national center for biotechnology information ( ncbi ) and used in the further analyses .
all 47 ligand molecules in the dataset , comprising 9 histamine agonists and 38 histamine antagonists , are shown in table 1 .
the molecular similarities between histamine and other 46 chemicals were calculated from smiles of the chemicals .
the structural similarity was calculated and represented as the tanimoto distance of each molecule from histamine .
the tanimoto coefficient for pairwise comparison of molecules is the most widely used measure of molecular structural similarity .
this coefficient is defined as tc = nab/(na + nb - nab ) , with nab being the number of common bits , na the unique bits in molecule a , and nb the unique bits in molecule b , using a molecular fingerprint . in this study ,
in order to calculate molecular vibrational frequency , the structure of a chemical must first be geometrically optimized .
since each provided theoretical 3-d conformer sdf is not at an energy minimum and may not represent the lowest energetic form in a vacuum , solvent , or a binding pocket , each sdf file of a ligand molecule underwent conversion to a single low - energy conformation using the general atomic and molecular electronic structure system ( gamess ) program package .
restricted hartree - fock ( rhf ) calculations using becke 's exchange and lee - yang - parr 's correlation functionals ( blyp ) density functional theory ( dft ) method with 6 - 31 g basis set were performed to optimize the geometries of the molecules .
each result was taken as the representative conformation of the molecule , although the calculation of molecular vibrational frequency has some dependence on conformation .
each geometry optimization result was subjected to the calculation step for the vibrational frequency with runtyp of hessian in the gamess program .
for the simplified molecular comparison , the calculated vibrational frequencies of a molecule were then sorted in increasing order and taken into the corrals , the step size of which was 5 .
the intensities of each frequency in the same corral were summed up as the representative of the corral in the frequency range of 0 - 5,000 cm . as a final outcome
, this potential molecular descriptor of each molecule was displayed in a 1-dimensional vector containing 1,000 elements .
finally , the similarity matrix , comprising the descriptors of 47 ligands of histamine receptor , was then subjected to hierarchical clustering in the agglomerative manner . in this study ,
the calculations of cimvf were performed by in - house scripts , written in python .
typically , a tanimoto coefficient > 0.85 is considered highly similar , and a coefficient > 0.75 is considered similar for the purpose of clustering molecules that may have similar biological activity profiles .
the highest tanimoto coefficient among agonists was 0.45 ( imetit ) , and this is not high enough to be considered a molecule that has potential agonist efficacy .
moreover , the lowest value of tanimoto coefficient among agonists was 0.08 ( dimaprit and skf91488 ) and is also a lower value as an antagonist .
it seems that there is no related pattern between tanimoto coefficients and the functional types of molecules in the case of histamine receptor agonists or antagonists . to search for a novel characteristic for the classification of histamine
receptor ligands , a kind of molecular calculation using agglomerative hierarchical clustering was adopted in this work .
the result of the hierarchical clustering of the similarity matrix from cimvf is shown in fig .
as shown in the figure , eight agonists were located nearby ( the part in the dotted circle ) , except impromidine , and all antagonists were clustered close to each other in the radial tree .
we can tell the regional difference between agonists and antagonists in the tree and also find that the information from the molecular vibrational frequency may play a role in the classification of agonists / antagonists for histamine receptor as a possible molecular descriptor .
for these methods , clustering with cimvf shows the more proper result in the case of histamine receptor ligands . with a more concentrated study on the relationship between the molecular vibrational frequency and pharmacological function of a ligand ,
the vibrational spectrum of a molecule may shed light on the field of ligand - receptor interaction mechanisms . | to learn the differences between the structure - activity relationship and molecular vibration - activity relationship in the ligand - receptor interaction of the histamine receptor , 47 ligands of the histamine receptor were analyzed by structural similarity and molecular vibrational frequency patterns .
the radial tree that was produced by clustering analysis of molecular vibrational frequency patterns shows its potential for the functional classification of histamine receptor ligands . |
neurologic complications from mycoplasma pneumoniae have been known since the 1940s.1 encephalitis is the most common complication , and occurs most often in children .
a review by lerer and kalavsky2 found that 53% of patients with central nervous system ( cns ) disease associated with m. pneumoniae were between the ages 6 years and 20 years .
it is estimated that between 5% and 10% of acute childhood encephalitis in europe and north america is attributable to m. pneumoniae.3 there is a spectrum of clinical manifestations such as aseptic meningitis , peripheral neuropathy , transverse myelitis , cranial nerve palsies , cerebellar ataxia , acute transverse myelitis , and acute disseminated encephalomyelitis.4 the incidence of these manifestations has been estimated to be between 0.06% and 0.1%,5 but cns symptoms are reported in up to 7% of patients in the hospital setting with confirmed m. pneumoniae infection.6 long - term neurological problems have been noted in 20%60% of cases , with severe disease resulting in mental retardation , brain atrophy , hydrocephalus , epilepsy , visual changes , and global neurologic deficits with brainstem dysfunction and cerebellar ataxa.7 two studies have shown that between 24% and 30% of patients require intensive care treatment , with mortality rates of between 8% and 10%.8,9
the index patient presented with a 2-week history of head - aches , fevers , and dry cough .
her examination revealed an ataxic gait with normal muscle power , reflexes , and skin sensation .
initial computed tomography imaging of her brain showed a single hypodense white matter lesion in the left frontal lobe . a subsequent magnetic resonance imaging scan ( mri ) of the brain demonstrated a corresponding t2 and fluid - attenuated inversion recovery ( flair ) hyperintense lesion in the left frontal lobe ( fig .
given the patient s age and symptoms , a diagnosis of multiple sclerosis was also considered .
other investigations revealed a positive total m. pneumoniae serology immunoglobulin ( ig)m antibody titer of > 1,280 .
the diagnosis of m. pneumoniae - related cns disease was made based on the patient s clinical symptoms , positive serology , and the mononuclear pleocytosis found in the csf .
this is typically in the range of 10200 cells/l for m. pneumoniae - associated cns infections.10 routine csf gram stain and bacterial cultures are characteristically negative .
one review of 58 cases showed a diagnosis was made in 84% of patients with positive serology , 14% with csf polymerase chain reaction ( pcr ) , and 2% with csf antibody determination.11 our laboratory was able to evaluate the csf for the open reading frame ( orf ) 521 nuclear capsid protein gene with pcr , which returned a negative result .
acyclovir was ceased 3 days into hospital admission , and the patient was prescribed a course of oral doxycycline .
the patient s symptoms resolved over a period of 1 week of observation in hospital .
further analysis of the csf with isoelectric focusing revealed oligoclonal bands of igg in the csf that was not present in the serum .
two subsequent cranial mri scans of the patient were obtained over a period of 3 months in the outpatient setting , and they showed the stable appearance of the left frontal lobe lesion .
the positive oligoclonal bands in the csf are likely related to the immunological phenomena that are commonly linked to m. pneumoniae infections .
it has been known since the 1970s that polyclonal b - cell activation and antibodies to brain tissue have been demonstrated in m. pneumoniae infections . 12 in 1996 ,
nishimura et al.13 reported three cases of m. pneumoniae encephalitis with associated anti - galactocerebroside antibodies . in this patient , a moderately positive anti - ganglioside gq1b antibody was found .
this supports the hypothesis that demyelination and neurologic dysfunction in this entity could be mediated by the immune system .
the pathogenesis of m. pneumoniae cns disease is unclear , but it may be related to direct invasion ( proven during brain biopsy culture15 or nucleic acid hybridization16 ) or autoimmunity ( immune complexes , cross - reacting antibodies , or a neurotoxin effect).17 this case highlights a rare cause of encephalitis in a patient presenting to a tertiary - level adult teaching hospital . | we report a rare cause of encephalitis that is not often described in the adult clinical setting . our case was a 16-year - old female who presented with a clinical picture of viral encephalitis ; however , magnetic resonance imaging showed a demyelinating lesion of the left frontal lobe . in this age group ,
differential diagnoses of acute demyelination encephalomyelitis and multiple sclerosis were entertained .
further investigations demonstrated positive mycoplasma pneumoniae serology . as a result ,
a diagnosis of mycoplasma pneumoniae - associated encephalitis was made based on a process of exclusion . |
the term " temporomandibular disorders " ( tmd ) refers to clinical alterations characterized by signs and symptoms involving the masticatory muscles or the temporomandibular joint ( tmj ) or both30 .
tmd have been studied since the beginning of the last century and became worldwide known when costen4 published an article in 1934 describing a set of craniofacial symptoms designated as the costen syndrome .
the great drive in epidemiologic studies on tmd came about with helkimo13,14 , who developed a clinical index and an anamnestic index that quantitatively measured the severity of tmd symptoms .
as the effectiveness of the assessment method used in epidemiologic studies was proved11 , an attempt was made to establish the prevalence of tmd in the population .
conti , et al.3 ( 1996 ) , in a sample of 310 young persons with mean age of 18.8 years , observed a prevalence of mild , moderate and severe tmd of 49.35% , 10.32% and 0.97% , respectively .
these authors concluded that , although tdm prevalence was relatively high , the need for treatment ( moderate and severe tmd ) in the surveyed population was 11.29% .
similar results were found by conti , et al.2 ( 2003 ) and valle - corotti , et al.28 ( 2003 ) , in a population of 200 patients with class i and ii malocclusion , with and without orthodontic treatment .
the results of these studies2,28 showed absence of tmd in 62.5% of the patients , mild tmd in 34% and moderate tmd in 3.5% , which indicates that a small portion of the surveyed population required treatment for the dysfunction .
differents aspects of functional occlusion was investigated in cases of tmd include : the deviation between the centric relation ( cr ) position and the habitual maximum intercuspation ( hmi ) position , contacts on the working and non - working sides , absence of lateral or protrusive guides and interference in the disocclusion guides . while assessing the functional occlusion , bell , et al.1 ( 2002 ) suggested that occlusal interference may be considered as an etiologic factor of tmd .
malocclusion has been associated with tmd , when it is believed that the alteration of form might cause alteration in the stomatognathic system function29 . with the intention of elucidating this relation ,
several authors have studied class i , ii malocclusion6,18 , posterior crossbite6 anterior open bite6,16 , horizontal overlap20 and vertical overlap22 , suggesting that these alterations are responsible for the onset of tmd symptoms .
orthodontic treatment has also been associated with tmd , and considered as a cause30 , cure29 or a preventive factor5 of dysfunctions for changing the patient 's occlusal pattern .
treatment of class iii malocclusion in adult patients may be compensatory or associated with orthognathic surgery and
the literature demonstrates that ortho - surgical management of class iii skeletal malocclusion may present favorable effects on tmd on mandibular function24,27 .
this improvement in tmj condition may be related to the type of osteotomy performed16 or to the type of fixation used10 .
the literature does not , however , refers to the tmd index in a population treated for class iii malocclusion .
the aims of this study were to compare the prevalence of temporomandibular disorders ( tmd ) in individuals submitted to either orthodontic or ortho - surgical class iii malocclusion treatment and to assess the influence of occlusal aspects on tmd severity .
the population studied of this study comprised patients treated by phd and master 's degree graduate students from bauru school of dentistry ( fob ) , university of so paulo ( n = 21 patients ) and patients treated by dr .
laurindo zanco furquim at his private clinic in the city of maring , pr , brazil ( n = 29 patients ) .
in both cases , initial patient selection was based of the review of clinical dental records .
the patients were fully informed on the objectives of the study and signed a written informed consent form , in compliance with the 196/96 resolution of the brazilian national health council .
the ortho - surgical cases were treated by the same two surgeons in both cities .
inclusion criteria were : bilateral class iii molar relationship and presence of all teeth up to the first molars as observed on the dental casts before treatment ; and presence of all teeth up to the first molars during the clinical exam ( except for the cases of extractions for orthodontic purposes ) .
the study population , which was assessed art least 1 year after completion of the orthodontic treatment , was assigned to 2 groups : group i : 25 young persons , submitted to orthodontic treatment to correct class iii malocclusion ; group ii : 25 young persons , submitted to ortho - surgical treatment to correct class iii malocclusion .
the patients filled out a chart containing personal information ( name , age , gender , address , telephone ) and questions about symptoms relative to tmd ( anamnestic questionnaire ) .
this questionnaire was developed on the basis of pre - existent charts3,79 and applied to the patients without the examiner 's interference , not to create an expectation , which could influence the results of the clinical exam to be performed .
the patients answered 10 questions relative to symptoms , which allowed classifying each case with respect to the degree of dysfunction .
the following possible answers were offered : " yes " , " no " or " sometimes " each " yes " was scored 2 , " sometimes " was scored 1 and " no " was scored 0 .
questions 4,6 and 7 received score 3 when the answer " yes " corresponded to bilateral or intense symptoms , score 2 if it corresponded to unilateral or mild , score 1 if it meant sometimes .
the sum of the scores obtained allowed classifying the sample as regards tmd , based on the following score scale , in accordance with conti , et al.3 ( 1996 ) scores from 0 to 3 - non - tmd patient ; scores from 4 to 8 : - mild tmd patient ; scores from 9 to 14 - moderate tmd patient ; scores from 15 to 23 : severe tmd patient .
tmj was examined by palpation of the lateral and posterior aspects ; maximum , lateral and protrusion mandibular movements ; and observation tmj noises .
the muscular exam consisted of bilateral palpation of the temporal muscles ( posterior , medial and anterior ) superficial masseter ( origin , medial and posterior ) deep masseter , sternocleidomastoid and trapezium ( superior ) muscles .
the dental and occlusal exams included assessment of overbite and overjet , maxillomandibular relationship ( cr and mhi ) , presence and type of lateral and anterior guides , interference on the non - working side and number of tooth contacts in mhi .
the non - parametric data were analyzed statistically by mann - whitney u test , which assessed the inter - group differences of the tmd index .
student 's t - test for parametric analysis assessed data referring to age , mouth opening , laterality , horizontal and vertical protrusion overlap .
chi - square test was applied to assess the association between the tmd index and the other conditions evaluated in this investigation .
the population studied of this study comprised patients treated by phd and master 's degree graduate students from bauru school of dentistry ( fob ) , university of so paulo ( n = 21 patients ) and patients treated by dr .
laurindo zanco furquim at his private clinic in the city of maring , pr , brazil ( n = 29 patients ) .
in both cases , initial patient selection was based of the review of clinical dental records .
the patients were fully informed on the objectives of the study and signed a written informed consent form , in compliance with the 196/96 resolution of the brazilian national health council .
the ortho - surgical cases were treated by the same two surgeons in both cities .
inclusion criteria were : bilateral class iii molar relationship and presence of all teeth up to the first molars as observed on the dental casts before treatment ; and presence of all teeth up to the first molars during the clinical exam ( except for the cases of extractions for orthodontic purposes ) .
the study population , which was assessed art least 1 year after completion of the orthodontic treatment , was assigned to 2 groups : group i : 25 young persons , submitted to orthodontic treatment to correct class iii malocclusion ; group ii : 25 young persons , submitted to ortho - surgical treatment to correct class iii malocclusion .
the patients filled out a chart containing personal information ( name , age , gender , address , telephone ) and questions about symptoms relative to tmd ( anamnestic questionnaire ) .
this questionnaire was developed on the basis of pre - existent charts3,79 and applied to the patients without the examiner 's interference , not to create an expectation , which could influence the results of the clinical exam to be performed .
the patients answered 10 questions relative to symptoms , which allowed classifying each case with respect to the degree of dysfunction .
the following possible answers were offered : " yes " , " no " or " sometimes " each " yes " was scored 2 , " sometimes " was scored 1 and " no " was scored 0 .
questions 4,6 and 7 received score 3 when the answer " yes " corresponded to bilateral or intense symptoms , score 2 if it corresponded to unilateral or mild , score 1 if it meant sometimes .
the sum of the scores obtained allowed classifying the sample as regards tmd , based on the following score scale , in accordance with conti , et al.3 ( 1996 ) scores from 0 to 3 - non - tmd patient ; scores from 4 to 8 : - mild tmd patient ; scores from 9 to 14 - moderate tmd patient ; scores from 15 to 23 : severe tmd patient .
tmj was examined by palpation of the lateral and posterior aspects ; maximum , lateral and protrusion mandibular movements ; and observation tmj noises .
the muscular exam consisted of bilateral palpation of the temporal muscles ( posterior , medial and anterior ) superficial masseter ( origin , medial and posterior ) deep masseter , sternocleidomastoid and trapezium ( superior ) muscles .
the dental and occlusal exams included assessment of overbite and overjet , maxillomandibular relationship ( cr and mhi ) , presence and type of lateral and anterior guides , interference on the non - working side and number of tooth contacts in mhi .
the non - parametric data were analyzed statistically by mann - whitney u test , which assessed the inter - group differences of the tmd index .
student 's t - test for parametric analysis assessed data referring to age , mouth opening , laterality , horizontal and vertical protrusion overlap .
chi - square test was applied to assess the association between the tmd index and the other conditions evaluated in this investigation .
group i ( orthodontically treated patients ) had a mean age of 24.7 years and group ii ( patients treated with orthodontics followed by orthognathic surgery ) had a mean age of 26.9 years .
gender distribution in the sample was 36% ( 18 ) men and 64% ( 32 ) women .
classification of the anamnestic index for the whole sample was performed by the sum of the scores of the answers to items of the anamnestic questionnaire : absent tmd , - 48% ; mild tmd - 42% ; moderate tmd - 10% .
the anamnestic index was also applied to each studied group with respect to the presence and/or severity of tmd ( figure 1 ) .
when the mann - whitney test was applied , no statistically significant difference ( p=0.40 ) was fund between the groups ( table 1 ) .
the only variable that showed a statistically significant association with the tmd index was " interference in the non - working side ( right and left ) " ( p=0.02 ; chi - square test ) , which was characterized by the impossibility of performing lateral disocclusion guide ( figure 2 ) .
the study of the effects of orthodontic treatment on tmd began to attract greater attention from orthodontists when the appearance of tmd signs and symptoms to orthodontic treatment , were filed30 . in the present study ,
the treatment of dental and skeletal alterations was studied in accordance with the different therapeutic approaches ( either orthodontic and/or surgical management ) .
the study population was divided into two groups : one treated orthodontically and the other treated with orthodontics followed by orthognathic surgery , which enable comparing the different treatments and the associated presence and/or severity of tmd signs and symptoms . the anamnestic questionnaire applied in this study
the tmd index ( absent , mild and moderate ) was obtained by the sum of the scores attributed to the answers to this questionnaire .
the reliability of 95% of tmd classification obtained by the anamnestic questionnaire was demonstrated by fonsca11 ( 1992 ) and confirmed by hesse , et al.15 ( 1997 ) , who reported a high correlation ( p=0.0003 ) among the answers to the questionnaire and the clinical findings .
this questionnaire represents a simplified form of assessment when compared to the clinical exam and may be incorporated to the initial orthodontic exam12 , complementing the orthodontic documentation , as its information is precise and extremely important to the orthodontist .
the results showed that in the 2 groups studied , 48% of the sample was found to be without tmd ; mild tmd was found in 42% and moderate tmd was diagnosed in 10% , whereas no severe tmd was found in any patient , irrespective of the group .
these values were obtained in accordance with the index proposed and modified by other authors11,13,14 . in terms of the need for treatment , it is accepted that only patients with moderate to severe tmd require direct intervention , while patients with light tmd predominantly need guidance and self - knowledge3,11 . in this study ,
the number of patients with some degree of tmd ( n=26 ) was similar to that of patients without tmd ( n=24 ) , and no statistically significant differences were found between these results . considering the entire sample ,
52% of the patients presented some degree of tmd , which demonstrates how common tmd signs and symptoms are and how neglected they might be by orthodontists because all individuals examined in this study had been treated for malocclusion and continued to present tmd .
the orthodontist must know how to diagnose the signs and symptoms of tmd , to explain them to the patient and to provide guidance , irrespective of the patient 's stage of treatment . due to the long duration of orthodontic treatments , 2 years on average ,
non - diagnosed or misdiagnosed signs and symptoms of tmd might bring displeasure to the orthodontist in addition to generating legal claims that would cause moral and financial damage . in this study ,
10% of the patients presented tmd , which is agreement with the findings of previous studies that found 10.5% of individuals with moderate and severe tmd29 and 10.3% with moderate tmd3 .
the prevalence of individuals with mild tmd ( 42% ) is close to outcomes reported by dahl , et al.5 ( 1988 ) , which were 43.1% prevalence among orthodontically treated patients and 40% among non - tmd patients ( control group ) .
these data demonstrate a similar prevalence among populations either submitted to orthodontic treatment or not .
the positive effects of orthognathic surgery as regards tmd have been mentioned in the literature16,19 . in this study
, the groups behaved in a similar manner in relation to the tmd index , and no advantages were observed in the surgically managed group .
however , this affirmation would have a sounder basis if a comparison were made with a group of non - treated individuals , or if the study were longitudinal . in this study , 60% of group 1 and 48% of group ii presented some degree of tmd .
although small , perhaps this difference might be attributed to a beneficial effect of orthognathic surgery .
the basis of this study was the use of an anamnestic questionnaire which , in a simplified manner , indicated the presence and severity of tmd .
although these professionals might not apt to treat tmd , they should at all times be at least capable of diagnosing its signs and symptoms .
though different occlusal characteristics were evaluated , only the interferences on the non - working side presented association with the tmd index , as in accordance with the literature , they are considered as potentially traumatic and capable of causing damage to the stomatognathic system21 .
table 1 and figure 2 show that the presence of interference on the non - working side differed significantly in relation to the tmd index . of the 10 patients with interference ,
9 presented with some degree of tmd ; however , only 2 individuals with moderate tmd required some type of treatment .
this association between occlusal interference and tmd is mentioned in the literature19 , but the mechanism of this relation remains unclear .
it can not be affirmed , however , that interference represents a determinant factor for developing tmd because patients in this investigation and in other studies23 who present occlusal interference and absence of tmd .
this non - association between occlusal interferences and tmd is perhaps explained by the masticatory system 's capacity of adapting to the occlusal situation17,18 .
occlusal interferences may , however , cause local damage , such as wear facets , localized overload in a single tooth and also individual periodontal alterations that cause tooth mobility .
these alterations may go unnoticed for years and become serious on a long - term basis .
the presence of parafunctional habits may be one of the factors that make patients with interferences develop tmd24 .
occlusal interferences must be corrected during orthodontic treatment by altering the torque or coordinating the arches , or even at the end of treatment , with the performance of oclusal adjustment , as the absence of this type of contact represents one of the requirements of the ideal occlusion15 .
based on the methodology used and the results obtained , it may be concluded that class iii orthodontic treatment was not associated with the presence of tmd signs and symptoms and the non - working side contacts can be occlusal factors of risk .
there was no significant difference in tmd prevalence between the studied groups ( orthodontically treated patients and patients treated with orthodontics followed by orthognathic surgery ) . | objective : the aims of this study were to compare the prevalence of temporomandibular disorders ( tmd ) in individuals submitted to either orthodontic or ortho - surgical class iii malocclusion treatment and to assess the influence of occlusal aspects on tmd severity.material and methods : the sample consisted of 50 individuals divided into two groups , according to the type of treatment ( orthodontic or orthodontic with orthognathic surgery ) .
the presence of signs and symptoms of tmd was evaluated by an anamnestic questionnaire and a clinical examination , including tmj and muscle palpation , active mandibular range of motion , joint noises and occlusal examination.results:based on the anamnestic questionnaire , 48% had no tmd , 42% had mild tmd and 10% had moderate tmd . the presence and severity of tmd did not show any relationship with the type of orthodontic treatment ( p>0.05 ) .
the chi - square test showed a positive association ( p<0.05 ) between tmd and non - working side occlusal interferences.conclusion:based on the methodology used and the results obtained , it may be concluded that class iii orthodontic treatment was not associated with the presence of tmd signs and symptoms and the non - working side contacts can be occlusal factors of risk .
there was no significant difference in tmd prevalence between the studied groups ( orthodontically treated patients and patients treated with orthodontics followed by orthognathic surgery ) . |
bridging conduits ( bc ) also termed thick tunneling nanotubes ( tnt ) are cellular processes that sustain long distance intercellular transport of rna , organelles , and receptors . in immunocytes , they enable antigen presentation and pro - inflammatory immune responses.(5 ) supported by both actin filament and microtubules , macrophage bc extend > 100 m in length ( our unpublished observations).(1 ) viruses , prions and bacteria may utilize the conduits to escape immune surveillance and as such accelerate infection . for the human immunodeficiency virus type 1 ( hiv-1 ) , such activities likely occur in mononuclear phagocytes ( mp , monocytes , macrophages , dendritic cells ) and cd4 + t lymphocytes and are targets , reservoirs , and vehicles for microbial dissemination .
indeed , direct cell - to - cell hiv-1 dissemination occurs 1000 faster than cell - free viral entry .
this can occur through the abilities of hiv-1 to bypass the rate - limiting step of receptor - mediated fusion shielding it from humoral and adoptive immune surveillance.(6 ) the underlying mechanisms of hiv-1 intercellular transfer through conduits remained unknown as the process is complex and seemingly convoluted .
first , hiv-1 enters cells through receptor - mediated endocytosis through clathrin - coated pits followed by fusion with endosomal intermediates where virus uncoating and content mixing can occur.(11)second , downstream fusion of clathrin - coated endosomes with a surface - accessible tubular network of the early endosome results in the accumulation of viral particulates in specific intracellular compartments.(12 ) in this manner , tubular endosome networks serve as intermediate station for downstream sorting of hiv-1 from early endosomes into multivesicular bodies ( mvb ) and rab9 late endosomes where virus can remain infectious for extended time periods.third , late endosomes can be further targeted to the trans - golgi network which functions as a sorting hub shuttling proteins to the plasma membrane.(15 ) sorting of hiv-1 from endosomes to the er and golgi can then only allow completion of viral life cycle.fourth , during productive infection hiv-1 constituents are shuttled to golgi and er and assemble into mature virions employing er membranes , golgi and post - golgi stacks and vesicles.lastly , prior to hiv-1 intercellular transfer through the bridging conduits ( our unpublished observations ) . in the present work we elucidated endocytic trafficking pathways that effect hiv-1 bc transport .
endocytic trafficking through er and golgi networks was found necessary for hiv-1 cell - to - cell transfer .
indeed , characterization of the proteome of isolated conduits revealed that these structures are highly enriched with endosome populations involved in hiv-1 endocytic entry and intracellular sorting to the er and golgi .
imaging showed a distribution of fluorescently labeled hiv-1 , hiv-1 envelope ( env , gp120 , gp160 ) and gag ( pr55 , matrix , p24 ) with these endocytic compartments and er - golgi . disruption of endocytic transport from endosomes ( early and late ) to the er - golgi impaired hiv-1 intercellular transfer .
these observations , taken together , provide new insights into intracellular viral processing routes that drive hiv-1 spread through bc serving as a potential mechanism for evasion of host immune surveillance .
rabbit ab giantin , tgn38 , rab9 , and mouse ab to calnexin were purchased from cell signaling technologies .
human and mouse abs to hiv-1 components including those to gag ( gag , p24 , p17 , matrix ) , gp120 , gp160 were obtained through the nih aids research and reference reagent program , division of aids , niaid , nih ( bethesda , md ) .
chicken antirabbit , goat antimouse alexa 488 , 594 , 647 , donkey antisheep alexa 488 , cholera toxin subunit b ( ctx - b ) alexa 594-conjugated , click it metabolic labeling reagents for proteins imaging kit , -galactosidase ( -gal ) staining kit , 1,1dioctadecyl-3 , 3,3 , 3-tetramethylindodicarbocyanine perchlorate ( did ) , 3,3dioctadecyloxacarbocyanine perchlorate ( dio ) , er - tracker green ( bodipy fl glibenclamide ) , bodipy fl c5-ceramide ( golgi - tracker ) , prolong gold antifading solution with dapi all purchased from molecular probes . brefeldin a ( bfa ) was purchased from sigma - aldrich .
experiments with human pbmc were performed in full compliance with the ethical guidelines of the national institutes of health and the university of nebraska medical center .
monocytes from hiv-1 , 2 and hepatitis seronegative human donors were obtained by leukophoresis , and purified by countercurrent centrifugal elutriation.(21 ) human monocytes in suspension ( teflon flasks ) were cultured in dmem ( invitrogen ) supplemented with 10% heat - inactivated human serum , 1% glutamine , 10 g / ml ciprofloxacin ( sigma - aldrich ) , and 1000 u / ml highly purified recombinant macrophage colony stimulating factor ( mcsf ) , a generous gift by pfizer inc . , ( cambridge , ma ) .
cells were allowed to differentiate to macrophages for 7 d at 37 c in a 5% co2 . at day 7
, mdm were exposed to the macrophage - tropic strain hiv-1ada at a multiplicity of infection ( moi ) of 0.5 infectious viral particles / cell for 24 h. prior to seeding in mixed cultures ( infected and uninfected mdm ) , hiv-1 infection was assessed by hiv-1 p24 immunostaining and flow cytometry .
all confocal imaging was performed in monocyte - derived macrophages ( mdm ) mixed ( 1:1 uninfected and infected ratio ) cultures unless stated otherwise .
mdm for imaging were grown in poly d - lysine - coated labtech ( bd biosciences ) chamber slides .
labeling of er and golgi in living cells was performed using er - tracker green ( bodipy fl glibenclamide ) and bodipy fl c5-ceramide ( golgi - tracker ) per manufacturer s instructions . for retrograde tracing from early endosomes to goli - er using ctx - b
, cells were incubated with 100 mm ctx - b in serum - free medium for 1 h. for disruption of endocytic sorting , mdm were treated with 0.5 mm bfa for 3 h. cells from all applications we washed three times with warm pbs and fixed with 4% paraformaldehyde ( pfa ) ph 7.4 for 30 min at room temperature .
cells were treated with blocking / permeabilizing solution ( 0.1% triton , 5% bsa in pbs ) and quenched with 50 mm nh4cl for 15 min .
cells were washed once with 0.1% triton in pbs and sequentially incubated with primary and secondary abs at room temperature .
slides were covered in prolong gold antifading reagent with dapi and imaged using a 63 oil lens in a lsm 510 confocal microscope ( zeiss ) .
cells were first maintained in teflon flasks and media changed every 2 d. cells were centrifuged at 300 g for 5 min and resuspended ( 2 10 cells / ml ) with minimal pipeting in growth media then seeded on a 9 mm thick polycarbonate filter ( neuroprobe ) containing 3 m in diameter pores separating the upper and the lower wells of a one - well boyden separation chamber ( neuroprobe ) .
bc were collected by exposing them to the uninfected mdm - conditioned medium ( lower well ) .
cell suspensions were added to the upper well and the chamber was placed in an enclosed humidifier box and incubated at 37 c and 5% co2 tissue culture incubator for 4 h. conduits were harvested in each filter which was immersed in 100% methanol for 15 s then placed firmly with migrated cell materials down ( attached to the undersurface of filters ) on a glass slide .
cells were wiped from the filter top with kimwipes and the filter was then gently peeled off the slide with forceps .
processes were solubilized in lysis buffer ph 8.5 [ 30 mm triscl , 7 m urea , 2 m thiourea , 4% ( w / v ) 3-(3-cholamidopropyl ) dimethylammonio)-1-propanesulfonic acid ( chaps ) , 20 mm dithiothreitol ( dtt ) and 1 protease inhibitor cocktail ( sigma ) ] by pipeting . the remainder of the lysate was allowed to crystallize on slides and harvested using razor blades . infected
mdm to be examined by sem were seeded on a polycarbonate filter membrane of a boyden chemotaxis chamber for 4 h to allow for migration of their processes through the pores .
filter membrane was removed washed in pbs and fixed in 2% glutaraldehyde and 2% pfa in 0.1 m sorensen s phosphate buffer .
samples were critical point dried , mounted on specimen stubs , sputter- coated with 40 nm of gold / palladium and observed with a fei quanta 200 sem ( fei company ) operated at 25 kv .
following bc isolation , proteins were solubilized in lysis buffer ph 8.5 [ 30 mm triscl ] , 7 m urea , 2 m thiourea , 4% weight / volume ( w / v ) chaps and 1 protease inhibitor cocktail ( sigma ) ] .
proteins were precipitated using a 2d clean up kit and quantified by 2d quant ( ge healthcare ) per the manufacturer s instructions .
samples underwent isoelectric focusing and second dimension electrophoresis as described.(22 ) proteins were fixed with 10% methanol , 7% acetic acid for 24 h and stained with syproruby at room temperature for 24 h. spots were automatically excised using an ettan spot picker ( ge healthcare ) followed by in - gel tryptic digestion ( 10 ng / spot of trypsin ( promega ) for 16 h at 37 c .
peptide extraction and purification c18 ziptip ( millipore ) were performed on the proprep protein digestion and mass spec preparation systems ( genomic solutions ) .
extracted peptides were fractionated on microcapillary rp - c18 column ( newobjectives ) and sequenced using esi
ms / ms system ( proteomex system with ltq - orbitrap mass spectrometer , thermo scientific ) in a nanospray configuration .
the acquired spectra were searched against the ncbi.fasta protein database narrowed to a subset of human proteins using sequest search engine ( bioworks 3.1sr software from thermo scientific ) .
the turbosequest search parameters were set as follows : threshold dta generation at 10000 , precursor mass tolerance for the dta generation at 1.4 , dta search , peptide tolerance at 1.5 and fragment ions tolerance at 1.00 .
proteins with two or more unique peptide sequences ( p < 0.05 ) were considered highly confident .
functional connectivity maps were generated using string 8.3 software at http://string-db.org ( table 1 ) .
metabolic labeling and detection of newly synthesized proteins in mdm were performed using click - it aha labeling kits per manufacturer s instructions ( invitrogen ) .
metabolic labeling with aha was extended to 24 h. string database accession number ( accessible at http://string-db.org/ ) .
accession numbers for uniprot ( accessible at http://www.uniprot.org/ ) . postulated subcellular localization ( accessible at http://locate.imb.uq.edu.au and http://www.uniprot.org/ ) as follows : plasma membrane ( pm ) ; secreted ( s ) ; endoplasmatic reticulum ( er ) ; ribosomes ( r ) ; cytoskeleton ( csk ) ; cytosol ( c ) ; mitochondria ( m ) ; endocytic vesicle ( ev ) ; lysosomes ( l ) ; golgi ( g ) ; hiv-1 ; not available ( n / a , in this group are included proteins with no postulated localization )
for immunoisolation of rab9 endosomes uninfected and infected mdm were seeded in mixed cultures and allowed to polarize ( > 90% of cell population connected by bc ) .
cells were then washed three times with 1x pbs , scraped in tg buffer ( 10 mm hepes - koh , ph 7.2 , 250 mm sucrose , 1 mm edta , and 1 mm mg(oac)2 and disrupted by 15 strokes in a dunce homogenizer .
nuclei and unbroken cells were removed by centrifugation at 800 g for 15 min at 4 c .
supernatant was layered on a 20% sucrose cushion and centrifuged at 100 000 g for 1 h. the pellet was resuspended in pbs and incubated with protein a / g paramagnetic beads ( 20 l of slurry ; millipore ) conjugated to rab9 and isotype control abs ( 10 g of ab ) for 12 h at 4 c .
endocytic compartments bound to beads were collected following three washes with 1 pbs on a magnetic separator ( invitrogen ) .
the tzm - bl cells were cultured in high glucose dulbecco s modified medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 100 units of penicillin and 100 g / ml of streptomycin .
cells were allowed to reach 70% confluence then detached by 25 mm trypsin / edta for 5 min at 37 c .
cells were then seeded onto 4 well poly - d - lysine labtech chamber slides for 2 d ( 40% confluence ) .
protein a / g paramagnetic beads - bound to isotype control and rab9 abs were reconstituted in supplemented dmem ( 20 l of slurry/500 l of medium / well ) and added to tzm - bl .
chamber slides were placed on a plate magnet for 24 h at 37 c and 5% co2 .
untreated , hiv-1-exposed and isotype ab - exposed tzm - bl were used as controls .
cells were maintained in culture medium for additional 48 h prior to -gal detection using -gal staining kit ( per manufacturer s instructions ) .
quantification of immunostaining was performed with imagej software , utilizing jacop plugins ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ) to calculate pearson s colocalization coefficients .
endosome size ( diameter ) was measured manually using lsm - browser software ( zeiss ) .
scatter plots and bar graphs were generated using prism ( graphpad software inc . ) .
one - tailed student s t - tests were used for all data and the error bars are shown as standard error of the mean ( s.e.m . ) unless noted otherwise .
to assess whether hiv-1 infection affects bc formation , bc connected cells were counted at 3 h in adherent single ( uninfected or infected alone ) or mixed cultures ( uninfected and infected mdm at 1:1 ratio ) .
bc frequency was increased in infected and mixed cultures compared to uninfected mdm in single culture ( figure 1a ) .
infected mdm in single and mixed cultures acquired elongated morphologies and formed bc that extended > 100 m ( figure 1b ) . single culture uninfected mdm showed round ruffled appearances . however when these cells were mixed with virus - infected mdm at 1:1 ratio uninfected cells
( a ) mdm connected by bc in single ( uninfected or infected ) or mixed ( uninfected and infected at equal ratios ) cultures at 180 min in adherence ( * p < 0.05 ; * * p < 0.001 ; n = 250 cells / group , error bars + s.e.m ) .
arrows indicate three long ( > 100 m ) tubular bc emanating from a single mdm toward neighboring cells ( scale bar , 20 m ) .
( c ) uninfected mdm labeled with dio lipophilic dye ( green ) in single culture .
note lack of cell polarity ( bc ) and direct cell - to - cell contact .
( d , e ) confocal images ( including differential interference contrast , dic images ) of uninfected ( dio - labeled , green ) and infected ( did - labeled , red ) mdm in mixed culture .
note both uninfected and infected mdm are capable of generating bc in mixed culture ( scale bar , 10 m ) .
hiv-1-infected mdm were seeded on a filter membrane separating a boyden chemotaxis apparatus into an upper and a lower chamber .
cells were allowed to polarize by extending their processes through the 3-m pores toward the conditioned medium from uninfected mdm ( lower chamber , figure 2a ) .
sem was performed to monitor cell polarization and migration of cellular protrusions through the pores ( figure 2b ) .
such bc extensions were detached from cell bodies ( figure 2c ) and collected from cells derived from five independent experiments .
these are shown in representative images of 420% gradient 2d gels ( figure 2d ) .
an average of 1100 spots were detected using decyder software and 275 proteins identified by mass spectrometry at high confidence ( p 0.05 and 2 or more unique peptides ; table 1 and s1 , supporting information ) .
ingenuity pathway analysis ( ipa ) separated the bc proteome into 3 major functional groups .
these included cellular morphology and migration ( actin cytoskeleton , cell adhesion and motion , leukocyte extravasation , tight junction and calcium signaling ; antigen and hiv-1 endocytic processing ( caveole- , clathrin- , and fc - dependent endocytosis , mechanisms of hiv-1 endocytic entry and exit , micropinocytosis ) ; metabolomic factors ( carbohydrate , protein and fatty acid synthesis ) .
functional connectivity analysis using protein interaction networks ( http://www.string-db.org ) revealed high levels of interaction among these clusters as shown by bold lines in the connectivity map ( figure s1a , supporting information).(23 ) interestingly , removal of highly abundant cytosolic and nuclear proteins residing in the cell body , uncovered low abundance proteins that were not identified in the whole cell and membrane preparations.(22 ) moreover , proteomic analysis comparisons between uninfected and uninfected mdm bc showed no major differences in protein i d ( data not shown ) .
seventy - nine proteins ( 30% of the overall proteome were organelles ) were 3% were hiv-1 components .
the organelle markers included endolysosomal compartments ( 14% ) , er ( 9% ) and golgi ( 4% ; figure 2e ) .
the excised spots corresponding to these proteins are shown in figure 4a and table 1 .
further functional subclassification and connectivity analysis of endocytic , er and golgi proteins showed that major protein clusters interact as regulators of post - translational modifications such as n - glycosylation , protein folding , viral / antigen endocytic uptake and release and cell - to - cell communication ( figure 4b d ) .
the data show that the er and golgi are in the conduits and support their roles in viral / antigen processing .
( b ) sem image of a human macrophage cultured on the upper chamber of a boyden chemotaxis apparatus , extending its processes through the porous membrane .
( c ) confocal image of bc adhered to glass slide postdetachment from cell bodies , immunostained for microtubules with / tubulin - alexa 488 ab ( fluorescent green , pseudocolored white ) .
( d ) representative image of 2d electrophoresis of bc lysate pooled from five independent cultures ( donors ) .
( refer to table 1 and s1 , supporting information , for protein i d ) .
major biological functions relevant to viral endocytic processing ( including post - translational modification ) and cytoskeletal rearrangements in bc .
( a ) classification by function of bc proteome based on higher statistical significance of protein groups [ log ( p value ) ; threshold set at 1.3 ] .
identification of er , golgi and endosome proteins and their functional relevance in viral traffic through bc .
( a ) representative 2d electrophoresis of bc lysate . isolated bc from five separate cultures and 500 g of pooled precipitated protein was applied to a ph gradient ( 310 ) strip and separated on the first dimension by isoelectric focusing .
the strip was loaded into a large format gradient gel ( 420% ) for second dimension separation of bc proteins .
gel was fixed and poststained with deep purple dye for positive detection of proteins spots .
encircled protein spots correspond to er ( red ) , golgi ( green ) , endosome ( black ) and hiv-1 ( green ) subproteome identified by nano - lc
( b , c ) ipa classification by biological and canonical function of er , golgi , endosome proteins with significant relevance [ log ( p value ) ] to hiv-1 bc trafficking . for classification by biological function only proteins above the set threshold ( 1.3 ) were grouped for analysis .
( d ) connectivity map of the bc subproteome displaying functional interaction between er , golgi , endosome and hiv-1 proteins .
protein ids corresponding to abbreviations used in the connectivity map are included in table 1 and s1 , supporting information .
mdm were immunostained for giantin ( golgi vesicle and membrane integral protein ) and calnexin ( er membrane protein ) .
in nonpolarized cells er and golgi were located in the perinulcear region and displayed known ruffled and stacked morphologies ( figure 5a , arrows ) .
in contrast , the golgi network acquired tubular morphologies ( figure 5a , b ; inset boxes 1 and 2 ) .
interestingly , a single golgi was observed between two connected cells ( figure 5b , yellow arrows ) .
confocal imaging showed gag and env compartments in bc that colocalized with giantin and calnexin ( figure 5a , b ) .
env and gag were also observed in 1.6 m calnexin and 1.2 m ( in diameter ) giantin endocytic vesicles in conduits ( figure 5c ) .
quantitation of fluorescence overlap using pearson s colocalization tests indicated 72 and 32% of env colocalized with er and golgi respectively ( figure 5d ) .
gag distribution with calnexin and giantin was measured at 50 and 22% respectively ( figure 5e ) .
distinct morphologies of er and golgi in bc - connected cells and their presence within the conduits .
( a and b ) confocal images of mdm in mixed culture connected by bc .
immunostaining for giantin ( golgi , green ) and calnexin ( er , purple ) demonstrates organelle tubular morphologies and their codistribution with hiv-1 env ( red ) and gag ( red ) within the conduits ( inset box 1 , 2 ) . arrows in panel b indicate a single tubular golgi equidistant from two connected cells ( scale bars , 10 m ; inset boxes , 5 m ) .
( c ) size distribution of gag and env endocytic vesicles carrying er ( n = 35 vesicles ) and golgi ( n = 28 vesicles ) markers in the bc ( error bars s.e.m ) .
pearson s colocalization coefficients indicate percent overlap of env and gag with calnexin and giantin ( error bars s.e.m ; n = 80 cells ) .
having observed that hiv-1 env and gag are within the bc and are associated with the er and golgi , we next investigated whether these were newly synthesized viral proteins or shuttled to these organelles . to explore associations between
newly synthesized viral proteins and the er and golgi , mdm were metabolically labeled with azidohomoalanine ( aha ; a methionine substitute ) in methionine - depleted medium .
maximum overlap of viral and metabolically labeled proteins was observed at the plasma membrane ( likely reflecting processes of progeny assembly ) but not the conduits ( figure 6a , b ; inset boxes 1 and 2 ) .
furthermore , minimal distribution of metabolically labeled protein with golgi was observed suggesting env and gag distributing with these organelles may be of endocytic origin ( figure 6c ) .
live tracking of fluorescently labeled hiv-1 ( dio ) with er / golgi ( er- and golgi - tracker dyes ) revealed accumulation of hiv-1 in er and golgi tubules extending into the conduits ( figure 6d , arrows ) .
mdm in mixed culture were labeled by metabolic incorporation of 50 m of azidohomoalanine ( aha ) into newly synthesized proteins for 24 h using methionine - free medium .
( a , b ) confocal imaging of metabolically labeled mdm immunostained for hiv-1 env and gag show overlap of these viral constituents with newly synthesized proteins at the plasma membrane but not bc .
( c ) co - immunostaining with giantin shows minimal distribution of aha - labeled proteins with the golgi tubule .
( d ) sequestration of endocytosed fluorescently labeled hiv-1 ( did dye , red ) into golgi and er ( golgi- and er - tracker dyes , green ) .
mdm in mixed culture were first labeled with a combination of bodipy fl glibenclamide ( er - tracker ) and bodipy fl c5-ceramide ( golgi - tracker ) for 1 h then exposed hiv-1-did for 1 h. excess dye and noninternalized hiv-1 were removed by washing and cells were observed by time - lapse confocal imaging .
arrows indicate distribution of endocytosed hiv-1 with er - golgi tubules in polarized mdm ( scale bars , 10 m ; inset boxes , 5 m ) .
next , we investigated the pathways for hiv-1 env and gag trafficking to the golgi network .
following endocytosis at the plasma membrane proteins destined for golgi can undergo retrograde sorting routes from early / recycling endosomes and late endosomes to the golgi network.(24 ) among the organelles of the secretory / recycling pathway trans - golgi has a central role as a site of protein sorting.(24 ) once proteins reach trans - golgi they face several sorting destinations : the extracellular space , different domains of the plasma membrane , and rab11 recycling endosomes.(24 ) therefore we questioned whether hiv-1 env and gag can undergo retrograde transport from early / recycling endosomes to the golgi network .
co - distirbution of env and gag with fluorescently labeled cholera toxin subunit b ( ctx - b ) was tested , since ctx - b has been previously documented to undergo retrograde sorting from early / recycling - endosomes to the golgi.(25 ) mdm in mixed culture were exposed to the ctx - b - alexa 594 and then stained for env and gag .
env / b - ctx and gag / b - ctx compartments were observed in bc ( figure 7a , b ) .
env and gag colocalizations with b - ctx were measured at 39 and 41% , respectively ( figure 7c ) .
these data suggested that a portion of the viral protein pool undergoes parallel sorting to ctx - b .
hiv-1 env and gag undergo retrograde transport from early and late endosomes to the er - golgi .
( a - c ) env and gag endocytic sorting parallels that of cholera toxin subunit b ( ctx - b ) .
mdm in mixed culture were exposed to ctx - b - alexa 594 conjugated ( red ) .
cells were fixed and immunostained for env ( green ) and gag ( green ) and imaged by confocal microscopy .
inset boxes show at a higher magnification bc - associated endocytic compartments transporting ctx - b , env and gag .
pearson s colocalization coefficients are indicated as percent overlap of ctx - b with env and gag .
( d , e , g ) distribution of env and gag with rab9 late endosomes and the trans - golgi along the bc .
mdm in mixed culture were immunostained for env ( red ) , gag ( red ) , tgn38 ( trans - golgi marker , green ) and rab9 ( late endosomes , purple ) .
distribution by size ( diameter ) of rab9 and tgn38 compartments positive for env and gag ( n = 65 endocytic compartments / group , error bars s.e.m ) .
confocal images of mdm showing colocalization of env and gag with rab9 and tgn38 compartments in the bc ( inset boxes 1 ; scale bar , 10 m ) .
( f , h ) quantitation of colocalization ( fluorescence overlap ) of env and gag with rab9 and tgn38 compartments in the bc ( n = 80 cells / group , error bars s.e.m ) .
in addition to retrograde transport from early endosome to golgi , a crucial step in the hiv-1 replication cycle is rab9-mediated retrograde sorting to the trans - golgi .
gene silencing of rab9 and other host factors ( tip47 , p40 ) that regulate late endosome to trans - golgi transport blocks hiv-1 replication.(16 ) since fluorescently labeled hiv-1 was detected in the er and golgi tubules , we tested the virulence of hiv-1 sequestered into rab9 endosomes and its distribution with the trans - golgi .
confocal imaging showed env and gag distribution with in rab9 vesicles and tgn38 ( trans - golgi membrane protein ) in the conduits ( figure 7d , f ) .
colocalization assays showed a 19 and 30% overlap of env with rab9 and tgn38 compartments .
eighteen and 24% of gag overlapped with rab9 and tgn38 ( figure 7e , g ) . to test
if hiv-1 present in rab9 endosomes were infectious , bc compartments were isolated by immunoaffinity chromatography .
protein a / g paramagnetic microbeads conjugated to rab9 or isotype abs ( used as control of binding specificity ) were incubated with homogenized cell lysate clarified from cellular debris ( plasma membrane and nuclei ) and collected by magnetic separation .
endocytic compartments bound to paramagnetic beads were then introduced to the tzm - bl cells using a magnet plate .
the tzm - bl reporter cell line expressed ss - gal under the control of the hiv-1 promoter . at 72 h postexposure ss - gal ( blue )
bright field images in figure 8a show ss - gal expression ( blue ) in tzm - bl exposed to free hiv-1 ( positive control ) and rab9 endosomes ( arrows ) .
quantitation of blue cells revealed that the contents of rab9 endosomes were infectious ( figure 8b ) .
together these data indicate that hiv-1 destined for intercellular transfer undergoes retrograde sorting from early and late endosomes to the trans - golgi .
rab9 endosomes carry infectious particles and disruption of er - golgi impairs hiv-1 transfer through the conduits .
( a ) rab9 endosomes were isolated by magnetic separation from mechanically ruptured polarized mdm using protein a / g paramagnetic beads conjugated to rab9 ab .
bead - captured endosomes were washed and were forced into tzm - bl reporter cell line by subjecting the cells to magnetic fields .
hiv-1 genome integration and expression in tzm - bl was measured at 72 h postexposure in the form of ss - gal production ( blue ) .
representative bright field images of uninfected tzm - bl ( negative control ) , cells exposed to beads conjugated to isotype ab ( control for specificity of binding ) , cell - free hiv-1 ( positive control ) and rab9 immune - isolated endosomes at 72 h postexposure .
( b ) quantiation of overall endosome infectivity in tzm - bl ( scale bars 100 m ; error bars , s.e.m . ,
( c , d ) confocal images of mdm connected by bc in mixed culture treated with fungal metabolite bfa and immunostained for hiv-1 env and gag .
arrows indicate accumulation of viral proteins in large vacuoles in the cell body ( scale bar , 10 m ; inset boxes , 5 m ) . next we questioned whether disruption of recycling and the er - golgi network would affect transfer of hiv-1 through the conduits .
incubation of cells with fungal metabolite bfa was found to inhibit er - to - golgi transport , resulting in the rapid redistribution of golgi markers into the er , collapsed trans - golgi and impair of rab11-mediated recycling.(26 ) exposure of polarized mdm to 0.5 mm of bfa resulted in the accumulation of env and gag in large tgn38 compartments ( figure 8c , d ; arrows ) .
these data suggest that integrity of the er and golgi - trans - golgi networks is necessary for viral protein trafficking through the bc .
employing functional proteomics and imaging assays we characterized the macrophage conduits and elucidated endocytic pathways that effect hiv-1 intercellular trafficking through bc . together with previous studies we show that following clathrin - dependent endocytosis , hiv-1 is sequestered into early and late endosomes and hitchhikes retrograde sorting pathways to reach the er and golgi . virus uses these organelles as a sorting station for hiv-1 prior to intercellular transfer . in parallel , hiv-1 induces cytoskeletal alterations enhancing the formation of a bc network.(32 ) the mechanisms of bc formation is incompletely understood . in t cells and mdm these protrusions are established as adjacent cells pull away from each - other following brief contact or by generation of the protrusion from one cell to another . since an increase in bc was observed in mdm mixed cultures .
this may be due to an increase in the metabolic and secretory activities associated with viral infection.(9 ) the er and golgi networks commonly located in the perinuclear region of cells extend within the conduits in polarized mdm .
what causes these to acquire such unique morphologies may be due to rearrangements of cytoskeletal and endocytic networks required for the formation and function of bridging conduits .
it is likely that during conduit formation microtubule bundling may also lead to rearrangement from golgi and er stacks to tubules .
alternatively , golgi and er may traffic between cells through conduits similar to mitochondria and lysosomes and their distribution within the bc may accelerate communication between connected cells .
indeed , er and golgi cellular distribution and morphologies have been linked to the dynamics of microtubule rearrangements .
these organelles attach to the growing plus end of the microtubules and travel on these tracks to divergent cellular locations .
we posit that in addition to sustaining organelle travel between cells , conduits may accelerate intercellular exchange by acting as sites of mrna translation and protein synthesis
. this may be similar to processes observed in the axon where synthesis and incorporation of neurotransmitters into presynaptic vesicles occurs in proximity of the synapse.(30 ) this kind of golgi / er rearrangements may accelerate hiv-1 intercellular spread as viral genome can be expressed in proximity to the uninfected target cell , rather than expressed in the cell body and then transported to the uninfected cell .
elongation and translation factors , transporters of mrna , and cellular and viral rna were identified in the bc ( our unpublished observations).(31 ) furthermore , presence of enzymes regulating protein , carbohydrate , lipid and metabolism further support the role of bc as metabolically active structures . we have previously demonstrated that formation of the conduits precedes syncytium formation in hiv-1 infected mdm.(32 ) therefore , er and golgi extending within the conduits are likely to be shared by the connected cells functioning as a unicellular entity .
indeed , in the current study we often observed single golgi tubules located halfway within the conduit between two cells . in multinucleated giant cells central accumulation of several golgi tubules surrounded by more than 50 nuclei and
what drives transport of hiv-1 to the golgi network and er and more importantly how does this affect targeting of the viral constituents to their final destination ( i.e. , bc intercellular transfer ) remain open questions . the need for post - translational processing may target viral constituents to er and golgi
. these in turn can also be used as address codes sending viral proteins to specific subcellular locations or bc for intercellular transfer .
proper folding and glycosylation of envelope proteins is an important evolutionary process that impacts viral infectivity and selection.(33 ) indeed , mutations in the glycan shield of hiv-1 may account in part for viral immune evasion persistence to neutralizing antibodies.(34 ) hiv-1 gp120 undergoes heavy o- and n - glycosylation and mannose trimming in the er - golgi network .
o - linked carbohydrates are attached to the hydroxyl group of serines and threonines residues of hiv-1 env after transport to golgi.(35 ) gag may also be targeted to the er following monoubiquitination , as a quality control measure.(36 ) ubiquitination seems to be responsible for viral budding within endocytic compartments such as mvb as well as gag maturation and infectivity.(37 ) regulators of the ubiquitin - proteosome pathway were readily identified in the bc proteome and it is likely that such processes may also drive intercellular transfer of active viral constituents . sorting of viral constituents through er and golgi may also be a survival mechanism hiv-1 adopts during infection - induced apoptosis or necrosis . at this time the virus survives by utilizing any available cellular membranes ( plasma and organelle ) for its assembly .
indeed , previous ultrastructural studies have shown hiv-1 progeny assembling on golgi and er membranes at the late stages of macrophage infection .
interestingly , recent studies have shown that immune cells such as natural killers ( nk ) use bridging conduits to transfer apoptosomes and other packed cellular fragments from a dying to healthy cells.(38 ) we have also often observed establishment of contact between multinucleated giant cells and single polarized macrophages at the late stages of infection ( our unpublished observations ) .
as such , it is likely that presence of viral proteins in er and golgi compartments along the conduits may reflect the ability of hiv-1 to survive death of the host and use the bc - mediated debris clearing activity to accelerate infection . at the early stages of infection associated with minimal cell death sorting of hiv-1 constituents through er and golgi can also be explained by bc - mediated antigen presentation .
it has been recently documented that antigen - presenting cells use bc in vivo to accelerate antigen presentation .
we have previously detected major histocompatibility complex class ii ( mhc ii ) hla - dr in rab11 endosomes and mvb transporting hiv-1 constituents through the macrophage conduits ( our unpublished observations ) .
hla - dr endocytic processing appears similar to hiv-1 as both undergo internalization and sorting through early endosomes to recycling endosomes and subsequently er , cis- and trans - golgi ( our unpublished observations).(39 ) therefore it is likely that a portion of the viral constituents parallels hla - dr intra- and intercellular traffic .
this would explain the dual role of bfa in disrupting hiv-1 transport through the conduits and antigen presentation.(39 )
using proteomic and imaging technologies new insights into how hiv-1 uses basic immune cell functions to its advantage was explored .
the virus hitchhikes from cell - to - cell using bc cytoskeletal and endocytic machineries to accelerate viral spread
. such mechanisms are likely relevant for preventative vaccination strategies as they proves a means for the virus to evade immune surveillance . | bridging conduits ( bc ) are tubular protrusions that facilitate cytoplasm and membrane exchanges between tethered cells .
we now report that the human immunodeficiency virus type i ( hiv-1 ) exploits these conduits to accelerate its spread and to shield it from immune surveillance .
endosome transport through bc drives hiv-1 intercellular transfers .
how this occurs was studied in human monocyte - derived macrophages using proteomic , biochemical , and imaging techniques .
endosome , endoplasmic reticulum ( er ) , golgi markers , and hiv-1 proteins were identified by proteomic assays in isolated conduits . both the er and golgi showed elongated and tubular morphologies that extended into the conduits of polarized macrophages .
env and gag antigen and fluorescent hiv-1 tracking demonstrated that these viral constituents were sequestered into endocytic and er - golgi organelles .
sequestered infectious viral components targeted the golgi and er by retrograde transport from early and rab9 late endosomes .
disruption of the er - golgi network impaired hiv-1 trafficking in the conduit endosomes .
this study provides , for the first time , mechanisms for how bc golgi and er direct cell cell viral transfer . |
in general , endodontically treated teeth are considered to have increased susceptibility to fracture than vital teeth .
the reasons attributed to this are the dehydration and loss of dentin caused during the endodontic therapy along with the pressure applied during obturation of the filling material as in lateral compaction technique .
the structural durability of the tooth following endodontic therapy is directly proportional to the remaining tooth structure present .
aggressive instrumentation of the root canal leads to loss of dentin which may structurally weaken the tooth .
bender and freedland have suggested that , the highest incidence of vertical root fracture occurs in endodontically treated teeth .
non - surgical retreatment techniques are the preferred choice over surgical management to reduce or eliminate the bacterial load , to contain infection and alleviate symptoms .
the goal of retreatment is to completely remove the filling material to enable thorough disinfection of the endodontic system prior to refilling .
various techniques have been advocated to remove obturating materials from the root canal system during retreatment .
the protaper universal retreatment system consists of three retreatment files , d1 , d2 and d3 , which are designed to be used in the coronal , middle and apical third of the root canal respectively .
the triangular cross section of these instruments is similar to the protaper shaping and finishing files .
the use of a solvent has been recommended to aid in removal of gutta - percha by softening it .
previous studies have suggested that bonded composite restorations can be considered as the first choice for coronal restorations as they play a vital role in increasing the fracture resistance of endodontically treated teeth .
furthermore , it has been shown that direct and indirect cusp coverage adhesive restorations can significantly increase the fracture resistance of endodontically treated teeth . to the best of our knowledge
no study has been carried out to assess the fracture resistance of teeth following endodontic retreatment .
thus , the purpose of this in vitro study was to assess the fracture resistance of teeth after endodontic retreatment with protaper universal retreatment system and measure the remaining dentin thickness with spiral computed tomography ( sct ) and to correlate the loss of remaining dentinal thickness with susceptibility to fracture .
a total of 30 extracted mandibular premolars with fully formed apices were used in this study .
the teeth were decoronated at the cemento enamel junctions to obtain standard root lengths of 14 mm using a diamond disk ( brassler dental products , savannah , ga . ) . in group
i , the working length was established by placing a size 15 k file into the canal until it was observed at the apical foramen , then decreasing the file length by 1 mm .
the canals were prepared using protaper universal niti rotary shaping and finishing instruments following manufacturer 's instructions in sequence from sx to f2 to obtain a final apical size of 25 with 8% taper using crown down technique . along with this instrumentation , canals were irrigated between the use of each succeeding file , by introducing 10 ml of 3% sodium hypochlorite ( prime dental products , india ) , with a 27-gauge needle .
after complete instrumentation , all specimens received final irrigation with 10 ml of 17% ethylenediaminetetraacetic acid ( edta ) ( prime dental products , india ) followed by 10 ml of sodium hypochlorite to remove the smear layer . following this ,
the canals were dried with sterile paper points . following the manufacturer 's instructions , the ah plus sealer ( dentsplydetrey gmbh , konstanz , germany ) was mixed and applied within the root canal .
a size 25% , 2% master cone ( diadent group international , korea ) was placed at the appropriate working length and apical tuckback was confirmed .
a spreader was used to laterally compact the 2% gutta - percha accessory cones coated with ah plus sealer .
excess gutta - percha was sheared off and condensed with a plugger 1 mm below the canal opening .
the opening of the canal was sealed with coltosol ( coltene - whaledent , cuyahoga falls , oh ) and the teeth were stored at 37c in 100% humidity for 2 weeks . in group ii , cleaning and shaping was performed in the same manner as for group i following which the teeth were subjected to sct ( light speed plus computed tomography scanner [ ge electricals , milwaukee , usa ] ) to assess the teeth at different sections of the coronal , middle and apical thirds respectively .
the remaining dentin thickness was then measured from the central canal to the outer periphery in buccal , lingual , mesial and distal directions .
following this , the teeth were obturated as stated in group i. in group ii , retreatment was performed after 48 h following obturation to allow for adequate time for the sealer to set with protaper universal niti rotary retreatment ( dentsply maillefer , ballaigues , switzerland ) instruments , which were used at 300 rpm and 3 n / cm torque .
the retreatment files were used in the recommended sequence - d1 file ( size 30 , 0.09 taper ) for removal of root filling in the coronal third , followed by d2 file ( size 25 , 0.08 taper ) for the middle - third and finally , the d3 file ( size 20 , 0.07 taper ) was used at working length .
final shaping of the apical third was performed with protaper instruments f2 ( size 25 , 0.08 taper ) and f3 ( size 30 , 0.09 taper ) .
after complete instrumentation , all specimens received final irrigation with 10 ml of 17% edta followed by 10 ml of sodium hypochlorite to remove the smear layer . following this , 10
after completion of cleaning and shaping procedures , the specimens were again assessed with sct for the remaining dentin thickness as mentioned earlier .
lateral compaction was done using a size 30% , 2% gutta - percha master point coated with ah plus sealer . following the manufacturer 's instructions ,
the master cone was placed at the appropriate working length and apical tuckback confirmed and a spreader was used to laterally compact the gutta - percha accessory cones coated with ah plus sealer .
excess gutta - percha was sheared off and condensed with a plugger 1 mm below the canal opening .
the opening of the canal was sealed with coltosol f and the teeth were stored at 37c in 100% humidity for 2 weeks .
all the teeth in both groups were mounted on a custom made acrylic blocks such that 9 mm of the root surface remained exposed while the rest was embedded in the acrylic resin ( digital products international products , india ) .
the temporary material was removed using a bur and the root canal access was shaped to accept the loading fixture .
the acrylic blocks were mounted with the roots aligned vertically on the instron testing machine one at a time .
the mode of application of the vertical loading force required to fracture the root specimens was similar to the technique proposed by sedgley and messer to test the brittleness of endodontically treated teeth .
the specimens were subjected to a constant load at a crosshead speed of 1.0 mm / min until the roots fractured . in this study , fracture was defined as the point at which a sharp and instantaneous drop was observed .
this point was verified by a computer attached to the instron testing machine ( model 3382 , instron , uk ) . for most specimens ,
the test was terminated at this point and the force applied was recorded and measured in newtons .
statistical analysis was performed using independent samples t - test for analysis of remaining dentin thickness using sct within group ii and paired samples t - test was used for assessment of fracture resistance between group i and ii ( p < 0.05 ) .
a total of 30 extracted mandibular premolars with fully formed apices were used in this study .
the teeth were decoronated at the cemento enamel junctions to obtain standard root lengths of 14 mm using a diamond disk ( brassler dental products , savannah , ga . ) .
in group i , the working length was established by placing a size 15 k file into the canal until it was observed at the apical foramen , then decreasing the file length by 1 mm .
the canals were prepared using protaper universal niti rotary shaping and finishing instruments following manufacturer 's instructions in sequence from sx to f2 to obtain a final apical size of 25 with 8% taper using crown down technique . along with this instrumentation , canals were irrigated between the use of each succeeding file , by introducing 10 ml of 3% sodium hypochlorite ( prime dental products , india ) , with a 27-gauge needle .
after complete instrumentation , all specimens received final irrigation with 10 ml of 17% ethylenediaminetetraacetic acid ( edta ) ( prime dental products , india ) followed by 10 ml of sodium hypochlorite to remove the smear layer . following this ,
the canals were dried with sterile paper points . following the manufacturer 's instructions , the ah plus sealer ( dentsplydetrey gmbh , konstanz , germany ) was mixed and applied within the root canal . a size 25% , 2% master cone ( diadent group international , korea )
a spreader was used to laterally compact the 2% gutta - percha accessory cones coated with ah plus sealer .
excess gutta - percha was sheared off and condensed with a plugger 1 mm below the canal opening .
the opening of the canal was sealed with coltosol ( coltene - whaledent , cuyahoga falls , oh ) and the teeth were stored at 37c in 100% humidity for 2 weeks .
in group ii , cleaning and shaping was performed in the same manner as for group i following which the teeth were subjected to sct ( light speed plus computed tomography scanner [ ge electricals , milwaukee , usa ] ) to assess the teeth at different sections of the coronal , middle and apical thirds respectively .
the remaining dentin thickness was then measured from the central canal to the outer periphery in buccal , lingual , mesial and distal directions .
in group ii , retreatment was performed after 48 h following obturation to allow for adequate time for the sealer to set with protaper universal niti rotary retreatment ( dentsply maillefer , ballaigues , switzerland ) instruments , which were used at 300 rpm and 3 n / cm torque .
the retreatment files were used in the recommended sequence - d1 file ( size 30 , 0.09 taper ) for removal of root filling in the coronal third , followed by d2 file ( size 25 , 0.08 taper ) for the middle - third and finally , the d3 file ( size 20 , 0.07 taper ) was used at working length .
final shaping of the apical third was performed with protaper instruments f2 ( size 25 , 0.08 taper ) and f3 ( size 30 , 0.09 taper ) .
after complete instrumentation , all specimens received final irrigation with 10 ml of 17% edta followed by 10 ml of sodium hypochlorite to remove the smear layer . following this , 10
after completion of cleaning and shaping procedures , the specimens were again assessed with sct for the remaining dentin thickness as mentioned earlier .
lateral compaction was done using a size 30% , 2% gutta - percha master point coated with ah plus sealer . following the manufacturer 's instructions ,
the master cone was placed at the appropriate working length and apical tuckback confirmed and a spreader was used to laterally compact the gutta - percha accessory cones coated with ah plus sealer .
excess gutta - percha was sheared off and condensed with a plugger 1 mm below the canal opening .
the opening of the canal was sealed with coltosol f and the teeth were stored at 37c in 100% humidity for 2 weeks .
all the teeth in both groups were mounted on a custom made acrylic blocks such that 9 mm of the root surface remained exposed while the rest was embedded in the acrylic resin ( digital products international products , india ) .
the temporary material was removed using a bur and the root canal access was shaped to accept the loading fixture .
the acrylic blocks were mounted with the roots aligned vertically on the instron testing machine one at a time .
the mode of application of the vertical loading force required to fracture the root specimens was similar to the technique proposed by sedgley and messer to test the brittleness of endodontically treated teeth .
the specimens were subjected to a constant load at a crosshead speed of 1.0 mm / min until the roots fractured . in this study , fracture was defined as the point at which a sharp and instantaneous drop was observed .
this point was verified by a computer attached to the instron testing machine ( model 3382 , instron , uk ) . for most specimens ,
the test was terminated at this point and the force applied was recorded and measured in newtons .
statistical analysis was performed using independent samples t - test for analysis of remaining dentin thickness using sct within group ii and paired samples t - test was used for assessment of fracture resistance between group i and ii ( p < 0.05 ) .
in group ii , the intra group comparison of the remaining dentin thickness in the coronal third reveals statistical significance , with a significant difference noted in the apical third [ table 1 ] .
remaining dentin thickness paired sample ( t - test ) results of the fracture resistance reveal a statistically significant difference ( p < 0.05 ) between groups i and ii with the mean fracture resistance of group i being higher than that of group ii [ table 2 ] . fracture resistance ( in newtons )
the prognosis of any endodontic therapy is directly correlated to the execution of its different phases appropriately .
the persistence of bacterial colonies within the intricacies of the root canal system leads to refractory apical periodontitis in endodontically treated teeth .
this has led to the choice of non - surgical management by retreatment of previously treated teeth by employing techniques for removal of obturating material followed by sufficient cleaning and reshaping procedures to provide adequate disinfection by appropriate irrigants .
it has already been established in the endodontic literature that the incidence of vertical root fracture is greater in root canal treated teeth , which often leads to extraction .
hence during the re - treatment procedures care should be exercised to prevent loss of dentin that may occur from excessive and aggressive re - instrumentation of root that can substantially weaken the structural integrity of the tooth making it more susceptible to fracture .
sct provides a means for non - invasive assessment without sample destruction and also provides a precise 3d reconstruction of the root canal system . in the present study
, sct has been used to assess the remaining dentin thickness after initial cleaning and shaping and also following retreatment to evaluate the loss of root dentin for the samples in group ii .
protaper universal retreatment system has been comprehensively designed to aid in removal of existing obturating material and to assist in cleaning and shaping to provide space for irrigation and disinfection and subsequent filling of the root canal .
d1 is used for removal of filling material the coronal thirds and d2 and d3 from the middle and apical thirds respectively .
the convex cross section of the d1 instrument along with its active tip facilitates its initial penetration into filling materials .
the removal of gutta - percha from the coronal portion of the root provides space for the introduction of solvents , which aids in further instrumentation procedures .
this system has been attributed to perform better due to their progressive taper , length of instrument ; specific flute design and rotary motion , which tend to cut and pull gp into file flutes and direct it toward the orifice .
various studies assessing the efficacy of instruments to remove filling materials from the root canal have postulated that filling residue is minimized when the enlargement in retreatment was bigger than the enlargement performed before the canal filling . in the present study ,
retreatment procedure was completed with the instrument used for enlargement , increased by only one size , before obturation .
the results of the present study reveal a significant difference with decreased fracture resistance in the retreatment group having a mean value of 280.6 n while the initial endodontic treatment group had a mean value of 448.6 n [ table 2 ] .
this statistically significant difference ( p < 0.05 ) in the fracture resistance of the two groups could be possibly due to a substantial difference in the remaining dentin thickness pre and post retreatment in the coronal and middle - third of the root canal [ table 1 ] .
this could be attributed to the use of d1 and d2 retreatment files , which has been found to cut superficial layer of dentin during root filling removal , which along with subsequent cleaning and shaping procedures may have resulted in the substantial loss of root dentin .
earlier investigations have reported that the filling material traced in the canal would be reduced when the enlargement in the retreatment was bigger than the enlargement performed before the canal filling .
the present study follows the protocol to increase the apical size following retreatment to one size larger than the initial treatment group , which may have accounted for the loss of root dentin from the apical thirds [ table 1 ] .
the sct analysis reveals the loss of root dentin in the pre and post retreatment samples in group ii .
this loss of root dentin during the course of retreatment procedure may have contributed to the reduced fracture resistance of the samples in group ii .
within the limitations of this study , it can be concluded that endodontically retreated teeth have shown significantly decreased resistance to fracture and this has a positive correlation to the increased loss of root dentin during the re - treatment procedures as has been confirmed by the aid of sct . | aim : to compare and assess the fracture resistance of endodontically treated teeth with those that have been subjected to endodontic retreatment.materials and methods:30 extracted mandibular premolars were decoronated at cementoenamel junction and randomly divided into 2 groups . in group i endodontic treatment was performed with protaper rotary system till size f2 and obturated . in group
ii , cleaning and shaping was done and teeth were subjected to spiral ct to assess the remaining dentin thickness and obturated .
later retreatment was done using protaper universal retreatment system and final shaping was performed till size f3 .
remaining dentin thickness was again assessed using spiral ct and then obturated .
all the specimens were subjected to fracture resistance using universal testing machine .
the results were statistically analyzed using independent samples t - test for analysis of remaining dentin thickness using sct within group ii and paired samples t - test was used for assessment of fracture resistance between group i and ii ( p < 0.05).results : in group ii , the intra group comparison of the remaining dentin thickness in the coronal third reveals statistical significance , with a significant difference noted in the apical third .
results of the fracture resistance reveal a statistically significant difference ( p < 0.05 ) between groups
i and ii with the mean fracture resistance of group i being higher than group ii.conclusion:endodontically retreated teeth have shown significantly decreased resistance to fracture and this has a positive correlation to the increased loss of root dentin during the retreatment procedures |
one of the reasons for the changes is concern for the patient 's safety .
to err is human , a landmark report released by the institute of medicine ( iom ) in 1999 estimated that medical errors cause injury to approximately 3% of hospital patients and results in a minimum of 44,000 and perhaps as many as 98,000 deaths per year in the united states .
another important finding came from the harvard medical practice study 1 , in which the authors reviewed over 30,000 randomly selected hospital records at new york state in 1984 as part of an interdisciplinary study of medical injury and malpractice litigation .
they found that injuries from adverse events occurred in 3.7% of hospital admissions , 27.6% of which were due to negligence and in which 13.6% led to death .
the annual cost attributable to all adverse drug events and preventable adverse drug events for a 700-bed american teaching hospital was estimated by one study as $ 5.6 million and $ 2.8 million repectively . though it is expected that such medical errors occur in saudi arabia , there are no studies on their extent in saudi hospitals .
calls for a change in the instructional methods have resulted in innovative medical curricula . the new curricula stress the importance of proficiency in several clinical skills by medical graduates rather than mere acquisition of knowledge .
as evidenced by their endorsement by many of the international bodies and medical schools , it is universally accepted that clinical skills constitute an essential learning outcome .
the acquisition of appropriate clinical skills is key to health education ; however , students sometimes complete their educational programs armed with theoretical knowledge but lack many of the clinical skills vital for their work . a major challenge for medical undergraduates
some medical schools in the middle east have changed their curricula and adopted such educational strategies as problem - based learning . also many medical schools have started to utilize clinical skills laboratories for training .
the objective of this narrative review article is to highlight the importance of simulation as a new teaching method for undergraduate and postgraduate education .
this is a narrative review of literature on a medical simulator and the use of simulation in medical education .
a literature search of medline / pubmed database for english - language publications and reference lists from relevant articles published between 1990 and august 2009 was conducted .
the main search terms were medical simulation , medical simulator , medical education , and clinical skills .
medical training programs should ensure that students have the necessary learning opportunities and assessed by the appropriate methods .
clinical skills competencies including communication skills , history - taking , professional attitudes , awareness of ethical basis of healthcare , physical examination , procedural skills , clinical laboratory skills , diagnostic skills , therapeutic skills , resuscitation skills , critical thinking , clinical reasoning , problem solving , team - work , organization skills , management skills , and information technology skills should be part of the core undergraduate curriculum .
traditionally , the acquisition and ongoing improvement of high level psychomotor skills required by future physician take place in an apprentice - style model of see one , do one , teach one. this apprentice - style of learning is no longer considered acceptable because of the increasing concern for the quality of patient care and safety and change in health care systems .
the pressure of managed care has shaped the forms and frequency of hospitalization and led to a higher percentage of acutely ill patients and shorter inpatient stays .
this has resulted in fewer opportunities for the medical learner to access a wide variety of diseases and physical findings .
relying on exposure to real hospital patients during training years may result in an ad - hoc method of learning clinical skills , as this depends on the availability of cases , and consequently to less than optimal development and performance of clinical skills .
there are many reports that indicate concerns for the level of skills medical graduates even in western countries possess .
the acquisition of expertise in clinical medicine requires the learner 's engagement in deliberate practice of desired learning outcomes . according to issenberg
et al ( 2002 ) deliberate practice involves ( a ) repetitive performance of intended cognitive or psychomotor skills in focused domain , coupled with ( b ) rigorous skills assessment , that provides learners ( c ) specific , informative feedback , that results in increasingly ( d ) better skills performance , in a controlled setting . concerns about patient safety and fewer available patients for learning , and many other factors have led to the introduction of simulation and the development of simulation centers and clinical skills laboratories in medical education .
aviation and aerospace industries have been using simulation as a teaching tool for many years .
simulators are now widely used in education and training in a variety of high risk professions and disciplines , including the military , commercial airlines , nuclear power plants , business and medicine .
recently , the inclusion of clinical skills training into the curricula of medical students has seen significant growth .
there are many examples of curricular reform that include clinical skills training , the use of simulators , and the creation of clinical skills centres .
simulation has been defined as a situation in which a particular set of conditions is created artificially in order to study or experience something that is possible in real life ; or a generic term that refers to the artificial representation of a real world process to achieve educational goals via experimental learning .
a simulator is defined as a device that enables the operator to reproduce or represent under test conditions phenomena likely to occur in actual performance . on the other hand , simulation based medical education can be defined as any educational activity that utilizes simulative aides to replicate clinical scenarios .
trainers can make mistakes and learn from them without the fear of distressing the patient .
experiential learning , which is a part of the definition of simulation , is an active process during which the learner constructs knowledge by linking new information and new experience with previous knowledge and understanding .
experiential learning or learning from experience during simulation based training sometimes involves the use of clinical scenarios as the bases of learning .
the practice of scenarios can be done individually , but it is mostly carried out by a team from the same or different specialties or professions in a simulated environment made to resemble the intended environment as closely as possible in order to immerse students in an experience closest to real life .
the practice of a scenario can be videotaped for immediate feedback to participants during the debriefing sessions .
video recording of the scenario is also used to initiate discussion and to make sure that all learning objectives were covered .
debriefing can focus both around the cognitive process involved in the recognition of the problem and the implementation of the management guidelines and the technical level at which the ability of the learner to apply rules and appropriate responses in a stressful situation is evaluated . during the full scale scenario - based training , the learner can acquire such important skills as interpersonal communication , teamwork , leadership , decision - making , the ability to prioritize tasks under pressure , and stress management .
however , training through simulation should be viewed as an adjuvant and not a replacement for learning with real patients .
simulation is not intended to replace the need for learning in the clinical environment , so it is important to integrate simulation training with the clinical practice during curriculum development .
simulators can be classified according to their resemblance to reality into low - fidelity , medium- fidelity and high - fidelity simulators .
example of a low - fidelity simulator is the intravenous insertion arm [ figure 1 ] and resusci - anne [ figure 2 ] .
moderate fidelity simulators give more resemblance of reality with such features as pulse , heart sounds , and breathing sounds but without the ability to talk and they lack chest or eye movement .
they can be used for both the introduction and deeper understanding of specific , increasingly complex competencies .
high fidelity simulators combine part or whole body manikins to carry the intervention with computers that drive the manikins to produce physical signs and feed physiological signs to monitors .
they can talk , breathe , blink , and respond either automatically or manually to physical and pharmacological interventions .
good examples of high - fidelity simulator is the meti human patient simulator ( hps ) which is model driven [ figure 4 ] and the noelle obstetric simulator which is instructor driven . in general , the higher the fidelity , the more expensive it is .
cardiology simulator meti human patient simulator ( hps ) virtual reality can also be incorporated into the simulators ( mostly part - task simulators ) to enhance learning .
virtual reality varies greatly according to its level of sophistication in its level of realism and of the user 's interaction with the virtual environment .
a common form of virtual reality involves the use of haptic ( touch ) feedback to produce a feeling of resistance when using instruments in a simulated environment .
high - fidelity and virtual reality simulations can bridge the gap between theory and practice by immersing the learner in a realistic , dynamic , complex setting .
the recreation of reality or fidelity is important for the success of simulation and for the participant .
since some simulators can be used to encourage independent or self - directed learning , they should be integrated into the overall curriculum .
the intended outcomes should be predefined and the training carried out in a controlled environment .
issenberg et al performed an excellent systematic review and identified ten features of high - fidelity medical simulation that can lead to effective learning .
the simulated scenarios are realistic enough to engage the students emotionally , thus providing a unique learning experience , where the high fidelity simulator patient
simulation can be adapted to accommodate the need of various medical specialties such as anesthesia , emergency medicine and trauma , intensive care medicine , obstetrics , pediatrics , and radiology as well as for the use of other professionals such as nurses , paramedics , and respiratory therapists .
. a single high - fidelity simulator with its monitoring system and other necessary equipment may cost up to $ 200 000 .
in addition , synthetic body fluids , replacement skins , bandages , syringes and other supplies are necessary to simulate the experience of treating real patients in a real hospital . the ability to practise without risk
simulation has many advantages , for it results in highly trained medical graduates who are less likely to make life - threatening or costly medical errors .
it has also proved to be effective in both undergraduate and post graduate education as well as faculty development .
simulation can be used in the primary health care setting to improve confidence in performing life - saving skills , clinical skills , communication skills , and the quality of care for patients with chronic diseases such as diabetes mellitus and bronchial asthma .
such simulators as part task trainers , computer - based systems , virtual reality and the haptic system , simulated patients , simulated environment , and integrated simulators have been also used effectively to assess and evaluate clinical skills .
the major challenge to medical simulation is the fact that evidence to date is weak in methodology .
the assumption that such learning is directly transferable to the clinical context is often untested .
only a few studies have shown a direct positive impact in the clinical outcome from the use of simulation for medical training .
advantages of simulation in conclusion , the promise of simulation - based medical training offers useful opportunities to reduce risks to patients and learners , improve learners competence and confidence , increase patient safety , and reduce health care costs in the long run .
however , robust research is needed to see if simulation training does actually improve patient outcomes . | one of the most important steps in curriculum development is the introduction of simulation- based medical teaching and learning .
simulation is a generic term that refers to an artificial representation of a real world process to achieve educational goals through experiential learning .
simulation based medical education is defined as any educational activity that utilizes simulation aides to replicate clinical scenarios .
although medical simulation is relatively new , simulation has been used for a long time in other high risk professions such as aviation .
medical simulation allows the acquisition of clinical skills through deliberate practice rather than an apprentice style of learning .
simulation tools serve as an alternative to real patients .
a trainee can make mistakes and learn from them without the fear of harming the patient .
there are different types and classification of simulators and their cost vary according to the degree of their resemblance to the reality , or
fidelity. simulation- based learning is expensive .
however , it is cost - effective if utilized properly .
medical simulation has been found to enhance clinical competence at the undergraduate and postgraduate levels .
it has also been found to have many advantages that can improve patient safety and reduce health care costs through the improvement of the medical provider 's competencies .
the objective of this narrative review article is to highlight the importance of simulation as a new teaching method in undergraduate and postgraduate education . |
autologous bone grafts remain the gold standard for the treatment of congenital craniofacial bone disorders , such as alveolar cleft [ 18 ]
. however , autologous bone grafts have potential problems , which include donor site morbidity and limitations to the amount of bone that can be harvested [ 913 ] .
porous beta - tricalcium phosphate ( -tcp ) , which is now commercially available , is known for its osteoconductive and biodegradable properties .
however , its use as a replacement for autologous bone grafts remains controversial [ 1418 ] .
according to recent studies , various growth factors exhibit osteogenic properties [ 8 , 1923 ] , such as bone morphogenetic protein 2 ( bmp-2 ) [ 2436 ] , basic fibroblast growth factor ( b - fgf ) [ 3742 ] , platelet derived growth factor ( pdgf ) [ 4346 ] , transforming growth factor - beta 1 ( tgf-1 ) [ 4750 ] , and vascular endothelial growth factor ( vegf ) [ 5154 ] . in general , growth factors administered in solution form
are readily diffused or degraded in vivo [ 26 , 50 , 55 , 56 ] .
thus , their enhanced and prolonged bioactivity at the target site is necessary to reduce bolus dosage , especially in pediatric patients .
recent studies suggest that granulocyte colony - stimulating factor ( g - csf ) promotes fracture healing or osteogenesis [ 5760 ] .
because g - csf is an essential drug most frequently used to treat neutropenia secondary to chemotherapy , it is widely administered not only to adults but also to pediatric patients [ 6169 ] .
accordingly , its biosafety is well established through extensive use in clinical contexts compared to other growth factors . commercially available
-tcp ( superpore , pentax , tokyo , japan ) was used in the present study as an osteoconductive scaffold and space - maintaining material . to investigate the bone regenerative properties of g - csf , topical supplementation either in solution or in sustained release form with a gelatin hydrogel system
the purpose of this study was to investigate whether g - csf with or without a controlled release system stimulates bone regeneration in combination with -tcp using a rat calvarial defect model [ 7073 ] .
the present study was approved by the institutional committee of animal experiments at hokkaido university ( institutional animal care and use committee protocol number 12 - 0017 ) .
fourteen wistar rats ( male , 13 weeks old ; weight , 250350 g ) were purchased from sankyo labo service corporation ( tokyo , japan ) . a total of 27 calvarial defects were randomly divided into nine treatment groups , with a total of three defects per treatment group . in solution - based treatment groups ,
defects were filled with a -tcp disc containing normal saline alone ( group a , control ) or 1 ( group b ) , 5 ( group c ) , or 20 g of g - csf ( group d ) . in controlled release groups ,
defects were filled with a -tcp disk with an overlaid gelatin hydrogel sheet incorporating normal saline alone ( group e ) or , 1 ( group f ) , 5 ( group g ) , or 20 g g - csf ( group h ) .
the remaining defects were left empty to measure spontaneous healing ( group i ) ( table 1 ) . commercially available porous -tcp blocks ( superpore )
blocks were cut into discs 5 mm in diameter and 1 mm thick using a fine surgical saw and round bur .
briefly , a mixed acidic gelatin - glutaraldehyde aqueous solution was cast into a polypropylene dish ( 80 80 mm ) and maintained at 4c for 12 hours .
the water content of gelatin hydrogels ( weight ratio of water present in hydrogel to wet hydrogel ) was 95 wt% .
gelatin hydrogels were designed so that degradation would be complete in approximately two weeks under in vivo conditions [ 29 , 39 , 49 , 74 ] .
hydrogel sheets were cut into discs 5 mm in diameter and 1 mm thick .
human recombinant g - csf was kindly supplied by kyowa kirin co. ( tokyo , japan ) . to prepare gelatin hydrogels incorporating g - csf , 20 l of normal saline solution containing 1 , 5 , or 20 g g - csf
similarly , 20 l of g - csf - free normal saline was dropped onto a freeze - dried hydrogel to obtain g - csf empty hydrogels .
animals were anesthetized by intraperitoneal administration of pentobarbital sodium ( 50 mg / kg ) .
subsequently , a skin incision was made and subperiosteal dissection was performed under a surgical microscope to raise the periosteal flaps . a bone defect 5 mm in diameter was then prepared on each side lateral to the sagittal suture using a fine surgical bur under copious sterile saline irrigation .
defects were filled with bone substitutes according to the groups described above ( table 1 and figure 1 ) .
periosteal flaps were repositioned using a 4 - 0 nylon suture , and the skin was closed with a running 4 - 0 nylon suture .
specimens were prepared for decalcified sectioning by immersing them in 10% ethylenediaminetetraacetic acid ( edta ) for four weeks .
embedded samples were then sectioned into 3 m slices parallel to the sagittal suture across the center of each calvarial defect using a microtome ( leica , sm2000r ) .
hematoxylin and eosin ( he ) staining was used for histological analysis and aniline blue staining was used for histomorphometric analysis .
each specimen was examined under a light microscope and digital photographs were obtained for histological evaluation of a region corresponding to the center of the calvarial defect ( figure 1(b ) ) .
high magnification images with aniline blue staining ( 1.001 mm or 1360 1024 pixels ) of the most - central area of the defect were quantified to measure the percentage of newly formed bone and remaining bioceramics using imaging software ( adobe photoshop cs5 ) .
statistical analysis was performed using kruskal - wallis one - way analysis of variance ( anova ) .
figure 2 shows low magnification images of decalcified specimens stained with he along the midline of each calvarial defect . no remaining gelatin hydrogel or surgical site infections were observed . in group
a ( control group ) , newly formed trabecular bone was observed focally but failed to occupy the entire defect . in groups
b d ( solution - based treatment groups ) and in group e ( g - csf - free gelatin hydrogel group ) , newly formed trabecular bone was observable but failed to fill the defect . in group f ( 1 g g - csf gelatin hydrogel group )
, newly formed bone tissue nearly bridged the calvarial gap , whereas residual -tcp was also present . in group g
( 5 g g - csf gelatin hydrogel group ) , most of the defect was occupied with newly formed bone tissue ; moreover , sparse residual -tcp was observed .
in contrast , group h ( 20 g g - csf gelatin hydrogel group ) showed focal formation of new bone surrounded by fibrous connective tissue at the superficial area of the defect with the presence of remaining biomaterials . in group
i ( untreated defect group ) , the defect was filled with fibrous connective tissue with hardly any newly formed bone .
figure 3 shows higher magnification images of groups e ( e ) and g ( g ) . in group g ,
newly formed bone was observed immediately below the periosteal flap and multinuclear giant cells were detected around the newly formed bone .
in contrast , in group e , the formation of fibrous tissue and blood vessels was significant compared with newly formed bone in the subperiosteal region .
figure 4 shows high magnification images of aniline blue staining in which matured bone tissue exhibits homogeneous dark blue and entrapped osteocytes .
figure 5 shows the percentage of newly formed bone and remaining -tcp per high - powered field . in groups a , b , c , d , e , and h , defects had a tendency to be occupied by more remaining -tcp compared to newly formed bone tissue . in group a ( control ) ,
the percentages of newly formed bone and remaining -tcp were 20.77% 25.44% and 35.01% 2.01% , respectively .
in contrast , in groups f and g ( 1 g and 5 g g - csf gelatin hydrogel groups ) , the percentage of newly formed bone ( 54.84% 9.46% and 69.53% 5.35% for groups f and g , resp . ) conspicuously exceeded values of remaining -tcp ( 20.47% 2.89% and 14.76% 7.36% for groups f and g , resp . ) .
the values were significantly higher in groups f and g compared to the control group ( p < 0.01 ) .
there was no significant difference between groups a ( control ) and i ( empty defect ) .
values corresponding to groups b , c , d , e , and h showed no significant difference compared to that of group a.
figure 7 shows the percentage of remaining -tcp , which was used to evaluate biodegradability in vivo .
there was no significant difference between groups a , b , c , d , e , f , and h. in contrast , only in group g ( 5 g g - csf gelatin hydrogel group ) the percentage was significantly lower compared to group a ( 14.76% 7.36% versus 33.53% 0.80% , p < 0.05 ) .
this result indicated a prominent enhancement of the biodegradable properties of -tcp , which was further accelerated by 5 g g - csf in sustained release form .
in the present study , we demonstrated that the controlled release of low - dose ( 1 g and 5 g ) g - csf significantly enhanced bone regeneration when combined with a -tcp disc .
moreover , administration of 5 g g - csf using a controlled release system significantly promoted the biodegradable properties of -tcp .
according to our results , this tissue - engineering approach combining -tcp and the sustained release of g - csf is potentially feasible and promising for clinical use . to our knowledge , this is the first report which demonstrates the bone regeneration properties of g - csf at membranous ossification sites . because systemic administration of 510 g g - csf / kg / day is commonly used for pediatric malignancies [ 64 , 6668 ] , the results shown here indicate that notably low doses of g - csf ( 15 g / defect/2 weeks ) with controlled release can promote osteogenesis . in this study , we used -tcp as an osteoconductive scaffold and space - maintaining material . in the present study ,
although the control group ( -tcp alone ) showed a greater tendency for bone formation compared to the untreated defect group , there were no significant differences between the groups .
furthermore , the defect in the control group had more residual -tcp than newly formed bone tissue .
these results suggest that -tcp alone implantation is not sufficient to fill the defect with regenerated bone in the craniofacial region .
some experimental studies have confirmed the osteoconductive properties of -tcp , which were comparable to autologous bone grafts [ 15 , 17 , 70 ] .
however , other groups have emphasized versatility by combining -tcp with autologous bone fragments [ 16 , 7779 ] , growth factors [ 45 , 46 , 8084 ] , simvastatin , or stem cells [ 18 , 85 , 86 ] in both experimental and clinical studies .
interestingly , ishida et al . reported that topical application of g - csf had bone regenerative properties via neovascularization and osteogenesis .
that study revealed a significant increase in cd34 cells an endothelial and hematopoietic progenitor - enriched cell population in capillaries corresponding to the bone defect site .
the study also showed that g - csf was responsible for mobilizing osteoblasts to the bone defect site .
in addition , recent studies demonstrated the promotion of fracture healing by cd34 cells [ 58 , 8789 ] .
kuroda et al . reported the first successful clinical case of a tibial nonunion treated with topically applied g - csf - mobilized cd34 cells .
some reports have shown that cd34 cells play an important role in releasing angiogenic factors , including vascular endothelial growth factor ( vegf ) , hepatocyte growth factor ( hgf ) , and fibroblast growth factor 2 ( fgf2 ) [ 58 , 90 , 91 ] .
moreover , the differentiation capacity of cd34 cells into osteoblasts has been shown in previous reports [ 59 , 92 ] . in the present study , the controlled release g - csf groups showed more newly formed bone immediately below the periosteum compared to the other groups .
on the other hand , rojbani et al . reported that osteoprogenitor cells differentiate from the dura mater .
presumably , the sustained release of g - csf may stimulate periosteal cells along an osteogenic lineage , resulting in enhanced bone formation . in the present study , we used gelatin hydrogel as a sustained release carrier of g - csf .
various growth factors have been shown to have bone regenerative properties , such as bone morphogenetic proteins ( bmps ) [ 2436 ] , b - fgf [ 3742 , 93 ] , pdgf [ 4346 ] , tgf-1 [ 4750 ] , and vegf [ 5154 ] .
bmp-2 has the strongest osteoinductive activity in promoting ectopic bone regeneration [ 26 , 29 ] and has been approved by the food and drug administration for use in orthopedics and oral surgery [ 30 , 34 , 35 ] . in general , growth factors administered in solution form are easily diffused or degraded prior to achieving full bioactivity [ 26 , 50 , 55 , 56 ] . therefore , commercially available bmp-2 in combination with a collagen sponge kit must contain milligram amounts of the growth factor ( 1.5 mg / ml ) [ 30 , 34 , 35 ] . potential risk for local inflammatory responses
should be taken into consideration after topical application . in order to reduce bolus dosage , enhanced and
one of the practical ways to control the in vivo release of growth factors is to use gelatin hydrogel , in which the growth factor is physicochemically immobilized and subsequently released in proportion to hydrogel degradation [ 74 , 94 ] . in the present study , the water content of gelatin hydrogels ( weight ratio of water present in hydrogel to wet hydrogel ) was 95 wt% .
the hydrogels were designed so that degradation would be complete in approximately 2 weeks under in vivo conditions [ 29 , 39 , 49 , 50 , 74 ] .
gelatin is commercially available and its biosafety is well established through its long clinical use as a plasma expander and drug ingredient . in the present study ,
the controlled release of 5 g g - csf ( group g ) significantly promoted the osteoconductive properties and biodegradability of -tcp . improved biodegradability compared to hydroxyapatite is a major characteristic of porous -tcp [ 71 , 9597 ] .
biodegradability is generally thought to occur in harmony with bone remodeling , in which -tcp allows tissue fluid dissolution and absorption by osteoclasts in vivo [ 72 , 95 ] .
reported that g - csf increased both osteoclast activity and bone resorption in the bone marrow , triggering an increase in the number of mesenchymal precursor cells in the bone marrow using a mouse model .
in another study , pdgf modified -tcp resorption , although the underlying mechanism was not provided .
some studies have shown that bmp-2 does not facilitate -tcp resorption [ 70 , 99 ] . in group g of the present study ,
we can speculate from the results that the controlled release of 5 g g - csf may stimulate the mobilization and differentiation of mesenchymal precursor cells in the periosteum as well as osteoclast activation .
in contrast , group h ( 20 g g - csf gelatin hydrogel group ) showed less new bone formation and -tcp resorption . this might be explained by the multidifferentiation potential of g - csf - mobilized progenitor cells , which is consistent with previously published reports [ 92 , 100102 ] .
interestingly , ishida et al . stated that topical application of 50 g g - csf did not induce bone regeneration according to preliminary data .
moreover , some reports have shown that sustained release of g - csf enhances tendon - bone integration with significantly more formation of sharpey 's fibers and microvessels .
these results led us to speculate that a prolonged high concentration of topical g - csf drives progenitor cells toward fibrous tissue formation rather than osteogenesis . therefore , sustaining relatively low concentrations of topical g - csf can play an important role in inducing balanced bone regeneration and -tcp resorption .
our findings suggest an optimal dose of 5 g per defect for controlled release of g - csf , which is consistent with previously published reports [ 59 , 103 ] .
first , the study was designed using small animals and a limited number per experimental group .
second , although some reports accept the calvarial defect rat model [ 7073 ] , the decortication procedure may not fully reflect clinical situations of congenital craniofacial anomalies , since some evidence suggests that fractures mobilize cd34 cells from the bone marrow into the peripheral blood [ 88 , 89 ] .
third , we used histomorphometric analysis to characterize newly formed bone and biodegradation of -tcp ; however , we did not identify cd34 cells or evaluate the activity of osteogenic cells at the bone defect site .
future studies should incorporate experimental models without decortication , larger animals , and immunohistochemical analysis . in conclusion , controlled release of 5 g g - csf using a gelatin hydrogel system
the present results indicate that the combination of g - csf slow - release and -tcp is feasible and promising for the treatment of congenital craniofacial bone defects . | autologous bone grafts remain the gold standard for the treatment of congenital craniofacial disorders ; however , there are potential problems including donor site morbidity and limitations to the amount of bone that can be harvested .
recent studies suggest that granulocyte colony - stimulating factor ( g - csf ) promotes fracture healing or osteogenesis .
the purpose of the present study was to investigate whether topically applied g - csf can stimulate the osteoconductive properties of beta - tricalcium phosphate ( -tcp ) in a rat calvarial defect model .
a total of 27 calvarial defects 5 mm in diameter were randomly divided into nine groups , which were treated with various combinations of a -tcp disc and g - csf in solution form or controlled release system using gelatin hydrogel .
histologic and histomorphometric analyses were performed at eight weeks postoperatively .
the controlled release of low - dose ( 1 g and 5 g ) g - csf significantly enhanced new bone formation when combined with a -tcp disc .
moreover , administration of 5 g g - csf using a controlled release system significantly promoted the biodegradable properties of -tcp . in conclusion ,
the controlled release of 5 g g - csf significantly enhanced the osteoconductive and biodegradable properties of -tcp .
the combination of g - csf slow - release and -tcp is a novel and promising approach for treating pediatric craniofacial bone defects . |
prostate cancer is a significant cause of death among men in europe and the united states . in the united states ,
the incidence increased to 24.8 per 100,000 men in 2009 , with a two - fold increase over that in 2008 .
however , all available treatments , including prostatectomy , may significantly impact the patient 's quality of life .
therefore , clinicians must attempt to determine at the time of diagnosis which patients might do well with active surveillance , who should receive immediate local treatment , and who will require aggressive multimodal therapy . in recent years , several nomograms have been developed to assist clinicians in the prediction of patient outcome following these different treatments .
however , most are limited by their complex nature or poor predictive value . in 2005 ,
cooperberg et al . derived the cancer of the prostate risk assessment ( capra ) score to preoperatively predict biochemical recurrence - free survival ( rfs ) after radical prostatectomy in patients with localized prostate cancer .
its high accuracy was verified through analysis using search data with multicenter enrollment . in the present study
, we evaluated the validity of the capra score at a single institution in korea .
under institutional review board supervision at inje university busan paik hospital , patients who underwent prostatectomy without adjuvant therapy as the first - line treatment for localized prostate cancer were retrospectively analyzed from january 2008 to june 2013 .
a total of 115 patients met all inclusion criteria to calculate the capra score and were included in the study group .
the same variable definitions as those in the original capra score design were used in this study .
biochemical recurrence after radical prostatectomy was defined as two consecutive prostate - specific antigen ( psa ) values of 0.2 ng / ml at any time postoperatively or any additional treatment more than 6 months after prostatectomy .
the patients were divided into two study groups : the seven - group model ( each capra score sum ) and the three - group model ( low , intermediate , and high risk ) .
the 3- and 5-year rfs rates were evaluated with kaplan - meier analysis for probabilities .
the concordance index for the capra scoring system was calculated with logistic regression and cox regression to determine the predicted survival probabilities .
logistic regression analyses were performed , and odds ratios ( ors ) were calculated for the occurrence of positive surgical margins , extracapsular extension , and seminal vesicle invasion .
3.01 ( r foundation for statistical computing , vienna , austria ) were used for the statistical analyses , and p<0.05 was considered statistically significant .
the mean age of the patients was 66.4 ( standard deviation [ sd ] , 6.5 ) years .
the mean preoperative psa level was 12.7 ( sd , 13.2 ) ng / ml , and 15 patients ( 13% ) developed recurrence at a median of 13 ( sd , 12.1 ) months .
the patient characteristics are listed in tables 1 , 2 and are compared with those from the capsure dataset , german data , and japan data . the number of patients with a high preoperative psa level ( > 10 ng / ml ) was similar to that of the german multicenter data ( 41.7% ) . however , the present study showed higher biopsy gleason scores than those in the german multicenter data .
these rates decreased as the capra score increased , but the trend was not statistically significant .
however , in the three - group model , the 3- and 5-year rfs rates significantly decreased as the risk increased ( p=0.011 ) . for patients with low ( score of 0 - 2 ) , intermediate ( score of 3 - 5 ) , and high risk ( score of 6 - 10 )
, the 3- and 5-year rfs rates were 93.5%/93.5% , 92.2%/85.6% , and 68.1%/61.3% , respectively .
the concordance indices of the capra score for the 3- and 5-year rfs rates were 0.74 and 0.77 , respectively .
the capra scores of both the three- and seven - group models predicted that the probability of recurrence generally increased with the actual probability of recurrence of the 3- and 5-year rfs .
however , in all situations , the capra score tended to underestimate the risk of recurrence ( fig .
2 ) . with the exception of the preoperative serum psa level and clinical t stage , most predictor variables for the capra score ( i.e. , age , gleason sum , and percentage of positive biopsies )
the strongest predictor variable was the gleason sum with a hazard ratio of 11.6 ( p=0.018 ) , and the most reliable variable was the percentage of positive biopsies with a hazard ratio of 5.6 ( p=0.002 ) .
we found significantly higher rates of positive surgical margins , extracapsular extension , and seminal vesicle invasion coinciding with higher capra scores or risks in the three - group model ( table 3 ) .
positive extracapsular extension occurred in 13.3% of patients with a capra score of 0 to 1 and in 66.7% with a capra score of 7 to 10 ( p=0.003 ) . in the three - group model , positive surgical margins occurred in 16.3% of patients in the low - risk group and in 45.2% in the high - risk group ( p=0.008 ) .
these events result not only in an increased incidence of cancer - related morbidities , but also in a heightened level of anxiety for patients and clinicians . therefore , well - established predictors of the risk of disease progression should help clinicians and patients to choose and assess the effectiveness of the various therapeutic options and should especially help with postoperative disease assessment .
many nomograms for preoperative risk assessment have recently been introduced . the d'amico risk classification and the kattan nomogram
the d'amico risk classification performs well ; however , there is significant overlap between intermediate- and high - risk patients .
the kattan nomogram was introduced with better integration of multiple risk factors , but it is difficult to apply without the paper forms of the instruments at hand .
however , in low - risk patients in a community setting , the rfs is overestimated .
the capra score originated from the capsure database , so it performs well in community - based cohorts , comparable with the kattan nomogram . in a previous study ,
the c - indices for the three systems were 0.63 ( 0.55 - 0.72 ) for the d'amico classification , 0.65 ( 0.56 - 0.75 ) for the kattan nomogram , and 0.66 ( 0.57 - 0.75 ) for the capra score . in a us multicenter survey using the search database ,
however , a higher prediction ability for recurrence was obtained in a german multicenter study ( 0.78 and 0.81 ) . in japan ,
the prediction ability of the 3-year rfs rate is variable among systematic reviews and meta - analyses .
furthermore , the capra score is reasonably accurate for predicting metastasis ( c - index=0.78 ) and cancer - specific mortality ( c - index=0.80 ) .
the c - index for the 3- and 5-year rfs in the present study was 0.74 and 0.77 , respectively .
these rates are higher than those of the original development set and us multicenter survey , but lower than the german multicenter survey and previous systematic reviews .
loeb et al . evaluated the capra score in a single - surgeon radical prostatectomy series , similar to our circumstances ; they calculated a c - index of 0.764 for 5-year rfs , similar to our result .
the short follow - up period and the more censored data than in other large - scale studies were probably the reasons for the differences .
in addition , the preoperative characteristics of the patients differed from those in other studies . in the present study ,
the proportion of patients with a high preoperative psa level ( > 10 ng / ml ) was higher than that in the capsure database when the capra score was first introduced ( 41.7% vs. 19% ) , but similar to that in the german data .
furthermore , the proportion of patients with a primary gleason score of 4 was higher than that in the capsure and german multicenter data ( 36.5% vs. 9% vs. 14% , respectively ) .
generally , lower - risk patients with prostate cancer have been included in us studies .
the only significant relationship was seen in the three - group model between the 3- and 5-year rfs and increasing risk ; otherwise , in the seven - group model , the trend of rfs was the same as that in the three - group model , but there was no statistical significance . in both the seven- and three - group models ,
the capra score predicted probabilities of recurrence that were generally higher than the actual probabilities of recurrence .
this is similar to the findings of the german multicenter study . in the german study ,
the probabilities showed a slight underestimation of the risk of recurrence within the medium - probability range of recurrence and an overestimation of the risk of recurrence within the high - probability range of recurrence . however , our study showed a general underestimation of the risk of recurrence in both the three- and seven - group models for the 3- and 5-year rfs .
pathologic outcomes were not available for the development set of the capsure study , but the search study analyzed the prediction ability of the capra score for pathologic outcomes .
it showed a statistically significant relationship between pathologic outcomes ( i.e. , positive surgical margin status , extracapsular extension , seminal vesicle involvement , and lymph node involvement ) and an increasing capra score .
seminal vesicle involvement was particularly striking , with a near doubling of risk ( or , 1.80 ; 95% confidence interval [ ci ] , 1.6 - 2.02 ) .
our results were the same : the capra score showed an increasing probability in terms of the margin status , extracapsular extension , and seminal vesicle invasion .
in addition , in the seven - group model analysis , seminal vesicle invasion was the most predictable result with an increasing capra score ( or , 2.35 ; 95% ci , 1.2 - 4.7 ) .
only three patients were diagnosed with lymph node invasion as a pathologic outcome in the present study . in the three - group model , the three pathologic outcomes were also significantly related to increasing risk , and seminal vesicle invasion was also the most predictable ( or , 5.62 ; 95% ci , 1.34 - 23.5 ) .
this validity of the capra score was also proven by the german multicenter study , and no organ confined with advanced disease was the most predictable ( or , 2.11 ; 95% ci , 1.9 - 2.3 ) .
first , few patients were studied compared with other large cohorts ( i.e. , capsure , search ) ; therefore , this study is not representative of all korean prostate cancer patients . however , it is the first study in korea on a new method for predicting prognoses and results after radical prostatectomy .
second , because of the short follow - up period , more detailed results regarding rfs could not be obtained .
clinicians must decide on treatments after considering the individual traits of patients , such as socioeconomic factors , preferences regarding quality of life , and even genetic factors .
thus , it must be understood that the capra score , like other recurrence - prediction instruments , can not replace clinical decision - making in patients with localized prostate cancer .
this instrument might help clinicians and patients in planning and counseling at the time of a localized prostate cancer diagnosis , especially when prostatectomy is planned .
in addition , characteristics such as convenient use , the scoring system ( seven - group model ) , and the ease of risk stratification ( three - group model ) may support its widespread use .
however , additional assessments of its validity are needed in korea , which is experiencing an increasing incidence of prostate cancer , through multi - institutional studies involving large cohorts . | purposeto evaluate the validity of the cancer of the prostate risk assessment ( capra ) score , a newly developed nomogram for preoperative prediction of recurrence after radical prostatectomy , in a single institution in korea.materials and methodswe retrospectively studied 115 men who had undergone radical prostatectomy as the first treatment for localized prostate cancer .
the validity of the capra score for the prediction of recurrence - free survival ( rfs ) and pathologic outcome was evaluated by using kaplan - meier analysis and a proportional hazards regression model . a seven - group model and a three - group model
were used for the results.resultsnone of the variables of the capra score was favorable compared with the previously reported data .
the three - group model was significantly related with 3- and 5-year rfs ( p<0.05 ) , but the seven - group model was not .
the concordance indices of the capra score were 0.74 and 0.77 . of four components excluding the clinical t stage , three independently predicted rfs ( age , gleason sum , and percentage of positive biopsies ) .
the capra score was significantly related to the margin status , extracapsular extension , and seminal vesicle invasion in both the seven- and three - group models . in the three - group model ,
pathologic outcomes were more strongly related , especially a higher risk of seminal vesicle invasion.conclusionsthe capra score showed high accuracy for predicting rfs .
in particular , the three - group model was more useful for predicting rfs and pathologic outcomes .
therefore , the capra score may be a useful prediction model for risk stratification and may help clinicians to develop localized prostate cancer treatment . |
brca1 and brca2 are tumour - suppressor genes located on chromosomes 17q21 and 13q12 , respectively .
functional brca proteins are involved in the maintenance of genome stability through repair of dna double - strand breaks ( dsbs ) by homologous recombination ( hr ) , cell growth regulation and control of cell division .
individuals carrying monoallelic germline pathogenic mutations in brca1 or brca2 ( brca1/2 ) are at higher risk of developing a variety of cancers , particularly breast and/or ovarian cancer .
meta - analyses have indicated a mean cumulative breast cancer risk at age 70 years to be 57% ( 95% ci 4766% ) for patients carrying the brca1 pathogenic mutations and 49% ( 95% ci 4057% ) for patients carrying the brca2 pathogenic mutations .
the equivalent mean cumulative ovarian cancer risk is 40% ( 95% ci 3546% ) for patients carrying the brca1 pathogenic mutations and 18% ( 95% ci 1323% ) for patients carrying the brca2 pathogenic mutations .
a prospective epidemiological study ( embrace ) showed that carriers of brca1 and brca2 pathogenic mutations have a mean cumulative risk of breast cancer at age 70 years of 60% ( 95% ci 4475% ) and 55% ( 95% ci 4170% ) , respectively .
the equivalent mean cumulative ovarian cancer risk is 59% ( 95% ci 4376% ) and 16.5% ( 95% ci 7.534% ) , respectively .
tumourigenesis in germline brca1/2 pathogenic mutation carriers generally follows a two - hit hypothesis , the first hit ' owing to the inherited pathogenic mutation of one brca allele and the second hit ' owing to the somatic inactivation of the second - wild - type allele .
increasing evidence suggests that other types of breast and ovarian cancers share genomic and phenotypic similarities with tumours associated with germline and somatic brca1/2 pathogenic mutations .
such cases may be sensitive to the same emerging targeted therapies as tumours associated with germline brca1/2 pathogenic mutations .
methods for the detection of brca1 and brca2 pathogenic mutations are now widely accessible . until now
, the principal aim of brca1/2 pathogenic mutation testing has been to enable risk assessment to permit early diagnosis and cancer prevention .
however , it is increasingly apparent that knowledge of brca status has prognostic utility that can affect treatment decisions and may improve survival .
this review highlights the biological role of brca1/2 pathogenic mutations and other associated defects in dna damage repair in breast and ovarian cancer , with particular focus on implications for clinical management strategies .
dna repair mechanisms also allow cancer cells to survive the dna injury imposed by chemotherapy or radiation .
an elaborate network of genome surveillance systems and dna repair mechanisms exist to repair dna lesions and ensure the integrity of the genome and hence cell fitness and viability .
dna dsbs , in which both strands of the double helix are severed , are the most dangerous type of dna lesion ; if left unrepaired , or repaired incorrectly , dsbs may result in massive loss of genetic information , genomic rearrangements or cell death .
two different mechanisms exist for the repair of dsbs : non - homologous end joining ( nhej ) and hr .
nhej is an intrinsically error - prone pathway , which modifies the broken dna ends , and ligates them together with little or no homology , generating small deletions or insertions . in contrast , hr is a highly conserved pathway that provides accurate repair of dsbs in the late s and g2 phases of the cell cycle using the intact sister chromatid as a template to repair the break and maintain sequence integrity .
brca1 and brca2 are key components of the hr pathway , and cells that lack these proteins are unable to repair dsbs by hr .
brca1 appears to have an early and broad role in the promotion and regulation of hr .
brca1 has been shown to colocalise at sites of dna damage with rad51 , another key protein involved in hr , while brca1/2-deficient cell lines lack rad51 foci .
brca1 appears to regulate hr , at least in part , through a modulatory role in the palb2-dependent loading of brca2-rad51 repair machinery at dna breaks .
a central role for brca2 in hr was first suggested by evidence showing the acquired chromosomal abnormalities of brca2-deficient cell lines to be similar to those seen in fanconi 's anaemia .
furthermore , cell lines derived from some patients with fanconi 's anaemia were shown to carry biallelic pathogenic mutations in brca2 , which led to brca2 also being referred to as fancd1 .
brca2 knockout cells sustain spontaneous aberrations in chromosome structure that accumulate during division in culture . in the absence of dna damage ,
rad51 is sequestered by brca2 , prohibiting rad51 nucleation onto double - stranded dna ( figure 1 ) .
following dna damage , brca2 relocalises to the site of dna damage and enables rad51 nucleation onto single - stranded dna .
pathogenic mutations in several other genes involved in hr - mediated dna repair have been shown to be associated with breast and ovarian cancer predisposition ( figures 2 and 3 ) . in the cancer genome atlas research network analysis of high - grade serous ovarian adenocarcinomas , genomic alterations in 26% of hr genes other than brca1/2
were observed , including amplification or pathogenic mutation of emsy ( 8% ) , promoter methylation of rad51c ( 3% ) , pathogenic mutation of atm / atr ( 2% ) and pathogenic mutation of fanconi 's anaemia genes ( 5% ) ( figure 2 ) .
in addition , focal deletion or pathogenic mutation of pten ( 7% ) has been observed ; however , the role of pten in hr or as a surrogate of hr deficiency remains to be determined .
brcaness ' if they exhibit similar dna repair defects to those seen in brca1/2-deficient cells .
monoallelic germline pathogenic mutations in palb2 , brip1 and atm have been shown to be associated with an increased breast cancer relative risk of approximately 23 .
biallelic pathogenic mutations in palb2 and brip1 have been observed in patients with fanconi 's anaemia , resulting in their alternative names of fancn and fancj , respectively .
more recently , brip1 pathogenic mutations have also been demonstrated in patients with ovarian cancer .
the frequency of germline palb2 pathogenic mutations in ovarian cancer cases does not appear increased as compared with the general population .
palb2 encodes the partner and localiser of brca2 protein , which stabilises the brca2 protein and anchors it to structures within the nucleus , allowing the brca2 protein to mediate dna repair .
encodes brca1-interacting protein - terminal helicase 1 , a dna helicase that influences the dna repair ability and tumour - suppressor function of brca1 .
atm is a protein kinase with a key role in sensing dna dsbs and monitoring their repair .
the rad51 paralogues , rad51c and rad51d , also have an integral role in the repair of dna dsbs through hr ( figure 3 ) . to date , germline pathogenic mutations in rad51
have not been observed in patients with breast or ovarian cancer , suggesting that they are lethal .
however , rad51c pathogenic mutations have been identified in up to 2.9% of highly penetrant breast and ovarian cancer families who previously screened negative for brca1/2 pathogenic mutations .
ovarian cancer occurrence in families with rad51c pathogenic mutations shows major similarities with families carrying brca1/2 pathogenic mutations .
in addition , as these families show apparent segregation of the pathogenic mutation with the cancer phenotype , the penetrance of rad51c pathogenic mutations is predicted to be comparable to that of brca2 pathogenic mutations ( antoniou , unpublished data ) .
however , the mean age at onset for ovarian cancer observed in women with rad51c pathogenic mutations is approximately 60 years , which is older than in brca1 pathogenic mutation carriers ( 51 years ) .
the paralogues rad51b , rad51d and xrcc2 are also associated with an increased ovarian cancer risk .
additional studies are required to better define their contribution to breast and ovarian cancer predisposition .
two genes are said to be synthetically lethal if a mutation in either gene alone is compatible with cell viability but simultaneous mutation of both genes causes cell death .
this suggests that inhibition of an additional dna damage repair pathway is likely to be synthetically lethal in cells lacking hr , whether through germline or somatic brca1/2 pathogenic mutations , posttranslational changes of brca or abnormalities of other genes involved in hr . the base excision repair pathway , in which poly - adp ribose polymerase ( parp ) has a major role , is important for the repair of certain kinds of dna damage , particularly in the absence of hr .
loss of parp function results in the accumulation of single - strand dna breaks , which are subsequently converted to dsbs by cellular transcription and replication .
these dsbs , which are typically repaired by hr or nhej in normal cells , would accumulate in hr - deficient cells , leading to subsequent cell death .
loss of parp1 function induces the formation of nuclear rad51 foci as a result of the increased formation of dna lesions that need to be repaired by the hr pathway .
two pivotal preclinical studies demonstrated loss of these rad51 foci in brca1- and brca2-deficient cells after parp inhibitor exposure . sensitivity to parp inhibition has also been observed in cells with defects in hr other than brca deficiency .
olaparib is a potent oral parp inhibitor that has been shown to induce synthetic lethality in brca1/2-deficient tumour cells .
antitumour activity of olaparib has been demonstrated in patients with brca - mutated breast and ovarian cancer in proof - of - concept trials . in a phase
ii trial , maintenance therapy with olaparib was shown to significantly prolong progression - free survival in patients with platinum - sensitive relapsed serous ovarian cancer compared with placebo , with the greatest benefit seen in patients with a pathogenic brca mutation .
an 82% reduction in risk of disease progression with olaparib compared with placebo was seen in patients with pathogenic brca mutations ( figure 4 ) .
retrospective exploratory analyses of time to subsequent treatment or death and time to second subsequent treatment or death ( indicators of the postprogression efficacy of olaparib ) also showed significant advantages in favour of olaparib over placebo in both the overall population and in patients with pathogenic brca mutations .
in addition to potential utility for the treatment of tumours harbouring a pathogenic brca1/2 mutation , parp inhibition might also be a useful therapeutic approach for the treatment of a wider range of tumours bearing a variety of deficiencies in the hr pathway and thus displaying properties of ' brcaness ' .
methods to identify patients most likely to benefit from these emerging therapies are therefore required .
at present , the primary goal of genetic testing for pathogenic mutations in brca1 and brca2 is identification of women at the greatest risk of developing breast and ovarian cancer to enable appropriate preventative strategies to be implemented .
however , now and in the near future , testing for pathogenic brca mutations or brca - like defects is likely to have a key role in patient care ; for example , in the identification of patients who are most likely to benefit from emerging therapies targeting dna repair mechanisms , such as parp inhibitors . in unselected breast cancer ,
the reported pathogenic brca mutation frequency is 15% , whereas in unselected ovarian cancer the reported frequency is higher at 616% .
it should be noted that these ranges exclude studies in male breast cancer and ashkenazi jewish women .
higher prevalence is associated with factors such as a positive family history , young age at onset , male breast cancer and multiple tumours in the same patient or certain histological characteristics . in an australian population - based , case control study , 1001 women with newly diagnosed histologically confirmed , non - mucinous , invasive epithelial ovarian , peritoneal or fallopian tube cancer were screened for point mutations and large rearrangements in brca1 and brca2 .
pathogenic brca1/2 mutations were identified in 14.1% of patients ( 141 mutations in 1001 women ; 95% confidence interval 11.9 , 16.3 ) . of these 141 pathogenic mutations ,
88 were in brca1 ( 62.4% ) and 53 were in brca2 ( 37.6% ) .
brca1/2 pathogenic mutations were seen in 16.6% of serous tumours , 16.8% of high - grade tumours and 17.1% of all high - grade serous tumours . of note , 44% of women with brca1/2 germline pathogenic mutations in this study had no previous family risk factors . in a study of 489 high - grade serous
ovarian adenocarcinomas , 64/316 ( 20.3% ) tumours with sequenced exomes ( n=316 ) were found to harbour pathogenic brca1 or brca2 mutations ; two tumours were observed to harbour both brca1 and brca2 mutations .
the majority of detected brca1/2 pathogenic mutations were germline in origin ( 71% ) , corresponding to a germline pathogenic variant frequency of 14.6% and a somatic pathogenic variant frequency of 6.0% .
accompanying heterozygous loss was observed with 81 and 72% of brca1 and brca2 pathogenic mutations , respectively , indicating inactivation of both alleles , as predicted by knudson 's two - hit hypothesis for a tumour - suppressor gene .
in addition to brca1/2 pathogenic mutations , 34/316 ( 10.8% ) of tumours had lost brca1 expression through brca1 promoter methylation .
epigenetic silencing of brca1 was shown to be mutually exclusive of germline brca1/2 pathogenic mutations ( figure 2 ) .
survival analysis based on brca status revealed divergent outcomes , with longer overall survival for brca - mutated cases compared with brca wild - type and brca1 epigenetically silenced cases , respectively ( figure 2 ) .
these data would suggest that a broader range of patients with ovarian cancer should now be tested , perhaps offering brca testing to all but patients whose tumours have mucinous histology .
so far , the low frequency of heterozygotes for brca pathogenic mutations relative to the high incidence of breast and ovarian cancer , coupled with the high costs of brca testing , has rendered exhaustive testing of all patients with breast and ovarian cancer impractical .
however , the evolution of multiple gene panel sequencing and the decreasing cost of genetic testing are allowing health - care budgets to offer pathogenic brca mutation testing to broader populations without a significant impact on cost .
the estimated population frequency of pathogenic brca1/2 mutations is 1:8001:1000 per gene ; however , the prevalence of brca1/2 germline pathogenic mutations varies considerably between different ethnic groups and geographical areas .
brca1 and brca2 pathogenic mutation frequency in patients with breast and ovarian cancer unselected for family history or age at onset is generally low . at present ,
the selection of appropriate candidates for testing is typically based on country - specific guidelines or by larger international societies .
widely accepted clinical criteria for referral include family history and age at cancer onset . based on these criteria , pathogenic mutations in brca1 or brca2
guidelines in some countries require a 1020% probability of detecting a brca1/2 pathogenic mutation within a family before mutational analysis is considered .
a number of predictive models and scoring systems have been developed to assess the probability of a pathogenic brca1/2 mutation in a given individual dependent on their family history , with varying degrees of validation .
methods that compute carriage probabilities are the most predictive but require specific computer software and data entry for all family members can be time - consuming .
scoring systems avoid data entry , are a good proxy to probability computations and can generally be easily modified to include new relevant predictive factors .
the manchester scoring system allocates a score to each affected individual in a family and computes the sum of the scores in the maternal and paternal lineage to determine whether or not a genetic test is recommended .
the manchester scoring system is empirical as the scores have been determined to fit the observations made in a group of selected families tested for pathogenic brca1/2 mutations .
it does not take into account information on unaffected family members , the presence of which is expected to decrease the probability of mutations , and gives the same weight to all affected family members whatever their degree of kinship .
an alternative system has been recently developed , which has been shown to be superior to the manchester scoring system .
the new scoring system is based on the conditional probability p that a proband is a carrier , given all relevant predictive information in the family .
parameters taken into account include pathogenic brca1/2 mutation frequencies in the population , as well as breast and ovarian cancer risks in carriers and non - carriers .
the performance of the new scoring system was evaluated using a simulation of 10 million families , built from women affected with breast or ovarian cancer at different ages . at a score threshold of 5 , where positive predicted value ( ppv ) ( 15% ie , percentage of carriers among tested individuals ) and specificity ( 87% ie , percentage of non - tested individuals among non - carriers ) are similar to the manchester scoring system with a pathogenic mutation probability of 10% , sensitivity ( ie , percentage of tested individuals among carriers ) with the new scoring system was higher than the manchester scoring system ( 77 vs 72% ) .
the improved performance of the new scoring system compared with the manchester scoring system was attributed to accounting for unaffected family members and for the degree of kinship of relatives with the proband .
pathogenic brca mutation testing on a broader population is likely to result in increased numbers of relatives screened , which may subsequently reduce the future incidence of ovarian cancer , as those relatives who are shown to harbour a pathogenic brca mutation can be started on a cancer prevention programme . despite mutation detection threshold ( ppv ) lowering , there remains a need to broaden brca1/2 testing criteria in order to optimise use of brca testing to guide treatment decisions .
one approach to broadening brca1/2 testing criteria is by introducing individual criteria , such as including women with triple - negative ( tn ) breast cancer ( ie , negative for oestrogen receptor , progesterone receptor expression and her2 amplification ) and women with high - grade ovarian cystoadenocarcinoma .
in one recent analysis , probabilities for the tn status of a breast tumour were obtained from the proportion of tn tumours among women tested for brca1/2 in studies without any selection on morphological characteristics .
a bayesian model was developed to calculate the probability of a pathogenic brca1 mutation according to age at diagnosis , assuming the rate of tn disease to be 68% among women with a pathogenic brca1 mutation and 13% among women with no pathogenic brca1 mutation ( including those with a pathogenic brca2 mutation ) .
results showed the probability of a pathogenic brca1 mutation to be high at 23% in women diagnosed with tn breast cancer at < 35 years , compared with 9.2% in women diagnosed at 3539 years , 5.5% at 4049 years , 4.3% at 5059 years and
thus tn disease status appears to be a good marker for pathogenic brca1 mutations especially in young women with breast cancer . including individual criteria specifically , ovarian cancer before age 61 years ( except borderline , mucinous tumours ) , breast cancer before age 36 years , tn breast cancer before age 51 years and male breast cancer at any age into one predictive model increased sensitivity to 77% ( a gain of 13% compared with use of french family criteria alone ) , with slight reductions in ppv ( 11% ) and specificity ( 82% ) .
however , the cost ' of introduction of individual criteria was to increase the number of tests by 49% . in order to maximise the potential of emerging therapies , such as parp inhibitors and agents targeting other proteins
involved in dna repair mechanisms , identification of patients with germline pathogenic mutations in other genes or biallelic somatic inactivation of genes involved in dna repair is likely to become increasingly important .
there is increasing interest in confirming the pathogenicity of many sequence variants in the brca1/2 genes whose pathogenicity is currently unknown , as well as other genes in the hr - pathway .
three groups have recently reported similar genomic scars ' , which appear to be potential surrogate markers of hr deficiency and potential sensitivity to dna - damaging agents .
these include a hr deficiency score based on genome - wide loss of heterozygosity , a telomeric allelic imbalance score or large - scale genomic instability .
the potential clinical application of genomic scars ' include facilitating the use of parp inhibitors and platinum - based chemotherapy in breast , ovarian and other cancers ; identifying cancer patients likely to benefit from treatments targeting defective dna repair ; and easing the challenge of selecting patients for genetic testing or recruitment to clinical trials of novel emerging therapies that target dna repair deficiencies in cancer .
such genomic scars ' would appear to merit further study as potential biomarkers of response to emerging targeted therapies ; however , direct testing of brca / hr - associated genes by next - generation sequencing ( ngs ) without investigating for such markers may be more practical .
the evolution of genetic testing may allow a more accessible option for broader testing strategies .
ngs is an efficient and cheaper alternative to sequential testing , allowing simultaneous screening of multiple cancer susceptibility genes .
assessing multiple gene panels will allow individualised testing dependent upon the risk for the individual from factors such as age , ethnicity or likelihood of inherited predisposition by family history of disease . in the future
, it is anticipated that more panels of genes linked to hr defects and cancer risks will be available at a lower cost .
in addition , the use of such gene panels directly on tumours in order to identify biallelic somatic events is an important issue and a technological challenge .
it is of course important , whatever the strategy used , that patients are kept well informed of the testing process .
it is increasingly apparent that the hr pathway for dna repair is not only disrupted by germline and somatic pathogenic brca1/2 mutations but also by pathogenic mutations in other genes involved in hr .
the presence of brca - like defects in patients with breast and ovarian cancer can be used to inform clinical management decisions . as such
, there is a clear need to broaden the criteria for brca1/2 germline genetic testing , as well as to expand testing to include identification of germline pathogenic mutations in other genes that may be involved in dna damage repair by hr dsbr .
furthermore , tumour markers of brcaness ' may have utility for the identification of patients most likely to respond to emerging targeted therapies , such as parp inhibitors . with the rapid evolution of multi - panel genetic sequencing , coupled with the decreasing cost of genetic testing | brca1 and brca2 are tumour - suppressor genes encoding proteins that are essential for the repair of dna double - strand breaks by homologous recombination ( hr ) .
cells that lack either brca1 or brca2 repair these lesions by alternative , more error - prone mechanisms .
individuals carrying germline pathogenic mutations in brca1 or brca2 are at highly elevated risk of developing breast and/or ovarian cancer .
genetic testing for germline pathogenic mutations in brca1 and brca2 has proved to be a valuable tool for determining eligibility for cancer screening and prevention programmes . in view of increasing evidence that the hr dna repair pathway can also be disrupted by sequence variants in other genes , screening for other brca - like defects
has potential implications for patient care . additionally , there is a growing argument for directly testing tumours for pathogenic mutations in brca1 , brca2 and other genes involved in hr - dna repair as inactivation of these genes may be strictly somatic
. tumours in which hr - dna repair is altered are most likely to respond to emerging targeted therapies , such as inhibitors of poly - adp ribose polymerase .
this review highlights the biological role of pathogenic brca mutations and other associated defects in dna damage repair mechanisms in breast and ovarian cancer , with particular focus on implications for patient management strategies . |
intraventricular tendons was first observed 115 years ago.1 they are usually thin , fibrous or fibromuscular structures that traverse the left ventricular cavity.2 they may be single or multiple and have no connection to the valvular cusps.2 false tendons can be an isolated finding or can be associated with cardiac abnormalities.3 loukas et al4 examined 200 formalin fixed , adult hearts and divided false tendons into five categories : in type i the false tendon is located between the posteromedial papillary muscle and the ventricular septum . in type ii the false tendon is located between the two papillary muscles . in type iii the false tendon
iv the false tendon is located between the ventricular septum and the free wall . in type v the false tendons appear weblike and have three or more points of insertion . after careful histological examination abdulla et al3 suggested that false tendons may be intracavitary radiations of the bundle of his and that their clinical significance may be more relevant than previously believed and merits further study .
recently , various physiological effects due to a newly observed thick , subaortic muscular band have been described.57 the purpose of this study was to describe the incidence of this peculiar new type of muscular band in a cardiovascular clinic .
a total of 4900 adult echocardiograms , performed between 2004 and 2010 for various clinical indications ranging from hypertension to cardiac murmurs and dyspnoea were reviewed and analyzed for the presence of a left ventricular muscular band in the subaortic position .
a total of 376 adult echocardiograms clearly demonstrated the presence of a muscular structure , located between the left ventricular inferior wall and the subaortic interventricular septum ( see figure 1 and 2 and movie clip 1 and 2 ) . in all of these cases
a striking local ventricular hypertrophic response was present at the site of septal implantation . in some of these cases the local hypertrophic response
a peculiar muscular structure , too thick in appearance to be called a tendon , extending between the subaortic portion of the interventricular septum and the inferior left ventricular wall is identified in this study .
a striking feature is a localized area of ventricular hypertrophy at the site of subaortic implantation of the muscular structure . among these 376 patients with the muscular subaortic band present ,
the following physiological effects have been observed : st - segment elevation of the anterior electrocardiographic leads , mimicking acute myocardial infarction,4 echocardiographical mimicking of hypertropic cardiomyopathy5 and right bundle branch block.6 it has been proposed before that this muscular , subaortic band may represent a sixth type of ventricular false tendon.8 the current clinical implication of the presence of this peculiar intracardiac structure is that it may mimic cardiac disease , such as myocardial infarction or brugada syndrome in the case of st segment elevation,5 it may lead to the erroneous diagnosis of hypertrophic cardiomyopathy if the echocardiographer is not vigilant6 and recently it has been described that a third type of right bundle branch block may result due to the presence of such a subaortic muscular band.7 | intraventricular tendons are structures that was identified more than a hundred years ago .
it has been suggested that they represent intracavitary radiations of the bundle of his and that they may be an isolated finding or be associated with structural cardiac abnormalities.loukas et al divided these structures into five categories and recently a sixth type have been added.various physiological disturbances have been observed due to the sixth type of tendon , such as st segment elevation and right bundle branch block .
it has been noted that this peculiar structure appears too thick to be called a tendon , thus the term band.this retrospective analysis analyzed the incidence of the thick , subaortic ( type 6 ) muscular band in a cardiovascular clinic . |
supplementary material is available for this article at 10.1007/s13659 - 011 - 0001 - 0 and is accessible for authorized users .
| 6/7-seco rearranged spiro - indolone alkaloids , meloyunines a ( 1 ) and b ( 2 ) and a monoterpenoid quinoline alkaloid meloyunine c ( 3 ) together with its possible intermediate 14,15-dehydromelohenine b ( 4 ) , and their precursor 14-vincamenine ( 5 ) were isolated from melodinus yunnanensis .
all structures were elucidated based on nmr , ftir , uv , and ms spectroscopic data .
the isolation of monoterpenoid indole , quinoline , and its immediate from the same plant chemically supported the biosynthesis of quinoline from indole .
compound 2 was cytotoxic against several human cancer cell lines .
electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 011 - 0001 - 0 and is accessible for authorized users . |
multiple myeloma ( mm ) is a clonal b - cell dyscrasia characterized by the accumulation of monotypic paraprotein - secreting cells in the bone marrow ( bm ) , in close contact with adjacent cells in its microenvironment .
a recent study reported that angiogenesis plays a significant role in the pathogenesis of mm .
plasma cells secrete a well - known angiogenic cytokine , vascular endothelial growth factor ( vegf ) , in response to stimulation of inflammatory factors , such as interleukin ( il)-6 , il-10 , il-17 , and il-20 [ 47 ] ; on the other hand , microvascular endothelial cells ( ecs ) and bm stromal cells secrete other potent growth factors for malignant plasma cells , thus regulating vegf stimulation .
another angiogenic cytokine , angiopoietin-2 ( ang-2 ) , prevents tie-2 binding and leads to vessel instability , associated with sprouting angiogenesis .
these accumulated data suggest that angiogenesis is tightly controlled by angiogenic cytokines and inflammatory factors in mm .
il-37 protein is associated with plasma cells and constitutively expressed in the cytoplasm of monocytes and peripheral blood mononuclear cells .
il-37 was reported to markedly inhibit the migration and proliferation and promote apoptosis in renal cell carcinoma .
moreover , il-37 prevented the pathogenesis of malignant b - cell neoplasms and nrasv12-mediated oncogenesis .
the aim of this study was to measure serum levels of il-37 in patients in different stages of mm and to correlate these levels with vegf and ang-2 in order to investigate their clinical significance .
the original research was approved by the medical ethics committee of our hospital , and every patient provided written informed consent .
we excluded individuals with hypertension , those with diabetes , and those who received any other surgery .
the stage of mm was classified according to the international staging system ( iss ) .
all participants in this study were from binzhou people s hospital ( china ) , since january 2011 .
all blood samples were collected , mixed with edta , centrifuged at 3000 rpm for 15 min at room temperature , then stored at 80c and analyzed at the end of the collection .
the detection of il-37 , vegf , and ang-2 in the serum was performed by elisa ( r&d systems , ca , usa ) , according to the operating manual .
the tube formation of vascular - like structures was determined by human umbilical vein ecs ( huvecs ) on matrigel ( bd biosciences , franklin , nj , usa ) . onto a 48-well plate coated with 150 l matrigel , 410 huvecs were planted .
cells were treated with recombinant human il-37 ( rhil-37 ; 100 ng / ml ) or phosphate buffer solution and then were maintained at 37c , 5% co2 , for 6 h. tubes were defined as straight cellular extensions joining 2 cell masses ; 3 random digital images ( 200 ) were counted for each well .
one - way analysis of variance , followed by t test , was used to examine differences between stages .
the original research was approved by the medical ethics committee of our hospital , and every patient provided written informed consent .
we excluded individuals with hypertension , those with diabetes , and those who received any other surgery .
the stage of mm was classified according to the international staging system ( iss ) .
all participants in this study were from binzhou people s hospital ( china ) , since january 2011 .
all blood samples were collected , mixed with edta , centrifuged at 3000 rpm for 15 min at room temperature , then stored at 80c and analyzed at the end of the collection .
the detection of il-37 , vegf , and ang-2 in the serum was performed by elisa ( r&d systems , ca , usa ) , according to the operating manual .
the tube formation of vascular - like structures was determined by human umbilical vein ecs ( huvecs ) on matrigel ( bd biosciences , franklin , nj , usa ) . onto a 48-well plate coated with 150 l matrigel
cells were treated with recombinant human il-37 ( rhil-37 ; 100 ng / ml ) or phosphate buffer solution and then were maintained at 37c , 5% co2 , for 6 h. tubes were defined as straight cellular extensions joining 2 cell masses ; 3 random digital images ( 200 ) were counted for each well .
one - way analysis of variance , followed by t test , was used to examine differences between stages .
serum levels of il-37 decreased in active patients with mm compared with healthy control subjects , whereas vegf and ang-2 increased , as shown in table 2 .
table 3 shows the values in each iss stage ; it also shows that vegf and ang-2 increased and il-37 decreased in parallel with disease stage ( p<0.001 for all cases ) .
significant negative correlations were found between serum levels of il-37 with vegf ( r=0.337 ; figure 1 ) and ang-2 ( r=0.306 ; p<0.05 ; figure 2 ) , and a positive correlation was found between vegf and ang-2 ( r=0.6458 ; p<0.001 ; figure 3 ) .
compared with the control ( 1.000.24 ) , rhil-37 pretreatment significantly decreased tube formation in huvecs ( 0.670.12 ) and significantly reduced supernatant vegf levels in huvecs ( figure 3c ) .
more and more evidence indicates that bm angiogenesis is crucial to mm pathogenesis and that the angiogenic and antiangiogenic switch contributes to mm progression .
the identification and specific contribution of proangiogenic molecules to be used as therapeutic targets and/or reliable angiogenesis biomarkers in mm is still unresolved . in this article
, we show that il-37 correlates significantly with important angiogenetic factors , suggesting that il-37 may be a biomarker for mm disease stage and angiogenesis processes .
we first determined serum il-37 , vegf , and ang-2 levels in patients with mm at different stages of disease and in healthy control subjects .
we found that , compared with healthy control subjects , serum levels of il-37 decreased in patients with mm , whereas vegf and ang-2 levels increased , depending on iss stage . these data support a putative value of these molecules in the mm disease process .
il-37 is a newly identified member of the il-1 family , which are proinflammatory cytokines demonstrating various anti - inflammatory and immunosuppressive properties in response to infection and inflammation .
recently , il-37 emerged as a fundamental anti - inflammatory cytokine , favoring tumor escape from immune surveillance .
high expression of il-37 , which may mediate antitumor immunity by regulating natural killer cell activity , was associated with better overall survival in hepatocellular carcinoma .
the ectopic selection of il-37 preserved the function of b - cell progenitors and prevented nrasv12-mediated oncogenesis in aging .
these results all suggest the essential role played by il-37 in the tumor microenvironment . through this research
, we verified that serum il-37 levels in patients with mm were lower than in healthy control subjects and that levels decreased with advancing disease . upon activation by mm plasma cells and mesenchymal stromal cells
, macrophages can release growth factors , proteolytic enzymes , cytokines , and inflammatory mediators that promote plasma cell growth and survival .
indeed , these macrophages are essential for the induction of an angiogenic response through vasculogenic mimicry , and this ability proceeds in step with progression of the plasma cell tumors .
observed reduced systemic levels of il-10 and local interferon - gamma gene transcripts in keratin-14 vegf - a transgenic mice treated with plasmid coding human il-37 sequence - formulated cationic liposomes . in mm , plasma cells secrete various angiogenic cytokines , such as vegf , matrix metalloproteinase-2 , and fibroblast growth factor-2 , which in turn cause inflammatory cells to secrete other angiogenic factors .
all these factors enlist and activate mm - associated macrophages to cooperate with other cells in angiogenesis .
bm sera with high ang-2 levels specifically contributed to ec activation , suggesting that ang-2 produced in the bm may contribute to mm angiogenesis . in our study
, we found that both vegf and ang-2 were significantly higher in patients with mm than in healthy control subjects and that these values increased in parallel with disease progression .
we also confirmed elevated il-37 levels in mm significant negative correlations with vegf and ang-2 . in view of these facts
, we further investigated whether angiogenesis could be weakened by the increased expression of il-37 .
we found that the tube formation of huvecs , which was also suppressed by rhil-37 pretreatment , may be involved in vegf secretion .
these results suggest that il-37 may play an important role in angiogenesis during mm progression .
our results show that in patients with mm , serum vegf and ang-2 levels increase and il-37 level decreases with disease progression .
these results suggest that serum levels of il-37 play a part in the pathophysiology of mm progression .
therefore , il-37 could serve as a biomarker of the disease state and angiogenesis processes in mm . | backgroundangiogenesis plays a significant role in complex inflammatory and angiogenic processes and is also involved in multiple myeloma ( mm ) pathogenesis .
il-37 is a proinflammatory cytokine in antitumor activity .
our purpose was to evaluate the il-37 clinical significance on mm.material/methodswe measured serum levels of il-37 in 45 patients with different stages of mm and 30 healthy control subjects and correlated il-37 with numerous cytokines , such as angiogenesis factors including vascular endothelial growth factor ( vegf ) and angiotensin-2 ( ang-2 ) .
we also measured the tube formation of human umbilical vein endothelial cells ( huvecs ) after pretreatment with recombinant human il-37 ( rhil-37).resultsserum il-37 level was lower in the patients with mm than in the healthy control subjects , whereas vegf and ang-2 levels were higher , depending on international staging system stage .
serum il-37 level had a negative correlation to vegf and ang-2 levels , and vegf had a positive correlation to ang-2 level .
the tube formation of huvecs was suppressed by the rhil-37 pretreatment.conclusionsour results indicate that serum level of il-37 plays a part in the pathophysiology of mm progression .
therefore , il-37 serum level may be a biomarker for disease stage and angiogenesis processes . |
the vast majority of women in prison have experienced some sort of interpersonal or sexual violence in their lives , with estimates as high as 90% ( commonwealth of ma , 2005 ; women in prison project , 2006 ) . yet , ironically , women , especially those who were homeless , drug addicted or living with dangerous partners prior to incarceration , are statistically safer from victimization in prison than they were prior to incarceration .
for example , the estimated prevalence of sexual assault in us prisons , based on the most recent bureau of justice statistics inmate survey , is about 4.4% ( beck & harrison , 2010 ) . yet
, for women on college campuses , the estimated prevalence of sexual assault is from 20 to 25% ( bureau of justice statistics , 2009 ; youth violence and suicide prevention , 2004 ) .
studies have also shown that incarcerated women with posttraumatic stress disorder ( ptsd ) report a much higher rate of witnessing violence than the female population in general and that many of them designate such witnessing as their most serious trauma ( hackett , 2009 ; hodes , 2006 ) .
some women express a feeling of safety and relief during intake at women 's prisons ( loper , 2002 ) . escaping homelessness , sex work , violent partners , dealers , and pimps
may also contribute to a new awareness of the level of danger with which they have lived ( blackburn , mullings & marquart , 2008 ) .
perhaps that small moment of relief upon entry provides the requisite psychic safety that allows these women to begin to identify their trauma symptoms and triggers .
theories of women 's pathways to criminal behavior include the harmed and harming woman , who has been seriously sexually abused and neglected , and is at high risk for anger and hostility , which is associated with increased rates of institutional difficulty and recidivism ( brennen , 2007 ) .
hurt people hurt people , which speaks to the repetitive nature of the trauma experience and how it is transmitted to others ( bloom & farragher , 2011 ) .
a history of sexual abuse is recognized as a risk factor for crime in both males and females . in general , people who have been sexually abused are more likely to be arrested ( hubbard , 2002 ) , and to experience future trauma ( council of state governments , 2005 ; najavits , 1997 )
. for women offenders , sexual violence , defined as the combined prevalence of adult and child sexual abuse and assault , is by far the most commonly reported type traumatic experience , followed by intimate partner violence ( battle , zlotnick , najavits , gutierrez , & winsor , 2002 ; zlotnick , 1997 ; zlotnick , najavits , rohsenow & johnson , 2003 ) . for incarcerated women , rates of sexual victimization across the lifespan are highest in childhood ( blackburn et al . , 2008 ;
clements - nolle , wolden & bargmann - losche , 2009 ; raj et al . , 2008 ) .
research suggests histories of sexual abuse interfere with female inmates ability to benefit from programs , perhaps in part , due to the disorientation and disconnection that trauma creates ( saakvitne , 2000 ) .
if the offender has limited coping skills and can not control her dissociative symptoms in the face of multiple triggers , it interferes with her engagement in cognitive - behavioral therapy ( cbt ) interventions ( hubbard , 2002 , van voohris , 1997 ) .
therefore , the principles of trauma- informed care , along with interventions aimed at trauma stabilization , are a priority for women offenders .
this theory is supported by research on women 's risk classification and security levels , which has shown that a history of childhood physical and/or sexual abuse is associated with increased institutional difficulty ( gilfus , 2002 ; van voorhis , salisbury , wright & bauman , 2008 ) . in studies of male prisoners ,
the most commonly reported trauma is witnessing someone being killed or seriously injured ( sarchiapone , carlia , cuomoa , marchettia & roy , 2008 ) , followed by physical assault ( johnson et al . , 2006 ) , and childhood sexual abuse ( weeks & widom , 1998 ) .
overall , higher rates of trauma and earlier age of trauma onset is associated with increased violence and victimization in prison ( komarovskaya , 2009 ) .
studies of male veterans in prison ( saxon et al . , 2005 ) , inmates in rural prisons and jails
( powell , holt & fondacaro , 1998 ) , and men in substance abuse treatment have repeatedly shown high rates of physical and/or sexual abuse histories and ptsd .
for example , an estimated 38% of men in substance abuse treatment have ptsd ( najavits , 2006 ) .
studies of childhood sexual abuse among male inmates vary , but most suggest a much higher rate of childhood sexual abuse than in the general male population ( bureau of justice statistics , 1999 ; johnson et al . , 2006 ; fondacaro et al . , 1999 ; weeks & widom , 1997 ) .
however , unlike women , men are rarely safer behind prison walls than prior to incarceration .
their risk of sexual assault increases exponentially when they enter a prison , ( national prea commission , 2009 ; testimony of hennessey , 2005 ) compared to the risk for males in the general population .
they face an increased threat of lethal violence in male facilities that may trigger more externalizing trauma responses ( i.e. , outward - directed aggression ) and high levels of arousal that can endanger staff and inmates ( freedman & hemenway , 2005 ) .
limits on confidentiality include knowledge of escape plans , contraband or knowledge of the introduction of contraband , and knowledge of intent to commit a crime , in addition to the limits that apply to all clients ( bartol & bartol , 2008 ) .
inmates may not trust clinical staff with disclosure of their trauma histories ( grella & greenwell , 2007 ) .
they may also consider any disclosure of trauma that was perpetrated by intimate partners or family members as ratting them out to the authorities , a violation of social codes both in and outside of prison .
both males and female may under - report sexual violence and symptoms of sexual trauma , even to researchers , either because they do not consider them to be out of the ordinary ( moses , reed , mazelis & glover , 2003 ) , or because of guilt and self - blame , or , especially for males , a sense of shame and fears about their sexual identity .
male and female inmates also learn not to report troubling psychiatric symptoms and to manage them on their own as a way to maintain a greater degree of autonomy during institutional stays and limiting vulnerability .
this is especially true for male inmates with histories of past victimization , who have been shown to be at a significantly greater risk for prison sexual assault than men without such a history ( beck & harrison , 2010 ; national institute of corrections , 2007 ; testimony of hennessey , 2005 ) .
inmates with any type of traumatic history may have symptoms of ptsd that increase difficulties within correctional institutions , both for the inmate , other inmates , and for staff responsible for their supervision ( saxon et al . , 2001 ) .
by taking a trauma - informed approach , correctional staff gain the opportunities to minimize such concentric harm and to reduce mental health and security costs for institutions ( bloom , 2003 ) .
good correctional practice requires environments that are highly structured and safe , with predictable and consistent limits , incentives and boundaries , as well as swift and certain consequences such that inmates are treated fairly and equally ( council of state governments , 2010 ) .
these same practices can provide the type of stability trauma survivors need to learn new information and skills that promote trauma recovery
. vicarious trauma ( a sense of identification with trauma that may result in staff experiencing trauma - type symptoms ) can affect workplace decision making .
boundaries may erode and cynicism and hopelessness can increase ( headington institute , 2010 ) . in the absence of
effective trauma - informed tools , prison systems may develop institutional trauma , becoming highly reactive and reliant on management - by - crisis .
the more the system responds with authoritative measures , the more deeply the dynamics are repeated and reinforced .
staff may have experienced direct exposure to trauma from witnessing prison violence , on the job injuries , during prior military service or in their personal lives .
they may have learned to function at the workplace in a state of constant hyper - vigilance or in numb detachment .
they also have important security concerns that may trump the need to focus on trauma , making organizational and cultural change exceedingly slow .
for example , there is empirical evidence that criminal sanctions and authoritative and punitive measures , without treatment , are the least effective means of reducing future criminal behavior ( andrews et al . ,
are more likely to be a successful means of reducing recidivism than surveillance and enforcement ( gendreau , goggin , cullen & andrews , 2000 ; latessa , cullen & gendreau , 2002 ) .
yet security staff may be reluctant to reduce the use of sanctions and surveillance when they perceive these as critical enforcement tools .
trauma - informed principles such as increasing empathy , compassionate care , grounding , and de - escalation may be perceived as weak , pandering , or
ineffective and risky , even though , ironically , they may be more likely to help to create security and a stable environment . even experienced staff who have developed good capacities to respond well to traumatized inmates typically lack formal training , and may be subject to countertransference ( emotional reactivity ) and burn out .
combined with the challenge of managing a system that is perpetually under - resourced , the result is staff who may feel as overwhelmed as the inmates in their care .
correctional staff training is starting to include more information about trauma and techniques to respond to effectively to trauma symptoms .
it can be helpful to give security staff information on the nature of the clinical services available , the trauma recovery approach and the structure and goals of trauma - specific interventions ( mccown , 2006 ) . when clinical and non - clinical staff members understand their common goals
, most offender interactions can focus on safety , supporting good coping skills and reinforcing treatment gains .
it is also key to recognize gender differences in how much attention is given to trauma ( gilfus , 2002 ; covington , 2008 ) .
trauma is far more likely to be addressed in female than in male inmates . in male facilities
females and traumamales and traumatypical trauma : childhood sexual abusetypical trauma : witnessing violencemore likely to develop ptsd when exposed to violencemore likely to be exposed to violence , but less likely to develop ptsdrepeated exposure to sexual and violent victimization from intimates beginning in childhoodexposure to violence from strangers and adversaries ; sexual abuse and coercion outside familyinternalizing : self - harm , eating disorders , addiction , avoidanceexternalizing : violence , substance abuse , crime and hyper - arousallikely to get mental health treatment rather than substance abuse treatmentlikely to get substance abuse treatment rather than mental health treatmenttreatment needs to emphasize empowerment , emotion regulation and safetytreatment needs to emphasize feelings , relationships and empathy
although training on gender specific issues is of critical importance , educating officers on both male and female trauma at the same time has several advantages .
staff may better engage the material and gain a deeper understanding of both the differences and commonalities of male and female reactions to trauma .
when staff is familiar with gendered responses , they are better prepared to deal with a range of reactions , whether an inmate 's response is characteristically masculine or feminine .
( battle et al . , 2002 ; fallot , 2008 ; henigsberg , folnegovi - malc & moro , 2001 ; merwin , rosenthal & coffey , 2009 ; sarchiaponea et al . , 2008 ;
the exploration of male trauma dynamics and trauma recovery can evoke compassion , curiosity and capture the attention of male staff .
when examples , exercises and role plays include scenarios that men can relate to , ticc can begin to be understood as a safety issue rather than the abuse excuse or something that only applies to female offenders .
the most effective tool for developing in - service training is ensuring that seasoned correctional officers and other staff take a lead role . despite limited resources , fears for their own safety , vicarious trauma , and conflict between enforcement responsibilities and compassion ,
it is important to reinforce staff intuition and compassion and to recognize and build on strategies that have been successful before introducing new information and skills.shift commanders and chiefs of security should assist with training content and be visible as co - trainers and in role plays and demonstrations .
training that highlights stress management , self - care and remedies for burn out tends to engage more experienced staff .
three essential training elements and examples are listed below ( and more resources for training and information appear at the end of this article ) .
controlling costs of healthcare , close custody and staff turnovercontrolling the high cost of secure mental health housing unitsreducing seclusion and restraint and de - escalating critical incidentseffective behavior management , safer facilities and job satisfaction
controlling costs of healthcare , close custody and staff turnover controlling the high cost of secure mental health housing units reducing seclusion and restraint and de - escalating critical incidents effective behavior management , safer facilities and job satisfaction trauma - related symptoms , behaviors , adaptations and their functiongrounding role plays and demonstrationspracticing verbal trauma de - escalation promptsdemonstrating talking inmates through pat downs and searchesredirecting offenders and inmates who bring up trauma details trauma - related symptoms , behaviors , adaptations and their function grounding role plays and demonstrations practicing verbal trauma de - escalation prompts demonstrating talking inmates through pat downs and searches redirecting offenders and inmates who bring up trauma details
panels of veterans in trauma recoveryvideos and films of stories of trauma healingstories of offenders overcoming victimizationsigns of vicarious trauma , supporting co - workers
panels of veterans in trauma recovery videos and films of stories of trauma healing stories of offenders overcoming victimization signs of vicarious trauma , supporting co - workers the best training approaches include buy - in from security and administration , skill demonstration and rehearsal , and establishing a human connection to trauma . a caveat , however , is that inmate stories and films that speak to healing from the effects of trauma are well - received , but details of extreme and horrifying experiences are not a desirable training tool .
the principle , headlines , not details is apt ( i.e. , trauma can be mentioned as a phrase , such as child abuse , rape , etc . , but there is no need to ever go into details of trauma ) .
this helps to reduce vicarious trauma and triggering of staff 's own personal trauma memories .
also , by modeling this in training , staff can then implement this approach with inmates . setting ground rules for keeping details of traumatic experiences to a minimum to avoid triggering effects
this can be approached as a means of ensuring safety and supporting self - care for both security staff and inmates , while clinical staff members perfect their skill at redirecting group members and reinforcing boundaries .
controlling costs of healthcare , close custody and staff turnovercontrolling the high cost of secure mental health housing unitsreducing seclusion and restraint and de - escalating critical incidentseffective behavior management , safer facilities and job satisfaction
controlling costs of healthcare , close custody and staff turnover controlling the high cost of secure mental health housing units reducing seclusion and restraint and de - escalating critical incidents effective behavior management , safer facilities and job satisfaction
trauma - related symptoms , behaviors , adaptations and their functiongrounding role plays and demonstrationspracticing verbal trauma de - escalation promptsdemonstrating talking inmates through pat downs and searchesredirecting offenders and inmates who bring up trauma details trauma - related symptoms , behaviors , adaptations and their function grounding role plays and demonstrations practicing verbal trauma de - escalation prompts demonstrating talking inmates through pat downs and searches redirecting offenders and inmates who bring up trauma details
panels of veterans in trauma recoveryvideos and films of stories of trauma healingstories of offenders overcoming victimizationsigns of vicarious trauma , supporting co - workers
panels of veterans in trauma recovery videos and films of stories of trauma healing stories of offenders overcoming victimization signs of vicarious trauma , supporting co - workers the best training approaches include buy - in from security and administration , skill demonstration and rehearsal , and establishing a human connection to trauma . a caveat , however , is that inmate stories and films that speak to healing from the effects of trauma are well - received , but details of extreme and horrifying experiences are not a desirable training tool .
the principle , headlines , not details is apt ( i.e. , trauma can be mentioned as a phrase , such as child abuse , rape , etc . , but there is no need to ever go into details of trauma ) .
this helps to reduce vicarious trauma and triggering of staff 's own personal trauma memories .
also , by modeling this in training , staff can then implement this approach with inmates . setting ground rules for keeping details of traumatic experiences to a minimum to avoid triggering effects
this can be approached as a means of ensuring safety and supporting self - care for both security staff and inmates , while clinical staff members perfect their skill at redirecting group members and reinforcing boundaries .
the effectiveness of cognitive - behavior interventions , or cbi , in prisons is well documented in the correctional literature ( andrews & bonta , 2003 ) .
prison systems have developed policy and procedure manuals mandating their use ( cbi standard operating procedures , 2001 ) .
the national institute of corrections supports training in several evidenced - based curricula on restructuring criminal thinking and developing pro - social coping skills .
features that make them the approach of choice for working with prisoners are that they can be facilitated by correctional staff and that they target observable behaviors ( van voohris , branwell & lester , 2009 ) .
moreover , meta - analyses have consistently shown cognitive - behavioral interventions to be effective at reducing not only offender recidivism , but also substance use and mental health problems ( andrews et al . , 1990 ; clark , 2010 ; landenberger & lipsey , 2005 ) .
unlike medical care and mental health services , rehabilitation programming , is driven by a risk assessment that measures factors associated with re - offending , rather than clinical diagnoses .
these factors are known as criminogenic risks ( andrews , bonta & hoge , 1990 ) .
rehabilitation programs are aimed at correcting criminal thinking , with a focus on changing present - day and future behavior ; hence , increasing public safety .
substance abuse , criminal thinking and anti - social personal attitudes and characteristics , such as impulsiveness , anger , and hostility are among the major risk factors for recidivism ( andrews , bonta & hoge , 1990 ) . for women ,
substance abuse has been identified as a key driver of increasing rates of incarceration ( grella & greenwall , 2007 ) and substance involvement is the primary reason they return to prison ( macdonald , 2008 ) .
additional women 's recidivism risk factors include mental health symptoms and past trauma and abuse ( van voorhis , salisbury , wright & bauman , 2008 ) .
although clinical staff may regard prison as an unsafe environment for clinical work that addresses trauma , the potential benefits of implementing trauma - specific interventions may be substantial .
since childhood physical and sexual abuse is associated both with institutional difficulties and low program engagement , prisoners may have difficulty benefiting from even the most effective cognitive - behavioral rehabilitation programming until the impact of childhood trauma is addressed ( brennen , 2007 ; covington , 2008 ; gilfus , 2002 ; hubbard , 2002 ; komarovskaya , 2009 ; van voorhis et al . , 2008 ) .
prison can also be a golden teachable moment in that the inmate 's incarceration may heighten awareness of needing new coping skills and addressing trauma impact .
it also provides a time apart from outside life on the streets and undermining influences that can distract or derail them from a recovery focus .
as correctional policy shifts its focus to decreasing recidivism , the advantages of evidenced - based counseling approaches are becoming more apparent to prison administrators ( taxman , 1999 ) . the use of present - focused , cognitive - behavioral , and coping skills treatments with strong educational components have helped stabilize inmates with ptsd and substance abuse problems ( zlotnick et al .
these approaches tend to be compatible with the correctional culture of responsibility , consistency , accountability and behavioral change , and are relevant to a range of trauma impacts ( e.g. , substance abuse , depression ) and a variety of new coping skills that promote rehabilitation .
manualized , present - focused approaches to trauma , such as seeking safety ( najavits , 2002 ) , have been effective with offenders without causing distress or decompensation that requires attention from prison mental health staff ( najavits , 2006 ) . by addressing trauma in terms of current impact , symptoms , related problems ( e.g. , substance abuse ) ,
building psychoeducation , and increasing safe coping skills , such a model can address trauma directly but without requiring the client to explore distressing memories .
best of both worlds approach ( targeting trauma and related impacts , but in present - focused safe ways ) can be ideal in prison settings .
past - focused models , such as exposure therapy ( foa , hembree & rothbaum , 2007 ) , may be evidence - based models for ptsd , but have a real risk of emotionally destabilizing inmates who are already vulnerable . the security response to such destabilization
can set the cycle of re - traumatization in motion . in the current climate ,
prison environments are likely best suited to present - focused approaches , given the unmet need for more mental health training , staffing limitations , and the typical lack of funding for additional formally trained and supervised staff required for past - focused ptsd treatments such as exposure therapy .
past - focused therapies may never be embraced as safe by staff , nor are all inmates likely to acquire the requisite level of readiness and trust of prison clinical staff to benefit from them .
present - focused approaches such as seeking safety can be done by a far broader range of staff , do not necessarily require formal training , are extremely low - cost to implement , and are known to be safe .
there is sometimes great reluctance to open the trauma can of worms given the prison environment and the limited clinical resources available . yet
, trauma - informed correctional care and staff training can go a long way toward creating an environment conducive to rehabilitation and staff and institutional safety .
trauma - informed principles are helpful regardless of whether the institution chooses to make trauma - specific clinical intervention available to inmates .
however , it can be noted that trauma - specific interventions have been found to be more powerful than trauma - informed interventions ( morrissey et al . , 2005 ) , and thus ideally making both available will result in greater success in prison settings .
it is important to remember that prison staff is legally responsible for medical care for inmates and must provide appropriate treatment , including mental health services .
the use of ticc can provide a contextual foundation that strengthens the prison setting to provide effective help in increasing pro - social coping skills , creating a calm and safe prison environment , reducing adverse events , and aiding staff morale , all of which can lead to better offender rehabilitation outcomes .
| backgroundrates of posttraumatic stress disorder and exposure to violence among incarcerated males and females in the us are exponentially higher than rates among the general population ; yet , abrupt detoxification from substances , the pervasive authoritative presence and sensory and environmental trauma triggers can pose a threat to individual and institutional stability during incarceration.objectivethe authors explore the unique challenges and promises of trauma - informed correctional care and suggest strategies for administrative support , staff development , programming , and relevant clinical approaches.methoda review of literature includes a comparison of gendered responses , implications for men 's facilities , and the compatibility of trauma recovery goals and forensic programming goals.resultstrauma-informed care demonstrates promise in increasing offender responsivity to evidence - based cognitive behavioral programming that reduces criminal risk factors and in supporting integrated programming for offenders with substance abuse and co - occurring disorders.conclusionsincorporating trauma recovery principles into correctional environments requires an understanding of criminal justice priorities , workforce development , and specific approaches to screening , assessment , and programming that unify the goals of clinical and security staff . |
tho tho cattle ( bos indicus ) is a local indigenous bovine species of north eastern hilly ( neh ) region especially in nagaland state of india .
the bull is half of the herd in animal husbandry , which indicates that the sire is one of the parents of all the calves in the herd .
since the bull has more genetic influence ( 8090% ) of calves it sires , fertile bull selection can be the most powerful method for improvement of the herd .
the demand for sperm from outstanding sires has increased with the development of frozen semen technology and the growth of large artificial breeding organizations .
methods to predict sperm production potential and particularly to identify the bulls with high sperm output potential at an early age are important , which is related with testicular parameters and scrotal circumference . traditionally , tho tho bulls have been selected on the basis of growth , rather than on reproductive traits . however , reproduction is one of the most important factors for the economics of livestock production .
generally , testicular measurements at early ages are very useful in selection of breeding sire to collect semen for artificial insemination or breeding purpose . the most important parameters are testicular diameter , testicular length , testicular volume , and scrotal circumference .
regression equations have revealed that testicular size was positively related to body weight and age .
positive correlations between prepubertal male hormone levels and subsequent testes size and mating frequency have also been reported .
these are the potentially useful indicators of reproductive traits and have been used to compose selection indices for tho tho bulls to use in breeding purpose .
these traits are easily measured and correlated with body weight and reproductive performance [ 46 ] .
testicular weight and body growth of large number of dairy bulls were measured to develop techniques which would give repeatable results and to determine which one was most highly correlated with scrotal circumference .
this study also was designed to provide the basis for investigation of parameters useful in predicting future reproductive performance and in establishing the extent to which differences in these parameters are varied .
measurement of scrotal circumference and testicular parameters is an essential part of breeding soundness evaluation of breeding bulls .
measurement of these parameters especially scrotal circumference has a great value on onset of puberty , total semen production , semen quality , pathological conditions of reproductive system , and the fertility or infertility status of breeding bulls .
moreover , testicular measurements have been utilized as the indicators for reproductive capabilities in the postpubertal period of bulls .
scrotal circumference is highly correlated with body weight and age and it is highly correlated with testis weight and testicular consistency is correlated with fertility .
testis size and body weight provides idea about the physical and physiological maturity of the bulls , its sperm output , and birth weight of its young ones .
there are various reports on the testicular growth and related to semen attributes in matured dairy bulls [ 1318 ] , buffaloes [ 1921 ] , ram , and mithun bulls .
if these reproductive traits are to be incorporated into management and breeding programs , it is desirable that their relationships to other traits are to be understood .
there are various factors involved in influencing scrotal circumference and testicular measurements such as age , breed of the bulls , and season of the year
. studies associating with body weight , testicular measurements , and scrotal circumference obtained during the different phases of growth of tho tho bovine species are scarce . in this respect
, it is not only necessary to determine how these traits respond to selection at different ages , but also to determine the magnitude of correlation between these traits .
therefore , the present study was designed to estimate the testicular parameters at different ages and correlate with body weight and age in tho tho bulls to provide data for the definite selection of tho tho bulls for future breeding purpose and conservation of this precious indigenous bovine species .
a total of thirty - two apparently healthy tho tho bulls with different age groups were selected from the herd of villages at around the national research centre on mithun ( icar ) , nagaland , india .
the study area lies between 255430 north latitude and 934415 east longitude and at an altitude range of 250300 mean sea level .
the animals were maintained under semi - intensive condition of grazing in the forest during day time and ting them in shed during night time .
the experimental animals in shed were fed daily with ad libitum quantity of locally available forages without providing concentrate .
the body weight of each bull was calculated according to shaeffer 's formula : l g/300 2.2 , where l = body length in inch and g = girth in inch . according to their age ,
the experimental bulls were equally divided into four groups , each of 8 bulls , namely , group i : 1824 months ( n = 8) , group ii : 2536 months ( n = 8) , group iii : 3748 months ( n = 8) , and group iv : 49 months and above ( n = 8) .
testicular parameters and scrotal circumference were measured with a caliper ( mitutoya digimatic caliper ) and a tape after restraining the bull in the chute .
testicular length was measured by placing the fixed arm of the caliper at the proximal end and the sliding arm at the distal end of the testes .
thickness or depth was measured by placing the fixed arm of the caliper at the anterior aspect and the sliding arm at the posterior aspect of the each testis , at the point of maximum depth .
width of each testis was measured by sliding the other testes up in the scrotum and placing one arm of the caliper at the medial aspect and the other at the lateral aspect , at the point of maximum width . for measurement of scrotal circumference ,
the testicle was pushed firmly into the bottom of the scrotum by placing the thumb and fingers laterally on the side of the neck of the scrotum and pushed ventrally .
a flexible cloth tape was formed into a loop and slipped over the scrotum and scrotal circumference was measured in centimeters by pulling the tape snugly around its greatest diameter .
testes volume was calculated by using the formula for volume of an ellipsoid , that is , 4/3abc , where a = thickness/2 , b = width/2 , and c = length/2 ( love et al .
weight of the testes was measured by multiplying volume with 1.038 which is the approximate density of testicular tissue in cattle .
means were analyzed by one way analysis of variance between the different age groups of tho tho bulls , followed by tukey 's post hoc test to determine significant differences between the groups using the spss / pc computer program ( version 15.0 ; spss , chicago , il ) .
correlation coefficient was estimated among the testicular parameters , scrotal circumference , age , and body weight of the bulls . differences at p < 0.05 and p < 0.01 were considered to be statistically significant .
the results of different testicular parameters , scrotal circumference , and body weight at different age of tho tho bulls were presented in table 1 , which indicated that these parameters were significantly ( p < 0.05 ) different between the age groups .
it has been reported that the average estimated testicular weight was 18.50 , 40.20 , and 51.25% greater in bulls aged 2536 , 3748 , and > 49 months of age than in 1824-month - old bulls .
similarly the average estimated testicular volume was 15.50 , 28.69 , and 33.85% greater in bulls aged 2536 , 3748 , and > 49 months of age than in 1824-month - old bulls . in case of scrotal circumference , the average estimated value was 6.76 , 10.58 , and 13.35% greater in bulls aged 2536 , 3748 , and > 49 months of age than in 1824-month - old bulls .
similarly as expected , various measurements of the testis were highly correlated with each other ( p < 0.01 ) as in exotic cattle , buffalo , and mithun .
body weight was more highly correlated with the various testicular measurements than age . but also according to age ,
the testicular parameters were increased significantly ( p < 0.05 ) in tho tho cattle bulls . moreover age ( figure 1 ) , testis weight ( figure 2 ) , and body weight ( figure 3 ) of tho tho cattle bulls
scrotal circumference was a simple measurement to obtain ; maximum circumference is reached at 5 - 6 years of age and remains relatively constant thereafter .
these results indicate that scrotal circumference is a useful indicator and is an important selection criterion to determine the testicular development in young bulls and it has highly correlation with testicular parameters .
in the present study , the results revealed that scrotal circumference of tho tho bulls have been highly correlated with other testicular parameters .
thus , measurement of scrotal circumference in tho tho bulls is very useful to predict the testicular parameters and can be used in breeding centre to select suitable breeding male for artificial breeding purpose .
there was no report on effect of scrotal circumference on testicular parameters in tho tho cattle and , to the best of our knowledge , this is the first report on the effect of scrotal circumference on testicular parameters in tho tho bulls .
analysis of various testicular parameters such as testis length , width , thickness , volume , and weight is important for extensive selection of tho tho bulls for future breeding and conservation program in the neh region especially in nagaland state .
correlation coefficients measured among the different testicular measurements and scrotal circumference for the 32 bulls were presented in table 2 .
body weight was somewhat more highly correlated with the various testicular measurements than with age . as would be expected the various measurements of testis size were highly correlated with each other .
all correlation coefficients were highly significant ( p < 0.01 ) . circumference of the testes
was highly correlated with other testicular measurements ( table 2 ) ; it was highly repeatable ( table 1 ) and it was a simple measurement to obtain and easy to interpret .
the correlations among this trait and other testicular measurements on a within - age group basis are not given , but they remained high .
the correlation of 0.94 between scrotal circumference and testis weight is similar to the values of 0.90 to 0.96 reported by other researchers for various comparisons between testicular dimensions and weight .
since circumference of the testes was easy to measure , the measurement had high repeatability and it was highly correlated with testis weight and volume and this measurement subsequently was taken on all bulls examined . in the present study ,
the correlation among the various testicular measurements were in agreement with the study in nigerian indigenous bulls , bangladesh indigenous bulls , sahiwal bulls , buffalo , and mithun bulls , but are at variance with another report on other bulls in which testes volume and length were not correlated with age and body weight . in our experience ,
tho tho bulls are indigenous animals , maintained under semi - intensive system and were sensitive to handle the testes and other reproductive organs .
. also in the earlier study of other cattle bulls , testicular volume was calculated by multiplying mean values for length , thickness , and total width of the testes . in our experiment ,
in this procedure , results were in higher side than those obtained by using the formula for estimating the volume of an ellipsoid which has a very high accuracy .
interestingly , in the present study , within each age group , the testicular measurement of tho tho bulls were lower than those reported for holstein bulls and higher than buffalo bulls , but maximum scrotal circumferences was reached at nearly the same age .
nevertheless , there appears to be ample scope for selecting tho tho bulls with large scrotal circumference since about 60 to 70% of bulls , within each age group had a scrotal circumference greater than the mean scrotal circumference for that age group . in young dairy and beef bulls , scrotal circumference and testes size
were highly correlated [ 18 , 32 ] and bulls designated questionable or unsatisfactory potential breeders have smaller scrotal circumference measurements . but the reports in other species like bubaline and bovine species showed that the spermatogenesis was higher in younger than older age . so that further studies with semen collection and more number of animals will help to pinpoint the exact age at which the mature rate of spermatogenesis is attained . in the present study ,
the mean value of scrotal circumference in group ii ( 2536 months ) was significantly ( p < 0.05 ) higher than group i ( 1824 months ) and group iv higher value than the other three groups .
it has been observed in the present study that sc was increased rapidly in young bulls and gradually in mature bulls .
but it has been reported that in old age this parameter is decreased due to senile atrophy . in the present study also , the proportion of increasing the scrotal circumference and testicular parameters was low in older bulls than in growing younger bulls . based on our results on the mean testicular circumference of 32 tho tho bulls ,
we recommend that , for optimum breeding potential and possible higher fertility , tho tho cattle bulls with a mean scrotal circumference of 27.97 , 30.00 , 31.28 , and 32.27 cm in the age groups of 1824 , 2536 , 3748 , and > 48 months , respectively , should be selected as the breeding sires .
scrotal circumference ( table 1 ) was increased with age and following a pattern similar with bw ( figure 1 ) , but testes tended to reach mature size more rapidly , as indicated by the linear relationship between sc and bw ( figure 2 ) .
high correlation was observed between testis size and scrotal circumference and based on this collective information , testicular diameter along with scrotal circumference are excellent indicators of spermatogenic function and this can be used for breeding soundness evaluation in tho tho bulls . in conclusion , present results indicate that as in exotic cattle and buffalo and mithun bulls , scrotal circumference in the tho tho bulls is a useful indicator of breeding soundness and should be used as an important criterion for selection of young bulls for breeding purpose .
however , estimates of changes in testicular development can be considerably improved by following the same bulls at different ages to avoid confounding between bulls and ages .
future , semen collection and the seminal parameters will be correlated with testicular and scrotal parameters are warranted to confirm the present findings . | the present study was undertaken to assess the relationship between the scrotal circumference and testicular parameters with body weight and age in tho tho bulls ( bos indicus ) , which were maintained at around the villages of national research centre on mithun ( icar ) , jharnapani , nagaland , india .
a total of 32 tho tho bulls were selected and divided into four groups according to their age and each group consisted of 8 bulls , namely , group i : 1824 months ( n = 8) , group ii : 2536 months ( n = 8) , group iii : 3748 months ( n = 8) , and group iv : 49 months and above ( n = 8) .
the scrotal circumference and testicular parameters were measured with caliper and tape and age of animals was calculated with dental formula .
the body weight of bulls was estimated with shaeffer 's formula .
result revealed that the scrotal circumference was highly correlated with testicular parameters and body weight compared to age .
compared to exotic cattle ( bos taurus ) , tho tho bull 's testicular parameters and scrotal circumference were lower .
the results of the present study in tho tho bulls revealed that scrotal circumference is a useful indicator and is an important selection criterion to determine the testicular development and breeding soundness in young bulls as it is highly correlated with tesicular parameters . |
leptin , an adipocyte derived cytokine , primarily was known to be involved in the regulation of food intake and energy expenditure ( dardeno et al . , 2010 ) .
later on studies showed the role of leptin in the immune system modulation ( lord et al . , 1998 ) .
the studies describing the role of leptin in diabetes are so conflicting and no one could classify it into either pro - inflammatory or anti - inflammatory groups ( lam and lu , 2007 ; weisberg et al . , 2003 ; xu et al . , 2003 ) .
similarly heat shock proteins ( hsps ) are endogenous highly conserved molecules produced by various cells , including macrophage , monocyte and polymorphonuclears when exposed to high temperature , inflammatory cytokines and oxidative stress ( kalmar and greensmith , 2009 ) .
the hsp genes are considered as vitagenes , which are involved in preserving cellular homeostasis during stressful conditions , which are induced by co - ordinating information flow along its convergent , divergent and multiply branched signaling pathways ( calabrese et al . , 2010 , 2011 ) .
more importantly both hsp70 and leptin are correlated with c reactive protein ( crp ) ( molvarec et al . , 2009 , 2011 ) .
the purpose of the present study was to study the correlation between serum hsp70 and leptin levels in patients with type 2 diabetes stratified according to gender .
we performed a cross sectional study on the established groups of patients with type 2 diabetes defined as 1 : patients with long standing diabetes for more than 3 years ; and 2 : patients with newly diagnosed diabetes within recent 6 months who were not on any glucose lowering treatment other than by dietary means alone and 3 : healthy controls .
patient recruitment was from the diabetes clinic of vali - asr hospital , affiliated with tehran university of medical sciences .
healthy controls were selected from those without any known disease including type 2 diabetes , hyperlipidemia , ischemic heart disease , and malignancy .
patients and controls were matched according to age , sex and body mass index ( bmi ) .
diabetes was diagnosed according to the criteria of the american diabetes association ( seo et al . , 2009 ) .
exclusion criteria were smoking , pregnancy , creatinine > 1.5 mg / dl or gfr < 70 cc / min , glomerulonephritis , congestive heart failure , use of antioxidant , statins , hormone replacement therapy and hospital admission in previous 6 months .
demographic and anthropometric data including age , sex , duration of diabetes , height , weight in light clothing , and blood pressure in sitting position were recorded .
the local ethics review committee of tehran university of medical science approved the study protocol .
blood samples were collected after 12 h of fasting , centrifuged and were kept at 70 c until analysis .
serum creatinine , fasting blood sugar ( fbs ) , total cholesterol , triglycerides , high - density lipoprotein- ( hdl ) cholesterol , low - density lipoprotein ( ldl ) cholesterol , and hba1c were measured for all participants .
glucose measurements ( intra - assay coefficient of variation [ cv ] 2.1% , inter - assay cv 2.6% ) were carried out using the glucose oxidase method .
cholesterol , hdl , ldl and triglycerides were determined using direct enzymatic methods ( parsazmun , karaj , iran ) .
measurements of hsp70 were performed using a quantitative sandwich elisa immunoassay ( eks-715 , stressgen , usa ) .
serum leptin concentration was determined by an enzyme - linked immunosorbent assay ( drg instruments gmbh , germany ) with an intra - assay cv of 5.96.9% and an inter - assay cv of 8.611.5% .
crp was assessed using a two - site , enzyme - linked immunosorbent assay ( elisa ) ( diagnostic biochem , london , ontario , canada ) .
the statistical package spss 17 for windows ( chicago , illinois , usa ) was used for analysis .
pearson 's correlation test was employed to study the correlation between leptin , hsp70 , crp , bmi and waist circumference .
partial correlation coefficient was employed to study the correlation between the studied variables after adjustment for bmi .
primary characteristics of the groups of patients and controls are presented in table 1 .
patients with long standing diabetes had higher leptin , hsp70 , hba1c and triglyceride than controls .
serum leptin levels were significantly lower in patients with newly diagnosed diabetes , compared to other groups ( table 1 ) .
women with newly diagnosed diabetes had higher leptin and crp levels compared to men ( table 2 ) .
table 2 presents the characteristics of patients with newly diagnosed diabetes , stratified according to gender .
we studied the correlation between serum hsp70 , leptin , bmi , and waist circumference ( table 3 ) . while hsp70 was significantly correlated in women with newly diagnosed diabetes ,
there were not such correlations in men with newly diagnosed diabetes , men and women in long standing diabetes and controls .
1 shows the correlation coefficient between leptin and hsp70 in the studied groups stratified according to gender .
the greatest difference exists between patients with newly diagnosed diabetes and patients with long standing diabetes ( fig . 1 ) .
the correlation was highest in women with newly diagnosed diabetes and was attenuated in women who were on treatment .
there was no correlation between leptin and hsp70 in men ( both newly diagnosed and long standing disease ) .
most of the studies which have shown a positive correlation are carried out in disease states . however , while some have shown a positive correlation , others have shown a negative one . in consistent with our findings
rong quan and collaborators showed that leptin activates hsp70 expression in a dose dependent manner in mcf-7 breast cancer cell line ( xue et al . , 2012 ) .
similarly bonior and collaborators showed that leptin administration stimulates the expression of hsp60 in pancreatic acinar and beta cell lines .
leptin protects pancreatic acinar cell line from the damage of acute pancreatitis ( bonior et al . , 2006 ) .
on the other hand , figueiredo et al . showed that leptin administration would decrease food intake and hsp70 levels in the liver and hypothalamus of chickens but not in the muscle .
the effect of leptin on hsp70 levels was independent of the inhibition of food intake ( figueiredo et al . , 2007 ) .
in another study , geranylgeranylacetone ( gga ) administration induced hsp70 expression in liver , which rendered physiologic protection against high fat diet in mice , while decreased leptin levels ( adachi et al . , 2010 ) .
sirotken et al . showed that leptin administration inhibited the transcription of hsp70 in ovarian follicular granulosa cells . heat stress , but not malnutrition can promote depletion of hsp70 in ovarian cells , which is partly reversed by leptin ( sirotkin and bauer , 2011 ) .
we hypothesized that the answer is the duality of hsp70 behavior in different inflammatory or non - inflammatory environments ( nakhjavani et al . , 2012 ) .
as stated , the positive correlation between leptin and hsp is observed in chronic inflammation such as type 2 diabetes ( our findings ) and breast cancer ; while all other studies , which have shown a negative correlation , are carried out in the normal cells , without any chronic inflammatory state .
we have previously shown the positive correlation of hsp70 with adma , a nitric oxide inhibitor , in chronic inflammatory states while it was negatively correlated to adma in normal physiology ( nakhjavani et al .
one could argue that this correlation is only present in women , while we do not observe such a correlation in men .
both leptin and hsp70 have been shown to be higher in women ( nakhjavani et al .
, 2010a , 2010b ; nakhjavani et al . , 2011 ; rossi et al . , 2012 ; samara et al . , 2010 )
earlier studies on leptin deficient mouse have shown marked reduced number of lymphocytes with impaired humoral response and thymic atrophy ( gruver and sempowski , 2008 ; mancuso et al . , 2002 ) .
lipoatrophy in hiv infected patients is assumed to be a result of reduced leptin in these patients ( papaevangelou et al . , 2007 ) .
serum leptin levels were significantly elevated in patients with multiple myeloma and chronic lymphatic leukemia ( pamuk et al . , 2006 ) .
serum leptin levels are increased in women with type 2 diabetes ( nakhjavani et al .
one explanation was that a greater adipose tissue in women is the underlying reason ( rossi et al . , 2012 ) .
on the other hand , studies have repeatedly shown that women with type 2 diabetes and metabolic syndrome have higher serum leptin levels , after adjustment for body fat composition ( meyers et al . , 2008 ; thomas et al . , 2000 ) .
the other explanation could be that an increased serum leptin level is associated with accelerated immune cell activation in women with type 2 diabetes .
interestingly we have previously shown that women with long standing diabetes have higher serum hsp70 levels ( nakhjavani et al .
rodrigues - krause and collaborators have suggested a role of bmi on extracellular hsp70 levels ( rodrigues - krause et al . , 2012 ) .
while intracellular hsp70 induction in response to pro - inflammatory stimuli can exert anti - inflammatory effects , extracellular hsps may signal danger , activating immune cells ( atalay et al .
since immune system is more active in women compared to men ( de leon - nava and morales - montor , 2006 ; grimaldi , 2006 ) , one would argue that the observed correlation between serum hsp70 and leptin implies a higher state of chronic inflammation .
in consistent with our findings zulkit et al . showed that food restriction induces leptin and hsp70 protein expression in female broiler chicken ( zulkifli et al . , 2002 ) .
the principal limitation of the present study is its cross sectional nature which precludes the determination of the direction of causality , however we took advantage of a relatively large sample size and close similarity between groups in most of the potentially confounding variables . in conclusion | in vitro and in vivo studies have shown conflicting results regarding heat shock protein ( hsp ) and leptin correlation .
more importantly both hsp70 and leptin are correlated with c reactive proteins .
the purpose of the present study was to study the correlation between serum hsp70 and leptin levels in patients with type 2 diabetes stratified according to gender .
we performed a cross sectional study on the established groups of patients with type 2 diabetes defined as 1 : patients with long standing diabetes for more than 3 years ; and 2 : patients with newly diagnosed diabetes within recent 6 months who were not on any glucose lowering treatment other than by dietary means alone and 3 : healthy controls . patients with long standing diabetes had higher hsp70 , hba1c and triglyceride than controls .
serum leptin levels were significantly lower in patients with newly diagnosed diabetes .
women with type 2 diabetes had higher leptin levels compared to men , both before and after treatment .
we showed a positive correlation between leptin and hsp70 levels in women with type 2 diabetes .
the correlation was strongest in women with newly diagnosed diabetes ( r = 0.59 ) and was attenuated in women who were on treatment ( r = 0.3 ) .
the significance of this correlation was only observed in women with type 2 diabetes .
there was no correlation between leptin and hsp70 in men .
the positive correlation between leptin and hsp is observed in chronic inflammation such as type 2 diabetes .
it could be hypothesized that the observed correlation between serum hsp70 and leptin implies a higher state of chronic inflammation . |
p73 is a member of the p53 family , although p73 ( and p63 ) are more closely related to the ancestral form of the protein than p53 .
these three mammalian genes encode transcription factors that play key roles as regulators of proliferation , differentiation , cell death , stem cell renewal , and cell fate commitment [ 24 ] .
all p53 family genes contain the same modular domain structure , including an amino - terminal transactivation domain ( ta ) , a dna - binding domain and a carboxy - terminal oligomerization domain .
the p73 gene contains 15 exons and transcription can be initiated from two n - terminal promoters ( fig . 1 ) .
transcription from the most upstream atg ( promoter 1 ) generates the transactivating ( ta ) isoforms , while the second promoter , situated within intron 3 , yields amino - terminal truncated proteins ( n isoforms ) . in general , ta and n isoforms display distinct biological activities .
the ta isoforms induce cell cycle arrest and apoptosis , and are therefore candidate tumour suppressors [ 5 , 6 ] while the truncated n isoforms are ( generally ) pro - survival and favour oncogenic transformation .
in addition , both tap73 and np73 transcripts undergo alternative c - terminal splicing , generating , in theory , up to seven different variants of each in normal cells , although not all these have been detected at the protein level [ 810 ] .
a genomic organisation of p73 and representation of different splicing variants that give rise to the isoforms of p73 .
the p1 promoter generates the ta isoforms , while the p2 promoter produces the n isoforms .
b schematic representation of the domains encoded by the different isoforms of p73 . on top
ta transactivation domain , dbd dna - binding domain , od oligomerization domain , sam sam domain , tid transactivation inhibitory domain p73 gene structure .
a genomic organisation of p73 and representation of different splicing variants that give rise to the isoforms of p73 .
the p1 promoter generates the ta isoforms , while the p2 promoter produces the n isoforms .
b schematic representation of the domains encoded by the different isoforms of p73 . on top , there are indicated the aminoacids included in each domain .
ta transactivation domain , dbd dna - binding domain , od oligomerization domain , sam sam domain , tid transactivation inhibitory domain it is , however , now generally accepted that n isoforms largely act as dominant negative inhibitors of the activity of ta isoforms , both by competing for consensus elements in promoter dna and by dimerisation [ 7 , 11 ] . however , this generalisation must be qualified since the longest np73 isoform contains a second c - terminal ta domain and can transactivate a set of genes distinct from that recognised by tap73 . moreover , the isoforms of both ta and
np73 contain a c - terminal transactivation inhibitory domain , which can compromise the transcriptional activity mediated by the n - terminal ta domain as a result of intramolecular interactions .
thus , to regard the output of p73 expression as the resultant of the ratio between ta and n isoform expression may be an oversimplification .
the human trp73 gene was identified in 1997 when it was localised within chromosome 1p36 , a region frequently deleted in tumours such as neuroblastoma and other late - stage human cancers [ 12 , 13 ] .
although genes other than p73 within the 1p36 region , such as chd5 , may also contribute to these phenotypes , these data suggest that p73 may have a role in neural development as well as in cancer .
this developmental role for p73 is substantiated by the phenotype of total p73 knockout mice .
unlike the tumour - susceptible p53 null mice , total p73 knockout mice do not develop tumours but show developmental defects in the central nervous system ( table 1 ) , including congenital hydrocephalus and hippocampal dysgenesis , with abnormalities in the ca1ca3 pyramidal cell layers and the dentate gyrus . in keeping with these anatomical abnormalities ,
the total p73 knockout mice also have defects in both embryonal and adult neurogenesis , suggesting that p73 isoforms may be survival factors for neural stem cells .
p73 mice also have a reduction in cortical thickness as a consequence of loss of mature cortical neurons .
table 1neuronal defects in p73 transgenic micep73 ( yang et al . , pozniak et al . )
tap73 ( tomasini et al . ) dnp73 ( wilhelm et al . ) anatomical defectcortexhypoplasianoneage - dependence hypocellularity and cortex thinninghippocampusdysgenesisabsence lower blade of dentate gyrusnoneventricularthird ventricle enlarged and hydrocephalusnoneenlarged ventricleothersloss of cajal - retzius neurons and reelin defective .
, wetzel et al . ) ( personal communication)(personal communication)youngagedyoungagedyoungagedburrowingntntreducedreducedntnormalclaspingpositivereducedpositivepositiventpositiveopen fieldreducedreducedreducedreducedntreducedbarnes mazereducedreducedntntntnthind griploosereducedntntntntlight dark boxpositiventntntntntyoung mice : 24 months old .
aged mice : 1517 months oldnt not testedthe behavioural experiments were performed in p73 mice neuronal defects in p73 transgenic mice young mice : 24 months old .
aged mice : 1517 months old the behavioural experiments were performed in p73 mice this neurological phenotype has been attributed primarily to the loss of np73 isoforms , since these are expressed in post - mitotic neurons and act as survival factors .
thus , ngf withdrawal or overexpression of p53 in sympathetic cervical ganglion cells leads to a reduction of endogenous np73 levels and apoptosis which is prevented by overexpression of np73 .
recently , the generation of np73 isoform - specific ko mice has confirmed the prosurvival role of np73 in differentiated mature neurons .
indeed , neuronal density in the motor cortex of np73 mice is significantly reduced after 10 months of age and progresses with evidence of neurodegeneration , although there were no striking hippocampal abnormalities .
in particular , it has been observed that the number of vomeronasal neurons and cajal - rezius cells was profoundly reduced , and that the choroid plexuses were atrophic .
however , the severity of the neurological defects observed in the np73-specific knockout is not as dramatic as that seen in the total p73 mice , suggesting that tap73 may also contribute to the development of the cns . while cortical thickness in selective tap73 null mice is normal , they show hippocampal dysgenesis with loss of the lower blade of the dentate gyrus similar to that seen in total p73 mice at p14 , before the complicating effect of ventricular enlargement further distorts the hippocampal architecture in the total knockout .
this anatomical phenotype is reflected in the reduction in neurogenesis in the subgranular zone of the dentate gyrus ( fig .
indeed , tap73 has been shown to regulate the negative bhlh hey2 which is known to sustain maintenance of neural precursors .
thus , the isoform - selective knockout studies suggest that tap73 and np73 contribute to cns development in ways that are only partially overlapping .
the dentate gyrus from day 7 after birth ( p7 ) of normal ( p73 ) and knockout ( p73 ) mice was stained with antibodies to glial fibrillary acidic protein ( gfap ) and nestin .
knockout mice show nearly half of gfap / nestin cells , indicating a very reduced stemness potential in these mice .
scale bars 50 m reduction of the putative stem cell in the dentate gyrus from p73 mice .
the dentate gyrus from day 7 after birth ( p7 ) of normal ( p73 ) and knockout ( p73 ) mice was stained with antibodies to glial fibrillary acidic protein ( gfap ) and nestin .
knockout mice show nearly half of gfap / nestin cells , indicating a very reduced stemness potential in these mice .
both embryonic and adult neural stem cells ( nsc ) are primary precursors that have the ability to differentiate into different cell types ( neurons , astrocytes and oligodendrocytes ) while retaining the capacity to produce identical nsc progeny ( self - renewal ) , and p53 and np63 have already been implicated in nsc biology [ 25 , 26 ] .
recently , four independent groups [ 16 , 23 , 27 , 28 ] have demonstrated that p73 is also a positive regulator of embryonic and adult nsc and some of this in vivo evidence has been discussed above .
neurospheres derived from p73 mice are smaller , with a reduced number of cells in s phase and an increase in the senescent population . at the molecular level
, it has been shown that nsc from p73 mice have transcriptional dysregulation of sox-2 , sox-3 , nanog , notch - i , notch-2 , hes-5 , jag2 and deltex , which are all components of signalling pathways involved in the regulation of proliferation and/or self - renewal , although further studies are required to address how p73 physiologically regulates these factors .
tap73 is the predominant isoform expressed in embryonic nsc , and endogenous expression of tap73 increases during differentiation of nsc .
further studies have shown that smaller numbers of neurons can be derived from p73 nsc which do not fully differentiate , with defects in arborization of the dendritic tree and in physical connectivity .
this is reflected in the anatomy of hippocampal neurons in the dentate gyrus of p73 mice in vivo , which show a reduced number of branches when compared to normal mice ( fig .
3hippocampal neuron morphology is altered in the dentate gyrus of knockout ( p73 ) mice .
golgi staining of dentate gyrus from normal ( p73 ) and p73 mice ( age=18 days after birth ) .
scale bars 100 m ( top panel ) and 50 m ( low panel ) hippocampal neuron morphology is altered in the dentate gyrus of knockout ( p73 ) mice .
golgi staining of dentate gyrus from normal ( p73 ) and p73 mice ( age=18 days after birth ) .
scale bars 100 m ( top panel ) and 50 m ( low panel ) p73 has also been implicated in oligodendrocyte as well as neuronal differentiation , and oligodendrocytic differentiation from p73 nsc is also impaired , with lower numbers and poorer quality than those derived from wild type nsc . however , dissociated p73 nsc retain the ability to differentiate into neurons , astrocytes and oligodendrocytes , indicating that loss of p73 does not affect the multipotency of nsc .
another , though perhaps less definitive model implicating p73 in neurogenesis is the terminal differentiation of neuroblastoma cells induced by retinoic acid ( ra ) and which is associated with an increased expression of tap73 .
in addition , ectopic expression of tap73 itself induces terminal neuronal differentiation . during ra treatment , tap73 isoforms regulate the n - cam promoter , while ectopic expression of tap73 led to a down - regulation of n - myc and an increased expression of prb , mimicking the ra effect on these two genes , which are crucial for neuroblastoma survival [ 32 , 33 ] . in conclusion , while recent work indicates a clear requirement for p73 , and particularly tap73 in the maintenance of stemness via a yet not fully identified mechanism both in vivo and in vitro [ 16 , 23 , 27 , 28 ] , other data suggest an involvement of p73 in neuronal and oligodendrocyte differentiation .
these multiple biological activities in the nervous system may reflect the molecular complexity of the 14 protein isoforms of p73 , their interactions with each other as well as their interplay with other p53/p63 family members , which are themselves expressed as multiple isoforms .
alternatively , since p73 appears to regulate between 1,000 and 2,000 genes , including 100200 transcription factors , several apparently divergent pathways could be simultaneously activated .
during the last 15 years , it has emerged that the p53 family has an important role in several neurodegenerative diseases .
of special interest to us , and on which we will focus , is their role in the most ( and increasingly ) prevalent form of neurodegeneration , alzheimer s disease ( ad ) .
ad exists as both familial and sporadic forms . the familial early onset ( eoad ) form , accounting for only a small percentage of cases , is inherited via mutations in either the -amyloid precursor protein ( app ) or one of the two presenilins ( ps ) , ps1 and ps2 .
in contrast , the greatest genetic risk for the sporadic , late onset ( load ) form is the 4 allele of apolipoprotein e ( apoe ) .
recently , genome - wide association studies have identified three additional risk genes , albeit with much weaker effects than apoe ; apoj / clu , picalm and cr1 [ 35 , 36 ] both sporadic and familial forms of the disease are characterised by two brain lesions : senile plaques
extracellular deposits of the -amyloid ( a ) peptide ; and neurofibrillary tangles ( nfts)intracellular aggregates of paired helical filaments ( phf ) composed of hyperphosphorylated forms of the microtubule associated protein , tau .
a is derived by proteolytic processing from the app , with the presenilins being a necessary component of the -secretase complex responsible for the final stage of this process .
the phosphorylation state of tau regulates its ability to bind microtubules ; in its hyperphosphorylated state tau is unbound and eventually polymerises into paired helical filaments , which then aggregate into nfts .
it is emerging that it is not these insoluble aggregates of tau , but rather some soluble form that is the toxic species responsible for neuronal dysfunction and eventually neuronal death . that mutations in app give rise to increased a production ( as do mutations in the presenilins , as subsequently observed ) , and as senile plaque appearance precedes that of nfts , the amyloid cascade hypothesis was formulated .
this hypothesis , which has not yet been refuted , holds that increased levels of a lead to the hyperphosphorylation of tau which then leads to neuronal dysfunction and eventually neurodegeneration .
attempts to model this cascade of events in mice has for the most part failed , as even when mutant transgenic forms of both app and ps1 are introduced , despite high levels of a production leading to extensive plaque formation , the aggregation of tau is not observed .
it is only when app , ps1 and human tau are concomitantly over expressed in triple transgenic mice that both plaques and tangles are observed .
the first indication that the p53 family may play a role in ad came in 1996 with the demonstration that intracellular a upregulates p53 in the brains of transgenic mice overexpressing just the a fragment of app .
that same year , the kosik group examined neurons from a p53 mouse , observing that p53 has a role in neuronal differentiation and in tau phosphorylation . in 2001 use of pifithrin- , an inhibitor of p53-dependent gene transcription was shown to protect neurons against a-induced apoptosis .
the p53 protein was also found to be upregulated in the brain of ad sufferers , a finding which we later confirmed . in 2004 , caricasole et al . showed that a activates the expression of the p53 target gene encoding the soluble wnt antagonist , dickkopf-1 ( dkk1 ) , and that knock - down of dkk1 in primary neurons almost completely blocked a-induced tau phosphorylation , implicating the p53 family in the amyloid cascade pathway . in the same year
, it was also reported that the p73 protein exhibits an altered subcellular distribution in ad brain . in hippocampal pyramidal neurons of control subjects ,
p73 immunoreactivity was predominately cytoplasmic , while in ad samples increased levels of p73 were found in the nuclei of pyramidal neurons and in dystrophic neurites .
we employed a simple cell model to show that transcriptionally active forms of p53 are able to induce tau phosphorylation at specifc phosphoepitopes , particularly the at8/tau-1 ( s199 , s202 and s205 ) and phf-1 ( s396 and s404 ) sites .
we found that this also holds true for p73 , with the ta forms increasing tau phosphorylation , while the n forms do not . in this system
, we also found that transcriptionally active forms of p63 activate tau phosphorylation , while np63 forms do not ( unpublished observations ) . in 2008 ,
the miller / kaplan group reported that in brains of aged ( 1618 months ) heterozygous p73 mice , there were substantial increases in tau phosphorylation levels together with filamentous aggregates of hyperphosphorylated tau with similarities to nfts . when they crossed these haploinsufficent p73 mice with mice harbouring a double mutant form of app ( the tgcrnd8 mouse ) , tau phosphorylation and tau filament formation occurred as early as 1.5 months .
this finding is especially remarkable given that a-based mouse models of ad do not manifest overt tau pathology .
it also lends yet further support to the amyloid cascade hypothesis , showing that a lies upstream of tau pathology and also that the cascade of events that a must set into motion , leading to the hyperphosphorylation of tau and subsequent neurodegeneration , involves the p53 family , and in particular p73 .
the miller / kaplan group went on to show that in primary cortical neurons generated from p73 ko mice the activity of c - jun n - terminal kinase ( jnk ) increased as the number of wt p73 alleles decreased , and that jnk inhibition decreased tau phosphorylation in these neurons .
they propose that , since np73 can bind and inhibit jnk , which the ta forms of p73 can not , it is the loss of the n forms of p73 that brings about tau phosphorylation and neurodegeneration .
the same group has previously demonstrated that np73 plays a neuroprotective role in the cns [ 17 , 50 ] and also claim that n forms of p73 predominate , at least in the postnatal mouse brain .
the loss of one copy of the entire p73 gene results in a reduction in np73 isoforms leading to an increase in jnk activity . in support of this claim
, there is an extensive literature implicating jnk in ad pathology , e.g. [ 5153 ] .
our group has shown that jnk directly targets tau and also plays a role in the regulation of app processing .
inhibiting jnk has also been shown to protect against a toxicity in an app / ps1 brain slice model of ad .
there exists one other knock - out mouse with a phenotype that includes neurodegeneration with accompanying aggregates of hyperphosphorylated tau filaments .
the deleted gene is prolylisomerase-1 , which is associated with a production and aggregation through its binding with phospho - thr688 of app , and tau phosphorylation through interaction with phospho - thr231 of tau . of interest , pin1
a has been shown to activate c - abl and increase p73 levels , while the swedish mutant form of app increases expression from both the ta and n promoters of the p73 gene , but results in an overall increase in the ta forms .
it appears then that p73 plays a key role in ad and , in particular , that the balance between the ta and n forms is crucial .
however , from the above , and other observations such as the fact that p53 transcriptional activity is regulated by the presenilins and that many of the components of the -secretase complex are themselves regulated by p53 , it would seem unlikely that p73 acts alone .
further investigations into p73 interactions with other p53 family members , in particular p53 itself , within nervous tissues will undoubtedly aid our understanding of its roles in both normal adult brain and in the pathogenic processes underlying alzheimer s disease .
from these studies , it emerges that p73 is the p53 family member with a fundamental role in central nervous system development and maintenance , and even in its degeneration . indeed , as shown in fig . 4
, the p73 gene plays a role throughout the neurogenesis process , from neural stem cells to mature postmitotic neurons .
although perhaps simplistic at this stage , these new findings suggest that tap73 is the isoform essential for neuronal differentiation and maintenance of neural stem cells .
however , the phenotype observed in isoform specific knock - out mice is milder than that in the full knock - out mice , indicating that , as in cancer , it is the interaction between p73 isoforms , which ultimately determines the phenotype .
functional neurons are generated from neural stem cells and then after maturation , integrated in neuronal circuits .
np73 plays a major role both as a survival mechanism as well as yet unknown pathways .
question marks indicate that molecular mechanism has not been fully investigated yet role of p73 in neurogenesis .
functional neurons are generated from neural stem cells and then after maturation , integrated in neuronal circuits .
np73 plays a major role both as a survival mechanism as well as yet unknown pathways . | p73 , a transcription factor of the p53 family , plays a key role in many biological processes including neuronal development .
indeed , mice deficient for both tap73 and np73 isoforms display neuronal pathologies , including hydrocephalus and hippocampal dysgenesis , with defects in the ca1-ca3 pyramidal cell layers and the dentate gyrus .
tap73 expression increases in parallel with neuronal differentiation and its ectopic expression induces neurite outgrowth and expression of neuronal markers in neuroblastoma cell lines and neural stem cells , suggesting that it has a pro - differentiation role .
in contrast , np73 shows a survival function in mature cortical neurons as selective np73 null mice have reduced cortical thickness .
recent evidence has also suggested that p73 isoforms are deregulated in neurodegenerative pathologies such as alzheimer s disease , with abnormal tau phosphorylation .
thus , in addition to its increasingly accepted contribution to tumorigenesis , the p73 subfamily also plays a role in neuronal development and neurodegeneration . |
rna - guided machineries
provide highly selective and rationally
programmable tools for the site - specific manipulation of nucleic acids .
several endogenous riboproteins are known that are steered toward
their endogenous targets by nucleic acid hybridization and that are
readily re - addressed toward new targets by expression or administration
of artificial external guidernas .
those include the snorna - guided
2-o - methylation and pseudo - uridinylation machineries
and the microrna - guided rna - induced silencing complex .
the harnessing
of the latter machinery , better known as rna interference , has developed into a standard tool in cell biology .
besides harnessing endogenous eukaryotic machineries , the engineering
of artificial riboproteins for the site - specific manipulation of nucleic
acids comes more and more into focus now .
tools are highly desired
that simplify genetic engineering and
that help to elucidate the role of point mutations and rna modifications . besides their application in basic biology research , such tools
have potential for translation into individualized medicine .
a highly
topical example is the re - engineering of the bacterial crispr - cas9
system for site - selective genome editing in eukaryotic cells .
endogenous riboproteins are typically assembled
by molecular recognition
between specific protein and rna structures .
the formation of a single covalent bond between an rna and a protein
component , however , is virtually unknown for that purpose .
nevertheless ,
we could recently demonstrate the assembly and functioning of highly
selective adenosine ( a)-to - inosine rna editing machineries inside
living cells following the latter approach .
since inosine is biochemically read as guanosine ( g ) , editing formally
creates a - to - g point mutations at the rna level .
if rna editing is
directed to the open reading frame , 12 out of the 20 canonical amino
acids can be substituted , including most
of the polar residues essential for enzyme catalysis , post - translational
protein modification , or signaling . furthermore , editing in the non - coding
part of the rna can interfere with translation initiation ( start codon ) ,
translation stop , microrna action , and splicing among others .
thus , the potential of site - directed rna editing for application
in basic biology research and medicine is evident .
we apply the snap - tag technology to
assemble the editing machinery via covalent bond formation .
this technology
requires the fusion of a snap - tag domain ( an evolved o6-alkylguanine - dna alkyltransferase ) with the c - terminal
catalytic domain of a human adar enzyme ( adenosine deaminases acting
on rna ) . at the rna component ,
the incorporation
of a small chemical moiety , o6-benzylguanine
( bg ) , is necessary .
the covalent bond is then formed in situ in a
single - turnover enzymatic reaction between the snap - tag and the bg
moiety with very fast kinetics ( kconjugation = 2.8 10 m s ) and high specificity ( figure 1a ) . recently , we demonstrated
the repair of a premature stop codon ( uag ) into a tryptophan codon
( uig ) in a fluorescent reporter gene in human cells ( 293 t ) .
notably , the repair reaction was strongly dependent
on the covalent attachment of the guiderna to the deaminase .
this
opens the appealing possibility of controlling the editing reaction
by triggering the assembly of the covalent rna
we decided
to apply light as a trigger , as it allows for the very precise and
fast control in time , space , and dosage .
to achieve the light - dependent
assembly of the covalent rna protein
conjugate , we masked the bg moiety chemically by installment of a
light - sensitive 6-nitropiperonyloxymethyl ( npom ) protection
group which absorbs broadly in the 330420
nm range . during synthesis
we obtained a separable 1:2 mixture of
regioisomers containing the npom group either at n7 or n9 position
of the guanine base . upon irradiation with 365 nm light on a common
uv - light table , both isomers , n7 and n9 , decay efficiently into free
bg and the respective nitroso acetophenone byproduct with similar
kinetics ( n7 isomer , figure 1b , t1/2 = 34 3 s ; n9 isomer , figure s13 , t1/2 =
47 4 s ) .
the decaging efficiency was determined
by comparison with a commercial standard ( dmnb - camp ) to be 2000
and 1500 m cm for
the n7 and n9 isomers , respectively , giving quantum yields
0.5 and 0.36 ( for details , see the supporting information ) .
( a ) concept of light - triggered site - directed rna editing .
assembly
of the guiderna deaminase conjugate requires release of the
npom - protected benzylguanine ( bg ) moiety and is a prerequisite for
the editing reaction .
( b ) first - order kinetic analysis ( via hplc )
of the photodeprotection of n7-bg at the small - molecule
level .
the hplc trace shows the product mixture after 60 s of 365
nm irradiation ( 75% conversion ) . the respective analysis for n9-bg
( c ) light - triggered conjugation reaction of fluorescein - labeled
npom - bg with snap - adar1 protein ( sds - page coomassie versus fluorescein
stain ) .
bg - fitc refers to the conjugate of bg with fluorescein isothiocyanate ,
and bg - fam refers to the conjugate with 6-carboxyfluorescein . to determine the reactivity of
the npom - protected bg with snap - deaminases
such probes were incubated with sub - stoichiometric amounts of
snap - adar1 either in the dark or in the presence of 365 nm light .
the conjugate formation was then determined by sds - page and fluorescence
analysis ( figure 1c ) .
it was clearly shown that full fluorescence labeling of the snap - deaminase
was readily accessible upon irradiation . however , some background
reactivity of the protected bg in particular of the n9 isomer with
snap - adar1 was visible .
the latter is coherent with the requirement
of the natural ancestor of the snap - tag to accept the desoxyribose
at the n9 position of the nucleobase .
we did not expect the low - level residual activity to play a role
under dilute conditions inside the living cell ; nevertheless , we continued
all further work with the pure n7 isomer of npom - bg . to study the assembly of the guiderna
deaminase
conjugate and its effect on in vitro rna editing , we attached the
n7-bg via a short linker to the 5-terminal aminolinker of a 17 nt guiderna that directs
the conjugate to codon 66 of the ecfp transcript in order to stimulate
the repair of a premature amber stop codon ( uag ) back to tryptophan
( figure 2a ) . via sds - page
we first characterized the light - dependent assembly of the riboprotein
( figure 2b ) .
conjugation
results in a readily detectable shift of the snap - deaminase toward
higher molecular weight . indeed ,
excellent control of the conjugate
assembly was achieved in a clearly light - dose - dependent manner , shifting
the snap - deaminase from non - conjugated to nearly complete conversion
following kinetics agreeing with the decaging kinetics of the bg precursor described above . to study the light - dependent
in vitro rna editing reaction ,
a master mix containing all components
was aliquoted in the dark into pcr tubes and aliquots were irradiated
individually with 365 nm light for 0 , 15 , 30 , 60 , or 180 s , respectively ,
prior to starting the editing reaction by incubation at 37 c .
a guiderna lacking the bg moiety served as a negative , and a guiderna
modified with authentic bg served as a positive control .
similarly , only very minor editing
above background was detectable in the non - irradiated sample with
the npom - caged bg - guiderna .
however , upon irradiation editing was
restored in a light - dose - dependent manner regaining an editing level
comparable to that of the positive control ( figure 2c , 75% with snap - adar1 ; figure s16 , 60% with snap - adar2 ) . plotting the intermediate
editing levels against the irradiation time resulted in first - order
kinetics ( figure 2c , t1/2 = 26 0.5 s ) very similar to those
obtained with the small - molecule precursor ( figure 1b ) .
( a ) n7-bg is included into an activated linker that
readily reacts with the aminolinker of commercially available rna
analogues to obtain the bg - guidernas .
( b ) the light - driven
conjugation reaction between npom - bg - guiderna and snap - adar1 is easily
monitored by sds - page ( coomassie stain ) .
( c ) in vitro site - directed
rna editing of the amber stop codon at position 66 in the ecfp gene .
the editing yield is clearly light - dose - dependent obeying first - order
kinetics .
sanger sequencing of the entire orf of the ecfp gene shows
no off - target editing ( figure s15 ) .
the
respective editing applying snap - adar2 instead of snap - adar1 is given
in figure s16 .
for further details , see
the supporting information . for the study of intracellular light - activated rna
editing , we incorporated
the n7-bg into a 19 nt antagomir - like chemically stabilized nucleic acid analogue that contained a gap of three natural ribonucleotides
around the editing site .
we applied antagomir - like chemistry to improve
the stability of the guiderna and the selectivity of the editing reaction ; this has been shown for rna interference before .
the guiderna targets a premature amber stop
codon ( uag ) at an egfp reporter ( w58amber ) , and successful editing
is indicated by turn - on of egfp fluorescence .
a guiderna with authentic
bg served as a positive and the same guiderna lacking the bg moiety
as a negative control .
further controls were done to test the necessity
of all components of the machinery .
snap - adar1 and the reporter gene
were transiently overexpressed from plasmids in hek293 t cells .
one
day after transfection of the plasmids , the respective guiderna was
lipofected into the cells .
four hours after lipofection , the medium
was changed and cells were irradiated with 365 nm light under high
control of dosage and wavelength by using a fluorescence microscope
equipped with a led light source .
one day later , the egfp fluorescence
was analyzed by microscopy before the rna was isolated , and the editing
yield was determined by sanger sequencing . compared to our previous
protocol
, we had to optimize the amounts and stoichiometry of snap - adar1
and guiderna in order to suppress some low - level ( 10% ) editing
caused by the npom - protected guiderna in the dark ( for details , see figures s19 and s20 ) .
the optimal amount of guiderna
used was 10 pmol/150 l and is in a range typical for sirna
duplexes . applying the nh2-guiderna ( negative editing control ) ,
only a very few cells developed a low - level gfp fluorescence and no
editing was detectable in the sequencing trace ( < 5% ) . however ,
transfecting bg - guiderna ( positive editing control ) gave brightly
fluorescent cells , similar to the transfection of functional wt egfp ,
and an editing yield of typically 45% was determined ( figures 3 and s17 ) .
furthermore , absolutely no editing was obtained at codon 58 by a chemically
stabilized , negative control guiderna that directs repair to codon
66 .
thus , the formation of the rna secondary structure directly at
the targeted codon is strictly required for site - directed rna editing
and is the major determinant of specificity .
the npom - protected guiderna
gave only very low editing yield over background ( < 5% ) and only
a small number of low - intensity fluorescent cells .
however , following
illumination , editing was switched on to a level similar to that of
the positive editing control , as indicated by fluorescence microscopy
but also by rna sequencing ( 45% editing yield ) .
intermediate editing
levels have been accessible by varying the light dose ( figure 3 ; more details can be found
in figure s17 ) .
the light dose applied
to photoactivate editing was well tolerated by the cells . in comparison
to the npom - guiderna , the editing yield of neither the positive nor
the negative editing controls was dependent on light ( figure s17 ) .
no unspecific off - target editing
was observed in the reporter gene , as indicated by sanger sequencing
( figure s18 ) .
snap - adar1 and
the reporter gene ( w58x egfp , or wt egfp ) are provided on plasmids ,
the guidernas are reverse - transfected , all as described in the supporting information .
shown is the fluorescence
microscopy analysis together with the respective sanger sequencing
traces 24 h post - transfection of the respective guiderna .
5-terminal ,
the guidernas are either carrying an aminolinker ( nh2 ) ,
the bg moiety ( bg ) , or the npom - protected bg moiety .
the stop66-guiderna
is a negative control bg - guiderna targeting the gfp gene around codon
66 instead of codon 58 .
as it requires massive genetic manipulation to switch the
expression of one isoform to another that differs only in a single
point mutation , site - directed rna editing might offer a practical
alternative
. a light - triggered variant
would be particularly attractive for developmental biology , as early
stages are often transparent , and the
spatiotemporal control of gene expression is of particular interest .
an emerging model system for developmental and neurobiology is the
marine annelid platynereis dumerilii(29 ) that is readily cultivated and easily manipulated at the one - cell zygote by microinjection . to test site - directed rna editing inside the
worm
, we injected two mrna transcripts encoding snap - adar1 and egfp
together with chemically stabilized 21 nt guidernas .
one day after
microinjection , when the zygotes were developing into trochophore
larvae , the fluorescence phenotype was analyzed by microscopy ( figures 4 and s22 ) .
a gfp - positive phenotype was only detectable
in the positive control ( wt gfp ) and in the editing sample ( figure 4a , f ) .
all negative
controls lacking parts of the machinery , such as the guiderna , snap - adar1 ,
or both , showed no green fluorescence ( figure 4b d ) . in the editing sample as well
as in the positive control , there was some heterogeneity of fluorescence
intensity that may result from the difficulty of precisely controlling
the injection volume . to determine the editing yield ,
a cohort of
trochophores ( each 80100 ) were lysed , and rna was extracted
and analyzed by sanger sequencing ( figure 4 ; for detailed analysis of all larvae , see table s23 ) .
editing was observed only when all
components were included and achieved 6070% over the entire
population .
no off - target editing was observed in the targeted transcript
( figure s24 ) .
the worms seem to develop
and behave normally over the first days and stages of development .
reporter
mrna ( egfp ) and snap - adar1 mrna were microinjected into one - cell zygotes ,
together with the respective bg / nh2-guiderna and rhodamine
are the fluorescence images of one
representative embryo 24 hours post fertilization ( hpf ) for each experiment
and the sequencing trace obtained from the rna of 80100 animals
per experiment : ( a ) positive control , ( b e ) negative controls
lacking single components of the editing machinery , and ( f ) editing
experiment . for details , see the supporting information .
deaminase
conjugate and fails when using the nh2-guiderna lacking
the bg moiety ( figure 4e ) .
in contrast to using the nh2-guiderna ( figure 4e ) lacking the bg
moiety , microinjection of the npom - protected guiderna resulted in
a small but significant number of faintly fluorescing trochophores
( 18% ) besides a large number of dark ones ( > 80% , figure 5a , table s23 ) . in accordance with this ,
rna sequencing of a cohort of
80100 animals revealed a low but significant residual editing
at the targeted stop codon ( 10% ) . in faintly fluorescent trochophore
larvae , the rhodamine signal was typically stronger ( figure 5a ) , indicating that the low - level
editing might be due to an undesirably high injection volume of the
editing components .
this low - level residual editing activity is reminiscent
of the situation described above for the light - dependent editing in
cell culture .
however , when the microinjected trochophores were treated
with 365 nm light on a uv trans - illuminator ( 5 min ) , half of the trochophores
developed a bright egfp signal ( figure 5b , table s23 ) .
the fluorescence
imaging was in agreement with an editing yield of 60% , as
determined by rna sequencing of 80100 animals ( figure 5b ) .
thus , irradiation allows
for activating rna editing to a yield nearly identical to that of
the positive editing control with an unprotected bg moiety ( figure 4f ) .
reporter
gene and snap - adar1 have been microinjected into one - cell platynereis zygotes as described in figure 4 , but now with a photoprotected chemically
stabilized bg - guiderna . within 1 h after microinjection ,
zygotes have been ( a ) kept in the dark or ( b ) treated with 365 nm
light ( 5 min ) .
fluorescence images and rna sequences ( 80100
animals / experiment ) are taken 24 hpf .
the sequence of the guiderna
is the same as in figure 4 . for further details ,
rna - guided
enzymes represent rationally programmable tools that
allow for the efficient and precise manipulation of nucleic acids
at specific sites in living organisms . here
, we further elaborate
a novel strategy for site - directed adenosine - to - inosine rna editing
( a ) by introducing photocontrol and ( b ) by applying the tool in developing platynereis dumerilii .
the presented approach is unique
in that the artificial rna - guided
editing enzyme is assembled via the formation of a single covalent
bond .
as covalent bond formation is essential
for the functioning , photocontrol is feasible by blocking the snap - tag - mediated
bond formation via installment of a single photoprotection group at
the o6-benzylguanine moiety .
specifically ,
we demonstrate the ready synthesis of npom - protected bg and its convenient
introduction into diversely chemically modified antisense oligomers
after their solid - phase synthesis .
this is in contrast to other strategies
that require the site - specific incorporation of ( often several ) photoprotected
nucleosides during solid - phase oligonucleotide synthesis to achieve
photocontrol of biochemical processes .
furthermore , our strategy needs less genetic engineering
compared to the introduction of photoresponsive groups into enzymes
by means of amber suppression or related strategies . in vitro
we could show that the attachment of the npom group
at
n7 , but not n9 , of the bg moiety blocks the conjugation reaction with
the snap - tag .
however , reactive benzyl guanine is readily released
upon 365 nm irradiation with high efficiency (
2000 m cm ) and allows for
the light - dose - dependent assembly of guiderna deaminase conjugates .
besides editing , the npom - protected bg will be applicable in other
approaches that rely on the snap - tag , like chemical inducers of dimerization . by controlling the assembly of the editase
,
we could trigger the in vitro editing of a purified mrna in a light - dose - dependent
manner covering the whole dynamic range from absence of editing in
the absence of light until full editing in the presence of light .
the desired action of our tool could be directly translated into mammalian
cell culture ; however , optimization was required to control low - level
residual editing by the photoprotected guiderna .
furthermore , we established site - directed
rna editing for the first time in a living organism .
specifically ,
we achieved the efficient and highly selective switch of a premature
stop into a tryptophan codon in developing platynereis dumerilii zygotes .
notably , no genetic engineering and livestock breeding
is required , thus circumventing time - consuming and cost - intensive
laboratory work .
as our editing tool is independent of any host - specific
factors , the technology should be transferable to any other organism .
in platynereis , the covalent assembly of the guiderna
deaminase
conjugate was again essential , and our simple photocontrol strategy
for site - directed rna editing was directly transferable . the tool
could now be further elaborated to achieve precise spatiotemporal
control of protein isoforms in cellular networks or in developing platynereis .
trifluoroacetamide
protected o6-benzylguanine
( bg , 120 mg , 0.33 mmol ) was solved in
dry dmf ( 1.2 ml ) under argon .
diazabicycloundecene ( 150
l , 153 mg ) was added at room temperature , and the solution
was stirred for 30 min .
npom chloride ( 0.5 mmol , 1.5 equiv ,
dissolved in 1.6 ml of dmf ) prepared in situ as described was added dropwise . after 2.5 h
, the reaction
mixture was diluted with etoac , washed with 1% citric acid ( 3 )
and brine ( 1 ) , and dried over na2so4 .
the evaporated crude product was cleaned via silica chromatography
( 24% meoh in dcm ) and yielded 24 mg ( 21% ) of npom - bg - tfa and 50 mg ( 42% ) of npom - bg - tfa . for full
characterization and assignment of the isomers and downstream synthesis ,
h nmr ( 600 mhz , dmso - d6 ) : = 9.99 ( t , j = 5.9 hz , 1h ) , 8.07 ( s ,
1h ) , 7.48 ( d , j = 8.1 hz ,
2h ) , 7.35 ( s , 1h ) , 7.28 ( d , j = 8.1 hz , 2h ) , 6.84
( s , 1h ) , 6.22 ( s , 2h ) , 6.16 ( s , 1h ) , 6.04 ( s , 1h ) , 5.54 ( m , 2h ) , 5.46
( m , 2h ) , 5.13 ( q , j = 6.2 hz , 1h ) , 4.39 ( d , j = 5.9 hz , 2h ) , 1.33 ( d , j = 6.2 hz ,
3h ) .
c nmr ( 151 mhz , dmso - d6 ) : = 164.1 , 159.8 , 156.4 ( q , j(c , f ) = 36 hz ) , 156.3 , 151.8 , 146.5 , 145.9 , 140.5 , 137.2 , 136.2 ,
135.5 , 127.9 , 127.5 , 116.0 ( q , j(c , f )
= 288 hz ) , 105.6 , 105.2 , 104.3 , 103.2 , 74.9 , 72.0 , 66.6 , 42.4 , 23.3 .
hr - esi - ms : [ m + h](theoretical ) = 590.16056
for c25h23f3n7o7 ; found 590.16118 .
h nmr ( 600 mhz , dmso - d6 ) : = 10.00 ( t , j =
6.0 hz , 1h ) , 7.80 ( s , 1h ) , 7.49 ( d , j = 8.1 hz , 2h ) ,
7.46 ( s , 1h ) , 7.31 ( d , j = 8.1 hz , 2h ) , 6.97 ( s ,
1h ) , 6.34 ( s , 2h ) , 6.15 ( s , 1h ) , 6.03 ( s , 1h ) , 5.435.49 ( m ,
2h ) , 5.40 ( d , j = 11.4 hz ) , 5.32 ( d , j = 11.4 hz ) , 5.21 ( q , j = 6.3 hz , 1h ) , 4.41 ( d , j = 6.0 hz , 2h ) , 1.38 ( d , j = 6.3 hz , 3h ) .
c nmr ( 151 mhz , dmso - d6 ) : = 160.8 , 157.2 ( q , j(c , f ) = 36 hz ) , 155.3 , 152.7 , 147.5 , 142.1 , 140.5 , 138.2 ,
137.0 , 136.6 , 129.6 , 128.3 , 116.9 ( q , j(c , f ) = 288 hz ) , 114.3 , 106.7 , 105.2 , 104.1 , 73.1 , 71.7 , 67.5 , 43.3 ,
24.1 .
hr - esi - ms : [ m + na](theoretical ) = 612.14250
for c25h22f3n7o7na ; found 612.14262 .
purified snap - adar1
( 170 nm ) , purified ecfp mrna ( 10 nm ) , and one of the respective guidernas
( 50 nm ) were prepared in buffer ( 25 mm trishcl , 0.75 mm mgcl2 , 75 mm kcl , 2 m heparin , and 640 u / ml murine rnase
inhibitor , 10 mm dtt , ph 8.3 ) in pcr tubes .
irradiation with 365 nm
light was performed on a uv trans - illuminator ( uvp tfl-40v , 25 w ,
intensity high ) for the indicated amount of time at room temperature .
subsequent editing was performed by incubation for 120 min while cycling
between 30 and 37 c .
reactions were stopped by heating to 70
c for 3 min and subsequent reverse transcription .
after pcr
amplification of the cdna , editing yields were estimated by the relative
height of the guanosine versus adenosine traces by sanger sequencing .
sequence of
the guidernas : ( npom)bg / nh2-ucg - gaacaccccagcacaga-3
( natural ribonucleotides ; 5-terminal modifications were introduced
via amino - linker , the 5-terminal three nucleotides serve as
linker and do not base - pair with the target ) .
cells ( 293 t : dsmz
code acc-635 ; 200 000 cells / well ) were seeded on 24-well plates
in full media ( dmem , 10% fbs , 1% penicillin / streptomycin , grown in
5% co2 , 37 c ) . at 6080% confluency ,
plasmid
pcdna3.1 vector ( life technologies ) carrying snap - adar1 ( 100 ng / well )
and pcdna3.1 vector carrying the respective egfp variant ( 500 ng / well ) were co - transfected with lipofectamine 2000
( 4 l/g ) .
after 24 h , the
cells were reverse transfected into 96-well plates ( 60 000 cells / well )
containing the respective guidernas ( 10 pmol / well ) pretreated with
lipofectamine 2000 ( 0.5 l / well ) .
four hours after reverse transfection ,
media was replaced with dmem without fbs and phenol red , containing
hepes ( 25 mm ) .
irradiation ( 365 nm ) was performed in a fluorescence
microscope ( zeiss cellobserverz.1 , equipped with a 365 nm colibri.2
led ) at 100% led power for the indicated amount of time .
twenty - four
hours later , the fluorescence phenotype was analyzed by fluorescence
microscopy ( zeiss cellobserverz.1 ) , and rna was extracted using the
rneasy minelute cleanup kit ( qiagen ) . after reverse transcription
and pcr amplification , the editing yield was estimated by sanger sequencing .
the sequence
of the w58x guidernas was ( npom)bg / nh2-usasu - gugucgg - cca - cggaascsasgsg-3 ;
the sequence of the stop66-guiderna was bg - uscsg - gaacacc - cca - gcascsasgsa-3 ( s = phosphothioate
linkage ; plain font indicates 2-methoxyribonucleotides , and
italic underlined indicates unmodified ribonucleotides ; the three
5-terminal nucleotides serve as a linker and do not base - pair
with the target ) . for
the in vitro transcription of stabilized mrnas of snap - adar1 and egfp
variants with the mmessage mmachine t7 ultra kit ( life technologies )
,
the respective genes were subcloned into the puc57-t7-rpp2 vector ,
resulting in 5-capped and 3-polyadenylated transcripts
additionally stabilized by a platynereis - specific
rpp2 5-utr , as described before .
fertilized zygotes were obtained from an in - house breeding
culture and were incubated at 14.8 c for 55 min .
prior to microinjection ,
the egg jelly was removed by rinsing the zygotes with natural seawater
( nsw ) in a 100 m sieve . to soften the vitellin envelope , a
1-min - long proteinase k treatment ( 70 g / ml )
100 zygotes
were embedded in the injection stage ( 2% agarose in nsw ) .
samples
were injected using femtotipps ii microcapillaries with a femtojet
express microinjector ( 700 hpa injection pressure , 0.1 s injection
time , 35 hpa compensation pressure ) in a cooled ( 14.8 c ) zeiss
axiovert 40 cl microscope equipped with a luigs and neumann micromanipulator
as described before .
injection started
1 hours post fertilization ( hpf ) and was stopped when the first cleavage
was detected ( ca .
irradiation at 365 nm was performed immediately
after microinjection for the indicated amount of time on a uv trans - illuminator
( uvp tfl-40v , 25 w , intensity high ) .
microinjected zygotes were bred
at 19 c in nunclon six - well plates containing 6 ml nsw .
twenty - four
hpf , healthy larvae ( early trochophore ) were separated from unhealthy
ones .
rna from 80100 healthy larvae ( two injection sessions )
was isolated 25 hpf by shock freezing ( liquid nitrogen ) , shear forces
( passing through 0.6 mm needle ) , vortexing ( 10 s ) , and subsequent
use of the rneasy minelute cleanup kit ( qiagen ) .
after reverse transcription
and pcr amplification , the editing yield was determined by sanger
sequencing .
injection samples contained 1.5 g/l rhodamine - dextran
( 10 kda mw , sigma ) for injection control , 250 ng/l of the respective
reporter mrna , 450 ng/l snap - adar1 mrna , and 25 m of
the respective guiderna .
sequence of the guiderna : bg / nh2-usasu - gugucgg - cca - cggaacasgsgscsa-3
( s = phosphothioate linkage ; plain font indicates 2-methoxy
ribonucleotides , and italic underlined indicates unmodified ribonucleotides ;
the 5-terminal three nucleotides serve as linker and do not
base - pair with the target ) . | site - directed
rna editing allows for the manipulation of rna and
protein function by reprogramming genetic information at the rna level .
for this we assemble artificial rna - guided editases and demonstrate
their transcript repair activity in cells and in developing embryos
of the annelid platynereis dumerilii .
a hallmark
of our assembly strategy is the covalent attachment of guiderna and
editing enzyme by applying the snap - tag technology , a process that
we demonstrate here to be readily triggered by light in vitro , in
mammalian cell culture , and also in p. dumerilii .
lacking both sophisticated chemistry and extensive genetic engineering
,
this technology provides a convenient route for the light - dependent
switching of protein isoforms .
the presented strategy may also serve
as a blue - print for the engineering of addressable machineries that
apply tailored nucleic acid analogues to manipulate rna or dna site - specifically
in living organisms . |
in the human gut , trillions of bacteria interact with the host 's systemic immune system in a complex balance between immune activation and tolerance .
pathogen recognition receptors ( prrs ) have been shown to play an important role in the differentiation between commensal and pathogenic bacteria .
the detection of pathogen molecules collectively known as pathogen - associated molecular patterns ( pamps ) by prrs activates the innate immune system , being involved in infection recognition and its consequent inflammatory response [ 2 , 3 ] .
the activation of prrs can lead to enhanced production of proinflammatory cytokines with a wide range of systemic and local effects . among them , interleukin ( il)-1 has been shown to be secreted in high amounts by colonic monocytes from patients with active inflammatory bowel disease ( ibd ) , and intestinal levels are consistently correlated with disease activity suggesting an important role of this cytokine in intestinal inflammation .
in addition , il-1 has also been implicated in the promotion of angiogenesis , tumor growth , and metastasis in experimental cancer models , being associated with more aggressive tumor biology [ 5 , 6 ] .
generation of il-1 requires the activity of caspase-1 , but the mechanism involved in the activation of proinflammatory caspases remained to be established until 2002 . in that year , the group of the late jrg tschopp reported the identification of the inflammasome , a multimeric molecular platform which triggers the activation of inflammatory caspases and processes pro - il-1 .
inflammasomes are cytosolic multiprotein complexes activated by specific prrs which are involved in infection recognition and inflammation [ 1317 ] . the structure of the inflammasome is assembled by intracellular nucleotide - binding - oligomerization - domain- ( nod- ) like receptors ( nlrs ) to initiate innate immune responses against invading pathogens activating caspase - l .
the subsequent activation of caspase - l leads to enhanced expression of il-1 and il-18 , recruitment and activation of immune cells , and triggering of pyroptosis , a caspase-1-dependent inflammatory form of cell death [ 14 , 1821 ] .
the importance of the inflammasome and the cell death programming associated with microbial invasion is to restrict pathogen growth and to activate and recruit immune cells to mediate host defense . as the activation of the inflammasome and the caspase-1 pathway leads to enhanced production of cytokines known to be upregulated in ibd and cancer , the role of this pathway in intestinal inflammation and colonic neoplasia has been the subject of intense research in recent years .
these multimeric platforms are found in a wide range of cell types including macrophages , dendritic cells , adipocytes , keratinocytes , and epithelial cells [ 2228 ] .
these complexes are activated either by nlr proteins nlrp1 , nlrp3 , nlrc4 , nlrp6 , and naip5 or by the dna - sensing complex of aim2 , a member of the interferon - inducible hin-200 protein family .
activation of these receptors by certain pamps leads to their oligomerization and subsequent interaction with the adaptor protein asc and the card domain of caspase-1 .
asc , as well , presents a card domain that works together with the card domain of procaspase-1 .
inflammasome - activated caspase-1 is then used for activation of the proinflammatory cytokines il-1 and il-18 , both belonging to the il-1 family .
these inflammatory cytokines enhance antimicrobial functions of phagosomes and promote protection against intracellular pathogens ( figure 1 ) .
crohn 's disease ( cd ) and ulcerative colitis ( uc ) are chronic immune - mediated inflammatory diseases of the gastrointestinal tract that result from a dysregulated mucosal immune response to bacterial antigens in the gut lumen of a genetically susceptible host [ 29 , 30 ] . in the gut - microbiota interplay related to ibd pathogenesis , several previous findings point towards the potential role of the inflammasome in the development of chronic intestinal inflammation .
the first evidence refers to the upregulation of inflammatory cytokines il-1 and il-18 in active ibd , and the discovery of il-18 gene polymorphisms associated with cd [ 3133 ] .
the second is the presence of a dysregulated il-1 production linked to cd and the association between the nlrp3 inflammasome and three rare autoinflammatory chronic disorders treated with canakinumab , a human monoclonal antibody targeted at il-1 [ 22 , 31 , 34 , 35 ] .
the third , and perhaps the most important evidence , is the association between the nlrp3 gene and cd in candidate - gene approach studies . genome - wide association studies ( gwas ) have tried to dissect the inherited element of ibd , identifying more than 70 cd and 40 uc susceptibility loci [ 36 , 37 ] .
these studies , however , do not explain the majority of the heritability related to ibd .
one interesting genomic region not associated with ibd in gwas , but pointed out in candidate - gene approach studies and gene expression analysis data , is the nlrp3 gene which encodes the nlrp3 or cryopyrin protein [ 3941 ] .
this protein is part of the nlrp3-inflammasome , and it has a pivotal role in the pathogenesis of other chronic inflammatory disorders as pseudogout , gout , and familial mediterranean fever [ 4244 ] . as a result of the genetic link between nlrp3 and cd ,
the nlrp3 inflammasome is the most studied caspase-1 inductor multimeric platform in the field of chronic intestinal inflammation . in two independent candidate - gene studies ,
the nlrp3 gene was associated with cd [ 39 , 40 ] , but there was no association in a posterior large study from the uk .
interestingly , the first study conducted by villani and coworkers also performed functional assays to evaluate the impact of these polymorphisms in nlrp3 expression and il-1 production . in this
regard , nlrp3 snps were associated with lower levels of nlrp3 m - rna expression in a loss - of - function fashion with homozygosis for the risk allele being associated with the lowest level of nlrp3 expression in peripheral blood cells and monocytes .
in addition , they observed an association between lower il-1 levels and the risk nlrp3 allele in cultured monocytes in the presence or absence of lipopolysaccharide . in both cases ,
homozygosis for the risk allele was associated with the lowest level of il-1. even though significantly higher il-1 levels were found in the ulcerated intestinal mucosa from human cd samples than in healthy controls , it was postulated that a dysregulated il-1 production might play a role in cd pathogenesis for patients bearing these snps .
the second study identifying an association between polymorphisms in the nlrp3 and cd included 498 cases and 794 controls , reporting that variants of nlrp3 conferred susceptibility to cd in swedish male individuals . even though an nlrp3 genetic susceptibility was found in this population , careful analysis of the results
show that , differently from the study by villani and coworkers , the nlrp3 snp associated with cd was a gain - of - function polymorphism , possibly promoting the production of mature il-1 with subsequent induction of caspase-1 activity .
the authors postulate that patients with this specific nlrp3 polymorphism might present an increased susceptibility to cd as a result of an increased il-1 production and not due to a dysregulation of the pathway .
in addition , the risk for developing cd in this study was exclusively associated with male patients bearing variant alleles in both nlrp3 and card8 genes . a third study evaluating the association between nlrp3 polymorphisms and ibd added more controversy to the topic .
lewis and coworkers raised questions about the previously reported association between cd and the nlrp3 locus supported by negative results based on control allele frequency data from large gwa studies . even though the role of the nlrp3 inflammasome in ibd is still a matter of debate , the mechanisms behind its function started to be recently unveiled .
nlrp3 can be triggered by bacterial constituents , synthetic purine - like compounds , endogenous urate crystals , and exogenous adenosine triphosphate ( atp ) [ 4648 ] .
of note , it was postulated that the passage of bacterial molecules into the host cytosol leading to nlrp3 inflammasome activation can be mediated by pannexin-1 and p2x7 receptor .
pannexin-1 constitutes a transmembrane hemichannel that associates with p2x7 receptor , a member of the atp - activated p2x purinergic receptors family , permeable to monovalent cations and anions , and capable of inducing the opening of a larger pore permeable to hydrophilic macromolecules .
in particular , the p2x7 receptors act as danger sensors in immune cells and have been implicated in different biological functions , including apoptosis and the production and release of proinflammatory cytokines . in this regard
, investigators have demonstrated that the cytosolic recognition of bacterial molecules resulting in the nlrp3 inflammasome activation is mediated by pannexin-1 activation .
these results seem to indicate that nlrp3 would function downstream of pannexin-1/p2x7 receptor in response to bacterial components to regulate caspase-1 activation ( figure 1 ) .
furthermore , the expression and site - specific modulation of p2x7 receptors was demonstrated on epithelial and immune cells of the gut , supporting the suggestion of purinergic signaling as an additional component of the innate immune circuits involved in the control of inflammation and cell fate in the gut and gut - associated lymphoid tissues .
in addition , in intestinal epithelial cells , the expression of p2x7 receptors was also found to be upregulated by interferon - gamma , a proinflammatory cytokine and a signature molecule of the th-1 type of immune response .
moreover , atp was shown to induce apoptosis and autophagy in human epithelial cells , possibly via reactive oxygen species production , through activation of the p2x7 receptor .
taken together , these findings appear to implicate p2x7 receptors associated with pannexin-1 and the consequent nlrp3 inflammasome activation in the pathogenesis of diseases based on the dysregulation of the immune response such as ibd . as the main downstream impact of the activation of the inflammasome is the upregulation of il-18 and il-1 ,
the knockout of these two important inflammatory cytokines as well as of other upstream regulators is pivotal to fully understand the role of the inflammasome in intestinal inflammation .
therefore , genetically modified mice lacking il-18 , il-18 receptor ( il-18r ) , il-1 receptor ( il-1r ) , nlrp3 , nlrp6 , asc , and caspase-1 were constructed . in general , the susceptibility of these animals to intestinal inflammation was tested using the dextran sulphate sodium ( dss ) experimental colitis model . in the context of dss colitis models ,
more recently , it has been shown that il-18 and il-18r knockout mice develop more severe inflammation compared to wild - type , which is not true for il-1r knockout mice [ 56 , 57 ] . in an infection mouse model with c. rodentium
, however , il-1r knockout was shown to present increase mortality with severe colitis characterized by intramural colonic bleeding and intestinal damage following infection .
consistent with these findings , most recent studies present clear data that mice lacking nlrp3 are more susceptible to develop colitis [ 26 , 5860 ] and asc and caspase-1-deficient mice present enhanced susceptibility to dss - induced inflammation .
however , other studies have shown exactly the opposite that transgenic or pharmacological blockage of il-1 converting enzyme ( ice ) or il-18 ameliorate dss colitis [ 6265 ] . in keeping with these results , bauer and coworkers
reported decreased sensitivity to dss in nlrp3 deficient mice . in that study , il-1 secretion was abrogated in macrophages lacking nlrp3 , asc , or caspase-1 confirming that dss activates caspase-1 via the nlrp3 inflammasome .
after administration of dss , nlrp3 knockout mice developed less severe colitis than wild - type mice and produced lower levels of proinflammatory cytokines in colonic tissue .
in addition , pharmacological inhibition of caspase-1 with pralnacasan achieved a level of mucosal protection equivalent to nlrp3 deficiency .
more recently , this protective role of nlrp3 against dss colitis was also demonstrated by yet another independent group . in any case , regardless of the still debated role of the nlrp3 inflammasome in dss colitis , it has been shown that nlrp3 , asc , and caspase-1 deficient mice do not develop colitis without dss treatment , implying that isolated inflammasome impairment does not result in spontaneous intestinal inflammation .
another inflammasome , nlrp6 , has been associated with ibd [ 11 , 12 , 67 ] .
consistent with the presumed role for nlrp6 in inflammasome signaling , chen and coworkers have shown that mice lacking nlrp6 present decreased levels of serum il-18 after dss treatment . these mice deficient in nlrp6 develop a colitis phenotype , and this is transmissible to cohoused wild - type mice , both early in postnatal life and during adulthood . upon injury ,
consistently , an analysis on a whole - genome expression profiling revealed a link between nlrp6 and self - renewal of the epithelium . the inability of mice lacking nlrp6 to repair damaged epithelium as efficiently as wt mice resulted in extended increase in epithelial proliferative activity .
recently , the role of the inflammasome in gut - related infection and sepsis has also been addressed .
for this purpose , it has been shown that mice treated with large - spectrum antibiotics before dss intervention show symptoms of sepsis , not colitis , due to translocation of a pathogenic strain of e. coli .
this particular model is very significant due to its resemblance to the common clinical scenario in which patients undergoing antibiotic and gut - damaging cytotoxic treatments develop septicemia . in this antibiotics - dss model ,
similarly , caspase 1 and il-1 deficient animals were protected from e - coli systemic inflammatory response showing that naip5-nlrc4 inflammasome signaling through il-1 is important for the development of gut - related sepsis .
locally , nlrc4-dependent il-1 production by intestinal phagocytes represents a specific response discriminating pathogenic from commensal bacteria and contributes to host defense in the intestine . upon infection with pathogenic bacteria ,
intestinal phagocytes produce mature il-1 through the nlrc4 inflammasome and mice deficient in nlrc4 or il-1 receptor are highly susceptible to intestinal infection [ 70 , 71 ] .
it seems , however , that the inflammasome does not only signal through il-1 or il-18 in systemic inflammatory responses .
it has been shown that systemic inflammasome activation by flagellin leads to loss of vascular fluid into the intestine and peritoneal cavity and death in mice , and this outcome depends on naip5 , nlrc4 , and caspase-1 signaling , but is independent of il-1 or il-18 . instead
, flagellin - related inflammasome activation results in a pathological release of signaling lipids , including prostaglandins and leukotrienes that rapidly initiate inflammation and vascular fluid loss .
the role of the inflammasome in cancer physiopathology is complex as it can either lead to inflammasome - dependent carcinogenic inflammation or play a role in the process of eliminating malignant precursors through programmed cell death .
not only the product of the inflammasome activation , caspase-1 , is associated with inflammation and carcinogenesis , but also it can stimulate immune responses against tumoral cells . in colonic tissue ,
the role of the inflammasome in colorectal cancer tumorigenesis was mainly explored using the azoxymethane ( aom ) dss model in which administration of dss after initiation with a low dose of aom exerts a powerful tumor - promoting inflammatory activity in colon in mice . using this inflammation - driven tumorigenesis model draws a parallel to the carcinogenic process that takes place in ibd - related intestinal neoplasia . in these models , it has been shown that the absence of inflammasome - related interleukins , mainly il-18 , can greatly impact carcinogenesis and tumor progression .
il-18-deficient mice , for instance , have increased inflammation and tumor development in a colitis - associated colon cancer model
. it seems , however , that il-18 can also influence epithelial growth by regulating the production of additional interleukins . in this
regard , activation of nlrp3 or nlrp6 inflammasomes leads to il-18-dependent downregulation of il-22 blocking protein ( il-22bp ) and higher expression of il-22 .
this il-22-il-22bp axis was shown to critically regulate intestinal tissue repair and tumorigenesis in the colon .
the main studies evaluating the role of the inflammasome in colitis - associated cancer using the aom / dss model are summarized in table 1 .
mice lacking nlrp3 were shown to be more susceptible to tumorigenesis in the aom - dss model in some studies [ 7 , 9 ] , but not in others . in studies that demonstrated a positive association , nlrp3 deficient mice presented more inflammation and higher tumor burden compared to controls . in these nlrp3 knockouts , colonic il-18 levels were shown to be lower than those of controls .
it was postulated , therefore , that il-18 might be associated with colon protection against tumorigenesis . in this regard , knockout mice for il-18 treated with aom / dss contained significantly more tumors than controls [ 7 , 8 ] .
importantly , recombinant il-18 was successfully used as rescue , being able to reverse disease progression perhaps through induction of ifn- and its antitumor signaling involving activation of the transcription factor stat1 . of note ,
il-18 uses myd88 as a downstream signal transduction effector and myd88 signaling has been shown to have a protective role in the development of aom / dss colitis .
it has been proposed that the increased susceptibility of il-18 deficient mice to colitis and cancer in the aom / dss model may be partially dependent on myd88-related mechanisms , although il-18 deficient mice present a milder phenotype compared with myd88 knockout mice ( less tumorigenesis ) implying that other myd88-related pathways might act with il-18 to minimize carcinogenesis . in the negative study
, there were no differences in tumor formation between nlrp3 deficient mice and controls after challenge with aom - dss .
in contrast , another inflammasome , nlrc4 , was found to be associated with tumorigenesis in this model . in this regard , nlrc4 knockout mice had significantly increased tumor numbers and tumor load compared to wild - type animals , though no differences in inflammation severity were noted . since nlrc4 is associated with p53-dependent apoptosis , it may provide a link to the increased tumorigenesis observed in caspase-1 deficient mice noted by three independent groups [ 7 , 9 , 10 ] .
caspase-1 has been shown to be associated with the regulation of colonic epithelial cell proliferation and apoptosis and not only inflammation per se . as a result ,
caspase-1 deficient mice show increased colonic epithelial cell proliferation in early stages of tumor formation and reduced apoptosis in advanced tumors .
hu and colleagues studied caspase-1 mrna expression levels in normal colon tissue and colon tumors from wt mice observing a significant reduction in caspase-1 mrna expression levels in tumors compared to normal colonic tissue , suggesting that lack of caspase-1 may play a role in tumor progression . similarly
to caspase-1 deficient mice , nlrc4 knockout mice features significantly enhanced proliferation in both steady state and the early phase of inflammation - induced tumor formation .
another inflammasome , nlrp6 , was also found to play a role in aom - dss tumorigenesis [ 11 , 12 ] . in this
regard , nlrp6-deficient mice developed significantly more tumors compared to wildtype mice after chemical induction .
the increase in tumors in these mice correlated with higher levels of intestinal epithelial proliferation , hyperplasia , and an increase in proinflammatory cytokines such as tnf , il-6 , and il-1. protection against tumorigenesis by nlrp6 is conferred specifically by hematopoietic cells rather than intestinal epithelial or stromal cells as irradiated wildtype mice that were transplanted with nlrp6 deficient bone marrow had similar numbers of tumors as nlrp6 deficient mice .
additionally , nlrp6 deficient recipients that received wildtype bone marrow were significantly protected against tumorigenesis to a similar extent as wildtype animals .
these findings suggest that deficiency in nlrp6 function in hematopoietic - derived cells is important for nlrp6-mediated protection against colitis - induced tumorigenesis . as sporadic and familial colorectal cancer tumorigenesis in humans
is often caused by wnt - activating mutations , normand and colleagues performed a transcriptional profiling of tumoral and nontumoral biopsies from nlrp6 deficient mice and controls treated with the dss - aom regimen .
within the set of 1,884 genes that were differentially expressed in nlrp6 deficient mice , a significant overrepresentation of paracrine actors of the p53 wnt and notch signaling pathways was observed , supporting the role of nlrp6 in regulation of intestinal crypt cell proliferation .
notably , the microarray analysis clearly revealed an overexpression of wnt - signaling pathway genes in tumor resection specimens of nlrp6 deficient mice , particularly the proto - oncogene mycl1 . in mice derived from animals treated with ethylnitrosourea
this mutation was later found to be located in the apc gene , the mouse homologue of the human apc gene responsible for human familial adenomatous polyposis .
the development of the apc deficient mice was one of the first spontaneous genetic animal models for bowel cancer .
in this model , affected mice develop multiple adenomas throughout the entire intestinal tract at an early age .
it has been shown that innate immune signaling has an important role in the intestinal tumorigenesis in this model . in this
regard , rakoff - nahoum and medzhitov have shown that myd88-dependent signaling controls the expression of several modifier genes of intestinal tumorigenesis in apcmin mice .
apcmin mice that are also deficient in myd88 have decreased number of polyps which are smaller in size than those in age - matched apcmin mice . in the inflammasome field
, there were attempts to evaluate the potential impact of caspase-1 signaling in the development of tumors in apcmin mice with disappoints results as the crossbreeding between apcmin and caspase-1 deficient mice does not impact the phenotype .
in summary , the activation of specific nlr inflammasomes was shown to be triggered by microbial molecules , whereas defects in nlrs determine innate immune system abnormalities and changes in the intestinal microbiota .
in particular , intestinal dysbiosis has been consistently linked to intestinal inflammation through defects of nlr family members . in conjunction , these data highlight the importance of the inflammasome in the innate intestinal immune response and the maintenance of intestinal homeostasis , with fundamental influence on barrier function and the efficient elimination of invading microorganisms .
therefore , the abnormal activation of the inflammasome , converging signals from the internal and external milieu , sensing diverse stressful and microbial elements , appears to position inflammasome as a critical mechanistic link in the context of chronic inflammatory disorders involving the gut . | the activation of specific cytosolic pathogen recognition receptors , the nucleotide - binding - oligomerization - domain- ( nod- ) like receptors ( nlrs ) , leads to the assembly of the inflammasome , a multimeric complex platform that activates caspase-1 .
the caspase-1 pathway leads to the upregulation of important cytokines from the interleukin ( il)-1 family , il-1 , and il-18 , with subsequent activation of the innate immune response . in this review , we discuss the molecular structure , the mechanisms behind the inflammasome activation , and its possible role in the pathogenesis of inflammatory bowel diseases and intestinal cancer . here
, we show that the available data points towards the importance of the inflammasome in the innate intestinal immune response , being the complex involved in the maintenance of intestinal homeostasis , correct intestinal barrier function and efficient elimination of invading pathogens . |
telomeres , unique structures at the physical ends of linear eukaryotic chromosomes , were first described almost 70 yearsago by hermann muller in his classic studies of the fruit fly drosophilia melanogaster ( 1 ) .
he noted that chromosomal inversions resulting from ionizing radiation ( ir)-induced double - strand breaks ( dsbs ) never involved the very end of a chromosome rejoining with some other part of a chromosome .
telomere , which comes from greek telos meaning end and meros meaning part based on this chromosome end protection phenomenon . shortly thereafter
, barbara mcclintock observed that while broken ends of maize chromosome fused , forming dicentric chromosomes , unbroken chromosomes rarely fused ; i.e. natural chromosomal termini are not
the absence of interstitial telomere sequence within ir - induced dicentrics was later verified in human cells ( 3 ) .
these studies demonstrate that normally cells accurately distinguish telomeric ends from random dsb ends and protect the former from illegitimate end - joining reactions .
how cells make this critical distinction continues to be an active area of research today , especially as the dividing lines between the two types of ends have become less , rather than more clear .
recent discoveries that certain dsb repair proteins act to preserve rather than to join the natural ends of mammalian chromosomes ( 47 ) , have provided impetus for the union of two seemingly disparate scientific fields , dna repair and telomere biology . here , we focus on the creation of dysfunctional mammalian telomeres in various repair deficient backgrounds that result from either the loss of end - capping structure or the loss of terminal sequence ( shortening ) , and the consequences of this loss of function .
telomeres serve multiple functions in preserving chromosome stability , including protecting the ends of chromosomes from degradation and preventing chromosomal end fusion .
the dna component of telomeres consists of tandem arrays of short , repetitive g - rich sequence [ ttaggg in vertebrates , ( 8,9 ) ] , oriented 5-to-3 towards the end of the chromosome ( 10 ) , ending in an essential 3 single - stranded overhang that ranges in length from 50 to 400 nt ( 1113 ) .
electron microscopy studies suggest this overhang can loop back and integrate into the duplex repeat tract , forming a
t - loop ( 14 ) , an attractive , although not necessarily exclusive , architectural solution to the end - capping dilemma .
telomeres gradually shorten in replicating cells due to end - processing and the end replication problem ( 1518 ) , so in order for continuous cell division to occur , they must be replenished .
the addition of telomeric repeats de novo is accomplished by the reverse transcriptase telomerase ( 19,20 ) .
thus , telomere length maintenance is a state of equilibrium between telomere loss and re - addition .
precisely how telomere length regulation occurs is unknown , but it does appear that the shortest telomeres are preferentially targeted for elongation by telomerase ( 21 ) .
telomere length is maintained in germ line cells , however , most human somatic cells do not express sufficient telomerase activity to prevent telomere loss as they divide . as a result , telomeres eventually shorten to the point where they initiate a cell cycle arrest in g1 , a state termed replicative senescence ( 22 ) .
telomere shortening can , therefore , limit the number of times somatic cells divide , contributing not only to aging phenotypes , but also providing an effective tumor suppressor mechanism .
cells that lose the ability to senesce because of mutations in p53 protein continue to divide , eventually entering
, cells that constitutively express telomerase can continue to divide almost indefinitely ( 23,24 ) .
consistent with the requirement for telomere maintenance as a step in carcinogenesis , most tumor cells express telomerase ( 25 ) .
however , an alternative mechanism for telomere maintenance has also been described ( 26,27 ) , which is observed in some tumors ( 28 ) and involves recombination ( 26,29 ) .
although telomerase is responsible for addition of telomeric sequence with every cell cycle , a plethora of other proteins also play important roles in the regulation of telomere length maintenance and in the formation of a protective end - cap that prevents chromosome fusion ( 30,31 ) .
proteins that directly bind the double - stranded telomeric repeats include the ttaggg repeat factors trf1 ( 32,33 ) and trf2 ( 34,35 ) .
pot1 ( protection of telomeres 1 ) specifically recognizes telomeric single - stranded dna , belongs to a family of oligosaccharide / oligonucleotide - binding ( ob)-fold - containing proteins and is highly conserved among eukaryotes ( 36,37 ) .
telomere - associated proteins that do not bind dna directly include tin2 ( trf1-interacting nuclear protein 2 ) , which associates with trf1 ( 38 ) and trf2 ( 39,40 ) .
the trf1 complex contains both tin2 and pot1 and acts to regulate telomere - length homeostasis .
tpp1 , the recently proposed name ( 31 ) for a pot1-interacting protein identified independently in three laboratories [ tint1 ( 41 ) , ptop ( 42 ) and pip1 ( 43 ) ] , recruits pot1 to telomeres and so is also involved in telomere length regulation .
rap1 ( human repressor activator protein ) ( 44 ) is recruited to telomeres by trf2 and has been shown to negatively regulate telomere length in vivo ( 45 ) .
six proteins have been proposed to form an essential , dynamic complex at human telomeres : trf1 , trf2 , pot1 , tin2 , tpp1 and rap1 .
this mammalian telomeric core complex serves to form and protect the telomere , and has been termed both the telosome ( 42 ) and alternately , shelterin ( 31 ) .
other proteins , many of which are more commonly associated with dna repair , are also found at telomeric ends [ reviewed in ( 46 ) ] .
examples include dna - pk ( ku70/ku86/dna - pkcs ) , the mrn complex ( mre11/rad50/nbs1 ) , parp1/2 , tankyrase 1/2 , atm , ercc1/xpf , rad51d , wrn and blm .
trf2 has been shown to bind to atm , blocking a damage response at telomeres ( 47 ) .
interestingly , it has recently been proposed that normally , functional human telomeres must be recognized as dna damage in the g2 phase of the cell cycle , in order to recruit the processing machinery necessary for formation of a functional telomere ( 48 ) .
the interplay between telomeres and dsbs may be better understood not by viewing the striking differences between them , but instead by viewing the obvious similarity both are dna ends , the very substrate telomere and damage response / repair proteins specifically recognize and bind .
functional telomeres are essential for continuous cellular proliferation , and therefore loss of chromosomal end - capping has consequences in both aging and carcinogenesis ( 49,50 ) .
we have shown that effective end - capping of mammalian telomeres requires the non - homologous end - joining ( nhej ) protein dna - dependent protein kinase ( dna - pk ) ( 4 ) .
mutation of any of the genes comprising dna - pk , i.e. ku70 , ku86 , or the catalytic subunit , dna - pkcs ( 51 ) , leads to spontaneous chromosomal end - to - end fusions that maintain large blocks of telomeric sequence at the points of fusion ( figure 1 ) .
dna - pk has since been shown to associate with human telomeric dna in vivo ( 52,53 ) .
events at the extreme terminus of the chromosome that normally serve to create a functionally protected telomere fail in the absence of dna - pk , resulting in inappropriate end - to - end fusion events of uncapped telomeres fusing not only to each other , but also to ir - induced dsbs ( 54 ) .
we have also shown that the kinase activity of dna - pkcs is required for effective telomere protection , just as it is for nhej ( 55,56 ) . however , as with nhej , the critical in vivo substrates are not known .
the in vivo consequences of inappropriate interstitial blocks of telomere sequence are also unknown , but they most likely affect chromatin stability , and in vitro studies have demonstrated they increase chromosomal instability ( 57 ) .
we find that telomere fusions contribute significantly to the background level of chromosomal aberrations , and that they occur despite the presence of ample telomere sequence .
thus , they are obviously not a consequence of telomere shortening , nor are they telomere associations [ defined as distinct telomere signals separated by less than approximately one - third the width of a chromatid , ( 58 ) ] .
banding studies demonstrated that any chromosome could be involved in the telomere fusions ( end - capping failure is not chromosome specific ) , and further , that some of the telomere fusions were clonal , suggestive of covalent linkages ( s. bouffler , unpublished data ) . utilizing the strand - specific co - fish technique ( 59,60 )
, we found that the telomere fusions in dna - pkcs - deficient cells exclusively involved telomeres synthesized via leading - strand dna synthesis , suggesting a crucial difference in the post - replicative protection of telomeres that is linked to their mode of replication ( 61 ) .
additional support for a model of end - specific differences in telomeric end - protection was provided by the demonstration of preferential loss of lagging - strand telomeres with wrn deficiency in human cell lines ( 62 ) .
it protects the 3 single - stranded g - rich overhang and is involved in t - loop formation , perhaps facilitating invasion of the 3 single - stranded overhang ( 14 ) .
inhibition of trf2 induces a dramatic telomere fusion phenotype ( 65 ) resulting from failure of end - protection preferentially at leading - strand telomeres ( 61 ) .
end - capping failure occurs after replication , as evidenced by the presence of numerous chromatid - type telomere fusions ( 61 ) ( figure 2 ) .
it has also been shown that dna damage foci form at telomeres uncapped by trf2 inhibition ( telomere dysfunction - induced foci ; tifs ) , and consistent with the cytogenetic results , uncapping of telomeres occurs in late s / g2 , i.e. after replication ( 66 ) .
in addition to loss of end - capping function , chromosomal fusion can also result from the absence of sufficient telomeric repeat sequences to form a functional telomere . the most obvious mechanism for the loss of telomeric repeat sequences is attrition due to the failure to compensate for the gradual loss of telomeric repeat sequences during cell division .
this failure to maintain telomere homeostasis can result from either insufficient telomerase activity or alterations in other telomere - associated proteins required for the recruitment of telomerase to the telomere .
the gradual loss of telomeric repeat sequence due to insufficient telomerase activity is exemplified by the telomere shortening that occurs in telomerase - deficient somatic cells with each cell division , although due to cell senescence or apoptosis , this does not normally result in chromosome instability .
in addition to gradual loss , telomeric repeat sequences can also be lost through stochastic processes , in which large blocks of telomeric repeat sequences are lost in single events .
stochastic events leading to telomere loss can occur through a variety of different mechanisms , the most obvious being large deletions involving recombination , problems encountered during dna synthesis or inefficient dna repair .
replication forks stall near telomeres in yeast , and require the rrm3 helicase , which promotes replication through regions of non - histone chromatin ( 67,68 ) .
mammalian telomeres may also pose problems for dna replication , since the mammalian telomere - binding proteins trf1 and trf2 can inhibit replication fork movement ( 69 ) .
studies in yeast have also demonstrated that telomeric regions are deficient in repair of dsbs .
the introduction of dsbs at different sites along a chromosome with the i - scei endonuclease demonstrated that dsbs near telomeres are not repaired efficiently by nhej , but instead result in complex chromosome rearrangements ( 70 ) .
mammalian telomeres have been shown to be deficient in repair of single - strand breaks ( 71 ) and get damaged from ultraviolet light ( 72 ) .
studies with mouse es cells ( 73,74 ) and human tumor cell lines ( j. p. murnane , unpublished data ) also show that a single dsb generated by i - scei often results in complex chromosome rearrangements not commonly observed at i - scei - induced dsbs at other locations within chromosomes ( 7577 ) .
cells containing mutations in various proteins known to be involved in telomere maintenance have provided valuable insights into the mechanisms of telomere loss .
one such protein is wrn , which is responsible for the human genetic disease werner syndrome ( ws ) , an autosomal recessive genetic disease presenting a wide range of phenotypic abnormalities , including characteristics of premature aging ( 78 ) .
wrn is a member of the recq dna helicase family and has both a 3 to 5 helicase activity and a 3 to 5 exonuclease activity ( 7981 ) .
cells isolated from individuals with ws exhibit shortened life span in culture ( 82 ) , as well as an increased rate of dna rearrangements , including translocations , deletions and dicentrics ( 8388 ) .
wrn binds to a number of proteins involved in recombination , including replication protein a ( rpa ) ( 89,90 ) , pcna and topoisomerase i ( 91 ) , dna polymerase delta ( 92 ) , and co - localizes with rad51 ( 93 ) and the mre11/rad50/nbs1 ( mrn ) complex in response to dsbs ( 94 ) .
consistent with these protein interactions , ws cells have been shown to have a defect in homologous recombination ( hr ) ( 95,96 ) .
in addition , the observations that ws cells have a prolonged s - phase ( 97 ) and wrn co - localizes with rpa in cells arrested in s - phase with hydroxyurea ( 98 ) , suggest that ws cells have a defect in resolving stalled replication forks ( 80,98,99 ) .
wrn has also been demonstrated to bind the dna - pk complex ( 94,100103 ) and fen-1 ( 104 ) , proteins involved in nhej .
moreover , the activity of wrn is influenced by its binding to the dna - pk complex ( 101,102 ) .
thus , in addition to a role in hr , wrn also appears to have a role in the nhej pathway for dsb repair .
the role of telomeres in cellular senescence initially led to the proposal that the shortened life - span of ws cells in culture might be due to accelerated telomere shortening ( 105 ) .
ws cells were found to have accelerated telomere shortening , although the premature senescence in ws cells was found to occur when telomeres were longer than in senescent normal cells ( 106 ) .
however , a subsequent study found that ws cells at senescence have telomeres that are similar in length to normal senescent cells ( 107 ) .
combined with the fact that ws cells can be immortalized by expression of telomerase ( 107109 ) , these results suggest that ws cells have a defect in telomere maintenance that leads to premature senescence .
although due to their long telomeres , mice deficient in wrn alone do not demonstrate the premature aging phenotype observed in humans with ws , later generations of telomerase - deficient mice with shortened telomeres demonstrate classic ws - like premature aging , accelerated replicative senescence , and genomic instability ( 110 ) .
the mechanism responsible for telomere shortening in wrn - deficient cells has yet to be determined .
one study found that cells expressing dominant - negative wrn showed an increase in chromosome ends without detectable telomeres even though no difference in average telomere length was observed ( 111 ) , indicating that telomere loss in ws is due to a stochastic process .
the association of wrn with the dna - pk complex involved in nhej would suggest that telomere loss in ws cells is due to a deficiency in repair of dsbs near telomeres .
however , the dna - pk complex also has a role in telomere capping ( 46,53,112,113 ) .
in addition , wrn has been found to bind to trf2 ( 114 ) , which is essential for maintaining the cap on the end of the chromosome ( 65 ) , and wrn is required for d - loop resolution regulated by trf1 and trf2 ( 114 ) .
based on these observations , another possible mechanism for telomere loss in wrn - deficient cells would involve t - loop deletions similar to those observed in cells deficient in trf2 ( 115 ) .
however , loss of end - capping structure would not lead directly to loss of telomeric repeat sequences .
a critical clue to the mechanism of telomere loss in ws cells comes from a recent study demonstrating the preferential loss of the lagging - strand of telomeres ( 62 ) , suggesting that telomere loss in ws cells is a result of the requirement for wrn in replication of the g - rich dna found in the lagging strand .
this model is consistent with the involvement of wrn in dna replication and/or resolution of stalled replication forks ( 80,98,99 ) , since failure to resolve stalled replication forks can result in dna dsbs ( 116,117 ) .
the importance of wrn in replication and/or recombination of telomeres is also apparent from its requirement in suppression of sister chromatid exchange ( sce ) specifically within telomeric dna ( t - sce ) ( 118 ) and activation of the alt pathway that involves recombination ( 119 ) , similar to that proposed for the yeast homolog for wrn , sgs1 ( 120122 ) .
mutations in nbs1 are responsible for the autosomal recessive disease nijmegen breakage syndrome ( nbs ) , which displays a wide range of phenotypic abnormalities , including premature aging , increased cancer incidence , chromosomal instability and sensitivity to ir ( 123,124 ) .
nbs1 is part of the mrn complex that also contains the mre11 and rad50 proteins ( 125,126 ) .
the mrn complex is a key player in the cellular response to dsbs in that association of the mrn complex with dsbs is required for the localization and activation of atm ( 127 ) , which in turn phosphorylates nbs1 ( 128130 ) . as a result ,
similar to cells deficient in atm , mammalian cells deficient in nbs1 lack the s - phase cell cycle checkpoint ( 131 ) and are sensitive to ir ( 132 ) . in addition to its roles in dna recombination and repair , the mrn complex also functions in telomere maintenance .
inhibition of nbs1 by rnai resulted in an increased frequency of telomere association ( 58 ) .
primary fibroblasts from individuals with nbs have shortened telomeres , which are proposed to play a role in the pathology of this disease ( 133 ) . in this respect
nbs is similar to at , where accelerated telomere shortening is observed in primary fibroblasts ( 134 ) , although no difference in telomere length is evident in immortal cells actively maintaining their telomeres ( 135 ) .
in fact , mice with combined knockouts in both atm and the rna component of telomerase show accelerated telomere loss and premature aging , leading to the hypothesis that telomere loss is the reason for some of the phenotypic abnormalities observed in at ( 136 ) . like wrn ,
similar to wrn , the mrn complex interacts with trf2 ( 137 ) , and therefore is likely to function in proper end - cap formation .
however , although mre11 and rad50 are found at the telomere throughout the cell cycle , nbs1 is associated with the telomere only during s - phase , suggesting that it is involved in telomere replication .
thus , like wrn , a defect in nbs1 may promote telomere loss through problems in dna replication or the resolution of stalled replication forks in telomeric regions .
in fact , wrn associates with mrn ( 94 ) , and both dominant - negative nbs1 ( 138 ) and wrn ( 111 ) have been found to cause similar increases in the rate of telomere loss with no change in average telomere length .
selectable marker genes adjacent to telomeres have been used to study the consequences of telomere loss in mammalian cells ( 139 ) .
this approach has the advantage of following the changes in individual chromosomes from the initial event , rather than attempting to reconstruct the sequence of events involved in the generation of complex rearrangements .
these marked telomeres contain a herpes simplex virus thymidine kinase ( hsv - tk ) selectable - marker gene to select for loss of the telomere , as well as an 18 bp recognition site for the i - scei endonuclease to introduce dsbs , which has been widely used to study dna repair and recombination in mammalian cells ( 7577 ) . using this system ,
the types of chromosome rearrangements resulting from telomere loss have been followed in both mouse es cells ( 73 ) and the ej-30 human tumor cell line ( 140142 ) . in both
the mouse es cells and ej-30 , telomere loss resulted in either a telomere added directly on to the end of the broken chromosome or inverted repeats resulting from sister chromatid fusion .
while the addition of a new telomere resulted in stabilization of the marker chromosome , sister chromatid fusion was followed by breakage / fusion / bridge ( b / f / b ) cycles and amplification of subtelomeric dna ( figure 3 ) .
these b / f / b cycles occur when the chromosome that has lost a telomere is replicated , and the sister chromatids fuse together at their ends .
the fused sister chromatids then form a bridge that breaks during anaphase when the two centromeres are pulled in opposite directions . following dna replication in the next cell cycle , the sister chromatids fuse once again , and therefore these b / f / b cycles continue until the marker chromosome acquires a new telomere . because neither direct telomere addition nor sister chromatid fusion have been observed at dsbs generated by i - scei at interstitial sites ( 7577 ) , these results suggest that there is something different about the processing of dsbs occurring near telomeres .
consistent with this conclusion , dsbs generated by i - scei are poorly repaired by nhej near telomeres in yeast , and result in complex chromosome rearrangements ( 70 ) . moreover ,
direct telomere addition on to the ends of broken chromosomes in yeast preferentially occurs near existing telomeric repeat sequences ( 143 ) .
one important difference between the mouse es cells and the ej-30 human tumor cell line is that while telomeres in mouse es cells are highly stable ( loss of the hsv - tk gene < 10 events / cell / generation ) , the telomeres in the human ej-30 tumor cell line are lost at a relatively high rate ( 10 events / cell / generation ) .
similar results were observed with other human tumor cell lines ( j. p. murnane , unpublished data ) .
this observation is consistent with other studies demonstrating that cancer cells commonly have telomere instability ( 144,145 ) .
therefore , although chromosomal rearrangements due to telomere loss in cancer cells are commonly thought to result from the extensive chromosome fusion that occurs during crisis ( 146,147 ) , a high rate of telomere loss is often observed even in human tumors and tumor cell lines that express telomerase ( 73,140,144,148,149 ) , suggesting that many tumor cells have a fundamental defect that promotes telomere loss .
another important difference between the mouse es cells and human tumor cell lines is that while the direct addition of a telomere at the site of the break is a common event it mouse es cells ( 74 ) , telomere loss in human tumor cell lines often results in sister chromatid fusion followed by b / f / b cycles ( 140 ) . in addition , while b / f / b cycles last only a few generations in mouse es cells ( 73,74 ) , in ej-30 they can last for many cell generations ( 73,140,141 ) .
this inability to terminate b / f / b cycles is likely to contribute to the chromosome instability resulting from telomere loss in human tumor cells .
the prolonged b / f / b cycles in the ej-30 human tumor cell line results in extensive dna amplification and terminal deletions of dna on the end of the marker chromosome that lost its telomere ( 141 ) .
the fused sister chromatids most often break within 1 mb of the site of fusion , which was confirmed by a subsequent study that found that anaphase bridges formed by sister chromatid fusions most often break near their center , regardless of length ( 150 ) . as a result ,
the region amplified by b / f / b cycles without selection is most often relatively small ( i.e. < 1 mb ) .
however , some breaks also occur far from the site of fusion , resulting in large duplications and deletions ( 73,140,141 ) .
this type of break is involved in the amplification of selectable marker genes located far from the end of the chromosome , and has been shown to occur at the location of fragile sites ( 151,152 ) .
thus , the loss of a telomere can result in the amplification of genes anywhere on the arm of a chromosome .
b / f / b cycles end when the chromosome acquires a telomere and again becomes stable .
the most common mechanism for telomere acquisition in both the mouse es cells and the ej-30 tumor cell line was through translocation of the ends of other chromosomes ( 139,142 ) . in ej-30
these translocations were either non - reciprocal ( nrt ) or involved duplications , as determined by the status of the donor chromosome , i.e. with nrts , one of the homologs of the donor chromosome is missing part or all of an arm and its telomere , whereas with duplications , both homologs are intact . both types of events have important consequences for the genome as a whole .
the translocations involving duplications commonly involve large portions of the arms of other chromosomes , and therefore generate allelic imbalances involving a large number of genes . on the other hand
, nrts result in the loss of a telomere on the donor chromosome , resulting in its instability and the eventual acquisition of a telomere through translocations from other chromosomes .
in fact , in one cell this transfer of instability was found to involve six different chromosomes , demonstrating that the loss of a single telomere can result in instability involving multiple chromosomes .
therefore , in addition to amplification of dna at the end of a chromosome that has lost a telomere , once initiated , b / f / b cycles can result in other rearrangements , not only involving the chromosome that initially lost its telomere , but other chromosomes as well . in view of the fact that b / f
/ b cycles can continue for many cell generations , the loss of even a single telomere can generate a wide variety of chromosomal changes in the cell population . taken together , the above results demonstrate that loss of telomeric function whether due to loss of sequence or loss of structure and the ensuing instability and chromosomal rearrangements , can be expected to have a dramatic impact on the stability of the genome .
therefore , in view of increasing evidence implicating genomic instability in carcinogenesis ( 153,154 ) , telomere loss is likely to be a significant contributing factor .
a role for loss of functional telomeres in the chromosome instability commonly associated with cancer is supported by the large increase in human - like carcinomas in mice deficient in both telomerase and p53 , and the presence of chromosome rearrangements typical of b / f / b cycles in these tumors ( 146,147,155,156 ) .
understanding the mechanisms of telomere maintenance and the various factors that promote telomere instability should therefore provide valuable insights into both human genetic disease and cancer .
strand - specific co - fish detection of leading- ( red ) and lagging- ( green ) strand telomeres demonstrating chromosomal telomere - telomere fusion in a dna - pkcs deficient background , indicative of end - capping failure owing to loss of structure , not loss of sequence .
co - fish detection of chromatid - type telomere fusions in trf2 deficient cells demonstrating preferential failure of end - capping at leading - strand ( red ) telomeres .
b / f / b cycles as a mechanism for chromosome instability resulting from telomere loss .
b / f / b cycles are initiated when sister chromatids fuse following the loss of a telomere .
owing to the presence of two centromeres , the fused sister chromatids break when the cell attempts to divide up its sister chromatids at anaphase . because the break does not occur exactly at the site of the fusion ,
one daughter cell will receive a copy of the chromosome with an inverted repeat at its end , while the other daughter cell will have a copy of the chromosome with a terminal deletion . owing to the lack of a telomere
, these chromosomes will again undergo sister chromatid fusion after dna replication , resulting in additional amplification and terminal deletions .
the location of telomeres ( squares ) , centromeres ( circles ) and orientation of the subtelomeric sequences ( horizontal arrows ) are shown . | telomeres are composed of repetitive g - rich sequence and an abundance of associated proteins that together form a dynamic cap that protects chromosome ends and allows them to be distinguished from deleterious dsbs .
telomere - associated proteins also function to regulate telomerase , the ribonucleoprtotein responsible for addition of the species - specific terminal repeat sequence .
loss of telomere function is an important mechanism for the chromosome instability commonly found in cancer .
dysfunctional telomeres can result either from alterations in the telomere - associated proteins required for end - capping function , or from alterations that promote the gradual or sudden loss of sufficient repeat sequence necessary to maintain proper telomere structure .
regardless of the mechanism , loss of telomere function can result in sister chromatid fusion and prolonged breakage / fusion / bridge ( b / f / b ) cycles , leading to extensive dna amplification and large terminal deletions .
b / f / b cycles terminate primarily when the unstable chromosome acquires a new telomere , most often by translocation of the ends of other chromosomes , thereby providing a mechanism for transfer of instability from one chromosome to another .
thus , the loss of a single telomere can result in on - going instability , affect multiple chromosomes , and generate many of the types of rearrangements commonly associated with human cancer . |
extensive epidemiological investigations have indicated that the incidence of type 1 diabetes mellitus ( t1 dm ) in children and adolescents is increasing steadily year by year and is presumably to be doubled by 2020 [ 15 ] . however , the etiology and pathogenesis of t1 dm are complex and have not been fully elucidated .
additionally , long - term insulin injection and acute and chronic clinical complications such as ketoacidosis , nephropathy , neuropathy , and retinopathy could cause significant physical and mental trauma on the children with this disease .
therefore , exploring the pathogenesis of t1 dm and developing novel intervention strategies have become top priorities in the field of endocrine research .
pancreatic beta cells secrete insulin to regulate blood glucose levels within a relatively narrow range .
t1 dm is an organ - specific autoimmune disease characterized by chronic and progressive apoptotic destruction of pancreatic beta cells , resulting in severe insulin deficiency .
autoimmunity against the beta cells is likely triggered by environmental risk factors that act in genetically susceptible individuals [ 79 ] .
once stimulated , various immune cells , including t or b cells , macrophages , and dendritic cells , infiltrate the islets to induce beta cell death via mechanisms such as reactive oxygen species , perforin / granzymes , fas / fasl , nitrogen species , and proinflammatory cytokines , among which cytokines have been recognized to play key roles in the regulation of autoimmunity and beta cell loss .
prolonged exposure to immune cell - secreted proinflammatory cytokines such as interleukin- ( il- ) 1 , tumor necrosis factor- ( tnf- ) , and interferon- ( ifn- ) is highly cytotoxic to pancreatic beta cells , which impairs insulin secretion and ultimately induces beta cell loss by apoptosis [ 1013 ] . during this inflammatory process
, proinflammatory cytokines affect the expression of many gene networks and modulate pro- and antiapoptotic pathways [ 14 , 15 ] . to date , research has mainly focused on protein - coding genes , though recent studies have shown that protein - coding genes account for less than 2% of the actively transcribed genome , suggesting that the majority of transcripts are noncoding rnas ( ncrnas ) .
ncrnas can be subdivided by length into small ncrnas ( shorter than 200 nucleotides ) and long ncrnas ( lncrnas ) , which are greater than 200 nucleotides in length , with little or no protein - coding capacity . in recent years ,
research in the field of rna biology has mainly focused on small ncrnas , largely ignoring lncrnas . for a long time
, more and more evidence suggests that lncrnas play significant roles in genome regulation , such as in x chromosome inactivation , genomic imprinting , chromatin modification , transcription , splicing , translation , degradation , and transport processes that are involved in the regulation of the growth and development of individual cells in apoptosis , proliferation , differentiation , and other cellular activities [ 17 , 1922 ] .
the dysregulation of these lncrnas has been associated with many human diseases , including different types of cancers , neurodegenerative diseases , and other disorders . with the development of lncrna microarrays , bioinformatics , and high - throughput sequencing , thousands of lncrnas
currently , some studies have examined the roles of lncrnas in diabetes , and the emerging evidence indicates that lncrnas may be involved in maintaining pancreatic beta cell function and insulin signal transduction , which may affect diabetes development . in this study , we examined the expression profiles of lncrnas and mrnas in min6 cells after exposure to a combination of il-1 , tnf- , and ifn- using microarray technology .
we found that the expression levels of many lncrnas and mrnas changed in response to proinflammatory cytokines .
some of the interested lncrnas with remarkable regulation were further verified by qrt - pcr , and the gene ontology ( go ) and pathway analyses were used to analyze the biological roles of these differentially expressed lncrnas and mrnas .
coding - noncoding gene coexpression networks were used to predict the potential interactions between these lncrnas and mrnas .
pancreatic min6 -cells were grown in high - glucose dulbecco 's modified eagle medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , 100 g / ml penicillin , and 50 m -mercaptoethanol at 37c in an atmosphere of 5% co2 .
min6 cells were seeded in 6-well plates to approximately 80% confluence and were then treated with or without 5 ng / ml il-1 , 25 ng / ml tnf- , and 25 ng / ml ifn- for 24 h. total rna was extracted from the min6 cells using trizol reagent ( invitrogen , usa ) and was then purified using an rneasy mini kit ( qiagen , hilden , germany ) according to the manufacturer 's protocol .
an affymetrix genechip mouse transcriptome array 1.0 , which is designed for measuring a broad range of expression changes across the whole mouse transcriptome , was used to profile the ncrnas and protein - coding genes .
the lncrnas were obtained from authoritative databases , including ensembl , refseq , ucsc known genes , vertebrate genome annotation ( vega ) database , mgc mammalian gene collection , mgi , noncode , and others .
the sample preparation and labeling , microarray hybridization , and washing were performed using a genechip wt terminal labeling and controls kit , an ambion wt expression kit , and a genechip hybridization , wash , and stain kit , respectively , according to the manufacturers ' standard protocols .
briefly , 1 g total rna , which was primed with primers containing a t7 promoter sequence , was used to synthesize single - stranded cdna .
then , the single - stranded cdna was transformed into double - stranded cdna , which served as a template to synthesize and amplify the antisense rna ( complimentary rna ) from an in vitro transcription reaction .
rnase h was used to hydrolyze the crna template , leaving single - stranded cdna .
after hydrolysis , the 2nd - cycle single - stranded cdna , which was purified to remove salts , enzymes , and unincorporated dntps , was fragmented and labeled .
the labeled cdnas were hybridized onto the affymetrix genechip mouse transcriptome array 1.0 using a genechip hybridization oven 645 . after washing the hybridized arrays ,
affymetrix expression console software ( version 1.3 ) was used to analyze the microarray data .
the significant differentially expressed lncrnas and mrnas between the two groups were selected if the fold changes of the threshold values were 2.0 or 2.0 ( p0.05 ) .
the three - step robust multichip analysis ( rma ) algorithm , which includes ( 1 ) background adjustment , ( 2 ) quantile normalization , and ( 3 ) summarization , was used to analyze the raw data .
reaction cdna was synthesized using the primescript rt master mix ( perfect real time , takara , japan ) according to the manufacturer 's recommendations .
reverse transcription of lncrnas was performed using the sybr green method in an applied biosystems 7500 system ( life technologies , us ) .
the gene expression levels were quantified based on the cycle threshold ( ct ) values and were normalized to the internal control gene -actin .
go , which describes gene and gene product attributes , contains three key domains named biological process , cellular component , and molecular function .
the kyoto encyclopedia of genes and genomes ( kegg ) database , a bioinformatics resource for deciphering the genome , was used to identify the significant pathways of the targeted genes .
an lncrna - mrna coexpression network , which can identify the interactions between the differentially expressed lncrnas and mrnas , was constructed according to the normalized signal intensities of specifically expressed genes and lncrnas .
the pearson correlation was calculated for each lncrna - gene , gene - gene , or lncrna - lncrna pair , and significant correlation pairs were selected to construct the network .
the random variance model t - test was employed to identify the differentially expressed genes and lncrnas between the control and cytokine - stimulated groups .
an affymetrix genechip mouse transcriptome array 1.0 was designed to profile all mouse lncrnas and protein - coding transcripts . according to the microarray expression profiling data ,
723 differently regulated lncrnas were identified in the cytokine - stimulated group compared with the control group with a set filter fold - change 2.0 , and 2180 differently regulated mrnas were identified with a set filter fold - change 1.5 . additionally , 444 upregulated and 279 downregulated lncrnas were detected and presented in table ( in supplementary material available online at http://dx.doi.org/10.1155/2016/9275106 ) .
the result of the hierarchical clustering shows the lncrna and mrna expression patterns ( figures 1(a ) and 1(b ) ) . to further explore the potential functions of dysregulated lncrna in the cytokine - stimulated group
the length distribution showed that the deregulated lncrnas were mostly less than 500 bp or longer than 5000 bp ( figure 2 ) .
the chromosomal distribution showed that the upregulated lncrnas were mostly located on chromosome 1 and that the downregulated lncrnas were mostly located on chromosome 2 ( figure 3 ) . based on our go analysis ,
the following top 10 enriched go terms were associated with the upregulated lncrnas : ( 1 ) innate immune response , ( 2 ) inflammatory response , ( 3 ) apoptotic process , ( 4 ) positive regulation of i - kappa b kinase / nuclear factor- ( nf- ) kappa b cascade , ( 5 ) defense response to virus , ( 6 ) biological process , ( 7 ) response to virus , ( 8) immune response , ( 9 ) positive regulation of transcription from rna polymerase ii promoter , and ( 10 ) negative regulation of endopeptidase activity ( figure 4(a ) ) .
the following top 10 enriched go terms were associated with the downregulated lncrnas : ( 1 ) transport , ( 2 ) cell cycle , ( 3 ) oxidation - reduction process , ( 4 ) nucleosome assembly , ( 5 ) cell division , ( 6 ) mitosis , ( 7 ) metabolic process , ( 8) biological process , ( 9 ) protein transport , and ( 10 ) ion transport ( figure 4(b ) ) . based on our pathway analysis ,
the following top 10 enriched pathways were associated with the upregulated lncrnas : ( 1 ) cytokine - cytokine receptor interaction , ( 2 ) nf - kappa b signaling pathway , ( 3 ) toll - like receptor signaling pathway , ( 4 ) nod - like receptor signaling pathway , ( 5 ) mapk signaling pathway , ( 6 ) rig - i - like receptor signaling pathway , ( 7 ) apoptosis , ( 8) jak - stat signaling pathway , ( 9 ) chemokine signaling pathway , and ( 10 ) hippo signaling pathway ( figure 5(a ) ) . additionally , the following top 10 enriched pathways were associated with the downregulated lncrnas : ( 1 ) metabolic pathways , ( 2 ) viral carcinogenesis , ( 3 ) protein processing in endoplasmic reticulum , ( 4 ) cell cycle , ( 5 ) n - glycan biosynthesis , ( 6 ) transcriptional misregulation in cancer , ( 7 ) purine metabolism , ( 8) insulin secretion , ( 9 ) biosynthesis of unsaturated fatty acids , and ( 10 ) maturity onset diabetes of the young ( figure 5(b ) ) .
based on the correlations between the differentially expressed lncrnas and mrnas with significant correlation pearson coefficients , we developed a profile between the lncrnas and coding mrnas in the cytokine - stimulated and control groups ( figures 6(a ) and 6(b ) ) .
the coexpression network showed that one lncrna was associated with one to dozens of mrnas and lncrnas . in a network analysis
, degree centrality is used to measure a gene or lncrna centrality within a network , determining its relative importance . from the two networks
, we identified the core regulatory lncrnas and mrnas , which had the largest degrees ( tables 2 and 3 ) .
we randomly selected 15 interested lncrnas to validate the microarray analysis data using qrt - pcr .
the results of the qrt - pcr were largely consistent with the data from the microarray ( figure 7 ) .
during the initial phases of t1 dm , cytokines and other inflammatory mediators released by immune cells progressively infiltrate islets , contributing to the functional suppression and death of beta cells . in nonobese diabetic ( nod )
these observations led to the concept that proinflammatory mediators produced by infiltrating cells play key roles in inducing beta cell death .
accordingly , prolonged exposure to proinflammatory cytokines is highly cytotoxic to pancreatic beta cell function [ 1013 ] and affects the expression of many gene networks [ 14 , 15 ] .
little information exists on the lncrnas that regulate beta cell apoptosis and contribute to t1 dm development [ 2729 ] .
compared with these studies , we use a new and different gene chip , expand the lncrna expression profile , and update the database . here
, we report the lncrna expression profiles of min6 cells that have been exposed to the proinflammatory cytokines il-1 , tnf- , and ifn-. comprehensive analysis shows that lncrnas are generally expressed at lower levels compared with protein - coding genes and are most likely to display tissue - specific expression patterns [ 30 , 31 ] .
the affymetrix genechip mouse transcriptome array 1.0 , which contains both lncrna and mrna probes , was employed to determine the expression profiles of lncrnas and mrnas in min6 cells . in this study
, we identified 723 differently regulated lncrnas and 2180 differently regulated mrnas in the cytokine - stimulated group compared with the control cells , among which 444 upregulated and 279 downregulated lncrnas were detected with a set filter fold - change 2.0 .
qrt - pcr was used to validate some of the microarray analysis data , and the results were consistent with the microarray data .
the mostly upregulated lncrna , nonmmut036704 , was found to exhibit sense overlap with the lipocalin 2 ( lnc2 ) , which is also known as neutrophil gelatinase - associated lipocalin ( ngal ) .
found that il-10 could mediate ngal expression in breast cancer cells , which might contribute to tumor progression .
in addition , ngal could create the local and systemic proinflammatory environment for atherosclerosis by induction of proinflammatory mediators . and ,
ngal was overexpression in subcutaneous adipose tissue in overweight women with gestational diabetes mellitus ( gdm ) , which suggests that it may play a role in the development of insulin resistance in gdm . currently , there are no studies about ngal in the apoptosis of cells .
but in view of its role in the parenchyma cells , we surmise that lncrna nonmmut036704 may play an important role in the development of t1 dm through the regulation of ngal .
this transcript was found to sense overlap cd274 antigen ( cd274 ) , which is also known as programmed death-1 ligand-1 ( pd - l1 ) .
a recent study shows that forced expression of pd-1 in transgenic mice significantly decreased the incidence of autoimmune diabetes .
another study also shows that pd-1 or pd - l1 blockade rapidly precipitated diabetes in prediabetic female nonobese diabetic ( nod ) mice regardless of age .
pd - l1 deficiency could increase susceptibility to diabetes via a direct effect on pathogenic t cells , while regulatory t cells and b cells prevented autoimmune diabetes via a mechanism independent of pd-1/pd - l1 pathway .
therefore , we surmise that lncrnanonmmut034373 may contribute to the development of t1 dm via regulating pd - l1 .
studies have reported that there are susceptible genes to t1 dm in some chromosomes , such as ptpn22 on chromosome 1 , ctla4 on chromosome 2 , ifih1 on chromosome 2 , and il2ra on chromosome 10 [ 3841 ] . in this study , compared with the other chromosomes , chromosome 1 had a higher percentage of upregulated lncrnas , and chromosome 2 had a higher percentage of downregulated lncrnas .
this finding suggests that we may have a higher chance to find the susceptible lncrnas that contribute to beta cell apoptosis on chromosomes 1 and 2 . to evaluate the potential biological functions of the differentially expressed lncrnas , go enrichment and pathway analyses were used to analyze the differentially expressed protein - coding genes associated with these lncrnas . in our existing data
, we found that the main biological processes that were enriched for the dysregulated lncrnas were innate immune response , inflammatory response ,
apoptotic process , and positive regulation of i - kappa b kinase / nf - kappa b cascade .
these findings are in agreement with one current concept that immune - mediated inflammation may serve as a pathogenic mechanism of type-1 diabetes .
the released inflammatory cytokines bind to their receptors on beta cells and could activate the transcription factors p38 , c - jun n - terminal kinase ( jnk ) , signal transducer and activator of transcription-1 ( stat-1 ) , and nf - kappa b and the mitogen - activated protein- ( map- ) kinase signaling pathways , provoking endoplasmic reticulum stress and leading to functional impairment and ultimately apoptosis .
here the pathway analysis showed that the identified lncrnas are mainly involved in the nf - kappa b signaling pathway , toll - like receptor signaling pathway , mapk signaling pathway , and jak - stat signaling pathway , indirectly indicating that these differentially expressed lncrnas play important roles in cytokine - induced islet cell injury .
pathway analysis also showed that the identified lncrnas are involved in the hippo signaling pathway , rig - i - like receptor signaling pathway . the hippo signaling pathway plays critical roles in organ size control and tissue regeneration by inhibiting cell proliferation and promoting apoptosis .
after partial hepatectomy ( ph ) in rats , the hippo signaling pathway is inhibited early during liver regeneration . in addition , inactivation of the hippo pathway or activation of its downstream effector , the yap transcription coactivator , improves cardiac regeneration .
therefore , we assumed that activation of the hippo signaling pathway may induce cells apoptosis and inactivation of the hippo signaling pathway may induce cells proliferation .
rig - i - like receptors are key cytoplasmic pathogen recognition receptors that are involved in the recognition of viruses , including functioning as major sensors of rna viruses , and promoting recognition of some dna viruses .
although there are no evidence showing that rig - i - like receptors are associated with type 1 diabetes , environmental factors such as virus infection have been thought to trigger t1 dm .
therefore , we speculate that activation of rig - i - like receptors may cause innate immune - related inflammatory response to promote cells apoptosis , resulting in t1 dm .
our current study reveals many differentially expressed lncrnas and their related mrnas in cytokine - induced min6 cell apoptosis using a microarray assay .
these differentially expressed lncrnas may play key or partial roles in cytokine - mediated beta cell dysfunction .
understanding the functions of these lncrnas could help us to identify new diagnostic and therapy targets for t1 dm .
thus , further studies to identify the contributions of some interested targets in the pathogenesis of t1 dm based on current study would be important and valuable . | type 1 diabetes mellitus ( t1 dm ) is an organ - specific autoimmune disease characterized by chronic and progressive apoptotic destruction of pancreatic beta cells . during the initial phases of t1 dm
, cytokines and other inflammatory mediators released by immune cells progressively infiltrate islet cells , induce alterations in gene expression , provoke functional impairment , and ultimately lead to apoptosis .
long noncoding rnas ( lncrnas ) are a new important class of pervasive genes that have a variety of biological functions and play key roles in many diseases .
however , whether they have a function in cytokine - induced beta cell apoptosis is still uncertain . in this study , lncrna microarray technology was used to identify the differently expressed lncrnas and mrnas in min6 cells exposed to proinflammatory cytokines .
four hundred forty - four upregulated and 279 downregulated lncrnas were detected with a set filter fold - change 2.0 . to elucidate the potential functions of these lncrnas , gene ontology ( go ) and
pathway analyses were used to evaluate the potential functions of differentially expressed lncrnas . additionally , a lncrna - mrna coexpression network was constructed to predict the interactions between the most strikingly regulated lncrnas and mrnas .
this study may be utilized as a background or reference resource for future functional studies on lncrnas related to the diagnosis and development of new therapies for t1 dm . |
nurses must be aware of and consider implementing novel strategies to ensure that all community members healthcare needs , which are a basic human right , are addressed .
clear data are available on refugees and migrants who take formal routes to gain asylum and residency .
more than 625,000 asylum applications were submitted in the european union in 2014 ( bitoulas , 2015 ) .
the makeup of asylum applicants in 2014 was 1834 years of age ( 53.7% ) , 3563 years of age ( 19.9% ) , 013 years of age ( 18.8% ) , 1417 years of age ( 6.7% ) , 65 years of age and over ( 0.8% ) , and unknown ( 0.1% ) , with the majority being men ( bitoulas , 2015 ) .
irregular migration is defined as the movement of persons to a new place of residence or transit that takes place outside the regulatory norms of the sending , transit and receiving countries , and an irregular migrant is defined as a person who , owing to irregular entry , breach of a condition of entry or the expiry of their legal basis for entering and residing , lacks legal status in a transit or host country
european union member countries have less access to data on irregular migrants and generally base estimates on sanctions against employers for employing irregular migrants ( european migration network , 2011 ) .
this type of data collection does not include irregular migrants who are present without employment or who are employed with employers who have not been sanctioned .
the most recent data estimates on irregular migrants in europe were collected by the clandestino project , commissioned by the european union , which was terminated in 2009 ( clandestino , 2012 ) .
estimates of irregular migrants , both working and nonworking , from data collected from 2007 to 2009 , were between 1.9 million and 3.8 million for the 27 member states of the european union ( eurostat , 2011 ) .
irregular migrants are at higher risk for health impairments because they do not have valid authorization to reside in the country where they live and therefore try to make a living in jobs that are typically dangerous or degrading while experiencing challenges in accessing health care , education , and housing ( european union agency for fundamental rights , 2011 ) .
not all european union states recognize the rights of irregular migrants to claim compensation for accidents in the workplace or provide a means to seek judicial redress when discriminatory or abusive practices occur in the workplace ( european union agency for fundamental rights , 2011 ) .
access to health care for irregular migrants is variable between european union states , and even in states that provide healthcare services there are obstacles of unawareness of entitlements and fear of information exchange between service providers and immigration enforcement authorities ( european union agency for fundamental rights , 2011 ) .
education access for irregular migrant children is complicated by rules that require documentation for enrollment or to receive diplomas ; as education levels increase , access becomes increasingly restrictive ( european union agency for fundamental rights , 2011 ) .
housing is also problematic for irregular migrants who lack appropriate documentation to secure housing or lack sufficient financial resources to maintain a place of residence ( european union agency for fundamental rights , 2011 ) .
international human rights should be applicable to every person , as a consequence of being human , regardless of immigration status ( united nations , 1948 , 1966a , 1966b ) .
the universal declaration of human rights states that everyone is entitled to the rights and freedoms set forth in the declaration , without distinction of any kind , such as race , colour , sex , language , religion , political or other opinion , national or social origin , property , birth , or other status ( united nations , 1948 , article 2 ) .
recommendations for european union member states from sources such as the universal periodic reviews and the european center for disease prevention and control have focused on assurance of access to basic social services for irregular migrants , which includes health care ( european center for disease prevention and control , 2015 ; european union agency for fundamental rights , 2011 ; united nations , 2015 ) .
spain , where this public health project took place , had a unique process within the european union for irregular migrants .
irregular entry was not considered a crime in spain ( european union agency for fundamental rights , 2011 ) .
spain was the only european country that allowed and fostered the registration of irregular migrants through a registration process called padrn , which gave irregular migrants access to free medical care and public education on the same basis as spaniards or regular migrants ( clandestino , 2009 ) .
many south american immigrants , african , and eastern europeans entered the country as false tourists and used irregular migration as part of the process to gain residency or citizenship ( clandestino ) .
irregular migrants in spain could access other basic human needs , in addition to health care , via a broad charitable organization called critas , which is the official charity group of the catholic church in spain .
food , clothing , and other necessary supplies are distributed to those in need through offices located within catholic churches ; the physical address of a person or family corresponds to a specific church or dioceses where the person or family must go to receive services ( critas espaola , 2009a , 2009b ) .
the requirement to have a physical address , verified by a utility bill or government paper , to locate and receive services may be a barrier to irregular migrants who do not have access to stable housing , are not listed on utility bills , or do not receive government papers .
due to an extended economic crisis , new legislation real decretoley 16/2012 , 2012 was passed on april 20 , 2012 , that restricted access of irregular migrants , as well as other sections of the population , to the national healthcare system ( gobierno de espaa , 2012 ) . in response , a nongovernmental organization ( ngo ) , yo s sanidad universal , was formed to denounce and work on the retraction of the law through civil disobedience by accompanying community members , including irregular migrants , who are excluded from healthcare services to seek assistance within the national healthcare system ( yo si sanidad universal , 2015 ) .
the groups of accompaniment are most often organized and enacted with spanish speakers ( european commission , 2012 ) , which can be a barrier for irregular migrants who do not speak spanish .
the right of the independent european states to enforce immigration law may have a negative indirect impact on the ability of migrants to access basic rights in the host country and may discourage irregular migrants from accessing support services from ngos or charity organizations for fear of arrest ( european union agency for fundamental rights , 2011 ) . a common method of policing for irregular migrants within the european union are routine identity checks that are often carried out on routine traffic stops , on public transportation , or in public spaces ( european union agency for fundamental rights , 2011 ) .
identity checks carried out near schools , health centers , or religious buildings have an indirect effect of discouraging migrants from accessing charitable services and can be frightening , humiliating , or traumatic for the juxtaposition of seeking charitable services and being suspect at the same time ( european union agency for fundamental rights , 2011 ) .
irregular migrants live with persistent fear and distrust because they reside in a society that does not recognize them ( skibaking , 2016 ) . enforcing immigration law at points of charitable service or resources for irregular migrants only deepens the fear and distrust while additionally building barriers to receiving fundamental basic rights .
social capital has been linked to health outcomes in public health literature ( islam , merlo , kawachi , lindstrom , & gerdtham , 2006 ; murayama , fujiwara , & kawachi , 2012 ) .
the concept of social capital has continued to evolve to include the nuances of bonding ,
bonding and bridging social capital are both horizontal in nature ; bonding social capital involves trusting relationships between a network of people ( e.g. , family , friends ) who view themselves as similar , while bridging social capital is based on mutual respect and support between group members who may not be alike in social identity or demographic ( e.g. , banks , police ; putnam , 2000 ) . linking social capital is a vertical strategy that links individuals in positions of high social capital with those individuals who lack social capital to open up and provide access to services or relationships that were previously inaccessible ( woolcock , 2001 ) . linking social capital is important in the health of poor communities where access to formal institutions , such as hospitals and clinics , must be based on trust and respect in order to improve access and health outcomes ( szreter & woolcock , 2004 ) . although linking social capital can be led by disadvantaged community members , trusting relationships
are usually successfully formed when those with greater social capital reach out to those with less social capital ( gittell & thompson , 2001 ; noguera , 2001 ) .
concisely , the burden resides with community members who have social capital to both recognize and create trusting relationships with those who lack social capital .
together , the action of high social capital community members initiating and interacting with vulnerable community members embodies the application of linking social capital to create change .
meeting the needs of irregular migrants in the communities in which they live is an essential component of community health .
nurses , through the nurse patient relationship , are well prepared to meet the needs of irregular migrants ( skibaking , 2016 ) .
nurses are prepared academically to be change agents who can enter communities of irregular migrants and work as mediators to provide linking social capital to the unseen members of society through the intentional creation of trusting relationships .
individuals are only able to change their lifestyle habits and improve health if they are able to learn from honest and truthful individuals ( afzali , shahhosseini , & hamzeghardeshi , 2015 ) . linking social capital
must be carefully constructed in order to create feelings of mutual respect and shared goals between group members and minimize the effects of the inherent power differential between those with high social capital and those with low social capital ( szreter & woolcock , 2004 ) .
irregular migrants are outside the formal systems of society and lack social capital , which may impair them from seeking assistance through ngos or charity organizations out of fear of deportment or lack of understanding due to low literacy , low numeracy , or insufficient language skills .
nursing 's role in providing care to society 's most vulnerable members through linking social capital has been clearly demonstrated since lillian wald and mary brewster began the henry street settlement in new york 's lower east side in 1893 and pioneered public health nursing ( yost , 1955 ) . after a random , brief encounter with the living conditions of the immigrants inhabiting the lower east side of new york city ,
wald became an activist to improve the health and wellbeing of those who were on the fringe of society through public health initiatives and advocacy ( wald , 1915/2014 ) .
she noted during her first encounter with a family living in poverty , squalor , and social exclusion that they [ the immigrants ] were not without ideals for the family life , and for society , of which they were so unloved and unlovely a part
she linked the immigrant family 's current condition with a lack of knowledge that could be rectified through education to help them avoid the natural consequences of their ignorance and promote health ( wald , 1915/2014 ) . in order to meet the needs of those at risk , wald and brewster moved into the henry street settlement house in the lower east side to live near and directly attend to the issues of those living there ( wald , 1915/2014 ; yost , 1955 ) .
wald and brewster navigated successfully to link social capital between privileged , educated , highstatus women and immigrants living in squalid conditions to build trust and construct mechanisms to meet community needs , which then improved health for both the immigrant community members and the larger community around them .
an analogous public health nursing approach was employed as the foundational structure to identify , address , and assist a community of irregular migrants attending englishspeaking worship services at a multicultural church in central madrid .
adult males make up 70% of the population , with 30% being women and children .
all irregular migrants attending worship services report being christian ; no data were collected to differentiate between catholic and protestant faiths .
the irregular migrant community socializes routinely amongst themselves before and after church services as well as outside of church services ( bonding social capital ) .
the influential members of their community , such as spiritual leaders and tribal chiefs , interact with the religious leader of the church and the church 's council members ( bridging social capital ) .
interaction between church members of both regular and irregular status occurs during church services , religious education classes , bible study classes , language classes , and a coffee hour after church . through these formal and informal activities ,
the church has become a place for spiritual , social , and emotional support as well as a location to access food and clothing for irregular migrants through critas .
when spanish law banned irregular migrants from healthcare services in the national healthcare system , the church community witnessed the negative effect of limited healthcare access .
irregular migrants are part of the social community of the church , so being unable to access health care posed a threat to not only the irregular migrants health but the church community 's health as well .
therefore , the decision was made to use linking social capital to improve access to healthcare services for irregular migrants to maintain community health .
work for the program began with bridging social capital to identify civic groups for collaboration .
the parish nurse scheduled meetings with local ngos ( e.g. , yo si sanidad universal ) , humanitarian organizations ( e.g. , red cross ) , and charity organizations ( e.g. , critas ) to identify systems already in place .
conversations addressed available services , distribution of services , and perceived barriers that kept targeted populations from accessing services . following the use of bridging social capital at the professional level , bridging social capital was employed again at the church level between the church leaders and the irregular migrant leaders .
gaining entrance into the community and creating a trusting relationship were the main objectives of these meetings .
discussions involved the perceived needs of the irregular migrant community , which services were most valued by the community ( primary , secondary , or tertiary care ) , and perceived barriers in accessing the services .
meetings were continued until an agreed process , location , time , and access point were mutually determined by all parties .
the religious leader of the church , who evokes trust and respect among the irregular migrants , was a significant gate keeper , allowing access to the spiritual and tribal leaders as well as access to a physical location to distribute services .
the bridging social capital meetings informed and directed further meetings to assess strategies and resources to meet the needs of the communities involved .
main themes included ways to address and eliminate or reduce the barriers identified by the service providers and the irregular migrant community .
based on these conversations , an inventory of resources available , the volunteers present , and anticipated financial needs were collected .
a charitable foundation was legally structured , according to local law , to manage the financial and legal issues related to the program .
initial ideas for the program included : ( a ) appointments on sunday mornings at the church , before worship services to lower transportation costs ; ( b ) an appointment sheet posted in a private area where interested parties could sign up for 10min appointments slots ; ( c ) a private office space for appointments with an external entrance to allow private entrance and exit ; ( d ) an office space located next to a restroom for access to water ; ( e ) no financial handouts for medication or supplies would be distributed , but the physical distribution of necessary medication and supplies would be given directly to the client ; and ( f ) intentional and specific collaboration with ngos , humanitarian agencies , and charities would begin on the bridging social capital level and move towards linking social capital as trust and respect increased between the organization and individuals seeking assistance .
the program provides direct assessment for basic health ailments that can be managed on an outpatient basis with immediate distribution of medications or supplies for symptomatic treatment .
additionally , health promotion seminars , which are adapted to the needs of lowliteracy and lownumeracy learners , are created and implemented on a regular basis .
after the seminar , individual bags containing supplies referenced in the seminar and a lowliteracy , lownumeracy teaching sheet are distributed to participants . finally , through linking social capital , the program acts as a triage and referral to link irregular migrants with ngos , humanitarian groups , and charity organizations .
after trust and mutual respect have been cultivated between the nurse and client , the suggestion to access appropriate services and charities is considered safe by the irregular migrant .
as skibaking ( 2016 ) clearly wrote , when nurses listen to the . . .
undocumented migrant , we are establishing caring connections we are creating new stories to flood the community ( p. 325 ) .
culturally , there was a huge divide between the white , educated women providing services and the irregular migrant men seeking services .
a white coat was worn by providers and a desk separated the provider from the client to discuss the issue of concern before the examination .
confidentiality was reinforced at each visit , truth keeping was a requirement , as was honoring the needs of the client before our assessment of needs by listening and seeking understanding .
twenty percent of visits each week are to address chronic conditions or are followup visits .
males make up 64% of the clients ; 76% of the clients are adults , 5% adolescents , and 19% infants and children .
the majority of visits are related to common ailments such as gastrointestinal concerns , respiratory infections , hypertension , and skin infections .
the nature of visits by gender and age are described in figures 2 and 3 , respectively .
irregular migrants are referred to outside resources for more severe health impairments , such as suspected broken bones , abnormal growths , uncontrolled hypertension , severe lacerations , sexually transmitted diseases , unwanted pregnancy , birth control , and psychiatric issues .
a specific process is employed when clients are referred to outside resources because most irregular migrants do not have sufficient language skills to communicate their needs .
additionally , there can be fear and distrust of seeking services beyond the church . to begin
, a note is written at the sixth grade english level and read to the client so they understand what information they are carrying .
then , this information is rewritten in spanish to assist the person receiving the client at the referred location .
finally , an email is sent to the receiving organization to make them aware that a client will be arriving with limited language skills , but carries a letter from our program to help them access the services he or she needs .
this is possible because formal lines of communication were created during the development of the program through bridging social capital .
the last step is to inform the irregular migrant of the exact location of the service provider and specific directions on how to use the public transportation system to arrive at the location at the right time and on the right day .
a followup visit is encouraged with the client after his or her referral to learn more about his or her experience and to provide information to improve the process .
most clients are happy to participate in the improvement process and share positive experiences readily with their community members through bonding social capital .
not all initial ideas worked due to cultural issues . the signup form for appointments was never used .
when irregular migrant community members were asked why the signup sheet did not work , two reasons were given : ( a ) not all members could write their english name clearly , and ( b ) there is a cultural norm of not showing weakness .
the irregular migrants did not want other members of their community to know they were seeking support .
the clients wanted the medications and supplies needed for treatment , but did not want others to see what they were receiving . having a pharmacy bag to put the medications in , for privacy reasons , was very important to the community .
beyond physical health needs , irregular migrant community members actively seek information on secondary health issues such as nutrition , child development and discipline , grief and loss , exercise and healthy behaviors , and clarifying culturally learned health behaviors ( i.e. , questions such as : how long do you have to wait between users of public restrooms to allow the air to change ? ) .
this prompted the need to connect with other health professionals to find appropriate resources and information .
one issue is the hierarchy between men and women ; women will be in the invisible line , but men will jump ahead of them .
the last meetings of the morning are always women and their children . additionally , there have been issues of trying to seek more medication than necessary , seeking medications for afflictions not being experienced , or bringing in an empty medication container and demanding the exact same medication to be distributed .
another challenge is guiding the expectations of the client . educating clients that health impairments are not always resolved with the first medical appointment or first therapy is critical to maintaining a trusting relationship and avoiding skepticism of the program .
correspondingly , followup can be difficult among a transient population , which can affect medication therapies , monitoring health conditions , or knowing if certain procedures were effective .
issues that influence volunteers are : ( a ) a lack of a shared native language between the nurse and client so there can be misunderstandings about what is being expressed ; ( b ) power differential can be uncomfortable when strong personalities interact ; ( c ) power struggles can arise ; ( d ) fairness is culturally assessed and clients can complain about services ; and ( e ) when a service is free there can be feelings of entitlement , which can be demoralizing for the providers .
the irregular migrant community reports feeling supported and is better able to access services it previously did not know were available or were uncertain how to access .
specific examples include : ( a ) two women and one man being successfully treated for sexually transmitted diseases from a foundation specializing in sexual health education ; ( b ) a woman was able to access services to address an unwanted pregnancy and obtain birth control by two distinct foundations providing women 's health services ; ( c ) a man was able to receive appropriate diagnostic testing , treatment , and followup for a cerebral vascular accident related to uncontrolled hypertension from a humanitarian organization .
this individual now has his daily medication purchased and distributed by the program as well as weekly followup to prevent a reoccurrence ; and ( d ) a man with an abdominal tumor was able to access an evaluation and treatment within the national healthcare system through the assistance of an ngo that accompanied him to his doctor visit and assured he was adequately evaluated . in all these cases ,
the irregular migrants were previously unaware of available services , were unable to access the services due insufficient language skills , or did not have sufficient trust to visit these services without the direct and repeated assurance of the program members . linking social capital was integral in helping the irregular migrants know , understand , and access services that are available in the community to meet their healthcare needs . additionally , the parish community is better protected from preventable communicable disease .
health promotion seminars on proper handwashing use and technique help lower the spread of gastrointestinal illnesses .
prompt and appropriate treatment of intestinal worms limits the spread between household members and other community members outside the household .
health promotion seminars on healthy eating are well attended by both irregular migrants and local community members to improve health and wellbeing . through these shared experiences and shared concern for each other , increasing networks of bonding and bridging social capital among church community members reinforce the benefits from providing linking social capital to the irregular migrant community .
this novel strategy of employing wald 's grassroots approach can be recreated by nurses in their local communities to start caring for the unseen .
the universal declaration of human rights ( united nations , 1948 , 1966a , 1966b ) clearly entitles all humans , regardless of status , to basic human rights , which includes health care .
violence , conflict , and extreme poverty continue to force migration globally ; migrants who choose irregular entry are at higher risk for health impairments due to lack of social capital ( european migration network , 2014 ; wermuth , 2003 ) .
nurses have the ability to build positive relationships with irregular migrants based on safe interactions in order to assist them in meeting their healthcare needs ( skibaking , 2016 ) .
the use of linking social capital between nurses and irregular migrants , stemming from a trusting and respectful nurse patient relationship , can improve access and healthcare outcomes for both the irregular migrant community and the larger community as a whole ( szreter & woolcock , 2004 ) .
furthermore , nurseled efforts to meet irregular migrant health needs and scholarly documentation of these efforts directly support the global nursing research priorities set by sigma theta tau international ( 2005 ) which include health promotion and disease prevention as well as advocacy and promotion of health of vulnerable and marginalized communities . | abstractpurposeto describe a novel strategy using linking social capital to provide healthcare access to irregular migrants with low literacy , low numeracy , and limited cultural assimilation in a european metropolitan area.organizing constructpublic data show numerous shortcomings in meeting the healthcare needs of refugees and irregular migrants surging into europe .
many irregular migrants living in european communities are unable to access information , care , or services due to lack of social capital .
an overview of the problem and traditional charity strategies , including their barriers , are briefly described .
a novel strategy using linking social capital to improve healthcare access of irregular migrants is explored and described .
information regarding the impact of this approach on the target population is provided .
the discussion of nursing 's role in employing linking social capital to care for the vulnerable is presented.conclusionsimmigration and refugee data show that issues related to migration will continue .
the novel strategy presented can be implemented by nurses with limited financial and physical resources in small community settings frequented by irregular migrants to improve health care.clinical relevancethe health and wellbeing of irregular migrants has an impact on community health .
nurses must be aware of and consider implementing novel strategies to ensure that all community members healthcare needs , which are a basic human right , are addressed . |
toxic epidermal necrolysis ( ten ) is a rare , life - threatening medical emergency in which there is extensive epidermal sloughing and mucositis .
it is caused by a variety of different drugs ( most commonly sulfonamides , non - steroidal anti - inflammatory drugs , anti - malarials , anticonvulsants and allopurinol ) .
early recognition allows prompt treatment and an immediate transfer to a burns unit or a specialized intensive care setting and better potential for successful recovery .
a 31-year - old caucasian male with a recent diagnosis of human - immunodeficiency virus ( hiv ) was started on co - trimoxazole , 960 mg twice daily for pneumocytis carinii prophylaxis .
he noticed a rash appearing on his hands 5 days after starting the treatment ; however , he continued taking the co - trimoxazole .
he presented to his genito - urinary medicine clinic 7days later with fever , skin pain , malaise , myalgia and an extensive erythematous rash , which covered the trunk and limbs ; he was sent promptly to the medical assessment unit .
a tender pink macular rash covered the majority of the surface of his limbs and trunk , with extensive bullous areas of epidermal sloughing .
there was also a diffuse mucosal ulceration of the mouth and the oropharynx [ figures 1 - 4 ] .
the score for ten ( scorten ) score , on admission was two , with patient scoring for tachycardia and having a detached body surface area of > 10% .
macular rash with bullous formation on anterior aspect of trunk and arms epidermal sloughing on back oral mucosal sloughing macular rash on both legs the offending drug was stopped and the patient was commenced on iv fluids , morphine analgesia and antibiotics .
an urgent dermatology opinion the same evening confirmed the proposed diagnosis of ten secondary to co - trimoxazole .
fluid and electrolyte balance was carefully monitored and he was given high calorie enteral feeds .
extensive debridement of the involved epidermis was followed by grafting with an artificial skin substitute called biobrane .
patient had the skin substitute placed on his trunk and limbs , covering a body surface area of approximately 22% .
the skin substitute stayed on for the next 2 weeks after which it was removed once the skin had healed .
it is important to recognize ten as it requires prompt treatment and immediate transfer to a burns unit or a specialized intensive care setting .
the mortality of ten varies between 30% and 50% , with the primary cause of death being infection and multi - system organ failure .
the risk of developing ten is 1000 times higher in patients with hiv and acquired immune deficiency syndrome than in the normal population .
therefore , in a patient with known hiv and new onset of an erythematous bullous rash after commencing a new treatment , it is important to include ten as a differential .
scorten is a severity - of illness score with which the severity of ten can be assessed by looking at seven independent risk factors .
patient is scored on age , associated malignancy , heart rate , serum urea , detached body surface area , serum bicarbonate and serum glucose .
the clinical features typically associated with ten include a prodrome of 2 - 3 days characterized by fever , cough , sore throat , severe skin pain and general malaise which is then followed by an acute macular rash .
there is a rapidly spreading necrosis of the mucus membranes , which is then followed by similar events in the epidermis .
nikolsky sign , which is characterized by epidermal separation induced by gentle lateral pressure on the skin surface is usually present .
mucus membranes , including conjunctival , pharyngeal , tracheal and esophageal , are also usually affected . as the cutaneous injury in ten is similar to a partial skin thickness
burn , the patient should be admitted to a burns unit , where meticulous wound care can be provided .
an urgent review by the dermatologist on call will usually allow a confident clinical diagnosis .
otherwise , biopsies of the skin or the bulla roof for immediate frozen histological sections can promptly differentiate between ten and staphylococcal scalded skin syndrome . at the time of admission
it is important to ensure good pain relief and a patient controlled analgesia pump may be useful . if large areas of necrotic tissue are present , sharp debridement in the operating room under anesthesia should be carried out .
wet dressings maintain a moist wound environment , speed re - epithelialization , minimize pain and decrease infection rates .
biobrane is a biosynthetic semi - permeable membrane designed to temporarily perform the functions of lost epidermis until re - epithelialization occurs .
it is a bi - layer material where the silicone outer layer acts like a protective epidermal barrier and the inner surface is composed of a three dimensional interwoven nylon filament structure upon which collagen peptides are boarded .
the material bonds firmly to the adequately prepared bed of an appropriate burn ( superficial partial thickness to mid - dermal injury ) until spontaneous detachment by re - epithelialization . in the management of ten
the benefits of high - dose corticosteroids and/or high - dose immunoglobulin infusions within 48 - 72h of onset of bulla formation remain unconfirmed . as blister fluid is rich in pro - inflammatory factors such as interleukins and the blister roof is necrotic , coverage with artificial skin substitutes such as biobrane provide optimal protection until the epidermis completely regenerates .
the use of such dressings on exposed dermis and the sensitive dermal nerve endings avoids frequent dressing change and minimizes pain and discomfort .
the risk of developing ten is a 1000 time higher in a hiv positive patient .
prompt diagnosis of ten avoids a delay in treatment and allows a speedy transfer to a burns unit , where fluid resuscitation , adequate analgesia and meticulous wound care can be provided .
the use of artificial skin substitute can reduce the risk of wound infection , sepsis , improve pain control and provide protection until the epidermis regenerates . | toxic epidermal necrolysis ( ten ) is a rare , but life - threatening medical emergency with significant morbidity and mortality .
current treatment standards for ten patients include stopping all possible drugs associated with the new onset of symptoms , prompt referral and treatment in a specialized center with fluid resuscitation , adequate analgesia and maintenance of nutritional needs .
extensive debridement of the involved epidermis followed by coverage with a skin substitute reduces the mortality from a skin infection and also improves the fluid and electrolyte balance and pain control .
this is increasingly considered an important part of the intensive care of these patients .
admitting physicians should be aware of this rare but life - threatening emergency , to allow prompt diagnosis and avoid delays in treatment . |
postpartum depression ( ppd ) is considered as an important health problem in modern societies .
the prevalence of pnd ranges from 7.6% to 39% in various areas of the world and differs according to the population tested and screening tools used [ 14 ] .
the diagnostic and statistical manual of mental disorders defines ppd as having five or more of the following symptoms for at least two weeks : insomnia / hypersomnia , psychomotor agitation or retardation , fatigue , appetite changes , feelings of hopelessness or guilt , decreased concentration , and suicidality .
risk factors that have been identified are poor marital relationship , prenatal depression , illness of the child , low socioeconomic status , low educational level , unwanted pregnancy , obesity , previous history of postpartum depression , and physical symptoms [ 610 ] .
some risk factors are merely seen in eastern communities such as sex of the infant [ 11 , 12 ] and grand multiparity .
the importance of ppd lies in the fact that it is associated with long - term effects on family and child .
women with ppd tend to discontinue breastfeeding and cognitive development of the child is also shown to be impaired due to insufficient maternal - infant interaction [ 15 , 16 ] .
therefore it is important to identify risk factors for ppd and to diagnose ppd in the early postpartum period to enable an immediate intervention .
it is possible to screen for postpartum depression using the self - administered edinburgh postnatal depression scale ( epds ) which is easy to apply and evaluate .
although it is not a diagnostic tool , a score above 13 is predictive of ppd and its sensitivity and specificity were found as 61.5% and 77.4% , respectively , in a turkish validation study .
complicated labour resulting in an emergency procedure has been identified as a potential risk factor for ppd in some studies but there are conflicting results showing no association with mode of delivery and risk of ppd [ 19 , 20 ] .
the rate of elective cesarean section is rapidly increasing all over the world and it is important to identify whether the mode of delivery has an influence of maternal depression .
turkey 's social structure is a mixture of modern western european and traditional anatolian but family bonds are tight even in urban areas .
women are usually supported by their families and friends during the puerperal period . during pregnancy and the puerperal period ,
women are followed up at primary health care centers on behalf of the ministry of health .
doctors , nurses , and midwives working at these centers are the primary health professionals to contact puerperal women and identify ppd .
this study aims to investigate the risk of postpartum depression after six weeks of delivery according to mode of delivery and to evaluate other related risk factors in order to develop precautions .
the study was carried out at celal bayar university faculty of medicine hospital , department of obstetrics and gynecology , serving as a tertiary care facility .
approval of the local ethical committee and written informed consents from all participants were obtained .
exclusion criteria included multiple pregnancy , conception by assisted reproductive techniques , a previously diagnosed mental illness , antenatally diagnosed fetal anomaly , admission to the intensive care unit ( either maternal or fetal ) , and being a single mother . term delivery defined women who gave birth after the completed 37 weeks of gestational age .
patients were divided into three groups of vaginal delivery , planned cesarean , and emergency cesarean .
a planned cesarean was performed when a woman had elective cesarean due to recurrent cesarean , placenta previa , cephalopelvic disproportion , or presentation anomaly .
an emergency cesarean was performed to all kind of conditions threatening maternal or fetal life such as previously undiagnosed placental anomalies , fetal distress , preeclampsia , eclampsia , or fetal cord prolapse . on presentation to the hospital a form about sociodemographic parameters such as age , education , family type , education of husband , income level , working status , place of antenatal care was filled .
patient characteristics such as type of delivery , number of experienced pregnancies , births , and abortions , planning of current pregnancy , history of a medical condition during pregnancy , history of hyperemesis gravidarum and fear of delivery were recorded . at the sixth week
postpartum visit patients were asked to fill out the edinburgh postpartum depression scale ( epds ) .
the epds is a 10-item self - administered questionnaire developed to screen depressive symptomatology over the past seven days in the postpartum period .
epds consists of 10 items with 4 possible answers and sores from 0 to 3 .
a cut off value of 13 has been used to determine women at risk for depression .
descriptive data analysis for demographic variables were expressed as mean and standard deviation for continuous variables and number ( percentage ) for categorical variables .
subjects were categorised into two groups : epds scores 13 group at risk for depression and epds < 12 group unlikely to be depressed .
categorical data were analysed using pearson chi - square test and the differences between mean values of two groups were determined by parametric tests .
the mean age of the women was 27.74 5.00 years ranging between 18 and 43 . when categorised , most of the patients were under 35 ( 90.3% ) .
the majority of women were housewives ( 80.2% ) , lived in nuclear families ( 83.3% ) and in the city ( 71.4% ) .
the education level of women and husband was secondary education or less and high school or more in 63.8% , 36.2% and 59.4% , 40.6% , resp . ) .
income level was described as low or middle - high by 59.4% and 40.6% , respectively .
most of the patients were receiving antenatal care at the same hospital ( 58.5% ) , one third were sent from community hospitals due to medical indications ( 32.1% ) , and 9.4% had a private doctor . in 43 cases a complication during pregnancy
was recorded ( hypertensive disorder , gestational diabetes , thyroid disorder , urinary system infection , etc . ) .
only 5 of the women ( 1.6% ) declared being unhappy with the baby 's gender , of those 4 had a male baby .
mode of delivery was 33.0% for spontaneous vaginal , 33.6% for elective cesarean , and 33.3% for emergency cesarean .
the indications for women who had a planned cesarean section were : recurrent cesarean ( 21.4% ) , cephalopelvic disproportion ( 6.0% ) , breech presentation ( 2.2% ) , and other ( epilepsy , placenta previa , lumbar hernia , etc . ) .
emergency cesarean was mostly performed for nonprogressive labor ( 8.5% ) , fetal distress ( 7.2% ) , recurrent cesarean presenting with labour ( 6.6% ) , preeclampsia ( 2.8% ) and other ( cephalo pelvic disproportion , vulvar lesion , etc . ) . during the study period none of the women
the mean score for epds was 9.4 6.17 ( min : 0 , max : 28 ) among all women .
a history of hyperemesis gravidarum , depression , and being a housewife were significantly associated with the risk of postpartum depression .
the overall frequency of risk of depression was 31.4% among all the women . when calculated according to mode of delivery , it was found as 27.6% , 31.8% , 34.9% for vaginal , elective cesarean , and emergency cesarean , respectively , with no significant difference between them .
the other variables of sociodemographic parameters did not show a significant effect on postpartum depression scores at sixth week .
being a first time mother had a significant effect on anxiety scores ( p = 0.016 ) similarly did a history of depression ( p = 0.005 ) .
in this study the rate of ppd at six weeks postpartum ( with a cut - off of 13 for the epds ) was found as 31.4% among all the women .
this result is relatively high when compared to those reported in three other turkish studies conducted in manisa , izmir , and erzurum , where the rates were 14.0% , 16.8% , and 14.0% , respectively [ 2224 ] , but similar to the study by dindar and erdogan where the cut - off score was 12 and the prevalence was reported as 25.6% .
these differences may be due to the variations in the application of the tests ( postpartum one or six weeks , one year ) .
there are different results on the prevalence of ppd ranging from 8.4% to 39% [ 24 , 26 ] depending on the time of tests , sample size , design of the study ( prospective versus retrospective ) , cut - off values of epds , and even type of test ( epds ; beck depression inventory , etc . ) .
there was no association between postpartum depression risk and mode of delivery but we found three other varibles affecting mood . these variables were history of depression , history of hyperemesis gravidarum , and being a housewife .
prior history of depression has been found to be an important factor in many studies independent of where it was done . [ 3 , 4 , 24 , 25 , 27 ] and our results are consistent .
women prone to depression should be categorised as high risk patients during and after pregnancy and family physicians as well as obstetricians or midwives should be aware of the increased risk of ppd in this group .
complications during pregnancy , such as hyperemesis or premature contractions , were shown to be risk factors for pnd .
we have also found that a history of hyperemesis gravidarum ( hg ) in the current pregnancy increases scores of epds .
another study from northeastern turkey found history of emesis as a risk factor for ppd .
it is postulated that women who experience hg tend to be more depressive and a recent study shows that mood and anxiety disorders , and personality disturbances are frequently observed among women with hg .
women diagnosed with hg during antenatal care should be followed closely after delivery and screening for ppd should be performed .
other studies from greece and beirut show that ppd was more frequent among unemployed women [ 3 , 4 ] .
returning to social life after childbirth and earning her own money seems to be protective against mood disturbances may it be in a western or eastern community .
being stuck at home with the baby is a depressive factor for women in this study .
it is noteworthy that housewives are at risk of depression both during pregnancy and postpartum . keeping in mind that 61.2% of women are housewives in turkey
, health professionals should be alert in this patient group and provide counselling during antenatal care and after birth .
there are conflicting results about the effect of age on ppd . a study from eastern turkey and the prospective cohort study by sword et al .
specify age less than 25 as an important risk factor for ppd as well as a study by lanes et al . .
breese mccoy et al . however found no difference between age groups in regard to ppd . in the present study
the mean age of our participants was 27.7 and the mean age for turkish women to be a first time mother is 22.3 .
age may play an important role when older aged women conceive by assisted reproduction but in our study we excluded these patients .
the correlation between age and risk of ppd was found as very weak in our study group . in our study ppd
was not associated with level of education which differs from reports from canada and lebanon which indicate a relationship between low educational level and risk of ppd [ 3 , 26 ] .
educational level may be directly associated with household income which in turn may affect the women 's concerns about the costs of the infant .
however in turkey the primary care provider of the family is considered the husband and level of education is not necessarily related to household income .
additionally we have found no increase in ppd in women living in a low income family which also conflicts with the results of lanes et al . and tannous et al . [ 26 , 34 ] .
unplanned pregnancies , number of births or abortions , hospitalisation during pregnancy , and fear about delivery were not associated with ppd in this study .
a study from sweden showed no association between fetal gender and risk of postpartum depression in the mother 6 weeks or 6 months after delivery in fact birth of a baby boy increased the risk of postpartum blues within first 5 days .
studies from china and india report a significant increase in ppd in case of a female baby [ 12 , 20 ] .
a study from eastern turkey showed that even the number of daughters at home were important in developing ppd . in turkey the economic income in the eastern regions
manisa is a western city where gender discrimination is not as usual as the eastern parts of turkey [ 25 , 32 ] and in our study infant gender was not related to ppd . when the women were asked whether they were content with the baby 's sex , 98.4% answered yes .
we have found no increase in ppd in women living in nuclear families , villages , and who were followed up in community hospitals .
several studies have investigated the importance of marital relationships and family interactions but this was out of the scope of our study . in the present study we have found no difference between epds scores and mode of delivery . the risk for ppd
was found as 31.4% among all women , 27.6% , 31.8% , 34.9% for vaginal , elective cesarean and emergency cesarean , respectively .
there is no consensus on the effect of mode of delivery on postnatal mood or depression . in this study patients
were not included if they delivered prematurely , were admitted to intensive care unit ( mother or baby ) , had a diagnosed mental illness or antenatally diagnosed fetal anomaly , multiple pregnancy , conceived by assisted reproductive techniques in order to rule out definitive risk factors for pnd and focus on the effect of mode of delivery .
eisenach et al . showed that acute pain response during vaginal birth increased postpartum depression risk and proposed a careful pain control during delivery .
a study by goecke et al . showed that emergency cesarean lead to low control over delivery and women tended to be more depressive when compared to elective cesarean where the timing of birth is set .
mode of delivery was not independently associated with postpartum depression in a prospective cohort study by sword et al . where young maternal age , maternal hospital admission , non - initiation of breastfeeding , urinary incontinence , multiparity , low social status , and low physical health were predictive variables .
results by durick et al . indicate little cause for concern about the quality of mother - infant interactions following cesarean deliveries . a study that found cesarean to be a risk factor for ppd related the risk to younger age at childbirth .
another study by boyce and todd showed that when compared with women having spontaneous vaginal or forceps deliveries , women having an emergency cesarean section had more than six times the risk of developing postnatal depression at three months postpartum . in beirut ,
a significantly higher proportion of women who delivered vaginally was depressed after delivery compared with those who delivered by caesarean section ( 21% versus 7% ) .
this was explained by the fact that in lebanon the medical system is highly privatised , and women of higher socioeconomic status are more likely to receive caesarean sections and this group was less depressed .
our results of no association between postpartum depression and mode of delivery is consistent with other recent studies [ 2 , 41 , 42 ] .
studies that found a relationship between ppd and cesarean section are mostly conducted in the last two decades .
currently regional anaesthesia is widely used and women are involved in childbirth , immediate breastfeeding , and postoperative pain management after operative delivery and this helps them to bond to their child .
even though some studies reported increased risk of ppd after emergency cesarean [ 19 , 40 ] , we did not find such a result .
this may be due to turkish women 's attitude towards fate and not realising that an emergency delivery means risk for the baby . in this study
we have excluded cases of perinatal death or patients with need for intensive care units for the baby and/or mother because these conditions are causes for depression . by excluding these factors
we have tried to analyse the sole effect of emergency cesarean and have found it as ineffective in creating risk for ppd .
data were collected by a cross - sectional design , and therefore relations between different factors that may contribute to depression can not be determined accurately .
the epds is a screening test and needs diagnostic confirmation by structured or semistructured interview .
thus , it may not be possible to draw accurate conclusions . a prospective study including prebirth mood and longitudinal studies with diagnostic confirmation need to be carried out for this question . the overall prevalence of risk for postpartum depression can be evaluated by field research but we have included only patients who had access to be treated at a university hospital
we observed that mode of delivery had no significant impact on the development of postnatal depression ; however other factors may interact such as history of depression , hyperemesis gravidarum , and being a housewife . | there are various factors related to postpartum depression . in this study
we have aimed to determine the effect of mode of delivery on the risk of postpartum depression .
a total of 318 women who applied for delivery were included in the study .
previously diagnosed fetal anomalies , preterm deliveries , stillbirths , and patients with need of intensive care unit were excluded from the study .
data about the patients were obtained during hospital stay . during the postpartum sixth week visit edinburgh postnatal depression scale ( epds ) was applied .
there was no significant difference between epds scores when compared according to age , education , gravidity , wanting the pregnancy , fear about birth , gender , family type , and income level ( p > 0.05 ) .
those who had experienced emesis during their pregnancy , had a history of depression , and were housewives had significantly higher epds scores ( p < 0.05 ) .
delivering by spontaneous vaginal birth , elective cesarean section , or emergency cesarean section had no effect on epds scores . in conclusion healthcare providers should be aware of postpartum depression risk in nonworking women with a history of emesis and depression and apply the epds to them for early detection of postpartum depression . |
there is no question that obesity is a major health problem for developed countries , and has recently been identified more frequently in children .
it contributes to type 2 diabetes mellitus , arteriosclerosis ( including stroke and coronary artery disease ) , musculoskeletal disorders , and some cancers .
it is also a component of the metabolic syndrome comprised of obesity , elevated plasma lipids , arteriosclerosis , type 2 diabetes mellitus , and fatty liver disease .
further , it contributes to diminished mobility in the elderly and in those with musculoskeletal problems .
consequently , there have been many measures advocated to address the problem , including specific diets , exercise programs , dietary medications , and vitamins .
there has also been increasing attention to measures to alter the dietary habits in children and teenagers .
specific diets have also been advocated to promote general good health , often with little scientific credence or well - documented value demonstrated . before developing a set of recommendations that would be useful to address these issues ,
it is necessary to discuss the nature , physiology , and biochemistry of humans ; an understanding of how we ingest and absorb nutrients ; our ability to use nutrients to warm our bodies and permit locomotion ; the results from excess assimilated fuel and the meaning of energy balance ; the basic food stuffs that we ingest , and for what each is required ; and how we are related to other animals in our physiology and biochemistry . along these lines
finally , discussions of micronutrients , the scientific method to explore efficacy , some nutritional fallacies , and recommendations will be presented .
carbohydrates , proteins , and fats have to undergo digestion of the large molecules into small entities to permit passage through the lining cells into the blood stream .
enzymes are proteins present on the lining of the intestine or secreted into the gut lumen to promote digestion .
fats are converted into fatty acids and glycerol ; proteins into amino acids or small combinations of amino acids ( peptides ) ; and long chain carbohydrates into smaller carbohydrates ( such as starch into glucose ) .
the smaller molecules pass through the intestinal wall more easily and are often facilitated in this passage by carrier mechanisms present on the gut membrane .
there is significant adaptability of the enzymatic and carrier mechanisms to changes in the ingestion of nutrients .
for example , the enzyme responsible for splitting lactose , the sugar in milk , into glucose and galactose , is downregulated , and is in lower concentration , when less lactose ( milk ) is ingested .
in contrast , iron absorption is facilitated across the intestinal membrane when there is too little iron in the blood .
in addition , the avid absorption of most carbohydrates makes some of the claims that certain carbohydrates ( starch ) are preferred to its constituents ( glucose ) incredible .
measured blood glucose is identical following the ingestion of an equivalent weight of glucose or starch .
an understanding of the categories of nutrients is necessary to grasp concepts of energy balance and obesity .
humans evolved from creatures of the sea , necessitating water and the major minerals , sodium ( na ) and chloride ( cl ) . in a sense
the cells of our tissues require the other mineral macronutrients : potassium , bicarbonate , magnesium , calcium , and phosphate .
there are finely tuned mechanisms involving the gastrointestinal tract , the kidneys , the blood , the tissues , and the membranes of cells , assuring homeostasis .
these macronutrients do not contribute to energy balance or obesity , but they are required for survival .
the 3 nutrients contributing to energy balance are proteins , carbohydrates , and lipids ( fats ) .
the amino acids are transported into the blood stream and body organs , especially the liver , for new protein synthesis .
these synthesized proteins provide for our bodies structure in cells , tissues , and organs ; enzymes ; hormones ; and other circulating messengers .
carbohydrates and lipids provide energy for the functioning of the body , including maintenance of heat , movement , and the cellular chemical processes involved in homeostasis .
although there are some dietary carbohydrates and fats that are essential , and can not be synthesized from other nutrients , there is interchangeability among them .
the constituents of carbohydrates and lipids enter various intermediary metabolic processes , mostly in the liver , that allow for this interchange and synthesis of new molecules .
the metabolism of the constituents of carbohydrates and lipids generates energy for the above - enumerated functions .
digested proteins also enter the intermediary metabolic cycles as their constituent amino acids and can produce energy or can be synthesized into carbohydrates ( gluconeogenesis ) or fats .
these 3 moieties have specialized uses , their essential functions , which can not be performed by the other 2 , but they are exchangeable from the standpoint of energy production and fat storage .
the measurement used for this is the calorie which is the amount of heat required to raise the temperature of 1 g of water 1c , usually expressed in kilocalories ( kcals ) or capitalized , calories ( cals ) .
1000 calories equals 1 kcal . the oxidation ( burning ) of fats produce about 9 kcal / g ; proteins and carbohydrates somewhat < 4 .
one can see that eating an amount ( weight ) of fats will allow for over twice the energy production from an equivalent amount of carbohydrates and protein .
in addition , ingestion of fat promotes much more fat storage , when excessive , than equivalent amounts of proteins or carbohydrates .
the interchange among these 3 nutrients by means of cyclical metabolic mechanisms should not surprise us , as nature is replete with cyclical processes , such as the co2 , issued from the respiration of plants and animals , in the photosynthetic production of carbohydrates and oxygen by chlorophyll - containing plants .
another example of a cyclical process is the co2-calcium carbonate cycle in the ocean and land , important in climatology theory .
the interchangeability of our 3 basic food substances allows for thriving humans in polar regions , which might have 90% of their calories provided by fats ( polar bear or seal meat and blubber ) , and many asians with 90% of their calories carbohydrate ( rice ) .
many of the metabolic processes in human cells are also contained in all or most animals .
our earth has undergone many changes that have required adaptable metabolic machinery , and redundant metabolic processes to assist in dealing with the changes .
life is the most enduring characteristic of earth , expressed by living cells , which have enormous capacity to survive in varied environments , through evolutionary change and adaptability .
the need to store metabolic fuel is determined by the metabolic rate of the organism .
the metabolic rate is comprised of the basal energy expenditure ( basal metabolic rate ) and the energy required for any additional physical work .
the basal metabolic rate assures maintenance of our body heat and all of the physical processes involved in life .
there is mounting evidence that there are significant genetic determinations of obesity . in our daily observations
we are a varied and unequal species in many respects , including our body habitus .
although eating rituals may be established through acculturation , it is striking how much parents and their children resemble each other in physical appearance .
further , observations of eating habits of individuals who are obese compared with those who tend to be thin usually show that the obese are much more careful in selecting what and how much they eat than the thin .
the thin seem to be able to maintain thin - ness without involving any conscious control .
a trial of overfeeding prison inmates showed that their metabolism sped up during overfeeding , and allowed for rapid weight loss with the return to a regular diet.2 in contrast , those overfed who were prone to obesity did not shed their weight as easily with dietary restitution .
in addition , biologically identical twin studies show that the tendency to obesity persists in both the twins , regardless whether they are brought up in the same or different households.3,4 recently , these concepts have received more credence in studies implicating a satiety hormonal factor , leptin ; a hunger - stimulating hormone , ghrelin ; and the hypothalamus.2 the upshot of all of these investigations is that obesity may not be under as much conscious control as was originally thought .
it is to stress that those tending to obesity would have to involve more conscious control than those genetically tending to be thin .
the micronutrients are vitamins and minerals that are required for optimal expression of life , but are found in relatively small quantities in the body
fat - soluble vitamins , including vitamins a and d , are stored in the liver and can produce harm in excessive amounts .
water - soluble vitamins , such as vitamin c and the b vitamins are generally harmless , even when ingested in large quantities , as the excess is excreted in the urine .
however , these water - soluble vitamins , especially pyridoxine , can occasionally be detrimental , particularly when used chronically in high amounts or when associated with kidney failure .
calcium , magnesium , potassium , chloride , and sodium are macronutrient minerals , as they are abundant in the body .
obvious examples of harmful , mineral micronutrients include iron , copper , and manganese when ingested excessively .
british sailors in the 17th and 18th centuries developed bleeding gums and skin lesions , ascribed to diminished intake of vitamin c. they were eventually called limeys as the provision of limes , which have an abundance of vitamin c , prevented the development of scurvy when supplied on the ocean - going ships .
one can see the difficulties involved in determining the amount of vitamin c needed to prevent or treat the disease , and translate this into the amount of limes to ingest daily .
ideally , those affected with scurvy could enter a trial involving various daily amounts of vitamin c , and find out the least amount to rectify the problem .
of course , a suitable measurement , such as a bleeding time or a measurement of blood vitamin c , would be necessary to make the judgment objective .
finally , the amount of vitamin c contained in each lime would allow calculation of how much of the rda is contained in each lime .
this calculation would have to take into account the freshness of the limes , as the amount of vitamin c would probably be higher in the fresher limes . from an ethical standpoint ,
the trial could not be done , as it would be inappropriate to give the scorbutic individuals the low doses of vitamin c. similar problems arise in determining the rda for all micronutrients .
one approach to this has utilized blood levels for a micronutrient , relating this to some effect on the body .
one can screen a large , healthy , population and find the average and variance of the micronutrient , then compare this to a group that has some defect , presumably due to a diminished store of the micronutrient .
then a difficult translation to the amount of the micronutrient required to promote restitution of the blood level would be required .
the body has exquisitely controlled mechanisms for the absorption and excretion of nutrients , such as the efficient excretion of excess ( na ) and water - soluble vitamins ; and the variable absorption of iron ( fe ) , dependent upon bodily stores of fe . there may be similar control mechanisms involving all of the micronutrients .
further , most plant and animal cells that we ingest contain micronutrients , although in variable amounts , allowing for micronutrient assimilation from most things that we eat .
it is noteworthy that a baby can live on milk alone for over a year .
presumably , humans could live on eggs alone , which might contain all of the necessary micronutrients .
gluten is a general term for some of the proteins found in wheat , barley , and rye , and is responsible for celiac disease ( cd ) , a relatively common malady , affecting around 1% of those of northern european ancestry , and present worldwide at a lower incidence .
although its most common presentations currently are iron deficiency or nonspecific gastrointestinal complaints , it can present with neurological symptoms , severe diarrhea , weight loss , nutritional deficiencies , menstrual irregularity and infertility , liver abnormalities , and a skin disorder .
it is diagnosed by blood studies and by biopsy of the intestinal epithelium and is cured by the observance of a diet that avoids gluten .
our most common gastrointestinal disturbance is the irritable bowel syndrome ( ibs ) , which may have the same symptoms as cd .
current investigations are underway to see if a subgroup of ibs patients is intolerant to gluten - containing diets , without having cd .
this information has been expanded by many who have promulgated avoidance of gluten for a host of different symptoms with little or no evidence supporting the contentions .
studies of symptom relief from dietary gluten withdrawal in ibs are much more difficult to accomplish successfully when compared with studies in cd . in cd
further , cd is potentially life threatening , making the incentive to accomplish the study higher than in a similar study on ibs patients .
although ibs may be responsible for many symptoms , its improvement can not be gauged easily by objective measurements , and it does not pose a risk of death . when compared with our paradigmatic example of limes , vitamin c , and scurvy , one can realize that a reliable study of gluten withdrawal in ibs would be much more difficult to accomplish .
there are food intolerances in normal individuals , often more prominent in those with ibs .
many of these are idiosyncratic , and do not lend themselves to general therapeutic dicta . although food allergy has been suggested as being responsible for these intolerances , classic allergic mechanisms , presenting clinically with a skin rash or trouble breathing , are relatively uncommon .
it is not uncommon to read in the lay literature concerns about the use of aspartame , an artificial sweetener , much sweeter per amount than glucose , fructose , or sucrose .
some have claimed that allowing too much sweeteners in children has promoted excessive consumption of sweet foods to the exclusion of other more nutritionally replete foods .
the scientific evaluation of this proposal is discussed in the following section , but it should be stressed that aspartame is comprised of a dipeptide of 2 amino acids , aspartic acid , and phenylalanine , which are found in most proteins .
it is nearly completely digested into its component amino acids , although a small amount is absorbed as the dipeptide . there might be rare intolerances to the dipeptide , but the constituent amino acids are as safe as those present in all the proteins that we eat [ an exception is excessive phenylalanine ingestion in patients with the rare congenital illness , phenylketonuria ( pku ) , as phenylalanine is one of the 2 amino acids in aspartame ] . if one wants to do a trial to see if an intervention is therapeutically useful for a disease , the best type of investigation is a randomized , doubly blind trial .
that is one in which members of a population receive the questioned therapy or a placebo on a randomized basis , and neither the patient nor the treating and evaluating health workers know which treatment is given . if the chosen endpoint shows improvement in the condition in the group receiving the therapy versus the placebo , then the therapy is considered of value . in the scurvy example , if a trial of vitamin c is shown to cure scurvy when compared with placebo , the vitamin c is considered therapeutic .
things are more complicated with studies involving limes , as it would be difficult to get a lime placebo
. some studies of specific nutrients have been performed to see if the nutrient has a salutary role .
experimental studies have suggested that vitamins with antioxidant properties , such as vitamin a or vitamin e , might have a binding effect on free radicals , or reactive oxygen species , and might prevent or reverse cancer .
unfortunately , well - controlled randomized , double - blind studies have not shown efficacy .
for example , a scandinavian study of cigarette smokers did not show a reduction in the incidence of lung cancer in those given vitamins a and e.5 in looking at more complex problems , such as obesity , one can see other confounding influences .
if a trial of a high - fat versus a low - fat containing diet is entertained , various things need to be controlled to exclude their influence : the other components of the diets , for example protein content , should be equivalent ; there has to be assurance that the diets are rigorously followed ; a decision has to be made whether to keep the caloric content the same or to allow the subject to eat as much as he wants of the diet ; the amount of physical exercise must be the same for each group ; the 2 groups must be as close to identical as possible , usually achieved statistically by randomization ; and the duration of the study should be long enough to achieve meaningful results .
many of these characteristics can not be achieved in the outpatient setting , and , yet , it is outpatients who will be taking the advice derived from the studies .
most weight - loss studies comparing various diets have not controlled for calories and have shown efficacy in the short term , but not the long term .
this should not surprise us , as weight loss or gain is related to total calories ingested , not to the type of food ( fat , carbohydrate , or protein ) , due to the caloric interchangeability of these nutrients ; and to the amount of physical exercise performed .
perhaps the most popular diet along these lines is the dash ( dietary approaches to stop hypertension ) eating plan.6 this diet contains foods with less sodium chloride , calories , and cholesterol than that of the usual american diet . in addition , an exercise program is included and encouraged . the balance among fats , carbohydrates , and fats is similar to that of the american diet .
one can see the difficulties in doing a study to substantiate that the diet is an improvement over other diets for prolonged weight loss .
one would have to control for salt intake , exercise , and caloric input , and the study would have to be sufficiently long to show prolonged efficacy .
we already know that exercise and caloric restriction promote weight loss ; what has not been demonstrated is efficacy of one diet over another in the long term .
the difficulty in achieving prolonged weight loss in the long term is due to the challenge of prolonged restriction of calories .
as mentioned above , some have advocated avoidance of sweet foods in children as they might condition children to eat less nutritionally rich foods .
note that exclusion of fruits is not included in the proscription , even though many are quite sweet .
one can see the difficulties in doing a well - controlled study of this thesis , randomizing one group of children to have no artificial sweeteners and no sweetened beverages ( except natural fruit juices ) , the other with no restrictions , for a prolonged duration , perhaps a year , and measuring the effect on body mass index ( a measurement of body fat content )
. the most important chore would be the exclusion of sweetened beverages in our society and the onerous task of supervising the restrictions .
the body has many mechanisms in place that allow for varying absorption , synthesis , interconversion , elimination , and retention of nutrients to augment survival under varying circumstances .
it should not be surprising that specific diets to control , modify , or prevent obesity have not shown efficacy over simple caloric restriction , fulfilling scientific scrutiny . as the amounts of micronutrients and macronutrients are present in variable amounts among foods , the homely recommendation that diets should strive to achieve some balance is reasonable .
mother s nagging her youngsters to eat their vegetables sounds like good teaching .
individual food tastes determine much of what we eat , and our tastes vary widely , both among different cultures and as each of us goes through life s stages .
gustatory satisfaction is one of the pleasures of life , and many of our social engagements take place around food and liquid consumption .
programs to modify this behavior will usually fail , especially in the long run . therefore , governmental programs trying to force dietary changes , when there is no strong , scientific evidence supporting the measures , is an unwise restriction of liberty , by limiting the freedom to chose different foods and by the cost for its implementation ( a type of tax , which is a diminution of the freedom to use the money elsewhere ) .
obesity should be considered a characteristic , similar to other differences among members of our species ( height , skin color , eye color , intelligence , sociability , etc . ) and should not be an object of scornful criticism .
children chiding the obese child are similar to other bullying practices , which parents and other adults should abjure .
a thin person who does not have to be careful about what he eats should not be applauded for his thin - ness .
rather the obese person who is able to control his weight through caloric limitation and exercise should be recognized for his noble achievement .
the most effective strategy for weight control is to effect change on the other side of the metabolic equation , exercise .
weight gain is directly related to caloric input and inversely related to metabolic activity and exercise .
it would seem reasonable to have governmental influence on exercise in children , with mandatory gym and recess activities .
it is reasonable for governmental involvement in labeling contents of food that would specify harmful ingredients for specific diseases , such as gluten for cd and phenylalanine for pku .
in addition , it is reasonable to indicate the caloric content of foods , as the information might influence one s behavior in weight control programs .
further , listing na and k content of foods might assist consumers with heart and kidney disorders .
the cost involved in labeling all of the micronutrients for each food item , both for the manufacturer and the monitoring , and , eventually , to the consumer seems excessive .
the guidepost should be whether the labeling of a nutrient content is of proven use in preventing or ameliorating disease .
governmental labeling of nutritious foods , versus non - nutritious foods is not well supported by well - performed , scientific studies .
this should be eschewed , along with labeling sweet foods , those containing fats , or artificial sweeteners , except for aspartame in those with pku .
further , governmental involvement in the amount of na , fats , sugars , starch , etc . that is
unhealthy for the general population , but its healthiness is dependent upon a point of view .
for example , someone subject to heart failure or with failing kidneys might require salt restriction ( na ) , whereas na restriction for the general population might be harmful . listing some carbohydrates as healthy ( those in fruit juices ) , whereas others ( fructose in cola products ) as unhealthy , does not make sense as each carbohydrate eventuates in the same metabolic cycles within the liver , and are metabolically interchangeable .
the food and drug administration ( fda ) does a responsible assessment of drugs , and should be the judge of the safety of artificial sweeteners .
aspartame has been studied quite exhaustively and is deemed safe by the fda , which should be the final arbiter .
any new sweeteners should be subjected to the same scrutiny , and any ongoing information on sweeteners should be within the province of the fda .
claims for excessive use of vitamins , so - called megavitamin therapy , should be evaluated similarly to any drug product assessment , including evidence of risks . in general
the rda listed for vitamins , determined by fda evaluation , and included in multivitamins , is a reasonable guideline for general use .
specialized considerations , such as additional folic acid during pregnancy , or increased vitamin d intake in those shown to be deficient , should also be encouraged .
various dietary programs should undergo the same scientific evaluation that other treatment programs undergo . clearly , it is unjustified for governmental advocation of 1 dietary program over another , when there are no hard data .
simple measures to limit caloric intake , regardless of the source , should be the mainstay . for reasons specified above ; including adaptability , interchangeability among nutrients , and cultural and genetic predispositions ; it is unlikely that 1 specific dietary program will prove useful for weight control over another for the general population .
carbohydrates , proteins , and fats have to undergo digestion of the large molecules into small entities to permit passage through the lining cells into the blood stream .
enzymes are proteins present on the lining of the intestine or secreted into the gut lumen to promote digestion .
fats are converted into fatty acids and glycerol ; proteins into amino acids or small combinations of amino acids ( peptides ) ; and long chain carbohydrates into smaller carbohydrates ( such as starch into glucose ) .
the smaller molecules pass through the intestinal wall more easily and are often facilitated in this passage by carrier mechanisms present on the gut membrane .
the digestive process is extremely efficient . under normal circumstances , almost all of the ingested carbohydrate and protein and 95% of the fat are absorbed .
there is significant adaptability of the enzymatic and carrier mechanisms to changes in the ingestion of nutrients .
for example , the enzyme responsible for splitting lactose , the sugar in milk , into glucose and galactose , is downregulated , and is in lower concentration , when less lactose ( milk ) is ingested .
in contrast , iron absorption is facilitated across the intestinal membrane when there is too little iron in the blood .
in addition , the avid absorption of most carbohydrates makes some of the claims that certain carbohydrates ( starch ) are preferred to its constituents ( glucose ) incredible .
measured blood glucose is identical following the ingestion of an equivalent weight of glucose or starch .
an understanding of the categories of nutrients is necessary to grasp concepts of energy balance and obesity .
humans evolved from creatures of the sea , necessitating water and the major minerals , sodium ( na ) and chloride ( cl ) . in a sense
the cells of our tissues require the other mineral macronutrients : potassium , bicarbonate , magnesium , calcium , and phosphate .
there are finely tuned mechanisms involving the gastrointestinal tract , the kidneys , the blood , the tissues , and the membranes of cells , assuring homeostasis .
these macronutrients do not contribute to energy balance or obesity , but they are required for survival .
the 3 nutrients contributing to energy balance are proteins , carbohydrates , and lipids ( fats ) .
proteins are comprised of strings of constituent amino acids . following digestion , the amino acids are transported into the blood stream and body organs , especially the liver , for new protein synthesis .
these synthesized proteins provide for our bodies structure in cells , tissues , and organs ; enzymes ; hormones ; and other circulating messengers .
carbohydrates and lipids provide energy for the functioning of the body , including maintenance of heat , movement , and the cellular chemical processes involved in homeostasis . although there are some dietary carbohydrates and fats that are essential , and can not be synthesized from other nutrients , there is interchangeability among them .
the constituents of carbohydrates and lipids enter various intermediary metabolic processes , mostly in the liver , that allow for this interchange and synthesis of new molecules .
the metabolism of the constituents of carbohydrates and lipids generates energy for the above - enumerated functions .
digested proteins also enter the intermediary metabolic cycles as their constituent amino acids and can produce energy or can be synthesized into carbohydrates ( gluconeogenesis ) or fats .
these 3 moieties have specialized uses , their essential functions , which can not be performed by the other 2 , but they are exchangeable from the standpoint of energy production and fat storage . however , the amounts of energy generation from the 3 are not identical .
the measurement used for this is the calorie which is the amount of heat required to raise the temperature of 1 g of water 1c , usually expressed in kilocalories ( kcals ) or capitalized , calories ( cals ) .
1000 calories equals 1 kcal . the oxidation ( burning ) of fats produce about 9 kcal / g ; proteins and carbohydrates somewhat < 4 .
one can see that eating an amount ( weight ) of fats will allow for over twice the energy production from an equivalent amount of carbohydrates and protein .
in addition , ingestion of fat promotes much more fat storage , when excessive , than equivalent amounts of proteins or carbohydrates .
the interchange among these 3 nutrients by means of cyclical metabolic mechanisms should not surprise us , as nature is replete with cyclical processes , such as the co2 , issued from the respiration of plants and animals , in the photosynthetic production of carbohydrates and oxygen by chlorophyll - containing plants .
another example of a cyclical process is the co2-calcium carbonate cycle in the ocean and land , important in climatology theory .
the interchangeability of our 3 basic food substances allows for thriving humans in polar regions , which might have 90% of their calories provided by fats ( polar bear or seal meat and blubber ) , and many asians with 90% of their calories carbohydrate ( rice ) .
many of the metabolic processes in human cells are also contained in all or most animals .
our earth has undergone many changes that have required adaptable metabolic machinery , and redundant metabolic processes to assist in dealing with the changes .
life is the most enduring characteristic of earth , expressed by living cells , which have enormous capacity to survive in varied environments , through evolutionary change and adaptability .
the need to store metabolic fuel is determined by the metabolic rate of the organism .
the metabolic rate is comprised of the basal energy expenditure ( basal metabolic rate ) and the energy required for any additional physical work .
the basal metabolic rate assures maintenance of our body heat and all of the physical processes involved in life .
there is mounting evidence that there are significant genetic determinations of obesity . in our daily observations
we are a varied and unequal species in many respects , including our body habitus .
although eating rituals may be established through acculturation , it is striking how much parents and their children resemble each other in physical appearance .
further , observations of eating habits of individuals who are obese compared with those who tend to be thin usually show that the obese are much more careful in selecting what and how much they eat than the thin .
the thin seem to be able to maintain thin - ness without involving any conscious control .
a trial of overfeeding prison inmates showed that their metabolism sped up during overfeeding , and allowed for rapid weight loss with the return to a regular diet.2 in contrast , those overfed who were prone to obesity did not shed their weight as easily with dietary restitution .
in addition , biologically identical twin studies show that the tendency to obesity persists in both the twins , regardless whether they are brought up in the same or different households.3,4 recently , these concepts have received more credence in studies implicating a satiety hormonal factor , leptin ; a hunger - stimulating hormone , ghrelin ; and the hypothalamus.2 the upshot of all of these investigations is that obesity may not be under as much conscious control as was originally thought .
it is to stress that those tending to obesity would have to involve more conscious control than those genetically tending to be thin .
the micronutrients are vitamins and minerals that are required for optimal expression of life , but are found in relatively small quantities in the body . excessive ingestion of micronutrients can be stored or excreted from the body .
fat - soluble vitamins , including vitamins a and d , are stored in the liver and can produce harm in excessive amounts .
water - soluble vitamins , such as vitamin c and the b vitamins are generally harmless , even when ingested in large quantities , as the excess is excreted in the urine .
however , these water - soluble vitamins , especially pyridoxine , can occasionally be detrimental , particularly when used chronically in high amounts or when associated with kidney failure .
calcium , magnesium , potassium , chloride , and sodium are macronutrient minerals , as they are abundant in the body .
obvious examples of harmful , mineral micronutrients include iron , copper , and manganese when ingested excessively .
british sailors in the 17th and 18th centuries developed bleeding gums and skin lesions , ascribed to diminished intake of vitamin c. they were eventually called limeys as the provision of limes , which have an abundance of vitamin c , prevented the development of scurvy when supplied on the ocean - going ships .
one can see the difficulties involved in determining the amount of vitamin c needed to prevent or treat the disease , and translate this into the amount of limes to ingest daily .
ideally , those affected with scurvy could enter a trial involving various daily amounts of vitamin c , and find out the least amount to rectify the problem .
of course , a suitable measurement , such as a bleeding time or a measurement of blood vitamin c , would be necessary to make the judgment objective .
finally , the amount of vitamin c contained in each lime would allow calculation of how much of the rda is contained in each lime .
this calculation would have to take into account the freshness of the limes , as the amount of vitamin c would probably be higher in the fresher limes . from an ethical standpoint ,
the trial could not be done , as it would be inappropriate to give the scorbutic individuals the low doses of vitamin c. similar problems arise in determining the rda for all micronutrients .
one approach to this has utilized blood levels for a micronutrient , relating this to some effect on the body .
one can screen a large , healthy , population and find the average and variance of the micronutrient , then compare this to a group that has some defect , presumably due to a diminished store of the micronutrient .
then a difficult translation to the amount of the micronutrient required to promote restitution of the blood level would be required .
the body has exquisitely controlled mechanisms for the absorption and excretion of nutrients , such as the efficient excretion of excess ( na ) and water - soluble vitamins ; and the variable absorption of iron ( fe ) , dependent upon bodily stores of fe . there may be similar control mechanisms involving all of the micronutrients .
further , most plant and animal cells that we ingest contain micronutrients , although in variable amounts , allowing for micronutrient assimilation from most things that we eat .
it is noteworthy that a baby can live on milk alone for over a year .
presumably , humans could live on eggs alone , which might contain all of the necessary micronutrients .
gluten is a general term for some of the proteins found in wheat , barley , and rye , and is responsible for celiac disease ( cd ) , a relatively common malady , affecting around 1% of those of northern european ancestry , and present worldwide at a lower incidence .
although its most common presentations currently are iron deficiency or nonspecific gastrointestinal complaints , it can present with neurological symptoms , severe diarrhea , weight loss , nutritional deficiencies , menstrual irregularity and infertility , liver abnormalities , and a skin disorder .
it is diagnosed by blood studies and by biopsy of the intestinal epithelium and is cured by the observance of a diet that avoids gluten .
our most common gastrointestinal disturbance is the irritable bowel syndrome ( ibs ) , which may have the same symptoms as cd .
current investigations are underway to see if a subgroup of ibs patients is intolerant to gluten - containing diets , without having cd .
this information has been expanded by many who have promulgated avoidance of gluten for a host of different symptoms with little or no evidence supporting the contentions .
studies of symptom relief from dietary gluten withdrawal in ibs are much more difficult to accomplish successfully when compared with studies in cd . in cd
further , cd is potentially life threatening , making the incentive to accomplish the study higher than in a similar study on ibs patients .
although ibs may be responsible for many symptoms , its improvement can not be gauged easily by objective measurements , and it does not pose a risk of death . when compared with our paradigmatic example of limes , vitamin c , and scurvy , one can realize that a reliable study of gluten withdrawal in ibs would be much more difficult to accomplish .
there are food intolerances in normal individuals , often more prominent in those with ibs .
many of these are idiosyncratic , and do not lend themselves to general therapeutic dicta . although food allergy has been suggested as being responsible for these intolerances , classic allergic mechanisms , presenting clinically with a skin rash or trouble breathing , are relatively uncommon .
it is not uncommon to read in the lay literature concerns about the use of aspartame , an artificial sweetener , much sweeter per amount than glucose , fructose , or sucrose .
some have claimed that allowing too much sweeteners in children has promoted excessive consumption of sweet foods to the exclusion of other more nutritionally replete foods .
the scientific evaluation of this proposal is discussed in the following section , but it should be stressed that aspartame is comprised of a dipeptide of 2 amino acids , aspartic acid , and phenylalanine , which are found in most proteins .
it is nearly completely digested into its component amino acids , although a small amount is absorbed as the dipeptide .
there might be rare intolerances to the dipeptide , but the constituent amino acids are as safe as those present in all the proteins that we eat [ an exception is excessive phenylalanine ingestion in patients with the rare congenital illness , phenylketonuria ( pku ) , as phenylalanine is one of the 2 amino acids in aspartame ] .
if one wants to do a trial to see if an intervention is therapeutically useful for a disease , the best type of investigation is a randomized , doubly blind trial .
that is one in which members of a population receive the questioned therapy or a placebo on a randomized basis , and neither the patient nor the treating and evaluating health workers know which treatment is given . if the chosen endpoint shows improvement in the condition in the group receiving the therapy versus the placebo , then the therapy is considered of value . in the scurvy example , if a trial of vitamin c is shown to cure scurvy when compared with placebo , the vitamin c is considered therapeutic .
things are more complicated with studies involving limes , as it would be difficult to get a lime placebo
. some studies of specific nutrients have been performed to see if the nutrient has a salutary role .
experimental studies have suggested that vitamins with antioxidant properties , such as vitamin a or vitamin e , might have a binding effect on free radicals , or reactive oxygen species , and might prevent or reverse cancer .
unfortunately , well - controlled randomized , double - blind studies have not shown efficacy .
for example , a scandinavian study of cigarette smokers did not show a reduction in the incidence of lung cancer in those given vitamins a and e.5 in looking at more complex problems , such as obesity , one can see other confounding influences .
if a trial of a high - fat versus a low - fat containing diet is entertained , various things need to be controlled to exclude their influence : the other components of the diets , for example protein content , should be equivalent ; there has to be assurance that the diets are rigorously followed ; a decision has to be made whether to keep the caloric content the same or to allow the subject to eat as much as he wants of the diet ; the amount of physical exercise must be the same for each group ; the 2 groups must be as close to identical as possible , usually achieved statistically by randomization ; and the duration of the study should be long enough to achieve meaningful results .
many of these characteristics can not be achieved in the outpatient setting , and , yet , it is outpatients who will be taking the advice derived from the studies .
most weight - loss studies comparing various diets have not controlled for calories and have shown efficacy in the short term , but not the long term .
this should not surprise us , as weight loss or gain is related to total calories ingested , not to the type of food ( fat , carbohydrate , or protein ) , due to the caloric interchangeability of these nutrients ; and to the amount of physical exercise performed .
perhaps the most popular diet along these lines is the dash ( dietary approaches to stop hypertension ) eating plan.6 this diet contains foods with less sodium chloride , calories , and cholesterol than that of the usual american diet .
the balance among fats , carbohydrates , and fats is similar to that of the american diet .
one can see the difficulties in doing a study to substantiate that the diet is an improvement over other diets for prolonged weight loss .
one would have to control for salt intake , exercise , and caloric input , and the study would have to be sufficiently long to show prolonged efficacy .
we already know that exercise and caloric restriction promote weight loss ; what has not been demonstrated is efficacy of one diet over another in the long term .
the difficulty in achieving prolonged weight loss in the long term is due to the challenge of prolonged restriction of calories .
as mentioned above , some have advocated avoidance of sweet foods in children as they might condition children to eat less nutritionally rich foods .
note that exclusion of fruits is not included in the proscription , even though many are quite sweet .
one can see the difficulties in doing a well - controlled study of this thesis , randomizing one group of children to have no artificial sweeteners and no sweetened beverages ( except natural fruit juices ) , the other with no restrictions , for a prolonged duration , perhaps a year , and measuring the effect on body mass index ( a measurement of body fat content ) .
the most important chore would be the exclusion of sweetened beverages in our society and the onerous task of supervising the restrictions .
the body has many mechanisms in place that allow for varying absorption , synthesis , interconversion , elimination , and retention of nutrients to augment survival under varying circumstances .
it should not be surprising that specific diets to control , modify , or prevent obesity have not shown efficacy over simple caloric restriction , fulfilling scientific scrutiny . as the amounts of micronutrients and macronutrients are present in variable amounts among foods , the homely recommendation that diets should strive to achieve some balance is reasonable .
mother s nagging her youngsters to eat their vegetables sounds like good teaching .
individual food tastes determine much of what we eat , and our tastes vary widely , both among different cultures and as each of us goes through life s stages .
gustatory satisfaction is one of the pleasures of life , and many of our social engagements take place around food and liquid consumption .
programs to modify this behavior will usually fail , especially in the long run . therefore , governmental programs trying to force dietary changes , when there is no strong , scientific evidence supporting the measures , is an unwise restriction of liberty , by limiting the freedom to chose different foods and by the cost for its implementation ( a type of tax , which is a diminution of the freedom to use the money elsewhere ) .
obesity should be considered a characteristic , similar to other differences among members of our species ( height , skin color , eye color , intelligence , sociability , etc . ) and should not be an object of scornful criticism .
children chiding the obese child are similar to other bullying practices , which parents and other adults should abjure .
a thin person who does not have to be careful about what he eats should not be applauded for his thin - ness .
rather the obese person who is able to control his weight through caloric limitation and exercise should be recognized for his noble achievement .
the most effective strategy for weight control is to effect change on the other side of the metabolic equation , exercise .
weight gain is directly related to caloric input and inversely related to metabolic activity and exercise .
it would seem reasonable to have governmental influence on exercise in children , with mandatory gym and recess activities .
it is reasonable for governmental involvement in labeling contents of food that would specify harmful ingredients for specific diseases , such as gluten for cd and phenylalanine for pku .
in addition , it is reasonable to indicate the caloric content of foods , as the information might influence one s behavior in weight control programs .
further , listing na and k content of foods might assist consumers with heart and kidney disorders .
the cost involved in labeling all of the micronutrients for each food item , both for the manufacturer and the monitoring , and , eventually , to the consumer seems excessive .
the guidepost should be whether the labeling of a nutrient content is of proven use in preventing or ameliorating disease .
governmental labeling of nutritious foods , versus non - nutritious foods is not well supported by well - performed , scientific studies .
this should be eschewed , along with labeling sweet foods , those containing fats , or artificial sweeteners , except for aspartame in those with pku .
further , governmental involvement in the amount of na , fats , sugars , starch , etc . that is
unhealthy for the general population , but its healthiness is dependent upon a point of view .
for example , someone subject to heart failure or with failing kidneys might require salt restriction ( na ) , whereas na restriction for the general population might be harmful . listing some carbohydrates as healthy ( those in fruit juices ) , whereas others ( fructose in cola products ) as unhealthy , does not make sense as each carbohydrate eventuates in the same metabolic cycles within the liver , and are metabolically interchangeable .
the food and drug administration ( fda ) does a responsible assessment of drugs , and should be the judge of the safety of artificial sweeteners .
aspartame has been studied quite exhaustively and is deemed safe by the fda , which should be the final arbiter .
any new sweeteners should be subjected to the same scrutiny , and any ongoing information on sweeteners should be within the province of the fda .
claims for excessive use of vitamins , so - called megavitamin therapy , should be evaluated similarly to any drug product assessment , including evidence of risks . in general
the rda listed for vitamins , determined by fda evaluation , and included in multivitamins , is a reasonable guideline for general use .
specialized considerations , such as additional folic acid during pregnancy , or increased vitamin d intake in those shown to be deficient , should also be encouraged .
clearly , it is unjustified for governmental advocation of 1 dietary program over another , when there are no hard data . simple measures to limit caloric intake , regardless of the source , should be the mainstay . for reasons specified above ; including adaptability , interchangeability among nutrients , and cultural and genetic predispositions ; it is unlikely that 1 specific dietary program will prove useful for weight control over another for the general population . | the views expressed are those of the author and may not necessarily reflect the views of the editorial board.abstract : this essay is an opinion article addressed to the busy practitioner .
it provides information on nutrition , diet , nutritional science , and obesity to serve as a reference in teaching his patients on these issues .
it is composed by a gastroenterologist who has been engaged in clinical gastroenterology and nutrition , research , and teaching in an academic medical center for 35 years .
it also relates the information to conclusions on reasonable involvement of the national government in these topics .
finally , its audience might include the interested , well - educated , lay public .
hence , excessive scientific parlance and referencing have been avoided . |
cardiovascular diseases ( cvds ) are currently the major cause of mortality and morbidity around the world . among the cvds , coronary artery disease ( cad )
coronary angiography ( cag ) is the best tool and gold standard for diagnosis of cad .
cag is an invasive procedure which is routinely used for the assessment and diagnosis of cad .
cag is the insertion of a catheter to the heart by puncturing the groin site via the femoral artery . in this procedure ,
dye is injected and the extent and severity of stenosis of the coronary arteries are assessed .
review of literature and 5 years of clinical and research experience of the first author on working with these patients showed that most of those candidates for cag are unaware of this procedure .
identifying the expectations of cag patients could help the patients to have a better encounter with this procedure , as well as more satisfaction and lesser hospital stay . however , there is no published research on patients expectations about cag in iran . with such understanding of patients expectations related to cag
furthermore , identifying the patients expectations will help nurses and physicians to provide information for patients based on their needs .
the review of literature showed that some research has been conducted quantitatively , measuring the effectiveness of interventions on the cag patients outcomes .
the most common studies have attempted to find out the effectiveness of video information and changing patients position on patients outcomes such as anxiety , stress , back pain , fatigue , satisfaction , and recall .
several studies conducted qualitatively in this area are limited to fear and perspectives of patients regarding cag .
( 2007 ) found that the fear of patients arouse from lack of control on physical and psychosocial aspects of cag , as well as unknown prognosis and possible medical complications . a qualitative study carried out by beckerman et al .
( 1995 ) also showed that patients undergoing cag have feelings of loss of control of physical and personal self .
review of literature indicated that the previous studies have focused on only one aspect of cag and there is no holistic study on patients expectations about cag . regarding the lack of research on this topic , particularly with qualitative approach in iran , this study aimed at identifying and exploring patients expectations about cag .
this qualitative study was a part of a larger study which aimed to explore the patients expectations about cag .
this approach was conducted to reach a deep understanding of the patients expectations related to cag .
the patients aged > 25 years were recruited from three hospitals ( nemazi , faghihi , and kowsar ) affiliated to shiraz university of medical sciences , shiraz , southwestern iran .
we recruited patients using purposive sampling according to maximum variation approach from a wide age range ( 25 - 75 years ) , different genders , different cultures and socioeconomic status .
all patients who were candidate for cag were at first eligible to participate . in total ,
15 patients ( 7 men and 8 women ) participated in the study . for data collection ,
all the interviews were conducted by the first author between august 2011 and april 2012 , 1 day before and 1 day after cag .
after the interview with 12 patients , the researchers reached saturation . to complete and refine the coding framework ,
three more interviews with patients were conducted . in sum , final saturation occurred with interviewing 15 patients .
data were analyzed using conventional qualitative content analysis . in this procedure , each transcribed interview was read sentence by sentence and the text was broken up in units of meaning ; then , the phrases with the same meaning and content were labeled and categorized together .
this was refined and analysis continued until a coding framework was developed . in the next step , based on similarity and content , the subcategories were used to make the main categories .
the categories were then formulated into four themes . to ensure trustworthiness of data , several approaches were addressed . after coding of data by the researcher ,
furthermore , field notes were taken by the researcher to have a deep understanding of the patients expectations .
the aim of the study was explained to the patients and their written informed consent was obtained according to the declaration of helsinki .
furthermore , it was explained that the patients could withdraw from the study at any time .
in this study , 15 patients ( 7 men and 8 women ) with a mean age of 49.8 11.6 ( 28 - 70 years ) participated .
based on the results obtained from analysis of the interviews with patients undergoing angiography of coronary arteries regarding their expectations , four main categories were formed including expectations from angiography , expectations from the treatment team , and need for preparation for angiography , education , and training .
all participants stated that they had agreed to undergo angiography to improve their cardiac condition and reduce their pain .
for instance , some of the statements of the participants are as follows : at nights , my heart aches a little .
i said to myself maybe it would get better after angiography because the doctor said you have stenosis inside your cardiac vessels .
i thought it would be cured after angiography ( 47 years old , male ) .
not only the doctor , but anybody who knew i had this pain said you have to do an angiography , so we did it
i said let 's giv atient is in the hands of god and then teit a try and god 's willing , it might completely be treated ( 49 years old , female ) .
at first , the doctor said go and get an ekg , so i got it then he said get an echo , so i got that too then he said i ll give you some medication , if you get better then you re fine , if not come for an angiography
so , i used the medication and in fact got better somehow then it was back all day long , but now i only have a little pain at night
i said let 's do an angiography , the pain might go away altogether .
the next classification obtained through interviewing the patients is their expectations from the treatment team ( physicians and nurses ) .
this category consists of subcategories such as mental support , comprehensive care , and skillfulness .
all the participants in this study expected sympathy and mental support from nurses and physicians because of the invasive and stressful nature of angiography . in this
regard , one of the patients stated that : now that it 's over , we still expect the doctors and nurses to explain and sympathize with us and assure us that its nothing and our condition will get better and we ll soon be discharged .
we want them to say we ll get better in no time and it 's just a matter of 24 hours .
i guess it would be better if they sympathized with patients ( 50 years old , male ) .
their most important expectations from health professionals were mental support before and during the procedure .
besides mental support , the participants expected the treatment team , especially the nurses , to take care of them physically after angiography .
the participants perceived the presence of their clinical nurse as a factor for feeling safe and reassured .
when you feel that there is someone who supports you and consistently takes care of you , you re not scared anymore .
but if you feel that the person is passive and visits you once an hour , you feel stressed out ( 34 years old , male ) . here
, the patients stated that they need to be reassured and supported by health professionals , especially nurses .
another concept in this category raised by the participants was the physician 's skillfulness during the procedure of angiography .
the patients stated that they expect their doctor to be experienced and skillful . consequently , their anxiety would reduce and they would accept to undergo angiography . in this regard ,
i say , first god then the doctors and nurses to do whatever they can
i expect the doctor to be good at the patient is in the hands of god and then the doctors ( 53 years old , female ) .
some patients go to , for example , hospital x and say the doctors are young , they are students or they are inexperienced .
although i do n't believe in them , they say so ( 49 years old , female ) .
some of the participants believed that preparing the patient by the treatment team and giving simple explanations to the patient leads to his / her higher comfort and acceptance .
in this regard , some of the statements of the participants are as follows :
i wouldve asked people about it and i would have been more prepared as a patient who is unaware , i expect the doctors to explain about the procedure beforehand so i would be more relaxed ( 59 years old , male ) .
i think it would be more effective if they gave us a brochure or something , or for example talk about the pain and say it does n't hurt its like getting a shot
they should say what they want to do is really simple ( 47 years old , male ) .
accordingly , it can be concluded that the patients expect to be prepared for cag by educational programs and they need to be made fully aware of the procedure by health professionals .
one of the main and most emphasized categories in the patients statements was the need for training which consists of various aspects such as training regarding the angiography technique and training about its possible side effects .
all the participants emphasized the need for training and believed that training regarding the method of angiography and its related side effects can lead to better acceptance and decision making in the patient .
moreover , the patient 's stress and anxiety ( because of unawareness ) is reduced . in this regard ,
some of the statements of the participants are as follows : as the patient is undergoing angiography , they should explain how it is , and that there 's no problem , say it 's really good .
i mean completely clarify and say angiography is like this and that there would be no fear .
they explain thoroughly that there is nothing to be afraid of because fear leads to more pain and worry it 's this stress and worry that is important for the heart , right ( 28 years old , female ) . i think if someone explains step by step to the patient 5 minutes before angiography and say what they want to do , how long it lasts , what will happen afterwards , to the end , then explain it to the patient , the fear would be less ( 32 years old , female ) .
patients also need to be trained and made aware of the stages of cag as well as recovery period and the possible complications .
patients believed that training by nurses and physicians can cause better toleration and low fear related to this procedure .
we found that most participants expected full recovery after angiography . all participants stated that they had agreed to undergo angiography to improve their cardiac condition and reduce their pain .
for instance , some of the statements of the participants are as follows : at nights , my heart aches a little .
i said to myself maybe it would get better after angiography because the doctor said you have stenosis inside your cardiac vessels .
i thought it would be cured after angiography ( 47 years old , male ) .
not only the doctor , but anybody who knew i had this pain said you have to do an angiography , so we did it
i said let 's giv atient is in the hands of god and then teit a try and god 's willing , it might completely be treated ( 49 years old , female ) .
at first , the doctor said go and get an ekg , so i got it then he said get an echo , so i got that too then he said i ll give you some medication , if you get better then you re fine , if not come for an angiography
so , i used the medication and in fact got better somehow then it was back all day long , but now i only have a little pain at night
i said let 's do an angiography , the pain might go away altogether .
the next classification obtained through interviewing the patients is their expectations from the treatment team ( physicians and nurses ) .
this category consists of subcategories such as mental support , comprehensive care , and skillfulness .
all the participants in this study expected sympathy and mental support from nurses and physicians because of the invasive and stressful nature of angiography . in this
regard , one of the patients stated that : now that it 's over , we still expect the doctors and nurses to explain and sympathize with us and assure us that its nothing and our condition will get better and we ll soon be discharged .
we want them to say we ll get better in no time and it 's just a matter of 24 hours .
i guess it would be better if they sympathized with patients ( 50 years old , male ) .
their most important expectations from health professionals were mental support before and during the procedure .
then , they would like to be cared physically especially after the procedure . besides mental support , the participants expected the treatment team , especially the nurses , to take care of them physically after angiography .
the participants perceived the presence of their clinical nurse as a factor for feeling safe and reassured .
when you feel that there is someone who supports you and consistently takes care of you , you re not scared anymore .
but if you feel that the person is passive and visits you once an hour , you feel stressed out ( 34 years old , male ) . here
, the patients stated that they need to be reassured and supported by health professionals , especially nurses .
another concept in this category raised by the participants was the physician 's skillfulness during the procedure of angiography .
consequently , their anxiety would reduce and they would accept to undergo angiography . in this regard ,
i say , first god then the doctors and nurses to do whatever they can
i expect the doctor to be good at the patient is in the hands of god and then the doctors ( 53 years old , female ) .
some patients go to , for example , hospital x and say the doctors are young , they are students or they are inexperienced .
although i do n't believe in them , they say so ( 49 years old , female ) .
another main category regarding the patients expectations is the need for preparation for angiography . some of the participants believed that preparing the patient by the treatment team and giving simple explanations to the patient leads to his / her higher comfort and acceptance . in this regard ,
i wouldve asked people about it and i would have been more prepared as a patient who is unaware , i expect the doctors to explain about the procedure beforehand so i would be more relaxed ( 59 years old , male ) .
i think it would be more effective if they gave us a brochure or something , or for example talk about the pain and say it does n't hurt its like getting a shot
they should say what they want to do is really simple ( 47 years old , male ) .
accordingly , it can be concluded that the patients expect to be prepared for cag by educational programs and they need to be made fully aware of the procedure by health professionals .
one of the main and most emphasized categories in the patients statements was the need for training which consists of various aspects such as training regarding the angiography technique and training about its possible side effects .
all the participants emphasized the need for training and believed that training regarding the method of angiography and its related side effects can lead to better acceptance and decision making in the patient .
moreover , the patient 's stress and anxiety ( because of unawareness ) is reduced . in this regard ,
some of the statements of the participants are as follows : as the patient is undergoing angiography , they should explain how it is , and that there 's no problem , say it 's really good .
i mean completely clarify and say angiography is like this and that there would be no fear .
they explain thoroughly that there is nothing to be afraid of because fear leads to more pain and worry it 's this stress and worry that is important for the heart , right ( 28 years old , female ) . i think if someone explains step by step to the patient 5 minutes before angiography and say what they want to do , how long it lasts , what will happen afterwards , to the end , then explain it to the patient , the fear would be less ( 32 years old , female ) .
patients also need to be trained and made aware of the stages of cag as well as recovery period and the possible complications .
patients believed that training by nurses and physicians can cause better toleration and low fear related to this procedure .
the results obtained from this study present a new insight into the expectations of patients undergoing cag .
the identification and understanding of these expectations is highly important for all the members of the treatment team in order to present better and more effective care to patients .
moreover , many patients stated that they had accepted to undergo angiography in hope of complete recovery and treatment .
( 2007 ) , in a study on patients undergoing coronary catheterization , found that most of the patients believed that catheterization would cure their condition .
most of the patients in their study were not completely aware of the nature of cag until the time of referral to the hospital .
beckerman et al . ( 1995 ) also showed that the candidates for this procedure had wrong beliefs about the function of cag .
( 2011 ) found that most patients did not have sufficient information about cag , and this issue resulted in having wrong beliefs about the procedure . in a clinical trial ,
2008 ) also found that patients undergoing cag have little information about the procedure , which in turn increased their anxiety and reduced their satisfaction .
2009 ) found an inconsistency between the patients expectations and the reality of angioplasty and stated that the main reason for this inconsistency was lack of appropriate training by the medical staff .
our results were consistent with those of the previously mentioned studies which confirmed that these patients do not have adequate and correct knowledge about cag .
incorrect understanding and lack of consistency between the patients expectation and the reality ultimately leads to the patients psychological problems .
the participants in our study expected the nurses , as members of the treatment team , to support them mentally and physically .
( 2009 ) also found that the nurses consistent physical and mental care leads to feelings of calmness and security in patients .
mental support and physical care can lead to tranquility and better adaptability in patients , especially those who undergo invasive procedures .
moreover , besides mental and physical care , the patients in our study stated that the active presence of nurses was an important factor in reducing their stress and anxiety .
( 2010 ) stated that one of the most important aspects of care was a caring presence .
( 2011 ) showed that the physical presence of nurses for performing routine tasks alone can not be adequate for responding to the patients needs ; the nurses should pay attention to the patients psycho - emotional needs as well .
( 2011 ) also claim that the presence of nurses next to patients leads to feelings of assurance and security .
evidence shows that this presence has many positive effects on patients , such as mental peace , fear and stress reduction , assurance , and security .
besides expectations from nurses , the patients in our study also expected their physicians to have sufficient experience and skill for performing angiography .
( 2007 ) stated that the physician 's skill was one of the most important aspects of the patients expectations in coronary catheterization .
( 1995 ) found that the patients belief in the physician 's skill and experience in performing catheterization facilitated their acceptance and reduced the physiological problems .
( 2009 ) also found that patients expect physicians to be experienced and skillful in performing invasive procedures .
our findings confirm the need for preparing patients for angiography and sufficient training about this technique and its consequences as one of the patients most important expectations .
all the participants in our study confirmed the need for sufficient training about the technique and procedure of angiography .
1995 ) also stated that those patients who undergo coronary catheterization need to be trained about the technique before , during , and after the procedure .
2009 ) confirmed that patients need sufficient training by the medical team before coronary angioplasty in order to be more prepared and aware . in a study by astin et al .
( 2009 ) , the patients stated that the need for preparation and training about the technique and its side effects was one of their most important informational needs .
( 2001 ) found that gaining information and knowledge about invasive procedures leads to better adaptation to and confrontation with such procedures .
( 2009 ) found that informational needs before the procedure are highly important expectations of patients .
the new and probably different findings of this study were compared with those of other studies on expectation of recovery and treatment by cag .
it was revealed that there is a mismatch between reality and expectations of these patients , leading to many problems for them . to alleviate such problems
, there is a need for identifying patients expectations and then instructing them and providing them with some information about diagnostic cag . in brief ,
our findings highlighted many aspects of patient 's expectations related to cag and gave a new insight for nurses and physicians to help these patients have better experience related to this procedure .
therefore , we can not generalize the findings to other qualitative studies and to all the patients undergoing cag .
furthermore , as all of the patients in this study were muslims , there is a need to conduct a study on what are the expectations of other patients with different religious and cultural backgrounds about cag .
we aimed to evaluate the expectations of patients undergoing cag . considering the increasing prevalence of cad and , in turn , cag , for its diagnosis
, it is important for nurses and the medical team to pay attention to the patients expectations in order to provide better care .
we found that patients undergoing cag have diverse expectations from the angiography itself and the treatment team .
nurses , as the key members of medical teams , should include these expectations in their routine care program and assist the patients to have better adaptability .
sensitivity to the patients expectations and assisting them result in higher awareness and less psychological problems in patients . | background : coronary angiography is recognized as the gold test for diagnosis of coronary artery diseases . based on the literature review ,
little is known about patients expectations about this procedure .
understanding the patient 's expectations is an important way to achieve patient - centered care .
the purpose of this study was to explore the expectations of iranian patients undergoing coronary angiography.materials and methods : this descriptive exploratory qualitative study was carried out between 2011 and 2012 in three hospitals in shiraz , iran , with a purposive sample of 15 patients ( 7 men and 8 women , about 28 - 70 years of age ) .
data were collected using semi - structured interviews to explore the patients expectations .
interview data were analyzed using conventional qualitative content analysis approach.results:the interviews revealed that the participants had four main categories of expectations about coronary angiography .
the patient 's expectations were those from angiography , from the treatment team , and the need for preparation of angiography , education , and training.conclusions:in general , the results of this study revealed a new insight into the expectations of patients undergoing coronary angiography .
patient education and preparation should include information addressing the expectations .
moreover , an appropriate care program based on the patients expectations can promote the quality of care and satisfaction of patients . |
the nasopalatine canal ( npc ) originates from the incisive papilla in the oral cavity and opens in the floor of the nasal cavity .
the canal shape is classified based on different indices in the sagittal plane . a study by mardinger et al .
classified the canal shape into cylindrical , funnel , hourglass and banana shapes in sagittal view .
the canal contains the nasopalatine neurovascular bundle and is positioned in the midline of the palate at the rear of central incisors and approximately at the junction of palatine midline and incisive sutures . in panoramic images , the canal is usually observed between the roots of the central teeth going from the middle up to apical third levels .
the location of the foramen is also variable and it can be seen going from the apical portion of central incisors up to the adjacent alveolar crest . evaluating the size and morphology of the npc before surgical procedures such as implant surgery is very important .
knowledge of the position and the content of the canal during surgical implant insertion procedure are also quite critical .
the contact of the implant with the npc may disturb implant osseointegration or cause sensory disturbance .
there are several radio - anatomical studies on cone - beam computed tomography ( cbct ) images of iranian population .
some of them focused on canals and foramina that are available in jaw bones and are related to field of dentistry .
anatomical variations in the morphology and the diameter of npc , the lack of sufficient data about this landmark in different populations , the increasing need for implant surgery and rehabilitation of edentulous areas of the maxilla as well as the absence of a similar radio - anatomical study in an iranian population were the reasons to design this investigation to assess this anatomic structure in an iranian population .
thus , the purpose of this study was to determine the form , height and diameter of the npc using cbct in an iranian population .
in this cross - sectional study , volumetric cbct images of 198 patients ( mean age ; 46.5 13.8 years ) who had been referred to a private maxillofacial radiology in rasht , guilan during 1 year ( 2011 - 2012 ) for various reasons such as implant placement or evaluation of maxillary sinuses were chosen .
demographic information about the age , gender and pattern of dentations in the anterior maxillary area as being either dentate or edentulous were recorded .
moreover , we obtained written consent of the participating patients to use their volumetric data derived from cbct images for this study . a maxillofacial radiologist with more than 10 years of experience evaluated the form of the foramen on the palatal ( oral ) side of the npc as seen in axial images ( 1 mm thickness and interval ) as having either a single foramen or more [ figure 1 ] .
y - shaped nasopalatine canal and the method of measurement of the height and the diameter of the canal in crosssectional cone - beam computed tomography images in cross - sectional images , the shape of the canal ( hourglass , funnel , conical , cylindrical , banana - shaped , y - shaped or a reverse form ) was determined .
the cbct images that presented central lesions or fracture in the anterior part were excluded from the study .
all cbct images were taken by a newtom vg cbct ( qr srl company , verona , italy ) device . finally , in cross - sectional images ( 1 mm thickness and interval ) reconstructed in the buccopalatal dimension
, the height of the canal was measured from the opening of foramen on the palatal side up to the nasal side .
the diameter of the canal was measured at the palatal , middle and nasal levels .
furthermore , the perpendicular distances between the anterior wall of the npc and the buccal cortex in the palatal ( w1b ) , middle ( w2b ) and nasal ( w3b ) planes and the distance between the posterior wall of the canal and the palatal cortex in the palatal ( w1p ) , middle ( w2p ) and nasal ( w3p ) planes were measured .
all the measurements were performed using nnt software ( qr srl company , verona , italy ) and calculating with using single - space decimal notation . regarding y - shaped canals seen in buccopalatal cross - sectional images ,
the height of the canal was assessed as the mean value of the height of the two canals and the diameter of the canal at the nasal level was the total diameters of the two canals . regarding two parallel cylindrical canals , the final height of the canal was the mean of the height and the diameter of both canals .
the distance between the opening of the canal and the crest was measured on the oblique line that connected the aforementioned points .
the location of the opening of the canal was categorized as buccal , palatal or middle in relation to the buccal cortex .
the normal distribution of the age of the patients was checked by a one - sample kolmogorov - smirnov test .
a binomia test was used for the evaluation of the normal gender distribution of the patients . to compare the distribution of the shape of the npc
an epsilon greenhouse - geisser test was used for comparing the height of the canal in the male and female groups . repeated analysis of variance ( anova ) was used for comparing the process of the changes in the diameter of the canal between the male and female groups at three different levels .
an independent t - test was employed to compare the diameter of the canal at three different levels in males and females .
a fisher 's exact test was performed to evaluate the position of the opening of the canal .
in this cross - sectional study , 198 subjects that included 98 females ( 49.5% ) and 100 males ( 50.5% ) were studied for evaluation of the form , height and diameter of the npc observed in cbct images .
the age of our subjects according to a one - sample kolmogorov - smirnov test had a normal distribution pattern ( p = 0.614 ) .
the frequency distribution of different age ranges of patient approximately 81.8% of the subjects had a single foramen ; another 9.1% had two foramina and the remaining 9.1% had three .
the cylindrical form had the highest frequency ( 57.6% ) , whereas the banana form ( 1.5% ) had the least frequency [ figure 3 ] .
the statistical analysis test performed using a chi - square test showed a statistically significant difference between the frequency of various forms of the canal ( p < 0.001 ) .
the frequency of different forms of the canal the minimum and maximum heights of the canal were estimated to be 5.3 and 21.4 mm respectively .
the mean height of the canal in males was 13.45 2.82 mm and in females was 12.25 2.84 mm .
an epsilon greenhouse - geisser analysis test revealed that there was a significant difference between male and female groups ( p = 0.003 ) .
the mean diameters of the canal at the palatal , middle and nasal levels were 3.53 1.1 mm , 2.35 1.1 mm and 3.7 2.3 mm respectively .
there were significant differences in the diameters of the canal at all three levels ( p < 0.001 ) .
the comparison between the mean diameter of the canal in female and male groups revealed that significant differences in the process of the changes of the diameters of the canal in three different levels were not detected ( p = 0.439 ) [ figure 4 ] .
overall , there were significant differences between the diameters of canal observed in female and male groups according to repeated anova tests ( p = 0.009 ) .
the process of the change in the diameter of the canal at three different levels as detected in male and female groups according to an independent t - test , the diameter of the canal at the palatal level in female and male groups was significantly different ( p = 0.001 ) , but at the middle ( p = 0.120 ) and nasal levels ( p = 0.275 ) there was no difference .
the diameter of the canal in the two dental groups under investigation is shown in table 1 .
diameters of the nasopalatine canal at different levels in two distinct dental groups table 2 reveals the width of available bone on the buccal and palatal sides of the npc at three different levels ( crestal , middle and nasal ) .
bone width on the buccal and palatal sides of the canal at three levels an evaluation of the bone width on the buccal and palatal sides of the canal at three different levels between females and males as measured by an independent t - test showed that only w2p had a significant difference between the two gender groups ( p = 0.028 ) .
the bone width on the palatal side of the canal between the two dental groups at three different levels had a significant difference ( p value of w1p : 0.005 , w2p : 0.012 , w3p : 0.005 ) ; however , on the buccal side , it was not significant ( p value of w1b : 0.667 , w2b : 0.803 , w3b : 0.687 ) .
an independent t - test was performed to compare the distance between the opening and the crest between the edentulous ( 8.3 2.4 ) and dentate ( 9.7 2.1 ) groups , which was significant ( p = 0.004 ) .
these measurements were significant between males ( 9.1 2.4 ) mm and females ( 9.9 1.8 ) mm ( p = 0.009 ) .
the opening of the canal was 88.9% on the palatal , 9.5% on the buccal and 1.5% on the middle sides . according to the fisher 's exact test
, the opening of the canal between both female and male groups showed no significant difference ( p = 0.551 ) .
there was , however , a significant difference ( p = 0.001 ) between two different dental groups .
approximately 81.8% of the subjects had a single foramen ; another 9.1% had two foramina and the remaining 9.1% had three .
the cylindrical form had the highest frequency ( 57.6% ) , whereas the banana form ( 1.5% ) had the least frequency [ figure 3 ] .
the statistical analysis test performed using a chi - square test showed a statistically significant difference between the frequency of various forms of the canal ( p < 0.001 ) .
the minimum and maximum heights of the canal were estimated to be 5.3 and 21.4 mm respectively .
the mean height of the canal in males was 13.45 2.82 mm and in females was 12.25 2.84 mm .
an epsilon greenhouse - geisser analysis test revealed that there was a significant difference between male and female groups ( p = 0.003 ) .
the mean diameters of the canal at the palatal , middle and nasal levels were 3.53 1.1 mm , 2.35 1.1 mm and 3.7 2.3 mm respectively .
there were significant differences in the diameters of the canal at all three levels ( p < 0.001 ) .
the comparison between the mean diameter of the canal in female and male groups revealed that significant differences in the process of the changes of the diameters of the canal in three different levels were not detected ( p = 0.439 ) [ figure 4 ] .
overall , there were significant differences between the diameters of canal observed in female and male groups according to repeated anova tests ( p = 0.009 ) .
the process of the change in the diameter of the canal at three different levels as detected in male and female groups according to an independent t - test , the diameter of the canal at the palatal level in female and male groups was significantly different ( p = 0.001 ) , but at the middle ( p = 0.120 ) and nasal levels ( p = 0.275 ) there was no difference .
the diameter of the canal in the two dental groups under investigation is shown in table 1 .
table 2 reveals the width of available bone on the buccal and palatal sides of the npc at three different levels ( crestal , middle and nasal ) .
bone width on the buccal and palatal sides of the canal at three levels an evaluation of the bone width on the buccal and palatal sides of the canal at three different levels between females and males as measured by an independent t - test showed that only w2p had a significant difference between the two gender groups ( p = 0.028 ) .
the bone width on the palatal side of the canal between the two dental groups at three different levels had a significant difference ( p value of w1p : 0.005 , w2p : 0.012 , w3p : 0.005 ) ; however , on the buccal side , it was not significant ( p value of w1b : 0.667 , w2b : 0.803 , w3b : 0.687 ) .
an independent t - test was performed to compare the distance between the opening and the crest between the edentulous ( 8.3 2.4 ) and dentate ( 9.7 2.1 ) groups , which was significant ( p = 0.004 ) .
these measurements were significant between males ( 9.1 2.4 ) mm and females ( 9.9 1.8 ) mm ( p = 0.009 ) .
the opening of the canal was 88.9% on the palatal , 9.5% on the buccal and 1.5% on the middle sides . according to the fisher 's exact test
, the opening of the canal between both female and male groups showed no significant difference ( p = 0.551 ) .
there was , however , a significant difference ( p = 0.001 ) between two different dental groups .
in this investigation , a single nasopalatine foramen on palatal side of the canal was the most commonly detected form . in the present study ,
the cylindrical shape of the canal had the highest frequency ( 57.6% ) and the banana - shaped form had the lowest frequency ( 1.5% ) .
this result is in agreement with the findings of mardinger et al . , and tzm et al .
liang et al . in their study also reported that the cylindrical form of the canal was the most frequently detected canal form . in the mraiwa et al .
study the y - shaped form of the canal ( 22 out of 34 ) and then the cylindrical form ( 8 out of 34 ) were the most common forms ; however , the sample size of this study was not adequate .
bornstein et al . found that a single canal form of the canal was the most frequent form of canal ( 45 out of 100 ) , followed by the y - shaped canal form ( 40 out of 100 ) and then two distinct parallel canals ( 15 out of 100 ) .
the mean height of the canal in the present study was higher than that reported by mardinger et al .
our results were dissimilar to the aforementioned studies but concurred with the bornstein et al . and
the estimated height of the npc in males ( 13.45 2.82 mm ) was higher than in females ( 12.252.84 mm ) , which is in agreement with prior reports by asaumi et al . and bornstein et al .
in this study , the mean width of the canal at the nasal ( 3.7 mm ) and palatal levels ( 3.53 mm ) was greater than at the middle level ( 2.35 mm ) .
the diameters of the canals observed in this study were variable , ranging from 0.7 mm to 10.9 mm .
they also estimated the mean diameter of the npc on the palatal side ( 4.6 mm ) , which was greater than that observed in this investigation ( 3.53 mm ) .
the reported mean diameters of the canal at the palatal ( 2.93 mm ) and nasal ( 2.76 mm ) levels by liang et al .
the presence or absence of adjacent teeth has an effect on widening the diameter of the canal on the palatal side .
the absence of anterior teeth results in an increase in the diameter width of the canal on the palatal side .
mardinger et al . has described the significance of this widening effect on the canal diameter at the palatal , middle and nasal levels . in this study ,
the diameter of the canal in each level in males was greater than in females ; however , except for the palatal side , there was no significant difference between female and male groups .
found that the diameter of the canal in males was greater than that detected in females .
mraiwa et al . did not , however , focus on the effect of patient gender on the diameter of the canal . in the liang et al .
study , the canal diameter was reported to be larger in the male group . in this study ,
the diameter of the canal in the edentulous group ( 4.12 mm ) was greater than in dentate group ( 3.45 mm ) and this difference was significant .
this finding is in agreement with other studies , but is dissimilar to the liang et al .
report ( 3.6 mm vs. 3.5 vmm that did not show a significant difference ) .
the bone width from the crestal to the nasal levels on the buccal side was variable , ranging from 5.96 mm to 6.82 mm and on palatal side , it ranged from 2.06 mm to 8.7 mm .
study , the bone width anterior to the npc ranged from 2.9 mm to 13.6 mm . the mean bone width in the anterior portion of the canal was 5.9 mm reported by barkin et al . but was 7.17 mm according to the tzm et al .
overall , the reported mean bone width in three different levels was not different between male or female groups in this study .
the difference in width of available bone on the palatal side between the two dental groups , edentulous or dentate , was significant but on the buccal side was insignificant .
asuami et al . found that the available bone width in the level of posterior edge of incisive foramen in their edentulous group ( 4.9 mm ) at the palatal level was lower than in their dentate group ( 6.9 mm ) and it was significant ( p < 0.05 ) .
has emphasized the importance of the effect of the presence or absence of anterior incisor teeth on the length and diameter of the npc . in tzm
study , the available bone width on the buccal side in the dentate group ( 7.38 mm ) was greater than that detected in edentulous group ( 4.88 mm ) .
the findings of bornstein et al . also emphasized the influence of the absence of central teeth and the time of tooth loss on the thickness of buccal bone width .
the difference in distance between the opening of the canal to the crest was significant between two dental groups : edentulous ( 8.26 2.44 mm ) and dentate ( 9.69 2.1 mm ) . in comparison ,
liang et al . estimated this measurement as 9.4 2.1 mm on dry skull overall .
overall , anthropometric specification could be as the reason of the variations in the form , height and width of the npc in different studies .
in our studied population , the form of the canal was mainly cylindrical and contained a single opening foramen .
the mean length of the npc was 12.84 mm with greater measurements than that seen in other populations .
the diameter of the canal in the edentulous group was greater than that of the dentate group .
the diameter of the canal at the palatal ( oral ) level was different according to gender . | background : implant placement plays a vital role in oral rehabilitation following loss of the incisors .
thus , having knowledge of anatomical variations of adjacent neurovascular structures especially the nasopalatine canal ( npc ) is essential . due to the lack of basic information in iran about the morphology of this canal and the probability of its variety in different populations
, this study was designed on an iranian population.materials and methods : in this descriptive study , we selected cone - beam computed tomography images of 198 patients comprising of 98 males and 100 females in two dental groups ( edentulous or dentate ) .
the shape of the nasopalatine foramen and the form of the canal in axial views were assessed .
then , the canal height and its diameter at the palatal , middle and nasal levels in cross - sectional images were measured .
the available bone in the buccal and palatal sides of the canal was assessed .
data analysis was carried out using a chi - square test and an independent t - test ( p 0.05).results : the majority of the samples ( 81.8% ) presented a single foramen .
cylindrical shape ( 57.6% ) was the most frequently detected canal form .
the mean of the estimated canal height was 12.84 2.88 mm .
the canal diameter at the palatal level between the sexes and dental groups showed statistically significant differences.conclusion:in our investigated population , the npc form was mainly cylindrical with a single opening foramen .
the mean of the canal height was higher than that found in other populations .
furthermore , the canal diameter in the edentulous group was greater than that observed in the other group . |
navigation - guided procedure has been widely conducted in the neurosurgical filed to perform anatomy - oriented approach8 ) .
however , the approach can not reflect the real - time intracranial image due to differential spatial displacement caused by cerebrospinal fluid drainage , volume change after surgical evacuation or newly developed lesions such as aggravation of brain edema or increased hematoma14 ) .
therefore , intraoperative scanning including computed tomography ( ct ) , magnetic resonance imaging ( mri ) and ultrasound sonography have been adapted to obtain real - time findings for operators to minimize invasion of normal brain tissue . considering consumed time , mobility , without major reconstruction of current facility in operative room ( or ) or intensive care unit ( icu ) , objectivity and costs , mobile ct ( mct )
can be a more appropriate method13 ) . since the first introduction of mct as an intraoperative monitoring modality in our institution in late 2008
, four mct have been installed in korea . besides the established use of mct in the or
, this apparatus can allow neurosurgeons to make appropriate management decisions in the icu by obtaining relevant imaging data more safely and rapidly .
the purpose of this study was to report our 3-year experience with mct under various clinical circumstances focusing on indications and advantages .
the study was approved by institutional review board for the ethical committee for human research .
this retrospective analysis was performed in patients who had mct for evaluation of intracranial lesions in or and icu from january 2009 to december 2011 . their medical charts including diagnosis , clinical implications , further treatment , consumed times , image quality and result were reviewed .
mct has been used with the following criteria in our hospital : 1 ) decision - making for the assessment of the residual tumor or hematoma ; 2 ) reduction of uncertainty in the process of surgery including suspected catheter malplacement , close observation for the change of hematoma in opposite site or sudden brain swelling ; 3 ) prompt diagnosis and management in icu .
mct parameters were 120 kv , 7 ma , effective radiation dose of 1.7 msv , 10-mm - wide collimated beam and eight 1.25-mm - wide detectors with an acquisition time of 2 s5 ) .
with regard to enhancement , a total of 90 ml of contrast agent ( iopromide , ultravist ; schering ag , berlin , germany ) was injected through an anterior cubital vein with a 4 ml / s flow rate with a 30 s x - ray time delay .
the images shown as multiplanar reformation in the axial , coronal and sagittal views were reconstructed .
every mct was performed with the ceretom ( neurologica , danvers , ma , usa ) .
mct parameters were 120 kv , 7 ma , effective radiation dose of 1.7 msv , 10-mm - wide collimated beam and eight 1.25-mm - wide detectors with an acquisition time of 2 s5 ) .
with regard to enhancement , a total of 90 ml of contrast agent ( iopromide , ultravist ; schering ag , berlin , germany ) was injected through an anterior cubital vein with a 4 ml / s flow rate with a 30 s x - ray time delay .
the images shown as multiplanar reformation in the axial , coronal and sagittal views were reconstructed .
every mct was performed with the ceretom ( neurologica , danvers , ma , usa ) .
a total of 297 patients , with a male : female ratio of 1.47 : 1.0 , and a meansd age of 57.917.2 years , underwent 357 mct as an intraoperative or bedside scan .
data of 202 cases of mct ( or , n=192 , 95% ; icu , n=10 , 5% ) were included for review and analyses of the study after excluding post - operative routine follow - up in the or . with regard to intra - operative images , extent of tumor resection ( n=55 , 27.2% ) , degree of hematoma evacuation ( n=42 , 20.8% ) , confirmation of catheter placement ( n=91 , 45.0% ) and evaluation of complications ( n=4 , 2.0% ) were checked .
an additional 14 procedures were introduced after confirmation of residual tumor ( n=7 , 50% ) , hematoma ( n=2 , 14.3% ) , malplaced catheters ( n=3 , 21.4% ) and new developed intracranial events ( n=2 , 14.3% ) .
detailed information on use of bedside ct scanning among 10 patients included : ventilator ( n=8 , 80% ) , sedation fluid ( n=7 , 70% ) , vasopressor administration ( n=6 , 60% ) , post - operative drains ( n=7 , 70% ) and mean glasgow coma scale score of 7.4 ( range , 5 - 11 ) . all images were obtained within 15 min by three staff members with sterile preparations .
a 47-year - old female was referred to our institution for further management of diffuse astrocytoma .
axial t2wi magnetic resonance ( mr ) showed a diffuse hyperintense white matter mass with anterior hypointensity lesion in the left frontal lobe .
contrast - enhanced axial t1wi mr revealed focal enhancement ( white arrow ) and low signal lesion ( arrowhead ) , suggestive of cystic change ( fig .
intraoperative ct - mr image fusion revealed a residual mass ( white arrow ) located close to anterior corpus callosum ( fig .
fused ct - mr image demonstrated complete resection of remnant tumor ( arrowhead ) without damage to corpus callosum ( fig .
initial ct showed acute subdural hemorrhage on both side with midline shift to the left ( fig .
after mct scan , additional surgical evacuation of hematoma was performed on the left side due to brain shift towards the right ( fig .
just before leaving the or , the patient vomited and the patient 's condition deteriorated .
a 47-year - old female was referred to our institution for further management of diffuse astrocytoma .
axial t2wi magnetic resonance ( mr ) showed a diffuse hyperintense white matter mass with anterior hypointensity lesion in the left frontal lobe .
contrast - enhanced axial t1wi mr revealed focal enhancement ( white arrow ) and low signal lesion ( arrowhead ) , suggestive of cystic change ( fig .
intraoperative ct - mr image fusion revealed a residual mass ( white arrow ) located close to anterior corpus callosum ( fig .
fused ct - mr image demonstrated complete resection of remnant tumor ( arrowhead ) without damage to corpus callosum ( fig .
initial ct showed acute subdural hemorrhage on both side with midline shift to the left ( fig .
after mct scan , additional surgical evacuation of hematoma was performed on the left side due to brain shift towards the right ( fig .
a 74-year - old female presented with headache . intracranial ct revealed mixed density of subdural hemorrhages ( fig .
real - time monitoring of change in brain is particular crucial in decision - making when uncertainty is present in various clinical settings including residual tumor , malplacement of drainage catheter or combination with other intra - cranial lesions during the procedures , as well as increased intracranial pressure or deteriorated in conscious level in icu .
our experience with mct demonstrates that more complete and safe surgery can be achieved in or and prompt diagnosis and proper management in icu by obtaining relevant imaging data more safely and rapidly .
fifty - five ( 28.6% ) mct procedures were conducted for estimating tumor resection . among them , additional tumor removals for seven cases were performed .
the feasibility of mct for assessing residual suprasellar tumors such as macroadenoma or meningioma and cerebellopontine angle tumor including vestibular schwanoma has been described in our previous report5 ) . on the other hand
, mct has relative limitation for depicting poorly enhanced lesion such as low grade glioma5 ) .
however , with reconstruction of three - dimensional images from mct data , image fusion based on preoperative mri findings can provide more valuable information on tumor margin assessment ( fig .
hosoda et al.3 ) reported that surgical resection with intraoperative ct prolongs survival by acquiring more complete surgical resection , which was confirmed during or immediately after the procedure .
external ventricular drainage ( evd ) is a widely performed neurosurgical procedure under anatomic landmarks , but freehand evd does not show high accuracy1 ) .
saladino et al.9 ) described 26 cases ( 12.3% ) malplaced ventriculostomy catheters of evd and shunt ; five ( 2.4% ) of required additional adjustment . in our cohort
, three cases of incomplete intra - ventricular catheter apposition were retargeted . with the guidance of mct
, more precise operations such as unusual insertion to the cyst or ommaya reservoir placement can be attained5 ) .
not limited to the neurosurgical filed , mct can be used in other surgeries including reconstruction surgery of skull base , orbital wall12 ) and sinus surgery4 ) .
masaryk et al.7 ) suggested that the total economic benefit of mct for 5 years would be more than us$2 million dollars due to dedication of conventional ct for outpatients without unnecessary delay .
in addition , cost savings can be achieved by eliminating needless complications in transfer patients .
no data regarding the incidence of adverse events in transfer process for ct imaging has been reported in korea .
smith et al.11 ) described that untoward incidents can be observed in up to 71% of icu cases .
therefore , post - operative patients with unstable vital signs are more likely to have complications due to their multiple connections with intravenous lines and drains .
the average procedure times of conventional ct scanning from ordering to arrival at icu was 28 min ( range , 18 - 35 min ) in our institution .
mean transport time for ct image from operation room to icu was 16 min ( range , 14 - 27 min ) .
this result may agree with that of other university hospitals because of their similar location of icu , or and ct rooms .
in addition , considering the current status in korea , conveyance of patients for neuroimaging is usually conducted with an intern doctor or a physician assistant . if case critical life events happen during the transfer , appropriate management can not be done . in comparison with conventional intracranial ct , interruption for mct scanning
less manpower to shift the patients was presently needed for mct ( three staff in mct vs. four - to - six staff in routine ct examination ) .
our protocol comprised an effective radiation dose of 1.7 msv ( approximately 45 mgy ) , which was lower or comparable with conventional head ct scan of 60 mgy10 ) or 47 mgy of multi - detector ct scan in our hospital . because of the short source image distance , scattered radiation in mct can be within acceptable limits for radiation safety as other portable x - ray devices in or and icu .
three of them had cerebrospinal fluid ( csf ) leakage , and two did re - operation due to delayed hemorrhage .
korinek et al.6 ) reported that csf leakage and early re - operation were independent risk factors of infection after craniotomy .
thus , we could not confirm mct as a cause of infection since the patient had high risk factor such as csf leakage and re - operation .
thus , suboptimal enhancement of cerebral vascular structure can be obtained in cases of cardiac dysfunction or vascular stenosis , which affect cerebral blood flow velocity7 ) . and algorithm enhancing image quality of mct angiography needs to be investigated further .
our 3-year experience with mct shows that more safe and complete surgery can be achieved without the need for transfer in the or as well as faster diagnosis and proper management in the icu . | objectiveobtaining real - time image is essential for neurosurgeons to minimize invasion of normal brain tissue and to prompt diagnosis of intracranial event .
the aim of this study was to report our three - year experience with a mobile computed tomography ( mct ) for intraoperative and bedside scanning.methodsa total of 357 mct ( 297 patients ) scans from january 2009 to december 2011 in single institution were reviewed . after excluding post - operative routine follow - up , 202 mct were included for analysis .
their medical records such as diagnosis , clinical application , impact on decision making , times , image quality and radiologic findings were assessed.resultstwo-hundred-two mct scans were performed in the operation room ( n=192 , 95% ) or intensive care unit ( icu ) ( n=10 , 5% ) . regarding intraoperative images , extent of resection of tumor ( n=55 , 27.2% ) , degree of hematoma removal ( n=42 , 20.8% ) , confirmation of catheter placement ( n=91 , 45.0% ) and monitoring unexpected complications ( n=4 , 2.0% ) were evaluated .
a total of 14 additional procedures were introduced after confirmation of residual tumor ( n=7 , 50% ) , hematoma ( n=2 , 14.3% ) , malpositioned catheter ( n=3 , 21.4% ) and newly developed intracranial events ( n=2 , 14.3% ) .
every image was obtained within 15 minutes and image quality was sufficient for interpretation.conclusionmct is feasible for prompt intraoperative and icu monitoring with enhanced diagnostic certainty , safety and efficiency . |
mobile - bearing knee prostheses were designed to increase the congruity of the articular surface and decrease polyethylene wear and component loosening after total knee arthroplasty ( tka)1,2 ) .
restoration of flexion after tka is crucial for successful clinical outcome and patient satisfaction especially in eastern countries where people require the use of high flexion positions including the tailor position and kneeling3 ) .
mobile - bearing high - flexion knee prostheses can be more effective in this regard than fixed - bearing prostheses due to design characteristics and rotation of bearing during hyperflexion4,5 ) . in this study
, we retrospectively compared the clinical and radiographic results of tkas using the low contact stress ( lcs ; depuy inc . ,
warsaw , in , usa ) system and those using a high - flexion prosthesis , pfc sigma rp - f ( press fit condylar , sigma rotating - platform high - flexion , johnson and johnson professional inc . , raynham , ma , usa ) system under the hypothesis that the mobile - bearing high - flexion knee prosthesis would result in more favorable clinical outcomes .
between december 2006 and january 2010 , 68 patients underwent tka using either the rp - f or lcs system . of them ,
35 patients with a minimum follow - up of 2 years were enrolled in this study . in the rp - f group , there were 16 patients ( 24 cases ) , all of whom were female .
their mean age was 67 years ( range , 59 to 76 years ) and the mean follow - up period was 39.9 months . in the lcs group ,
their mean age was 68.8 years ( range , 60 to 77 years ) and the mean follow - up period was 25.6 months .
the surgery was performed by the same surgeon using a medial parapatella approach in all patients .
tibial resection was followed by femoral resection and the posterior cruciate ligament was removed in all cases .
the flexion / extension gap was measured intraoperatively using a gapper to achieve mediolateral balance .
joint exercises started from the 1st postoperative day using a continuous passive movement machine . from the 2nd postoperative day , partial weight - bearing using crutches or
age , gender , body mass index ( bmi ) , flexion contracture of the knee , further flexion , and genu varum angle were recorded preoperatively . for the evaluation of knee function ,
the knee society knee score ( ksks ) and knee society functional score ( ksfs ) were assessed preoperatively and postoperatively6 ) . in addition , the possibility of tailor position and kneeling and the presence of anterior crepitus and patellar clunk syndrome were examined . for radiographic assessment , radiologic component position and radiolucent line on the anteroposterior and lateral radiographs obtained at the last follow - up were evaluated according to the american knee society roentgenographic evaluation and scoring system7 ) .
the following variables were measured on the radiographs to assess the component position : the medial angle between the femoral articular surface and the femoral axis ( ) and the medial angle between the tibial implant and the tibial axis ( ) on the anteroposterior view and the acute angle between the line perpendicular to the femoral articular surface and the femoral axis ( ) and the posterior angle between the tibial articular surface and the tibial axis ( ) on the lateral view . for the assessment of the femorotibialb ( limb ) alignment ,
the femorotibial angle defined as the acute angle between the femoral axis and the tibial axis was measured .
the thickness of a radiolucent line was measured in units of mm and the added values were transformed into a score with 4 considered insignificant , 5 and 9 requiring follow - up , and 10 having a high probability of fixation failure regardless of the presence of clinical symptoms .
the insall - salvati ratio8 ) on the lateral view and the congruence angle and tilt angle9 ) on the merchant view were measured .
improvements in the range of motion ( rom ) , ksks , and ksfs from the preoperative to the postoperative period were assessed using the wilcoxon rank sum test . for comparison of the clinical results between the groups ,
the chi - square test was used to analyze the possibility of tailor position and kneeling and the radiolucent line score .
no statistically significant intergroup differences were observed in the preoperative variables including age , gender , bmi , flexion contracture , further flexion , genu varum angle , and ksks and ksfs ( table 1 ) .
the mean flexion contracture and further flexion were significantly improved between the preoperative and the last follow - up evaluations in both groups : from 5.2 to 0.7 ( p=0.005 ) and from 111.7 to 126.3 ( p=0.046 ) , respectively , in the rp - f group and from 5.2 to 1.7 ( p=0.026 ) and from 112.7 to 125 ( p=0.027 ) , respectively , in the lcs group . however , no statistically significant intergroup differences were noted regarding the flexion contracture ( p=0.149 ) and the further flexion ( p=0.986 ) ( table 2 ) .
the ksks and ksfs improved significantly between the preoperative and the last follow - up evaluations in both groups : from 48.2 to 91.4 ( p=0.000 ) and from 42.9 to 87.8 ( p=0.000 ) , respectively , in the rp - f group and from 40.2 to 92.2 ( p=0.000 ) and from 39.9 to 87.4 ( p=0.000 ) , respectively , in the lcs group .
however , no statistically significant intergroup differences were found regarding the ksks ( p=0.389 ) and ksfs ( p=0.722 ) ( table 2 ) .
tailor position and kneeling was possible in 19 cases ( 79.2% ) and 6 cases ( 25% ) , respectively , in the rp - f group and in 21 cases ( 80.7% ) and 4 cases ( 15.4% ) , respectively , in the lcs group .
the mean femorotibial angle was changed from a varus of 0.6 to a valgus of 5.9 in the rp - f group and from a valgus of 1.5 to a valgus of 5.8 in the lcs group . regarding the implant location ,
the mean value was 89.0 in the rp - f group and 89.6 in the lcs group and the mean value was 85.9 in the rp - f group and 82.8 in the lcs group ( table 3 ) . in the rp - f group , in all cases except for 1 case with severe osteolysis and early loosening that required a revision surgery ( fig .
1 ) , the radiolucent line score was 4 and most of the radiolucent lines were observed in the posterior femoral condyle ( zone 4 ) in 7 cases ( 29.2% ) . in the lcs group ,
the score was 4 in all cases except for a score of 5 in 1 case , and a 1 mm radiolucent line was observed in the posterior femoral condyle in 4 cases ( 15.4% ) , but no statistical significance was found ( p=0.134 ) ( fig .
the mean insall - salvati ratio , congruence angle , and tilt angle were 1.15 , -7.6 , and 5.1 , respectively , in the rp - f group and 1.19 , -11.9 , and 6.6 , respectively , in the lcs group .
there was no statistically significant intergroup difference in the insall - salvati ratio ( p=0.515 ) and congruence angle ( p=0.059 ) , except for the tilt angle ( p=0.026 ) ( table 3 ) . in the rp - f group , early loosening that required a revision arthroplasty
was observed in 1 case ( fig . 1 ) and painful patella crepitus was observed in 2 cases ( 8.3% ) . of the latter 2 cases ,
patellar clunk syndrome was observed in 1 case where pain and crepitus disappeared and rom increased after arthroscopic nodule excision ( fig .
no statistically significant intergroup differences were observed in the preoperative variables including age , gender , bmi , flexion contracture , further flexion , genu varum angle , and ksks and ksfs ( table 1 ) .
the mean flexion contracture and further flexion were significantly improved between the preoperative and the last follow - up evaluations in both groups : from 5.2 to 0.7 ( p=0.005 ) and from 111.7 to 126.3 ( p=0.046 ) , respectively , in the rp - f group and from 5.2 to 1.7 ( p=0.026 ) and from 112.7 to 125 ( p=0.027 ) , respectively , in the lcs group . however , no statistically significant intergroup differences were noted regarding the flexion contracture ( p=0.149 ) and the further flexion ( p=0.986 ) ( table 2 ) .
the ksks and ksfs improved significantly between the preoperative and the last follow - up evaluations in both groups : from 48.2 to 91.4 ( p=0.000 ) and from 42.9 to 87.8 ( p=0.000 ) , respectively , in the rp - f group and from 40.2 to 92.2 ( p=0.000 ) and from 39.9 to 87.4 ( p=0.000 ) , respectively , in the lcs group .
however , no statistically significant intergroup differences were found regarding the ksks ( p=0.389 ) and ksfs ( p=0.722 ) ( table 2 ) .
tailor position and kneeling was possible in 19 cases ( 79.2% ) and 6 cases ( 25% ) , respectively , in the rp - f group and in 21 cases ( 80.7% ) and 4 cases ( 15.4% ) , respectively , in the lcs group .
the mean femorotibial angle was changed from a varus of 0.6 to a valgus of 5.9 in the rp - f group and from a valgus of 1.5 to a valgus of 5.8 in the lcs group . regarding the implant location ,
the mean value was 89.0 in the rp - f group and 89.6 in the lcs group and the mean value was 85.9 in the rp - f group and 82.8 in the lcs group ( table 3 ) . in the rp - f group , in all cases except for 1 case with severe osteolysis and early loosening that required a revision surgery ( fig .
1 ) , the radiolucent line score was 4 and most of the radiolucent lines were observed in the posterior femoral condyle ( zone 4 ) in 7 cases ( 29.2% ) . in the lcs group ,
the score was 4 in all cases except for a score of 5 in 1 case , and a 1 mm radiolucent line was observed in the posterior femoral condyle in 4 cases ( 15.4% ) , but no statistical significance was found ( p=0.134 ) ( fig .
the mean insall - salvati ratio , congruence angle , and tilt angle were 1.15 , -7.6 , and 5.1 , respectively , in the rp - f group and 1.19 , -11.9 , and 6.6 , respectively , in the lcs group .
there was no statistically significant intergroup difference in the insall - salvati ratio ( p=0.515 ) and congruence angle ( p=0.059 ) , except for the tilt angle ( p=0.026 ) ( table 3 ) .
in the rp - f group , early loosening that required a revision arthroplasty was observed in 1 case ( fig . 1 ) and painful patella crepitus was observed in 2 cases ( 8.3% ) . of the latter 2 cases ,
patellar clunk syndrome was observed in 1 case where pain and crepitus disappeared and rom increased after arthroscopic nodule excision ( fig .
conventional fixed - bearing knee prostheses have been associated with component wear and loosening1,2 ) . however
, mobile - bearing prostheses can be advantageous in reducing wear and loosening theoretically because they are specifically designed to improve congruity and decrease stresses to the knee such as rotational stresses10 - 14 ) .
in addition , the femoral component design that alter the radius of femoral component allows for greater flexion , which eventually results in increased rom .
the lcs system , that has a wide contact surface include reduced contact stress and constrained force and increased rom due to the various radii of curvature of the femoral component1,15,16 ) .
the pfc sigma rp - f system , that reduces the radius of curvature of the posterior femoral condyles , increasing posterior femoral roll - back with a post - cam mechanism17 ) .
in addition , the increase in posterior condylar offset and rotation of the bearing surface improves internal rotation of the tibia for high flexion of the knee .
however , we could not find significant differences between the groups regarding the clinical scores and maximum rom during flexion in this study . according to ritter18 ) , the preoperative flexion range is a major determinant of the postoperative further flexion . in this study
, we could not find notable differences in the preoperative flexion range between the groups .
kurosaka et al.19 ) reported that patients with a weak extensor mechanism due to long - standing arthritis could have a reduced range of flexion under weight - bearing movement .
all the patients in our study had degenerative arthritis and were advanced in age with a mean of 68.1 years .
therefore , we considered that the postoperative rom was more related to the preoperative rom than the prosthesis design .
femoral component loosening after tka can be influenced by infection , implant design , malalignment , bone strength , cemented fixation technique , and excessive use of the knee joint .
factors that can cause osteolysis and bone loss include operative technique , patient characteristics , and implant design20 - 24 ) .
the compressive force applied to the posterior femoral condyles with the knee at 90 of flexion is three times greater than body weight17 ) .
based on the observation of 15 cases of femoral component loosening after 1,600 total knee arthroplasties , king and scott25 ) reported that when support for the implant is inadequate , the compressive force on the posterior femoral condyles is transmitted in the form of a shear force at the distal interface with the implant and lead to a tensile force at the anterior interface , and accordingly the distal posterior interface acts as a pivot point for component loosening .
han et al.26 ) encountered 27 cases ( 38% ) of femoral component loosening in 72 cases of total knee replacements and attributed this to extensive removal of the posterior femoral condyles that results in inadequate support and excessive movement of the femoral component in high flexion26 ) .
kim et al.27 ) could not find significant differences in radiographic results and survival rate in a comparison study of two mobile - bearing prostheses including a high - flexion design .
however , early loosening and osteolysis requiring a revision surgery was observed in 1 case in the rp - f group and radiolucent lines were observed in the posterior femoral condyles ( zone 4 ) in 7 cases ( 29.2% ) in the rp - f group compared to 5 cases ( 15.4% ) in the lcs group in this study . there were no differences between the groups regarding the factors that could influence the occurrence of osteolysis including diagnosis , infection , alignment , and rom .
in addition , we took care to inject cement in the dough stage in both groups and perform drilling in sclerotic bone lesions .
therefore , we believed that osteolysis 1 ) progresses from the posterior femoral condyles as king and scott25 ) reported , 2 ) can be caused by extensive posterior femoral condyle resection because the incidence was higher in the rp - f group where more resection was unavoidable due to the implant design compared to the lcs group and 3 ) can be influenced by stresses that are focused on the posterior femoral condyles and post because of activities requiring high degrees of flexion especially for people engaged in agriculture28,29 ) . in our opinion
, early loosening can be prevented with the use of a stem or lug in addition to the rp - f implant , as suggested by king and scott25 ) , and insall and scott30 ) , or proper cementing on the posterior condyle .
patella clunk syndrome was first described by hozack et al.31 ) and similar complications of tka were referred to as tethered patellar syndrome by thorpe et al.8 ) and synovial entrapment syndrome by pollock et al.32 ) .
although authors used different terms , these syndromes belong to the same disease entity : they occur when a fibrous nodule grows excessively and induce impingement between the component .
the cause of this syndrome reported that posterior stabilized prostheses , sharp anterior edge of the femoral component , and narrow shape and anterior position of the cam8,33,34 ) .
other possible causative factors include patella height , thickness , and abnormal patella tracking8,31,33,35 ) . in this study , there were 2 cases of painful patella crepitus in the rp - f group and 1 of these was patellar clunk syndrome that was treated with arthroscopic resection .
there were no factors that could affect the postoperative soft tissue and ligament balancing including infection , proximal tibial correction osteotomy , and revision surgery .
considering that the alignment and implant location were within the normal range , the patellar clunk syndrome can be attributable to the design characteristics of the rp - f implant that has box - cutting and is extended to the anterior aspect of the proximal femur , and has an edge that curves sharply anterior to the intercondylar notch27,33,34,36 ) .
no statistically significant difference was found in the congruence angle . the lcs group appeared to have better patella tracking
compared to the rp - f group because the lateral tilt was significantly smaller in the former group .
the short - term clinical and radiographic results of tka did not show significant differences between the pfc sigma rp - f system and the rotating platform lcs system . regarding the complications , severe osteolysis and early loosening
was observed in 1 case in the rp - f group and patella clunk syndrome was suspected in 2 more cases in the rp - f group compared to the lcs group .
we believe that further studies involving a larger number of patients and a longer follow - up period are necessary . | purposethis study compared the results of rotating - platform high - flexion ( rp - f ) total knee arthroplasty with low contact stress ( lcs ) for clinical and radiographical assessment after a short - term period.materials and methods68 total knee arthroplasties using a rp - f and lcs system were analyzed retrospectively .
thirty - five of the 68 were osteoarthritic knees and were followed - up for more than 2 years .
the clinical evaluation included range of motion ( rom ) , knee society knee score and function score ( ksks and ksfs ) , tailor position and kneeling .
the radiographic evaluation included femorotibial angle , position of implants , radiolucent line and position of patella.resultsthe postoperative rom , ksks , and ksfs improved statistically in both implants .
comparing rp - f with lcs there were statistically no differences in rom ( p=0.863 ) , ksks ( p=0.835 ) , ksfs ( p=0.535 ) and tailor position ( p=0.489 ) .
there were no significant radiographic differences.conclusionstotal knee arthroplasty with rp - f and lcs showed similar clinical and radiographic results ; it also showed excellent and predictable results at the short - term follow up .
however , in rp - f there was 1 case of early osteolysis , 1 case of patella clunk syndrome and 1 case of painful patella crepitus ; therefore , further case studies and follow - up are needed . |
charles bonnet syndrome ( cbs ) was first described by charles bonnet in 1769 in his 89 years old grandfather who started experiencing visual hallucinations of men , women , carriages and building after vision loss due to cataract and who was aware about the false nature of his perceptions .
it is characterized by vivid and complex visual hallucinations in a patient suffering from vision loss in the absence of any other psychiatric illness and the patient is usually aware that these perceptions are not real .
gold and rabins suggested the term may be useful when used in the strict sense of complex visual experiences with insight .
here , we are reporting an atypical case of cbs of a 72 years lady who presented with visual hallucinations , auditory hallucination with absent insight after she had vision loss .
her vision loss was due to diabetic retinopathy , ocular hemorrhage and cataract and her psychotic symptoms that were not responding to adequate antipsychotic medication resolved completely after bilateral cataract surgery .
a 72-years - old female was brought to psychiatry outpatient department , relatives reported that for last 1 month , she is complaining , she is not in her home as she often says that she should be taken to her own home .
she was not able to move about freely due to her impaired vision , weight and diabetic neuropathy .
she would often say that there are people around her whom she does not know .
she was also hearing voices of some persons crying and would ask her daughter - in - law about it .
she said she could see a few children eating from her plate when she is eating her meals and she would ask them to be removed .
she said there are 3 - 4 children of 6 - 8 years age who are eating from her plate .
she would say that she is being taken in an ox - cart , she could see the desert and ruins of a building .
she would repeatedly say that she can see many persons around her and they should be removed from there , or she should be taken to her own home .
, she also said that someone has carried out black magic on her , though on examination , it was not a firmly held belief . on mental status examination
she was on injection insulin twice a day 18 units and 12 units , morning and evening . in march 2009 , she had an ophthalmic examination .
she had complete vision loss since march 2009 ( 2 months ) and had to be helped for activities of daily living .
she was started on tablet aripiprazole 10 mg / day , later increased to 15 mg / day .
however , there was no improvement after 2 months and in june 2009 , tablet risperidone was started initially 2 mg / day , increased to 4 mg / day .
a computed tomography scan was suggested but due to her difficulty in walking and obesity , relatives expressed inability to get it done . in june 2010 , she had her ophthalmic checkup . she had mature cataract in both eye , it was not possible to examine her retina .
a decision was made to perform cataract surgery in both eyes simultaneously , as it was difficult to bring her to hospital repeatedly .
her risperidone was stopped ; she remained symptom free until her death in september 2011 .
for cbs the following diagnostic criteria are accepted by most authorities : ( 1 ) the presence of formed , complex , persistent or repetitive , stereotyped visual hallucinations .
( 2 ) full or partial retention of insight into the unreal nature of the hallucinations . ( 3 ) absence of hallucinations in other sensory modalities . ( 4 ) absence of primary or secondary delusions .
cbs can be easily confused with such psychiatric conditions as delirium , dementia , and psychosis , which are common in elderly patients and these conditions must be ruled out beforehand as they carry a different prognostic significance .
it has been claimed that cases of true cbs ( i.e. , complex visual hallucinations in the absence of neuropsychiatric disorder and with full insight ) are exceedingly rare and most cases described in the literature are cbs plus , i.e. , visual hallucinations in the presence of a neuropsychiatric disorder or with the sufferer totally lacking insight that the hallucinations are unreal . hallucinations in cbs are usually associated with bilateral vision loss due to macular degeneration , glaucoma , and cataract .
few case reports also described visual hallucinations in patients suffering from subarachnoid hemorrhage , pituitary adenoma , occipital lobe epilepsy , herpes simplex encephalitis .
its prevalence in patients suffering from vision loss ranges from 15% to 57% in different studies .
it has been postulated that visual hallucinations in a person can be the result of secretary phenomenon .
the visual cortex had complex connections and it was believed that visual hallucinations result from increased neuronal activity or from a complex interaction related to the disappearance of the cortical inhibition .
it was found that hallucinations in cbs correlate with cerebral activity in the ventral extra striate visual cortex .
although , hallucination can disappear with or without treatment and reassurance about relationship of these hallucinations with vision loss is all that is required in few cases , yet drugs are also tried in most of the cases and carbamazepine , gabapentin , cisapride , olanzapine , risperidone , ondansetron , and mirtazapine are all reported to be beneficial in stopping the hallucinations . in our case ,
patient had developed complex visual hallucinations , auditory hallucinations with absent insight after vision loss in both eyes .
her psychotic symptoms were unresponsive to adequate trial of antipsychotics , but resolved completely after cataract surgery .
cbs has been described in most cases where vision loss was irreversible . in this case ,
vision loss which initially appeared to be irreversible could be corrected and with restoration of vision the hallucinations improved completely . in older people who are visually impaired , developing visual hallucinations adds the burden of disease .
it can also become intimidating for care givers as well as was happening in the present case . keeping
the possibility of cbs is important in providing the care and reassurance to patient and care givers , who may start fearing insanity . | charles bonnet syndrome ( cbs ) is not uncommon disorder .
it may not present with all typical symptoms and intact insight . here , a case of atypical cbs
is reported where antipsychotics were not effective .
patient improved completely after restoration of vision . |
a 62-year - old man ( 169 cm in height and 69 kg in weight ) was admitted to hospital with dizziness and paresthesia of both hands .
he was diagnosed with cerebral infarction , and while doing additional examination , cerebral infarction was assumed to be due to infective endocarditis .
he was scheduled to have mitral valve replacement using mics due to tremendous risk of systemic embolism .
two months back , he was admitted to the department of neurosurgery in our hospital because of subarachnoid hemorrhage in left central sulcus and precentral sulcus , and blood spots on the lower limbs and thrombocytopenia ( 33,000 /mm ) were incidentally detected .
although in a bone marrow aspiration , pathologic finding was not observed and the platelet associated immunoglobulins was negative , itp was clinically diagnosed . however , steroid therapy was not started immediately and his platelet count was examined as needed .
because mics needed the use of heparin prior to cardiopulmonary bypass ( cpb ) , we decided to transfuse platelets after surgery .
for premedication , 0.2 mg of glycopyrrolate was injected intramuscularly 30 minutes before the surgery .
vital signs at the time of arrival at the operation room were blood pressure at 125/75 mmhg , heart rate at 98 beats / min , and oxygen saturation ( spo2 ) as 99% . for the induction of anesthesia , while 0.1 g / kg / min of remifentanil was infused for three minutes , 10 mg of etomidate and 10 mg of cisatracurium were also injected .
the stylet was removed immediately after the tip of dlt passed through the vocal cords .
when the tube was inserted at a depth of 29 cm to lips , resistance was felt .
tracheal cuff and the bronchial cuff were inflated with 6 ml and 2 ml of air , respectively . after making a judgment that the location of tube was appropriate by using bronchoscope ,
the bronchial cuff was deflated in order to prevent bronchial injury from postural changes , and there was no bleeding .
anesthesia was maintained with sevoflurane 1.5 vol% , oxygen 1.0 l / min , medical air 1.5 l / min and remifentanil 0.12 g / kg / min were continuously infused intravenously .
after the exchange of dlt using tube exchange catheter , we confirmed the location of the dlt by using bronchoscope , and there was no bleeding .
insertion of superior vena cava ( svc ) drainage catheter and central venous pressure catheter were performed via right internal jugular vein .
there was no sign of bleeding during those procedures . at the end of the procedure
the appearance of the bleeding was blood - tinged or frothy pink like pulmonary edema .
the bleeding was sucked via suction catheter and the amount of the bleeding was about 150 - 200 ml . due to the bleeding via the left side of dlt , we carried out lung separation immediately and decided to check the location of the bleeding with a fiberoptic bronchoscope .
the carinal region and the left and the right bronchus were examined with a fiberoptic bronchoscope , but the focus of the bleeding in the traveling direction from the left mainstem bronchus was not detected . because fiberoptic bronchoscopic view had poor visibility due to blood , we could not detect the focus of the bleeding and active bleeding from bronchus .
because we could not find the evidence of tracheobronchial laceration or rupture and the patient did not have any subcutaneous emphysema , and pneumomediastium , we excluded the possibility of mechanical injury of airway .
therefore , we suspected bronchial arterial injury ; mics was immediately stopped and bronchial artery embolization was scheduled in the angiographic room .
vital signs at that time , were blood pressure ( bp ) at 105/50 mmhg , heart rate ( hr ) at 115 beats / min , and spo2 89% . during the transportation of the patient to the angiographic room , gradual decrease in the bleeding was noted , and the patient 's vital signs in the angiographic room were stable ( bp at 110 - 120/50 - 80 mmhg , hr 100 beats / min , and spo2 99 - 100% ) .
angiography of the bronchial artery was performed , but focus of the bleeding was not found ( fig .
1 ) . with reference to the risk of re - bleeding from the injured bronchial artery and the possibility of uncontrolled bleeding in patient with itp
after finishing the embolization of bilateral bronchial arteries , the chest x - ray was checked , and total atelectasis of left lung was observed ( fig .
2 ) . there was no evidence of active bleeding , but blood clot was detected and removed using bronchoscope .
the patient was scheduled for mics 6 days later . due to lowered platelet count ( 54,000 /mm ) ,
a high - dose ivig therapy ( 35 g / day , 0.5 g / kg ) for 2 days was started .
we intubated with single lumen endotracheal tube and lung isolation was achieved with intermittent total lung deflation .
the surgery was done safely , and the outcome was favorable for the patient without any postsurgical decrease in platelet count or bleeding .
he was extubated on the postoperative day ( pod ) 2 , and transferred from the intensive care unit to the ward on pod 5 .
itp is an autoimmune coagulation disorder characterized by a decreased number of circulating platelets and a shortened platelet life span .
generally , platelet count is very low in conditions of itp , often 50,000 /mm or less .
although successful cardiac operations have been reported in itp patients , large quantities of platelets , or blood transfusions were necessary in the postoperative period .
platelet counts usually increase in the consecutive 5 to 7 days after administration of high - dose ivig and subsequently are restored to the previous level within a month 's time .
a published work reports on 23 patients with itp , who safely underwent a cardiac surgical procedure , moreover no platelets were transfused in 3 patients .
achieved mitral valve replacement in itp patient at redo operation using neither allogenic platelet nor blood transfusion throughout the whole perioperative course , by means of preoperative high - dose ivig and simultaneous splenectomy .
although platelet count was low by 63,000 /mm in the preoperative laboratory study in our patient , considering economic status of the patient and based on the koyanagi et al .
however , it is recommended to perform a high - dose ivig therapy , splenectomy , and platelet transfusion prior to cardiac surgery in itp patients to increase platelet count [ 5 , 6 ] .
consensus has not been reached concerning the selection of therapeutic method , but surgery may be performed when platelet count is increased following various therapies in case of elective surgery for a patient with stable pre - surgery hemodynamics . in the past several years , there has been an increase in operations using mics . because most of the mics uses a right thoracotomy ,
the most common techniques for achieving lung isolation are left - sided dlt or right - sided bronchial blocker placement .
once positioned , there are definite benefits of lung isolation as they are less likely to be dislodged than bronchial blockers and they allow for the application of continuous positive airway pressure and suction to the deflated lung
. therefore , most of the anesthesiologists use dlt for lung isolation in mics . however , dlt may be more challenging to place in certain patients and may be associated with airway trauma and bleeding .
there is no consensus associated with control of platelet count for reducing the airway trauma and bleeding in itp patients .
based on our experience , increasing the platelet count by high - dose ivig therapy or platelet transfusion is helpful to reduce the complication associated with airway in itp patient . also , olv is not mandatory during the conduct of mics .
ventilation can be held or made intermittent both prior to cpb or at its conclusion to permit exposure .
cpb can also be initiated prior to exposing the left atrium at the beginning of the operation , although this approach may unnecessarily increase the total cpb time .
if the anesthesiologists consider the risk of airway trauma or bleeding in itp patients , they could choose the ventilation method ; single lumen endotracheal tube with arndt bronchial blocker , univent tube under fiberoptic bronchoscope guidance or intermittent single - lung ventilation as per the need of the surgeon .
it is rare to identify frank arterial extravasation as the definite source of hemorrhage during angiography for hemoptysis .
it is difficult to be sure that the vessel that is embolized is actually the source of hemorrhage and theoretically every possible vessel contributing to bronchial artery supply should be embolized .
indications for bronchial artery embolization include massive hemoptysis , recurrent hemoptysis , and to control bleeding temporarily before surgery . in conclusion , we recommend performing a high - dose ivig therapy and platelet transfusion prior to cardiac surgery in patients with itp to increase platelet count .
if the platelet count is increased , it may reduce the complications of airway management .
also , in order to reduce the risk of bronchial arterial injury in itp patients , lung isolation can be achieved by single lumen endotracheal tube with arndt bronchial blocker or univent tube under fiberoptic bronchoscope guidance .
if olv is not mandatory for performing mics , ventilation can be held or made intermittent both prior to cpb or at its conclusion to permit exposure .
anesthesiologists must carefully observe the progress of patients after the operation , and if there are suspicious symptoms or signs , adequate diagnosis and quick treatment should be done considering the possibility of bleeding from airway . | minimally invasive cardiac surgery ( mics ) requires lung isolation .
lung isolation is usually achieved with double - lumen endotracheal tube ( dlt ) .
patients with idiopathic thrombocytopenic purpura ( itp ) have an increased risk of bleeding events .
we suspected endobronchial hemorrhage after exchange of dlt during induction of anesthesia for replacement of mitral valve in a 62-year - old man with a known itp .
the mics was stopped and bronchial artery embolization was performed in the angiographic room . in the present case , in order to reduce the risk of bronchial arterial injury in itp patient we intubated with single lumen endotracheal tube .
lung isolation led to achievement of intermittent total lung deflation .
based on the results , we recommend a high - dose intravenous immunoglobulin therapy and platelet transfusion prior to cardiac surgery in patients with itp to increase platelet count .
moreover , it is proposed that in order to clear the vision during the operation , ventilation can be held or made intermittent both prior to cardiopulmonary bypass or at its conclusion to permit exposure . |
upon myocardial infarction , cardiomyocytes are lost and scar tissue is formed . to compensate this loss , cardiac hypertrophy and morphological remodelling responses try to restore the cardiac output , but can ultimately lead to heart failure .
heart transplantation is currently the only treatment option , but the number of donor hearts does not meet the needs of all heart failure patients .
furthermore , rejection and the complications of immunosuppressive therapy limit the success of heart transplantation . in the last decade ,
cell transplantation therapy has emerged as a potential therapy to treat patients suffering from heart failure .
this approach is a promising strategy to regenerate cardiac tissue after myocardial infarction , thereby replacing dead and/or fibrotic tissue , to reduce or prevent adverse remodelling of the heart and improve cardiac function .
many different progenitor cells have been used in small animals , and in pre - clinical and clinical settings , thereby suggesting a beneficial effect of the presence of engrafted cells in the injured heart [ 13 ] . to regenerate the myocardium ,
this includes cardiomyocytes to restore contractile properties , and smooth muscle cells and endothelial cells to restore perfusion and stabilize the heart .
only a few cell types are particularly suitable as a cell source due to their differentiation potential into the cardiac lineages , as was demonstrated both in vitro as well as in vivo . from the perspective of differentiation , embryonic stem cells ( escs ) seem to be an ideal candidate for cell transplantation because of their pluri - potency , i.e. to form cells of all three lineages . however , besides ethical issues , escs need careful selection of fully differentiated cells because of the risk of teratoma formation by undifferentiated cells .
recently , other pluripotent cells have been described , termed induced pluripotent stem cells that are capable of full differentiation into all lineages .
although promising and having similar potential as escs , these reprogrammed fibroblasts need the introduction of viral- or transient expressed pluri - potency genes and long - term effects of these introductions are not clear yet .
other stem or progenitor cells , isolated from the blood , bone marrow or other tissues are also used for cardiac cell transplantation , but their full differentiation potential towards all cardiac lineages is disputed . since several years , different progenitor cells are isolated from the myocardium itself .
they are thought to be a more suitable cell type because of their origin of isolation , potential predisposition for cardiac purposes and their greater potential to differentiate towards cardiomyocytes compared to other adult stem cells .
these cells can easily be expanded in culture and are very promising as a source of cell therapy [ 911 ] .
these cells are able to differentiate into vascular structures and beating cardiomyocytes in vitro , without co - culturing with neonatal cardiomyocytes . in addition
the cmpcs can also form vasculature and cardiomyocytes upon injection into injured myocardium in immunodeficient mice .
upon injection of cells into the diseased myocardium , cardiac regeneration can potentially be achieved via multiple mechanisms .
although actual differentiation of progenitor cells and active contribution to cardiac function is the main goal , the engraftment of progenitor cells and the number of newly generated cardiomyocytes and vascular cells are in many cases too low to explain the improved cardiac function and morphology [ 1214 ] .
in addition to differentiation and active participation , it is suggested that transplanted progenitor cells fuse with endogenous present cells and that release of soluble factors contributes to cardiac repair by inducing cytoprotection , neovascularization and regeneration via endogenous resident cardiac stem cells ( cscs ) [ 13 , 15 , 16 ] .
although cardiac progenitor cells are a very promising cell source for therapy because of their differentiation potential , their release of paracrine factors might also have significant effects . in a study by chimenti et al .
, paracrine factors of cardiac - derived progenitor cells , so - called cardiosphere - derived cells ( cdcs ) , are investigated . upon injection of cdcs in the heart , improved cardiac output and reduced scarring was observed .
cdcs differentiated into capillaries and cardiomyocytes , thereby explaining 2050% of the observed increases in angiogenesis and myogenesis .
however , cdcs also attracted endogenous cells , thereby inducing blood supply and reducing apoptosis , this is suggestive for paracrine effects to be involved .
the collected secretions of cdcs in vitro , labelled as conditioned medium ( cm ) , was studied in detail .
the cm was shown to be able to protect neonatal rat ventricular myocytes from apoptosis under hypoxic conditions , and improve angiogenesis of human umbilical vein endothelial cells in a matrigel assay in vitro . in a model of chronic myocardial infarction in rats ,
tang et al . confirmed a role for paracrine factors by intracoronary infusion of rat c - kit cscs in a 4-week - old infarct . in some animals no engrafted cells
could be found , but still scar area was reduced and cardiac output improved as compared to the saline infused control animals .
moreover , they observed that more cardiomyocytes , vessels and endogenous cardiac progenitor cells were present in the csc - treated animals , but without the presence of engrafted transplanted cells .
this suggests that secreted factors , released by the transplanted csc , trigger proliferation and/or migration of endothelial cells and endogenous csc and thereby stimulate cardiomyocyte survival or csc myogenic differentiation .
upon transplantation of human cmpcs into the myocardium of infarcted mouse hearts , we could observe a reduced adverse remodelling of the heart in time .
this effect was sustained for at least 3 months and human cells , expressing markers for sarcomeric proteins , as well as smooth muscle and endothelial cells were still present .
although very promising , only few injected cells ( 34% ) were engrafted and could be observed at 3 months .
the positive outcome are partially because of direct differentiation of progenitor cells towards endothelial , smooth muscle cells and cardiomyocytes , but the data suggest that paracrine effects are also involved .
interestingly , increased vascular density was observed upon transplantation of vascular endothelial growth factor ( vegf ) producing cmpcs .
thus far , cell - based therapies to regenerate the injured heart are successful in reducing the infarct size and improving cardiac output .
however , true regeneration by engrafted cells is limited and suggests paracrine factors to be involved [ 13 , 16 ] .
this effect reflects the communication between different cells via molecules that are released , thereby affecting processes in neighbouring cells .
paracrine factors from progenitor cells are of major interest and have extensively been studied with respect to the release of growth factor , cytokines and chemokines .
many factors are described to be produced by different progenitor cell populations , including insulin like growth factor-1 , stem cell derived factor , monocyte chemoattractant protein-1 , hepatocyte growth factor and vegf .
several effects have been described on the survival , proliferation and differentiation of cardiac cells , including cardiomyocytes , smooth muscle cells , endothelial cells and endogenous progenitor cells .
ultimately , cm of progenitor cells has been applied in animal models in vivo and ex vivo . in a langendorf perfused rat heart , mouse esc - cm enhanced recovery of myocardial function upon global ischemia . upon injection of concentrated cm from akt - modified mesenchymal stem cells ( mscs ) in infarcted rat hearts , apoptotic cell numbers were reduced and consequently reduced infarct sizes were assessed .
furthermore timmers et al . demonstrated that injection of cm from esc - derived mscs leads to reduced infarct size and improved cardiac function in a pig model of ischemia reperfusion . in later experiments , exosomes within the cm
exosomes are small membrane vesicles with a lipid bilayer , secreted by many , if not all , cells .
these exocytosed internal vesicles of endosomal origin range in size between 50 and 100 nm and can be observed in a cup - shaped morphology by electron microscopy analysis after differential centrifugation .
they form by inward budding of the cell membrane to form endosomes , followed by invagination of the limiting membrane of late endosomes to from multivesicular bodies .
fusion of the multivesicular bodies with the plasma membrane results in the release of the internal vesicles , then called exosomes .
these vesicles contain cytosolic derivates and express the extracellular domain of membrane - bound receptors at the surface of the membrane .
exosomes contain many different proteins , including growth factors and cytokines , and coding and non - coding rna molecules [ 23 , 24 ] .
exosomes are involved in intercellular communication between different cell types , but the mechanisms of their effects are not clear . it is postulated that exosomes can influence target cell behaviour by receptor ligand binding , fusion and endocytosis .
exosomes are described to be involved in numerous processes , including immune modulating processes and angiogenesis and migration of endothelial cells to facilitate tumour growth and the formation of metastasis . in cardiovascular research ,
exosomes have been identified as the active component within the cm which was injected in a pig model of ischemia reperfusion injury , demonstrating reduced damage and improved outcome .
ultimately these exosomes , derived from mscs , have been injected in a mouse model of ischemia reperfusion leading to a reduced infarct size . to investigate whether cmpc - derived exosomes could play a role in cmpc transplantation therapy
, we studied the effect of these exosomes on endothelial cells migration in vitro and explore the mechanism involved .
cmpcs are isolated as previously described [ 911 ] . in short , human foetal heart tissue
was collected after elective abortion and individual permission using standard informed consent procedures and prior approval of the ethics committee of the university medical center utrecht were obtained .
small pieces of human foetal cardiac tissue were digested with collagenase and put through a cell strainer to obtain a single cell suspension .
the cells were then incubated with mouse anti - sca-1 coated magnetic beads to isolate cmpcs .
these cmpcs were cultured and grown to confluency in exosome - depleted medium for 3 to 4 days . from this cmpc - cm
, exosomes are isolated by differential centrifugation and visualized using whole - mount transmission electron microscopy ( fig .
exosome vesicles are described to float in a sucrose gradient at a density between 1.10 and 1.20 g / ml .
flotillin-1 was used as a marker protein to identify exosome - containing fractions and we observed that cmpc exosomes float round 1.101.12 g / ml ( fig .
( a ) exosomes were isolated from 34 days cm from cmpcs by differential centrifugation ; by an initial step at 10,000 g , floating cells and cell debris were removed from the cm and followed by a centrifugation step at 100,000 g .
( b ) exosomes are purified using a sucrose gradient , for which the exosomes are resuspended in 2.5 m sucrose . on top of the isolated exosomes , different molarities of sucrose ( from 2.0 m to 0.4 m ) are layered .
after overnight centrifugation at 200,000 g , 14 fractions with sucrose densities ranging from 1.06 to 1.27
the cmpc - derived exosomes float in a sucrose gradient at 1.101.12 g / ml .
because exosomes are described to be involved in angiogenesis and endothelial cell migration and based on the observation of increased capillary formation upon cmpc transplantation , we suggested that cmpc - derived exosomes enhance migration of endothelial cells .
the in vitro scratch wound assay was performed with human microvascular endothelial cells ( hmecs ) . upon stimulation of hmecs with cmpc - cm , wound closure of hmecs
is increased and , by diminishing the number of exosomes in this medium , this closure is reduced ( fig .
2a ) . adding exosomes from 1 10 cmpcs in serum - free medium ( basal medium ) , migration of hmecs
( a ) in a confluent monolayer of hmecs , a scratch was made and cells were incubated for 6 hrs with cm or exosome - depleted cm . three- to 4-day - old cmpc - cm , depleted from cells and cellular debris , is compared to exosome - depleted cm .
depletion of exosomes from the cm reduces the wound closure ( n= 7 ; error bars = s.e.m . ; not significant ) .
( b ) isolated exosomes from 1 10 cmpc , resuspended in the basal culture medium of hmec ( mcdb131 ) without serum additives , is incubated with the hmecs . in the presence of the exosomes the closure of the wound
is highly enhanced ( n= 7 ; error bars = s.e.m . * = p < 0.05 ) .
to explore the mechanism how exosomes might stimulate migration of endothelial cells , we studied the role of mmps . for cell migration , the breakdown and remodelling of matrix is required and this is mediated by mmps .
interestingly , mmps can be found in secreted vesicles , including exosomes . to identify the presence of mmp activity within cmpc - derived exosomes ,
, we observed the presence of gelatinase and collagenase activity , mainly the presence of inactive , intermediate and active mmp-2 ( fig .
3a ) . blocking mmp activity with marimastat , the migratory response of the hmecs upon exosomal stimulation
could completely be blocked ( data not shown ) , but because of the direct effect of marimastat on hmecs we could not exclude the inhibition of mmps that are released from these endothelial cells .
( a ) a zymogram was performed to detect gelatolytic mmp activity within the exosomes .
the most abundant bands identify mmp-2 in the pro , intermediate and active form being predominantly expressed .
( b ) using western blotting , emmprin ( 4755 kd ) is detected in the exosomes .
because a membrane - bound mmp activator , emmprin , has been demonstrated to be released via microvesicle shedding as well , we suggested that cmpc - derived exosomes might display emmprin and thereby stimulate hmec migration .
3b , cmpc - derived exosomes contain an enriched level of emmprin , as compared to total cell lysate .
emmprin is known to induce mmp and vegf release from neighbouring cells , thereby inducing angiogenic responses and cell migration . to elucidate the causal role of exosomal - expressed emmprin on hmec migration , we performed a scratch assay with exosomes incubated with an emmprin neutralizing antibody .
we observed that the enhanced closure of the wound via exosomes is completely blocked when the exosomes are treated with an emmprin neutralizing antibody ( fig .
exosomes , used to stimulate hmec cells in a scratch wound assay for 6 hrs , are treated with an emmprin neutralizing antibody ( fitzgerald ; 10r - cd147ahu ; 20 g / ml ) or with an appropriate isotype control .
excess neutralizing antibody is washed away by ultracentrifugation at 100,000 g . the beneficial exosome effect ( derived from 1 10 cmpcs )
is blocked when exosomes are treated with the emmprin antibody ( n= 4 ; error bars = s.e.m . ; * = p < 0.05 ) .
upon cmpc cell transplantation into the myocardium , the engrafted cells contributed to formation of new capillaries and cardiomyocytes by direct differentiation , which potentially can lead to improved heart function and morphology [ 1012 ] .
however , only few donor - derived cells could be detected after 3 months , suggesting a loss of transplanted cells but a stimulation of endogenous cardiac recovery .
the transplanted cmpcs most likely produce paracrine factors that enhance this endogenous regeneration potential , including cardiomyogenesis and angiogenesis . here ,
exosomes from cmpcs are able to enhance migration of endothelial cells via an emmprin - mediated mechanism .
this suggests that exosomes , released by cmpcs upon transplantation , might be involved in the activation of endogenous cells and thereby result in increased capillary density .
the role of mmps and their regulators are well established in migration and angiogenesis . here
, we show that cmpc - derived exosomes contain several mmps and this might suggest that exosomes themselves are able to breakdown the extracellular matrix or activate pro - active mmps .
additionally , emmprin , located on the exosomes , might activate target cell mmp release and/or activity .
the effect of cmpc - derived exosomes in other processes that are important for cardiac regenerative responses , such as survival , proliferation and differentiation , are unknown .
however , cm from progenitor cells , containing exosomes , can affect proliferation , survival and differentiation of cardiomyocytes , cardiac fibroblasts and cardiac progenitor cells [ 1720 ] .
moreover , lai and colleagues identified exosomes as the active component within msc - cm that resulted in cardiac protection upon reperfusion injury [ 20 , 21 , 25 ] .
the full capacity of cmpcs is of major interest because of its regenerative potential via direct differentiation and through the proposed enhanced activation of endogenous cells .
the potential of cmpc and other progenitor cell - derived exosomes in different processes , such as proliferation , differentiation and survival on cardiomyocytes , smooth muscle cells and endogenous progenitor cells , are subject to research .
| abstractpatients suffering from heart failure as a result of myocardial infarction are in need of heart transplantation .
unfortunately the number of donor hearts is very low and therefore new therapies are subject of investigation .
cell transplantation therapy upon myocardial infarction is a very promising strategy to replace the dead myocardium with viable cardiomyocytes , smooth muscle cells and endothelial cells , thereby reducing scarring and improving cardiac performance . despite promising results , resulting in reduced infarct size and improved cardiac function on short term , only a few cells survive the ischemic milieu and are retained in the heart , thereby minimizing long - term effects .
although new capillaries and cardiomyocytes are formed around the infarcted area , only a small percentage of the transplanted cells can be detected months after myocardial infarction .
this suggests the stimulation of an endogenous regenerative capacity of the heart upon cell transplantation , resulting from release of growth factor , cytokine and other paracrine molecules by the progenitor cells the so - called paracrine hypothesis . here , we focus on a relative new component of paracrine signalling , i.e. exosomes .
we are interested in the release and function of exosomes derived from cardiac progenitor cells and studied their effects on the migratory capacity of endothelial cells . |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.