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[ "osteoid osteoma is a benign bone tumor consisting of an osteoid nidus in a highly vascular connective tissue stroma .", "these tumors are exquisitely painful , and demonstrate characteristic findings on clinical and radiographic examinations .", "the major drawback of this traditional approach is prolonged surgery and hospital stay , weakening of the bone requiring a prophylactic fixation , and its subsequent removal and additional time off school due to open surgery . to overcome this drawback", ", different minimally invasive techniques have been described as alternative therapeutic options ( radiofrequency ablation , laser photocoagulation , and percutaneous resection ) .", "computed tomography ( ct ) guidance has become a very useful and easy method for the percutaneous treatment of these lesions .", "we started using ct guided radiofrequency ablation for osteiod osteoma 5 years ago and immediately noted many beneficial effects in terms of duration of hospital stay , morbidity , and overall patient comfort prompting us to do this study .", "we report our experience with ct - guided percutaneous radiofrequency ablation ( rfa ) of osteoid osteoma in common and technically challenging locations in 30 pediatric patients and evaluate technical and clinical results .", "this is a retrospective observational study of 30 pediatric and adolescent patients who underwent ct - guided percutaneous rfa of osteoid osteoma between june 2009 to may 2014 .", "all patients reported severe pain that usually increased at night and required nonsterioidal anti - inflammatory drugs for pain relief .", "the osteoid osteoma was diagnosed from clinical and imaging findings ( radiography , ct , and/or magnetic resonance imaging ( mri ) scan ) , demonstrating a nidus and other findings that are typical of osteoid osteoma .", "patients and their legal guardians were fully informed of the procedure as well as of the surgical and medical alternatives , and informed consent was obtained .", "all procedures were performed under general anesthesia in the ct room on six slice ct scanner ( philips brilliance , massachusetts , usa ) .", "a guidewire ( k - wire ) was drilled into the center of the nidus using either a hand drill ( aesculap inc .", "b braun , center valley , pa ) or a battery operated drill ( stryker corp . , kalamazoo , mi ) with a cannulated drill bit ( 2.54.5 mm ) ( zimmer , warsaw , in ) along the planned tract by the pediatric orthopedist ; keeping the cannulated drill bit in nidus of osteoid osteoma", ", k - wire was exchanged for the stiff end of the amplatz wire ; a 5f sheath which was cut near hub and a linear cut along its entire length was made to allow it to expand and peel off when rfa probe was inserted through ; subsequently , a 4 mm cannulated bone drill was exchanged for a 5f sheath over an amplatz wire .", "( 17 gauge 2 cm diameter , 12 cm long , three tines ) angiodynamics , inc .", ", ga , usa ] was inserted into the nidus of osteoid osteoma through the sheath .", "sheath was withdrawn approximately 2 cm back , and the tines were opened and confirmed to be within the lesion with check ct sections . using the radiofrequency waves from rf generator ( model 1500x ; angiodynamics , inc .", ", ga , usa ) , the tines were heated to a target temperature of 90c and power of 60100 w and the peak temperature was maintained for 6 min .", "after 6 min , the tines were withdrawn into the probe and then the rf probe was removed .", "the treatment success was evaluated in terms of pain relief before and after procedure ( 1 day , 1 month , and 3 months ) .", "patients were also contacted by telephone at 6 months for follow - up regarding complications or recurrence of pain .", "technical success was defined as the ability to localize the radiolucent nidus and placement of an electrode under ct guidance with ablation performed for the desired period .", "clinical success was defined as complete relief of pain without the use of oral pain medication within 1 month of the procedure .", "from june 2009 to may 2014 , 30 pediatric patients underwent ct - guided rfa of osteoid osteoma .", "there were 25 boys and 5 girls , with a male - to - female ratio of 5:1 .", "their age ranged between 4 years to 20 years with a mean age of 13.16 years .", "lesions were grouped into common and challenging location . among the common location ( n = 25 )", ", lesions were located in the femur ( n = 21 ) [ figure 1 ] and tibia ( n = 4 ) . among the challenging locations ( n = 5 ) , four were near articular surface ( one each at glenoid fossa of right scapula , head of right radius , talocalcalcaneal joint of right calcaneum [ figure 2 ] and left femoral head ) and one was in the left sacrum .", "radiography ( a ) and ct scan bone window axial image ( b ) show a radiolucent nidus and surrounding reactive sclerosis .", "( d ) ct scan post procedure shows rf ablation tract a 17-year - old boy with a right calcaneal osteoid osteoma near the talocalcaneal joint margin .", "( a ) magnetic resonance imaging ( stir ) sagittal image shows central nidus surrounded by hypointense reactive sclerosis .", "rest of the surrounding calcaneum appear hyperintense due to reactive edema due to osteoid osteoma .", "( b ) ct scan shows rfa probe in situ in nidus technical success was achieved in all patients ( 100% ) .", "primary clinical success was 96.66% ( 29 of total 30 patients ) , despite pediatric population and challenging location .", "one patient with an osteoid osteoma in the shaft of right femur had persistent pain after 1 month and was treated successfully with a second procedure ( secondary success rate 100% ) .", "one patient from the challenging location group with osteoid osteoma at right radial head had immediate post - procedure weakness of wrist and finger extension because the lesion was very close to posterior interosseous nerve , which slowly recovered with physiotherapy .", "since the promising results of rosenthal et al . , in the management of osteoid osteoma with rfa , a large number of studies evaluating rfa of osteoid osteoma have been reported in the literature .", "most of these studies found very high technical success rates ( 100% ) and good primary success rates with a single session of ablation ranging from 76% to 100% .", "hence , this minimally invasive technique has become the method of choice for treatment of osteoid osteomas , provided that the diagnosis is based on a typical clinical , scintigraphy , and ct presentation . to our knowledge", ", there are few reports in the literature regarding the role of percutaneous rfa in treating osteoid osteomas in children at atypical locations .", "our technical success rate was 100% , which is similar to most of the other studies ( 100% ) . in our study in the pediatric population , the primary and secondary clinical success ratings were 96.66% and 100% , respectively , which are comparable to success rates in most other studies among adults , where primary success and secondary success ranged from 76% to 100% and 87% to 100% , respectively . in a study done by donkol et al .", "on the efficacy of rfa of osteiod osteoma in children showed that the technical success , primary clinical success , and secondary clinical success rates were 91.3% , 78.2% , and 82.6% respectively .", "and vanderschueren et al . , in their studies , showed that lower age can be a risk factor for lower clinical success rate .", "this can be explained by the greater technical difficulty during ablation of osteoid osteoma in children due to the small body mass , difficulty in positioning and fixation of the needle , and shorter ablation time ( range : 26 min in their study ) for each procedure .", "our study showed that if technical success is 100% and if strict desired temperature ( 90c ) can be maintained for desired time ( 6 min ) using controlled power ( wattage ) delivery ( 60100 w ) then high clinical success can be achieved even in pediatric population similar to adult population . in our study ,", "this happened in a child with radial head ( technically challenging position ) osteoid osteoma who had developed an immediate post procedure weakness of wrist and finger extension as the lesion was very close to posterior interosseous nerve .", "there are few case reports of likely articular cartilage damage in weight bearing joints such as acetabulum following ct - guided percutaneous rfa of juxta - articular osteoid osteoma , however , it was not confirmed whether the articular cartilage was damaged only by head due to rfa or if it had also been weakened before by the osteiod osteoma .", "on rfa of intra - articular osteoid osteoma of the hip showed that there is good ossification and bone regeneration following rfa and that it is a safe and effective treatment even for intra - articular lesions .", "no delayed complications were observed in our study . in comparison to percutaneous ct - guided curettage of osteiod osteoma , during rfa there is lack of confirmation of histological diagnosis . however , there are other disadvantages of curettage technique that it is little more traumatic with complications of neuropraxia , skin abrasions , damage to blood vessels especially in femur leading to avascular necrosis , and frequently incomplete curettage .", "we suggest that when the clinical and radiological features are not typical , the technique should be supplemented with a core biopsy from the nidus area .", "this is specially indicated when the differential diagnosis is infection as management will be required .", "limitations of our study are that it is an observational study , lack of confirmation of histological diagnosis , and lack of imaging follow - up .", "percutaneous ct - guided rf ablation is a relatively safe , highly effective , and minimally invasive procedure for the treatment of osteoid osteoma in pediatric population despite atypical location ." ]

[ "<S> context : percutaneous radiofrequency ablation ( rfa ) of osteoid osteoma has a high technical and clinical success rate . however , there is limited data on its use in the pediatric population , especially in technically challenging locations.objective:to assess the safety and efficacy of computed tomography ( ct)-guided percutaneous rfa of osteoid osteoma in pediatric population.patients and methods : from june 2009 to may 2014 , 30 patients with osteoid osteoma were treated with ct - guided rfa in common ( 25 cases ) and technically challenging ( five cases : four near articular surface and one in sacrum ) locations . </S>", "<S> therapy was performed under general anesthesia with a three - array expandable rf probe for 6 min at 90c and power of 60100 w. the patients were discharged next day under instruction . the treatment success was evaluated in terms of pain relief before and after ( 1 day , 1 month , and 6 months ) treatment.results:technical success was achieved in all patients ( 100% ) . </S>", "<S> primary clinical success was 96.66% ( 29 of total 30 patients ) , despite the pediatric population and atypical location . </S>", "<S> one patient had persistent pain after 1 month and was treated successfully with a second procedure ( secondary success rate was 100% ) . </S>", "<S> one patient had immediate complication of weakness of right hand and fingers extension . </S>", "<S> no delayed complications were observed.conclusions:ct-guided rfa is relatively safe and highly effective for treatment of osteoid osteoma in pediatric population , even in technically difficult locations . </S>" ]

[ "Introduction", "Patients and Methods", "Results", "Discussion", "Conclusion", "Financial support and sponsorship", "Conflicts of interest" ]

[ [ "osteoid osteoma is a benign bone tumor consisting of an osteoid nidus in a highly vascular connective tissue stroma .", "these tumors are exquisitely painful , and demonstrate characteristic findings on clinical and radiographic examinations .", "the major drawback of this traditional approach is prolonged surgery and hospital stay , weakening of the bone requiring a prophylactic fixation , and its subsequent removal and additional time off school due to open surgery . to overcome this drawback", ", different minimally invasive techniques have been described as alternative therapeutic options ( radiofrequency ablation , laser photocoagulation , and percutaneous resection ) .", "computed tomography ( ct ) guidance has become a very useful and easy method for the percutaneous treatment of these lesions .", "we started using ct guided radiofrequency ablation for osteiod osteoma 5 years ago and immediately noted many beneficial effects in terms of duration of hospital stay , morbidity , and overall patient comfort prompting us to do this study .", "we report our experience with ct - guided percutaneous radiofrequency ablation ( rfa ) of osteoid osteoma in common and technically challenging locations in 30 pediatric patients and evaluate technical and clinical results ." ], [ "this is a retrospective observational study of 30 pediatric and adolescent patients who underwent ct - guided percutaneous rfa of osteoid osteoma between june 2009 to may 2014 .", "all patients reported severe pain that usually increased at night and required nonsterioidal anti - inflammatory drugs for pain relief .", "the osteoid osteoma was diagnosed from clinical and imaging findings ( radiography , ct , and/or magnetic resonance imaging ( mri ) scan ) , demonstrating a nidus and other findings that are typical of osteoid osteoma .", "patients and their legal guardians were fully informed of the procedure as well as of the surgical and medical alternatives , and informed consent was obtained .", "all procedures were performed under general anesthesia in the ct room on six slice ct scanner ( philips brilliance , massachusetts , usa ) .", "a guidewire ( k - wire ) was drilled into the center of the nidus using either a hand drill ( aesculap inc .", "b braun , center valley , pa ) or a battery operated drill ( stryker corp . , kalamazoo , mi ) with a cannulated drill bit ( 2.54.5 mm ) ( zimmer , warsaw , in ) along the planned tract by the pediatric orthopedist ; keeping the cannulated drill bit in nidus of osteoid osteoma", ", k - wire was exchanged for the stiff end of the amplatz wire ; a 5f sheath which was cut near hub and a linear cut along its entire length was made to allow it to expand and peel off when rfa probe was inserted through ; subsequently , a 4 mm cannulated bone drill was exchanged for a 5f sheath over an amplatz wire .", "( 17 gauge 2 cm diameter , 12 cm long , three tines ) angiodynamics , inc .", ", ga , usa ] was inserted into the nidus of osteoid osteoma through the sheath .", "sheath was withdrawn approximately 2 cm back , and the tines were opened and confirmed to be within the lesion with check ct sections . using the radiofrequency waves from rf generator ( model 1500x ; angiodynamics , inc .", ", ga , usa ) , the tines were heated to a target temperature of 90c and power of 60100 w and the peak temperature was maintained for 6 min .", "after 6 min , the tines were withdrawn into the probe and then the rf probe was removed .", "the treatment success was evaluated in terms of pain relief before and after procedure ( 1 day , 1 month , and 3 months ) .", "patients were also contacted by telephone at 6 months for follow - up regarding complications or recurrence of pain .", "technical success was defined as the ability to localize the radiolucent nidus and placement of an electrode under ct guidance with ablation performed for the desired period .", "clinical success was defined as complete relief of pain without the use of oral pain medication within 1 month of the procedure ." ], [ "from june 2009 to may 2014 , 30 pediatric patients underwent ct - guided rfa of osteoid osteoma .", "there were 25 boys and 5 girls , with a male - to - female ratio of 5:1 .", "their age ranged between 4 years to 20 years with a mean age of 13.16 years .", "lesions were grouped into common and challenging location . among the common location ( n = 25 )", ", lesions were located in the femur ( n = 21 ) [ figure 1 ] and tibia ( n = 4 ) . among the challenging locations ( n = 5 ) , four were near articular surface ( one each at glenoid fossa of right scapula , head of right radius , talocalcalcaneal joint of right calcaneum [ figure 2 ] and left femoral head ) and one was in the left sacrum .", "radiography ( a ) and ct scan bone window axial image ( b ) show a radiolucent nidus and surrounding reactive sclerosis .", "( d ) ct scan post procedure shows rf ablation tract a 17-year - old boy with a right calcaneal osteoid osteoma near the talocalcaneal joint margin .", "( a ) magnetic resonance imaging ( stir ) sagittal image shows central nidus surrounded by hypointense reactive sclerosis .", "rest of the surrounding calcaneum appear hyperintense due to reactive edema due to osteoid osteoma .", "( b ) ct scan shows rfa probe in situ in nidus technical success was achieved in all patients ( 100% ) .", "primary clinical success was 96.66% ( 29 of total 30 patients ) , despite pediatric population and challenging location .", "one patient with an osteoid osteoma in the shaft of right femur had persistent pain after 1 month and was treated successfully with a second procedure ( secondary success rate 100% ) .", "one patient from the challenging location group with osteoid osteoma at right radial head had immediate post - procedure weakness of wrist and finger extension because the lesion was very close to posterior interosseous nerve , which slowly recovered with physiotherapy ." ], [ "since the promising results of rosenthal et al . , in the management of osteoid osteoma with rfa , a large number of studies evaluating rfa of osteoid osteoma have been reported in the literature .", "most of these studies found very high technical success rates ( 100% ) and good primary success rates with a single session of ablation ranging from 76% to 100% .", "hence , this minimally invasive technique has become the method of choice for treatment of osteoid osteomas , provided that the diagnosis is based on a typical clinical , scintigraphy , and ct presentation . to our knowledge", ", there are few reports in the literature regarding the role of percutaneous rfa in treating osteoid osteomas in children at atypical locations .", "our technical success rate was 100% , which is similar to most of the other studies ( 100% ) . in our study in the pediatric population , the primary and secondary clinical success ratings were 96.66% and 100% , respectively , which are comparable to success rates in most other studies among adults , where primary success and secondary success ranged from 76% to 100% and 87% to 100% , respectively . in a study done by donkol et al .", "on the efficacy of rfa of osteiod osteoma in children showed that the technical success , primary clinical success , and secondary clinical success rates were 91.3% , 78.2% , and 82.6% respectively .", "and vanderschueren et al . , in their studies , showed that lower age can be a risk factor for lower clinical success rate .", "this can be explained by the greater technical difficulty during ablation of osteoid osteoma in children due to the small body mass , difficulty in positioning and fixation of the needle , and shorter ablation time ( range : 26 min in their study ) for each procedure .", "our study showed that if technical success is 100% and if strict desired temperature ( 90c ) can be maintained for desired time ( 6 min ) using controlled power ( wattage ) delivery ( 60100 w ) then high clinical success can be achieved even in pediatric population similar to adult population . in our study ,", "this happened in a child with radial head ( technically challenging position ) osteoid osteoma who had developed an immediate post procedure weakness of wrist and finger extension as the lesion was very close to posterior interosseous nerve .", "there are few case reports of likely articular cartilage damage in weight bearing joints such as acetabulum following ct - guided percutaneous rfa of juxta - articular osteoid osteoma , however , it was not confirmed whether the articular cartilage was damaged only by head due to rfa or if it had also been weakened before by the osteiod osteoma .", "on rfa of intra - articular osteoid osteoma of the hip showed that there is good ossification and bone regeneration following rfa and that it is a safe and effective treatment even for intra - articular lesions .", "no delayed complications were observed in our study . in comparison to percutaneous ct - guided curettage of osteiod osteoma , during rfa there is lack of confirmation of histological diagnosis . however , there are other disadvantages of curettage technique that it is little more traumatic with complications of neuropraxia , skin abrasions , damage to blood vessels especially in femur leading to avascular necrosis , and frequently incomplete curettage .", "we suggest that when the clinical and radiological features are not typical , the technique should be supplemented with a core biopsy from the nidus area .", "this is specially indicated when the differential diagnosis is infection as management will be required .", "limitations of our study are that it is an observational study , lack of confirmation of histological diagnosis , and lack of imaging follow - up ." ], [ "percutaneous ct - guided rf ablation is a relatively safe , highly effective , and minimally invasive procedure for the treatment of osteoid osteoma in pediatric population despite atypical location ." ], [ "" ], [ "" ] ]

[ "mouse dis3l2 was expressed in sf9 cells as an n - terminal strep - sumo - tev fusion protein from the pfl vector of the multibac baculovirus expression system .", "sf9 cells were infected with baculovirus in hyclone ccm3 media ( thermo scientific ) at 27c . following 60 hours of expression", "the cells were centrifuged at 1200 rpm and resuspended in wash buffer ( 50 mm tris ph 8 , 100 mm nacl , and 5 mm dtt ) , flash frozen in liquid nitrogen and stored at 80c .", "frozen cells were thawed , nacl concentration increased to 500 mm , and then lysed by sonication .", "the lysate was treated with 0.2% poly - ethylene imine ( pei ) to precipitate nucleic acids prior to ultracentrifugation at 35,000 rpm at 4c for 1 hour .", "the soluble fraction was incubated with 1 ml of strep - tactin superflow resin ( iba biotagnology ) per 10 ml of lysate for 1 hour on a rolling shaker .", "the resin was applied to a gravity flow column and washed extensively with wash buffer .", "the cleaved protein was diluted with an equal volume of heparin buffer a ( 25 mm hepes ph 7.5 , 5 mm dtt ) to a final nacl concentration of 50 mm .", "dis3l2 was loaded onto a hitrap heparin hp column ( ge life science ) equilibrated with 25 mm hepes ph 7.5 , 50 mm nacl and 5 mm dtt .", "a linear gradient between 0.05 m and 1 m nacl was used to elute dis3l2 at 0.25 m nacl .", "fractions that contained dis3l2 were analyzed by sds - page , pooled and concentrated to 2 ml and loaded onto a hiload 16/60 superdex 200 gel filtration column equilibrated with 10 mm tris ph 8 , 100 mm nacl , 2 mm mgcl2 and 5 mm dtt .", "fractions containing dis3l2 were pooled and concentrated to 25 mg / ml , flash frozen in liquid nitrogen and stored at 80c .", "mutants of dis3l2 were constructed by sequence and ligation - independent cloning ( slic ) using mutant primers .", "we expressed and purified a d389n active site mutant lacking the protease - sensitive loop within csd1 ( residues 148169 and 194221 ) , and both the n ( 136 ) and c ( 857870 ) termini ( extended data figure 2 ) . the truncated dis3l2 was incubated with a 1.2 molar excess of u13 rna for 30 minutes at 20c .", "crystallization was carried out by the hanging drop vapor diffusion method by mixing the dis3l2-u13 complex at 15 mg / ml with an equal volume of 100 mm tri - ammonium citrate ph 5.5 , 100200 mm ammonium chloride and 2022% peg 3350 .", "crystals appeared in 12 days at 18c and were placed directly in a freshly prepared solution composed of the crystallization condition supplemented with 20% glycerol and flash frozen in liquid n2 .", "x - ray diffraction data were collected to 2.95- resolution at the x25 beamline at the national synchrotron light source ( nsls ) at brookhaven national laboratory ( bnl ) ( extended data table 1 ) .", "we generated a molecular replacement ( mr ) search model by pruning yeast rrp44 ( pdb 2vnu ) with the sculptor utility .", "the pruned model was separated into its individual csd1 , csd2 , rnb and s1 domains .", "phasing was determined by mr with phaser , using each of the domains of yeast rrp44 as individual search models .", "phaser found solutions for csd2 , rnb and s1 domains , but failed to find a unique solution for csd1 .", "the initial mr phases were input into the autobuild routine in phenix , where the resolve and buccaneer option for density modification and model building , respectively , was critical for phase improvement and interpretation of initial electron density maps .", "the csd1 of yrrp44 was placed manually into the initial map and the autobuild routine repeated .", "the resulting phases were of sufficient quality to manually build protein and rna in coot .", "although a u13 homo - oligonucleotide was used in crystallization , interpretable electron density accounted for 14 nucleotides .", "this is readily explained by slipping of the homo - oligou into two conformations along the rna binding cleft .", "one conformation is fully threaded into the active site ( at the 0 position ) , and a second conformation occupies the 1 position .", "crystallographically , these two states are indistinguishable , and thus is best accounted for by modeling 14 nucleotides .", "the final model includes two copies of truncated dis3l2 with the exception of disordered residues ( 3748 , 119228 and 252258 ) , two u14 molecules , two mg ions and 6 water molecules .", "the structure was refined using automatically determined ncs restraints , tls and isotropic b - factor refinement as implemented in phenix to an r / rfree of 21/25 % .", "the final model was validated with molprobity , where 96.8 % of residues reside in the favored and 3.13 % in the allowed regions of the ramachandran plot ( extended data table 1 ) . the copy represented by chain", "a ( dis3l2 ) and chain c ( u14 ) had appreciably better electron density and was used for all structural analysis described in this manuscript .", "weak electron density , presumably due to disorder in the region surrounding u5 , precluded accurate modeling of the position of this nucleotide , so we carefully avoided over interpretation of the interactions observed in this region , though we are confident of its presence and general location .", "all structural figures were generated with the pymol molecular graphics system , version 1.6 schrdinger , llc . all exonuclease assays were conducted under multiple turnover conditions , where we measured the linear initial rate of rna degradation and calculated the specific activity ( nt degraded minute molecule of enzyme ) .", "the indicated amount of dis3l2 was incubated at 30c with 100 nm 5 ' radiolabeled rna substrate in reaction buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt and 100 um mgcl2 ) .", "a 50 l reaction was initiated and 5 l was removed and quenched at 0 , 0.5 , 1 , 2 , 4 , 8 and 16 minute time points in formamide loading buffer ( 0.025% sds , 95% formamide , 0.025% bromophenol blue , 0.025% xylene cyanol and 18 mm edta ) .", "quenched samples were heated to 95c for 2 minutes and resolved by denaturing urea page .", "the amount of substrate degraded ( fmol ) was plotted vs. time and linear regression was used to determine the initial rate ( slope ) of the linear portion of the curve with graphpad prism 6 .", "the initial rate was converted specific activity ( nt / min / molecule of enzyme ) by dividing the initial rate ( fmol substrate processed / min ) by the fmol of enzyme used in each assay and multiplying by n-3 ( where n is the number of nucleotides of the substrate and 3 is the length of the end - product ) .", "briefly , the rna coding sequence was flanked by a 5 ' hammerhead ribozyme and 3 ' hepatitis delta virus ribozyme ( hdv ) to ensure homogeneous ends .", "constructs were cloned into a prsf vector containing a t7 promoter and the rna produced by run - off transcription .", "the pre - let-7 sequence used in this study is pre - let7a1 : ugagguaguagguuguauaguuuuagggucacacccaccacugggagauaacuauacaaucuacugucuuuc transcribed rna was gel purified with denaturing page and resuspended in depc - treated water .", "a range of dis3l2 concentrations ; 05 nm for oligou , pre - let-7-u15 , deoxyu15 and pre - let-7 substrates and 0150 nm for a15 and c15 were incubated with 1 pm 5 ' radiolabeled rna for 1 hour in binding buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt , 50 g / ml bsa and 100 m edta ) .", "we determined that wild - type dis3l2 is inactive in the presence of edta and no magnesium , but binds to rna with the same affinity as the inactive d389n mutant .", "a slot blot filtration system was used ( biorad ) to capture dis3l2/rna complexes on the top nitrocellulose membrane and unbound rna on the bottom nylon membrane .", "the membranes were washed with 100 l of binding buffer , prior to applying 100 l of the binding reaction , followed by 100 l of binding buffer . the nitrocellulose and nylon membranes were dried and analyzed by phosphor imaging .", "all binding assays were conducted in triplicate , were quantified and fraction bound plotted vs. free protein concentration .", "mouse dis3l2 was expressed in sf9 cells as an n - terminal strep - sumo - tev fusion protein from the pfl vector of the multibac baculovirus expression system .", "sf9 cells were infected with baculovirus in hyclone ccm3 media ( thermo scientific ) at 27c . following 60 hours of expression", "the cells were centrifuged at 1200 rpm and resuspended in wash buffer ( 50 mm tris ph 8 , 100 mm nacl , and 5 mm dtt ) , flash frozen in liquid nitrogen and stored at 80c .", "frozen cells were thawed , nacl concentration increased to 500 mm , and then lysed by sonication .", "the lysate was treated with 0.2% poly - ethylene imine ( pei ) to precipitate nucleic acids prior to ultracentrifugation at 35,000 rpm at 4c for 1 hour .", "the soluble fraction was incubated with 1 ml of strep - tactin superflow resin ( iba biotagnology ) per 10 ml of lysate for 1 hour on a rolling shaker .", "the resin was applied to a gravity flow column and washed extensively with wash buffer .", "the cleaved protein was diluted with an equal volume of heparin buffer a ( 25 mm hepes ph 7.5 , 5 mm dtt ) to a final nacl concentration of 50 mm .", "dis3l2 was loaded onto a hitrap heparin hp column ( ge life science ) equilibrated with 25 mm hepes ph 7.5 , 50 mm nacl and 5 mm dtt .", "a linear gradient between 0.05 m and 1 m nacl was used to elute dis3l2 at 0.25 m nacl .", "fractions that contained dis3l2 were analyzed by sds - page , pooled and concentrated to 2 ml and loaded onto a hiload 16/60 superdex 200 gel filtration column equilibrated with 10 mm tris ph 8 , 100 mm nacl , 2 mm mgcl2 and 5 mm dtt .", "fractions containing dis3l2 were pooled and concentrated to 25 mg / ml , flash frozen in liquid nitrogen and stored at 80c .", "mutants of dis3l2 were constructed by sequence and ligation - independent cloning ( slic ) using mutant primers .", "we expressed and purified a d389n active site mutant lacking the protease - sensitive loop within csd1 ( residues 148169 and 194221 ) , and both the n ( 136 ) and c ( 857870 ) termini ( extended data figure 2 ) . the truncated dis3l2 was incubated with a 1.2 molar excess of u13 rna for 30 minutes at 20c .", "crystallization was carried out by the hanging drop vapor diffusion method by mixing the dis3l2-u13 complex at 15 mg / ml with an equal volume of 100 mm tri - ammonium citrate ph 5.5 , 100200 mm ammonium chloride and 2022% peg 3350 .", "crystals appeared in 12 days at 18c and were placed directly in a freshly prepared solution composed of the crystallization condition supplemented with 20% glycerol and flash frozen in liquid n2 .", "x - ray diffraction data were collected to 2.95- resolution at the x25 beamline at the national synchrotron light source ( nsls ) at brookhaven national laboratory ( bnl ) ( extended data table 1 ) .", "we generated a molecular replacement ( mr ) search model by pruning yeast rrp44 ( pdb 2vnu ) with the sculptor utility .", "the pruned model was separated into its individual csd1 , csd2 , rnb and s1 domains .", "phasing was determined by mr with phaser , using each of the domains of yeast rrp44 as individual search models .", "phaser found solutions for csd2 , rnb and s1 domains , but failed to find a unique solution for csd1 .", "the initial mr phases were input into the autobuild routine in phenix , where the resolve and buccaneer option for density modification and model building , respectively , was critical for phase improvement and interpretation of initial electron density maps .", "the csd1 of yrrp44 was placed manually into the initial map and the autobuild routine repeated .", "the resulting phases were of sufficient quality to manually build protein and rna in coot .", "although a u13 homo - oligonucleotide was used in crystallization , interpretable electron density accounted for 14 nucleotides .", "this is readily explained by slipping of the homo - oligou into two conformations along the rna binding cleft .", "one conformation is fully threaded into the active site ( at the 0 position ) , and a second conformation occupies the 1 position .", "crystallographically , these two states are indistinguishable , and thus is best accounted for by modeling 14 nucleotides .", "the final model includes two copies of truncated dis3l2 with the exception of disordered residues ( 3748 , 119228 and 252258 ) , two u14 molecules , two mg ions and 6 water molecules .", "the structure was refined using automatically determined ncs restraints , tls and isotropic b - factor refinement as implemented in phenix to an r / rfree of 21/25 % .", "the final model was validated with molprobity , where 96.8 % of residues reside in the favored and 3.13 % in the allowed regions of the ramachandran plot ( extended data table 1 ) . the copy represented by chain", "a ( dis3l2 ) and chain c ( u14 ) had appreciably better electron density and was used for all structural analysis described in this manuscript .", "weak electron density , presumably due to disorder in the region surrounding u5 , precluded accurate modeling of the position of this nucleotide , so we carefully avoided over interpretation of the interactions observed in this region , though we are confident of its presence and general location .", "all structural figures were generated with the pymol molecular graphics system , version 1.6 schrdinger , llc .", "all exonuclease assays were conducted under multiple turnover conditions , where we measured the linear initial rate of rna degradation and calculated the specific activity ( nt degraded minute molecule of enzyme ) .", "the indicated amount of dis3l2 was incubated at 30c with 100 nm 5 ' radiolabeled rna substrate in reaction buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt and 100 um mgcl2 ) .", "a 50 l reaction was initiated and 5 l was removed and quenched at 0 , 0.5 , 1 , 2 , 4 , 8 and 16 minute time points in formamide loading buffer ( 0.025% sds , 95% formamide , 0.025% bromophenol blue , 0.025% xylene cyanol and 18 mm edta ) .", "quenched samples were heated to 95c for 2 minutes and resolved by denaturing urea page .", "the amount of substrate degraded ( fmol ) was plotted vs. time and linear regression was used to determine the initial rate ( slope ) of the linear portion of the curve with graphpad prism 6 .", "the initial rate was converted specific activity ( nt / min / molecule of enzyme ) by dividing the initial rate ( fmol substrate processed / min ) by the fmol of enzyme used in each assay and multiplying by n-3 ( where n is the number of nucleotides of the substrate and 3 is the length of the end - product ) .", "briefly , the rna coding sequence was flanked by a 5 ' hammerhead ribozyme and 3 ' hepatitis delta virus ribozyme ( hdv ) to ensure homogeneous ends .", "constructs were cloned into a prsf vector containing a t7 promoter and the rna produced by run - off transcription .", "the pre - let-7 sequence used in this study is pre - let7a1 : ugagguaguagguuguauaguuuuagggucacacccaccacugggagauaacuauacaaucuacugucuuuc transcribed rna was gel purified with denaturing page and resuspended in depc - treated water .", "a range of dis3l2 concentrations ; 05 nm for oligou , pre - let-7-u15 , deoxyu15 and pre - let-7 substrates and 0150 nm for a15 and c15 were incubated with 1 pm 5 ' radiolabeled rna for 1 hour in binding buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt , 50 g / ml bsa and 100 m edta ) .", "we determined that wild - type dis3l2 is inactive in the presence of edta and no magnesium , but binds to rna with the same affinity as the inactive d389n mutant .", "a slot blot filtration system was used ( biorad ) to capture dis3l2/rna complexes on the top nitrocellulose membrane and unbound rna on the bottom nylon membrane .", "the membranes were washed with 100 l of binding buffer , prior to applying 100 l of the binding reaction , followed by 100 l of binding buffer . the nitrocellulose and nylon membranes were dried and analyzed by phosphor imaging .", "all binding assays were conducted in triplicate , were quantified and fraction bound plotted vs. free protein concentration .", "a , dis3l2 exonuclease assays were conducted with 5 ' radio - labeled rna substrates at 30c over a 16 minute time course and products resolved by denaturing urea page .", "two representative gels , one from an exonuclease assay with u15 that was used to quantify dis3l2 enzymatic activity and another for deoxyu15 , showing no detectable activity under the initial rate conditions tested .", "b , the initial rate plots for each substrate used to calculate specific activity as shown in fig .", "the concentration of enzyme used to measure the initial rate is indicated on each plot .", "the amount of substrate degraded ( fmol ) was plotted vs. time and the initial rate ( slope ) was determined with linear regression using graphpad prism 6 .", "dis3l2-rna complexes were captured on the top nitrocellulose membrane and unbound rna to the bottom nylon membrane .", "1b , was determined by plotting the fraction rna bound vs. the concentration of free dis3l2 and fit by non - linear regression with graph pad prism 6 .", "extended data figure 2 | sequence alignment of mouse and human dis3l2 , yeast rrp44 and human dis3 .", "dis3l2 is composed of two cold shock domains ( csd1 , residues 49240 , 1 , 15 and csd2 , residues 241324 , 610 , 2 ) at its n - terminus followed by a catalytic rnb domain ( residues 325765 , 317 , 1121 ) and flanked by an s1 domain ( residues 766856 , 2229 ) .", "the secondary structure elements deduced from the structure of mouse dis3l2 are shown on top of the sequences , color - coded by domain as in fig .", "the segments of mouse dis3l2 that were truncated to facilitate crystallization are outlined in a black box .", "blue diamonds indicate residues interacting with the backbone of u14 rna and red diamonds denote conserved active site residues .", "extended data figure 3 | substrate specificity of truncated dis3l2 and electron density map for u14 rna . a , comparison of specific activity ( nt min molecule ) of truncated dis3l2 construct resembling the one used in crystallization , but without the d389n mutation with the same set of substrates analyzed for wild - type dis3l2 .", "truncated dis3l2 had comparable levels of activity and displayed the same substrate preference as wild - type dis3l2 .", "the specific activity was calculated from the initial rate plots , in the same way as described in fig.1a and extended data fig .", "initial molecular replacement phases were improved by density modification with resolve and automated model building with buccaneer , as implemented by the autobuild wizard in phenix .", "the unbiased density modified electron density map prior to inclusion of rna is shown contoured at 1. despite the crystallization of dis3l2 in complex with u13 , unambiguous density allowed modeling of u14 ( details are discussed in the methods ) .", "clear electron density accounted for all 14 nucleotides , except some disorder contributed to weak electron density surrounding u5 , precluding its accurate placement in density .", "extended data figure 4 | dis3l2 domain interface analysis . a , analysis of the csd1-rnb interface .", "the conformation of csd1 is stabilized by two protein - protein interactions with the rnb ( k240 with d739 and d91 with t613 ) , and an rna mediated interaction with the rnb through u6-u7 and 11 .", "csd2 is intimately associated with csd1 , but also interfaces with the rnb through 3 ( s242 with e337 and k319 with e332 ) .", "s1 is part of a large hydrophobic interface with rnb ( 11 , 13 , 17 and 18 , 19 ) .", "the s1 domain also interfaces with the rnb through interactions with u8 ( also see fig .", "3d ) d , csd1 is further stabilized through an rna mediated interaction with s1 .", "a , specific activity ( nt min molecule ) of dis3l2 with increasing length oligou ( u9 , u11 , u13 and u15 ) .", "b , the initial rate plots for each substrate used to calculate specific activity in panel a. the amount of substrate degraded ( fmol ) was plotted vs. time and the initial rate ( slope ) was determined with linear regression using graphpad prism 6 .", "c , equilibrium dissociation constants ( kd ) for increasing oligou length . the kd s.d . determined from", "d , the equilibrium dissociation constant ( kd ) for each substrate in panel c was determined by plotting the fraction rna bound vs. the concentration of free dis3l2 and fit by non - linear regression with graph pad prism 6 .", "extended data figure 6 | dis3l2 has a conserved active site and an rna path that resembles rnase ii .", "conserved active site residues are shown as green sticks and u13 and u14 are shown as orange sticks . a single mg ion ( purple sphere )", "as proposed for rnase ii , dis3l2 may utilize a two mg ion mechanism during catalysis .", "dis3l2 side chains and the mg ion are colored the same as in panel a , and yrrp44 residues are shown as grey sticks .", "a wide - open funnel created by the csd lobe and s1 allows rna to access the top of the rnb .", "the path of rna in rnase ii more closely resembles that in dis3l2 , compared to yrrp44 , though narrow along its length , underscoring its ability to accommodate only single - stranded rna substrates .", "e , perpendicular side and top views of the electrostatic surface potential of dis3l2 ( contoured at 5 kt / e , white = neutral , blue = positive and red = negative ) .", "a much wider positively charged funnel on the top of the rnb supports the ability of dis3l2 to degrade structured rna substrates .", "f , electrostatic surface potential of the single - strand specific rnase ii , in the same configuration as panel c. a narrow rna binding channel can only accommodate single - stranded rna .", "extended data figure 7 | dis3l2 discriminates u over a and c. a , dis3l2 selects oligou - tailed substrates by way of an extensive u - specific interaction network along most of its binding path . in u - zone 1", "hydrogen bond pairs are shown as dashed lines and disrupted hydrogen bonds are shown as curved red lines .", "b , u - zone 2 , with the same layout as panel a. c , u - zone 3 . extended data figure 8 | initial rate plots of dis3l2 u - zone mutants . a , initial rate plots for", "assays were conducted under the same conditions as described for u15 in extended data fig . 1 . , where reactions contained 0.5 nm enzyme and 100 nm 5 ' radio - labeled u15 .", "b , initial rate plots for u - zone 2 , conducted as described for panel a. c , initial rate plots for u - zone 3 , conducted as described for panel a. extended data figure 9 | binding affinity of dis3l2 u - zone mutants a , slot blot filter - binding assay was used to measure the binding affinity of selected dis3l2 mutants with u15 . the equilibrium dissociation constant ( kd ) for u - zone 1 mutants as shown in fig .", "3 g , was determined by plotting the fraction rna bound vs. the concentration of free protein and fit by non - linear regression with graph pad prism 6 .", "b , the equilibrium dissociation constant ( kd ) for u - zone 2 mutants as shown in fig .", "3 g , and measured as described for panel a. c , the equilibrium dissociation constant ( kd ) for u - zone 3 as shown in fig ." ]

[ "<S> summary paragraphthe pluripotency factor lin28 inhibits the biogenesis of the let-7 family of mammalian micrornas14 . </S>", "<S> lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development5 , glucose metabolism6 and tissue regeneration7 . </S>", "<S> alternatively , lin28 overexpression is correlated with the onset of numerous cancers8 , while let-7 , a tumor suppressor , silences several human oncogenes5 . </S>", "<S> lin28 binds to precursor let-7 ( pre - let-7 ) hairpins9 , triggering the 3 ' oligo - uridylation activity of tut4/71012 . </S>", "<S> the oligou tail added to pre - let-7 serves as a decay signal , as it is rapidly degraded by dis3l213,14 , a homolog of the catalytic subunit of the rna exosome . </S>", "<S> the molecular basis of lin28 mediated recruitment of tut4/7 to pre - let-7 and its subsequent degradation by dis3l2 is largely unknown . to examine the mechanism of dis3l2 substrate recognition we determined the structure of mouse dis3l2 in complex with an oligou rna to mimic the uridylated tail of pre - let-7 . </S>", "<S> three rna binding domains form an open funnel on one face of the catalytic domain that allows rna to navigate a path to the active site different from its exosome counterpart . </S>", "<S> the resulting path reveals an extensive network of uracil - specific interactions spanning the first twelve nucleotides of an oligou - tailed rna . </S>", "<S> we identify three u - specificity zones that explain how dis3l2 recognizes , binds and processes uridylated pre - let-7 in the final step of the lin28/let-7 pathway . </S>" ]

[ "Methods", "Protein Preparation", "Crystallization", "Structure determination", "Specific Activity Determination", "Equilibrium Binding Assays", "Supplementary Material" ]

[ [ "mouse dis3l2 was expressed in sf9 cells as an n - terminal strep - sumo - tev fusion protein from the pfl vector of the multibac baculovirus expression system .", "sf9 cells were infected with baculovirus in hyclone ccm3 media ( thermo scientific ) at 27c . following 60 hours of expression", "the cells were centrifuged at 1200 rpm and resuspended in wash buffer ( 50 mm tris ph 8 , 100 mm nacl , and 5 mm dtt ) , flash frozen in liquid nitrogen and stored at 80c .", "frozen cells were thawed , nacl concentration increased to 500 mm , and then lysed by sonication .", "the lysate was treated with 0.2% poly - ethylene imine ( pei ) to precipitate nucleic acids prior to ultracentrifugation at 35,000 rpm at 4c for 1 hour .", "the soluble fraction was incubated with 1 ml of strep - tactin superflow resin ( iba biotagnology ) per 10 ml of lysate for 1 hour on a rolling shaker .", "the resin was applied to a gravity flow column and washed extensively with wash buffer .", "the cleaved protein was diluted with an equal volume of heparin buffer a ( 25 mm hepes ph 7.5 , 5 mm dtt ) to a final nacl concentration of 50 mm .", "dis3l2 was loaded onto a hitrap heparin hp column ( ge life science ) equilibrated with 25 mm hepes ph 7.5 , 50 mm nacl and 5 mm dtt .", "a linear gradient between 0.05 m and 1 m nacl was used to elute dis3l2 at 0.25 m nacl .", "fractions that contained dis3l2 were analyzed by sds - page , pooled and concentrated to 2 ml and loaded onto a hiload 16/60 superdex 200 gel filtration column equilibrated with 10 mm tris ph 8 , 100 mm nacl , 2 mm mgcl2 and 5 mm dtt .", "fractions containing dis3l2 were pooled and concentrated to 25 mg / ml , flash frozen in liquid nitrogen and stored at 80c .", "mutants of dis3l2 were constructed by sequence and ligation - independent cloning ( slic ) using mutant primers .", "we expressed and purified a d389n active site mutant lacking the protease - sensitive loop within csd1 ( residues 148169 and 194221 ) , and both the n ( 136 ) and c ( 857870 ) termini ( extended data figure 2 ) . the truncated dis3l2 was incubated with a 1.2 molar excess of u13 rna for 30 minutes at 20c .", "crystallization was carried out by the hanging drop vapor diffusion method by mixing the dis3l2-u13 complex at 15 mg / ml with an equal volume of 100 mm tri - ammonium citrate ph 5.5 , 100200 mm ammonium chloride and 2022% peg 3350 .", "crystals appeared in 12 days at 18c and were placed directly in a freshly prepared solution composed of the crystallization condition supplemented with 20% glycerol and flash frozen in liquid n2 .", "x - ray diffraction data were collected to 2.95- resolution at the x25 beamline at the national synchrotron light source ( nsls ) at brookhaven national laboratory ( bnl ) ( extended data table 1 ) .", "we generated a molecular replacement ( mr ) search model by pruning yeast rrp44 ( pdb 2vnu ) with the sculptor utility .", "the pruned model was separated into its individual csd1 , csd2 , rnb and s1 domains .", "phasing was determined by mr with phaser , using each of the domains of yeast rrp44 as individual search models .", "phaser found solutions for csd2 , rnb and s1 domains , but failed to find a unique solution for csd1 .", "the initial mr phases were input into the autobuild routine in phenix , where the resolve and buccaneer option for density modification and model building , respectively , was critical for phase improvement and interpretation of initial electron density maps .", "the csd1 of yrrp44 was placed manually into the initial map and the autobuild routine repeated .", "the resulting phases were of sufficient quality to manually build protein and rna in coot .", "although a u13 homo - oligonucleotide was used in crystallization , interpretable electron density accounted for 14 nucleotides .", "this is readily explained by slipping of the homo - oligou into two conformations along the rna binding cleft .", "one conformation is fully threaded into the active site ( at the 0 position ) , and a second conformation occupies the 1 position .", "crystallographically , these two states are indistinguishable , and thus is best accounted for by modeling 14 nucleotides .", "the final model includes two copies of truncated dis3l2 with the exception of disordered residues ( 3748 , 119228 and 252258 ) , two u14 molecules , two mg ions and 6 water molecules .", "the structure was refined using automatically determined ncs restraints , tls and isotropic b - factor refinement as implemented in phenix to an r / rfree of 21/25 % .", "the final model was validated with molprobity , where 96.8 % of residues reside in the favored and 3.13 % in the allowed regions of the ramachandran plot ( extended data table 1 ) . the copy represented by chain", "a ( dis3l2 ) and chain c ( u14 ) had appreciably better electron density and was used for all structural analysis described in this manuscript .", "weak electron density , presumably due to disorder in the region surrounding u5 , precluded accurate modeling of the position of this nucleotide , so we carefully avoided over interpretation of the interactions observed in this region , though we are confident of its presence and general location .", "all structural figures were generated with the pymol molecular graphics system , version 1.6 schrdinger , llc . all exonuclease assays were conducted under multiple turnover conditions , where we measured the linear initial rate of rna degradation and calculated the specific activity ( nt degraded minute molecule of enzyme ) .", "the indicated amount of dis3l2 was incubated at 30c with 100 nm 5 ' radiolabeled rna substrate in reaction buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt and 100 um mgcl2 ) .", "a 50 l reaction was initiated and 5 l was removed and quenched at 0 , 0.5 , 1 , 2 , 4 , 8 and 16 minute time points in formamide loading buffer ( 0.025% sds , 95% formamide , 0.025% bromophenol blue , 0.025% xylene cyanol and 18 mm edta ) .", "quenched samples were heated to 95c for 2 minutes and resolved by denaturing urea page .", "the amount of substrate degraded ( fmol ) was plotted vs. time and linear regression was used to determine the initial rate ( slope ) of the linear portion of the curve with graphpad prism 6 .", "the initial rate was converted specific activity ( nt / min / molecule of enzyme ) by dividing the initial rate ( fmol substrate processed / min ) by the fmol of enzyme used in each assay and multiplying by n-3 ( where n is the number of nucleotides of the substrate and 3 is the length of the end - product ) .", "briefly , the rna coding sequence was flanked by a 5 ' hammerhead ribozyme and 3 ' hepatitis delta virus ribozyme ( hdv ) to ensure homogeneous ends .", "constructs were cloned into a prsf vector containing a t7 promoter and the rna produced by run - off transcription .", "the pre - let-7 sequence used in this study is pre - let7a1 : ugagguaguagguuguauaguuuuagggucacacccaccacugggagauaacuauacaaucuacugucuuuc transcribed rna was gel purified with denaturing page and resuspended in depc - treated water .", "a range of dis3l2 concentrations ; 05 nm for oligou , pre - let-7-u15 , deoxyu15 and pre - let-7 substrates and 0150 nm for a15 and c15 were incubated with 1 pm 5 ' radiolabeled rna for 1 hour in binding buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt , 50 g / ml bsa and 100 m edta ) .", "we determined that wild - type dis3l2 is inactive in the presence of edta and no magnesium , but binds to rna with the same affinity as the inactive d389n mutant .", "a slot blot filtration system was used ( biorad ) to capture dis3l2/rna complexes on the top nitrocellulose membrane and unbound rna on the bottom nylon membrane .", "the membranes were washed with 100 l of binding buffer , prior to applying 100 l of the binding reaction , followed by 100 l of binding buffer . the nitrocellulose and nylon membranes were dried and analyzed by phosphor imaging .", "all binding assays were conducted in triplicate , were quantified and fraction bound plotted vs. free protein concentration ." ], [ "mouse dis3l2 was expressed in sf9 cells as an n - terminal strep - sumo - tev fusion protein from the pfl vector of the multibac baculovirus expression system .", "sf9 cells were infected with baculovirus in hyclone ccm3 media ( thermo scientific ) at 27c . following 60 hours of expression", "the cells were centrifuged at 1200 rpm and resuspended in wash buffer ( 50 mm tris ph 8 , 100 mm nacl , and 5 mm dtt ) , flash frozen in liquid nitrogen and stored at 80c .", "frozen cells were thawed , nacl concentration increased to 500 mm , and then lysed by sonication .", "the lysate was treated with 0.2% poly - ethylene imine ( pei ) to precipitate nucleic acids prior to ultracentrifugation at 35,000 rpm at 4c for 1 hour .", "the soluble fraction was incubated with 1 ml of strep - tactin superflow resin ( iba biotagnology ) per 10 ml of lysate for 1 hour on a rolling shaker .", "the resin was applied to a gravity flow column and washed extensively with wash buffer .", "the cleaved protein was diluted with an equal volume of heparin buffer a ( 25 mm hepes ph 7.5 , 5 mm dtt ) to a final nacl concentration of 50 mm .", "dis3l2 was loaded onto a hitrap heparin hp column ( ge life science ) equilibrated with 25 mm hepes ph 7.5 , 50 mm nacl and 5 mm dtt .", "a linear gradient between 0.05 m and 1 m nacl was used to elute dis3l2 at 0.25 m nacl .", "fractions that contained dis3l2 were analyzed by sds - page , pooled and concentrated to 2 ml and loaded onto a hiload 16/60 superdex 200 gel filtration column equilibrated with 10 mm tris ph 8 , 100 mm nacl , 2 mm mgcl2 and 5 mm dtt .", "fractions containing dis3l2 were pooled and concentrated to 25 mg / ml , flash frozen in liquid nitrogen and stored at 80c .", "mutants of dis3l2 were constructed by sequence and ligation - independent cloning ( slic ) using mutant primers ." ], [ "we expressed and purified a d389n active site mutant lacking the protease - sensitive loop within csd1 ( residues 148169 and 194221 ) , and both the n ( 136 ) and c ( 857870 ) termini ( extended data figure 2 ) . the truncated dis3l2 was incubated with a 1.2 molar excess of u13 rna for 30 minutes at 20c .", "crystallization was carried out by the hanging drop vapor diffusion method by mixing the dis3l2-u13 complex at 15 mg / ml with an equal volume of 100 mm tri - ammonium citrate ph 5.5 , 100200 mm ammonium chloride and 2022% peg 3350 .", "crystals appeared in 12 days at 18c and were placed directly in a freshly prepared solution composed of the crystallization condition supplemented with 20% glycerol and flash frozen in liquid n2 ." ], [ "x - ray diffraction data were collected to 2.95- resolution at the x25 beamline at the national synchrotron light source ( nsls ) at brookhaven national laboratory ( bnl ) ( extended data table 1 ) .", "we generated a molecular replacement ( mr ) search model by pruning yeast rrp44 ( pdb 2vnu ) with the sculptor utility .", "the pruned model was separated into its individual csd1 , csd2 , rnb and s1 domains .", "phasing was determined by mr with phaser , using each of the domains of yeast rrp44 as individual search models .", "phaser found solutions for csd2 , rnb and s1 domains , but failed to find a unique solution for csd1 .", "the initial mr phases were input into the autobuild routine in phenix , where the resolve and buccaneer option for density modification and model building , respectively , was critical for phase improvement and interpretation of initial electron density maps .", "the csd1 of yrrp44 was placed manually into the initial map and the autobuild routine repeated .", "the resulting phases were of sufficient quality to manually build protein and rna in coot .", "although a u13 homo - oligonucleotide was used in crystallization , interpretable electron density accounted for 14 nucleotides .", "this is readily explained by slipping of the homo - oligou into two conformations along the rna binding cleft .", "one conformation is fully threaded into the active site ( at the 0 position ) , and a second conformation occupies the 1 position .", "crystallographically , these two states are indistinguishable , and thus is best accounted for by modeling 14 nucleotides .", "the final model includes two copies of truncated dis3l2 with the exception of disordered residues ( 3748 , 119228 and 252258 ) , two u14 molecules , two mg ions and 6 water molecules .", "the structure was refined using automatically determined ncs restraints , tls and isotropic b - factor refinement as implemented in phenix to an r / rfree of 21/25 % .", "the final model was validated with molprobity , where 96.8 % of residues reside in the favored and 3.13 % in the allowed regions of the ramachandran plot ( extended data table 1 ) . the copy represented by chain", "a ( dis3l2 ) and chain c ( u14 ) had appreciably better electron density and was used for all structural analysis described in this manuscript .", "weak electron density , presumably due to disorder in the region surrounding u5 , precluded accurate modeling of the position of this nucleotide , so we carefully avoided over interpretation of the interactions observed in this region , though we are confident of its presence and general location .", "all structural figures were generated with the pymol molecular graphics system , version 1.6 schrdinger , llc ." ], [ "all exonuclease assays were conducted under multiple turnover conditions , where we measured the linear initial rate of rna degradation and calculated the specific activity ( nt degraded minute molecule of enzyme ) .", "the indicated amount of dis3l2 was incubated at 30c with 100 nm 5 ' radiolabeled rna substrate in reaction buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt and 100 um mgcl2 ) .", "a 50 l reaction was initiated and 5 l was removed and quenched at 0 , 0.5 , 1 , 2 , 4 , 8 and 16 minute time points in formamide loading buffer ( 0.025% sds , 95% formamide , 0.025% bromophenol blue , 0.025% xylene cyanol and 18 mm edta ) .", "quenched samples were heated to 95c for 2 minutes and resolved by denaturing urea page .", "the amount of substrate degraded ( fmol ) was plotted vs. time and linear regression was used to determine the initial rate ( slope ) of the linear portion of the curve with graphpad prism 6 .", "the initial rate was converted specific activity ( nt / min / molecule of enzyme ) by dividing the initial rate ( fmol substrate processed / min ) by the fmol of enzyme used in each assay and multiplying by n-3 ( where n is the number of nucleotides of the substrate and 3 is the length of the end - product ) .", "briefly , the rna coding sequence was flanked by a 5 ' hammerhead ribozyme and 3 ' hepatitis delta virus ribozyme ( hdv ) to ensure homogeneous ends .", "constructs were cloned into a prsf vector containing a t7 promoter and the rna produced by run - off transcription .", "the pre - let-7 sequence used in this study is pre - let7a1 : ugagguaguagguuguauaguuuuagggucacacccaccacugggagauaacuauacaaucuacugucuuuc transcribed rna was gel purified with denaturing page and resuspended in depc - treated water ." ], [ "a range of dis3l2 concentrations ; 05 nm for oligou , pre - let-7-u15 , deoxyu15 and pre - let-7 substrates and 0150 nm for a15 and c15 were incubated with 1 pm 5 ' radiolabeled rna for 1 hour in binding buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt , 50 g / ml bsa and 100 m edta ) .", "we determined that wild - type dis3l2 is inactive in the presence of edta and no magnesium , but binds to rna with the same affinity as the inactive d389n mutant .", "a slot blot filtration system was used ( biorad ) to capture dis3l2/rna complexes on the top nitrocellulose membrane and unbound rna on the bottom nylon membrane .", "the membranes were washed with 100 l of binding buffer , prior to applying 100 l of the binding reaction , followed by 100 l of binding buffer . the nitrocellulose and nylon membranes were dried and analyzed by phosphor imaging .", "all binding assays were conducted in triplicate , were quantified and fraction bound plotted vs. free protein concentration ." ], [ "a , dis3l2 exonuclease assays were conducted with 5 ' radio - labeled rna substrates at 30c over a 16 minute time course and products resolved by denaturing urea page .", "two representative gels , one from an exonuclease assay with u15 that was used to quantify dis3l2 enzymatic activity and another for deoxyu15 , showing no detectable activity under the initial rate conditions tested .", "b , the initial rate plots for each substrate used to calculate specific activity as shown in fig .", "the concentration of enzyme used to measure the initial rate is indicated on each plot .", "the amount of substrate degraded ( fmol ) was plotted vs. time and the initial rate ( slope ) was determined with linear regression using graphpad prism 6 .", "dis3l2-rna complexes were captured on the top nitrocellulose membrane and unbound rna to the bottom nylon membrane .", "1b , was determined by plotting the fraction rna bound vs. the concentration of free dis3l2 and fit by non - linear regression with graph pad prism 6 .", "extended data figure 2 | sequence alignment of mouse and human dis3l2 , yeast rrp44 and human dis3 .", "dis3l2 is composed of two cold shock domains ( csd1 , residues 49240 , 1 , 15 and csd2 , residues 241324 , 610 , 2 ) at its n - terminus followed by a catalytic rnb domain ( residues 325765 , 317 , 1121 ) and flanked by an s1 domain ( residues 766856 , 2229 ) .", "the secondary structure elements deduced from the structure of mouse dis3l2 are shown on top of the sequences , color - coded by domain as in fig .", "the segments of mouse dis3l2 that were truncated to facilitate crystallization are outlined in a black box .", "blue diamonds indicate residues interacting with the backbone of u14 rna and red diamonds denote conserved active site residues .", "extended data figure 3 | substrate specificity of truncated dis3l2 and electron density map for u14 rna . a , comparison of specific activity ( nt min molecule ) of truncated dis3l2 construct resembling the one used in crystallization , but without the d389n mutation with the same set of substrates analyzed for wild - type dis3l2 .", "truncated dis3l2 had comparable levels of activity and displayed the same substrate preference as wild - type dis3l2 .", "the specific activity was calculated from the initial rate plots , in the same way as described in fig.1a and extended data fig .", "initial molecular replacement phases were improved by density modification with resolve and automated model building with buccaneer , as implemented by the autobuild wizard in phenix .", "the unbiased density modified electron density map prior to inclusion of rna is shown contoured at 1. despite the crystallization of dis3l2 in complex with u13 , unambiguous density allowed modeling of u14 ( details are discussed in the methods ) .", "clear electron density accounted for all 14 nucleotides , except some disorder contributed to weak electron density surrounding u5 , precluding its accurate placement in density .", "extended data figure 4 | dis3l2 domain interface analysis . a , analysis of the csd1-rnb interface .", "the conformation of csd1 is stabilized by two protein - protein interactions with the rnb ( k240 with d739 and d91 with t613 ) , and an rna mediated interaction with the rnb through u6-u7 and 11 .", "csd2 is intimately associated with csd1 , but also interfaces with the rnb through 3 ( s242 with e337 and k319 with e332 ) .", "s1 is part of a large hydrophobic interface with rnb ( 11 , 13 , 17 and 18 , 19 ) .", "the s1 domain also interfaces with the rnb through interactions with u8 ( also see fig .", "3d ) d , csd1 is further stabilized through an rna mediated interaction with s1 .", "a , specific activity ( nt min molecule ) of dis3l2 with increasing length oligou ( u9 , u11 , u13 and u15 ) .", "b , the initial rate plots for each substrate used to calculate specific activity in panel a. the amount of substrate degraded ( fmol ) was plotted vs. time and the initial rate ( slope ) was determined with linear regression using graphpad prism 6 .", "c , equilibrium dissociation constants ( kd ) for increasing oligou length . the kd s.d . determined from", "d , the equilibrium dissociation constant ( kd ) for each substrate in panel c was determined by plotting the fraction rna bound vs. the concentration of free dis3l2 and fit by non - linear regression with graph pad prism 6 .", "extended data figure 6 | dis3l2 has a conserved active site and an rna path that resembles rnase ii .", "conserved active site residues are shown as green sticks and u13 and u14 are shown as orange sticks . a single mg ion ( purple sphere )", "as proposed for rnase ii , dis3l2 may utilize a two mg ion mechanism during catalysis .", "dis3l2 side chains and the mg ion are colored the same as in panel a , and yrrp44 residues are shown as grey sticks .", "a wide - open funnel created by the csd lobe and s1 allows rna to access the top of the rnb .", "the path of rna in rnase ii more closely resembles that in dis3l2 , compared to yrrp44 , though narrow along its length , underscoring its ability to accommodate only single - stranded rna substrates .", "e , perpendicular side and top views of the electrostatic surface potential of dis3l2 ( contoured at 5 kt / e , white = neutral , blue = positive and red = negative ) .", "a much wider positively charged funnel on the top of the rnb supports the ability of dis3l2 to degrade structured rna substrates .", "f , electrostatic surface potential of the single - strand specific rnase ii , in the same configuration as panel c. a narrow rna binding channel can only accommodate single - stranded rna .", "extended data figure 7 | dis3l2 discriminates u over a and c. a , dis3l2 selects oligou - tailed substrates by way of an extensive u - specific interaction network along most of its binding path . in u - zone 1", "hydrogen bond pairs are shown as dashed lines and disrupted hydrogen bonds are shown as curved red lines .", "b , u - zone 2 , with the same layout as panel a. c , u - zone 3 . extended data figure 8 | initial rate plots of dis3l2 u - zone mutants . a , initial rate plots for", "assays were conducted under the same conditions as described for u15 in extended data fig . 1 . , where reactions contained 0.5 nm enzyme and 100 nm 5 ' radio - labeled u15 .", "b , initial rate plots for u - zone 2 , conducted as described for panel a. c , initial rate plots for u - zone 3 , conducted as described for panel a. extended data figure 9 | binding affinity of dis3l2 u - zone mutants a , slot blot filter - binding assay was used to measure the binding affinity of selected dis3l2 mutants with u15 . the equilibrium dissociation constant ( kd ) for u - zone 1 mutants as shown in fig .", "3 g , was determined by plotting the fraction rna bound vs. the concentration of free protein and fit by non - linear regression with graph pad prism 6 .", "b , the equilibrium dissociation constant ( kd ) for u - zone 2 mutants as shown in fig .", "3 g , and measured as described for panel a. c , the equilibrium dissociation constant ( kd ) for u - zone 3 as shown in fig ." ] ]

[ "the knowledge about the role of cytokines in psoriasis has developed in the last several years .", "initially , only th1 cells and cytokines secreted by these cells , like tnf- , ifn- , and il-2 , have been associated with the development and maintenance of chronic inflammatory diseases , such as psoriasis .", "th1 cells differentiate from naive cd4 + cells in the presence of il-12 , il-18 , and ifn and . it is well known that th1 cytokines have strong inflammatory effects in activating macrophage , neutrophil , and cd8 + cytotoxic t cells . in the 1990s ,", "th17 cells were described as a new t - cell population that produces il-17 , il-6 , il-21 , il-22 , and tnf . transforming growth factor ( tgf)-1 , il-6 , il-23 , and il-15 stimulated initial th17 differentiation from naive t cells .", "tgf-1 is secreted by activated t cells and it initiates t cell and fibroblast activation , as well as angiogenesis and neovascularization [ 37 ] .", "il-6 , secreted by macrophages , endothelial cells , and epithelial cells , is responsible for augmentation of keratinocyte hyperplasia and invasion of macrophages and t cells [ 3 , 8 ] .", "il-15 , produced by monocytes , macrophages , dcs , and t cells , can appear to induce angiogenesis , immune cell recruitment , and activation of keratinocytes [ 4 , 9 , 10 ] .", "cytokines produced by th17 cells were found to initiate acanthosis , hyperkeratosis , and parakeratosis .", "th17 cells demonstrated involvement in neutrophil and monocyte chemotaxis , t - cell migration and activation , and neovascularization .", "th22 cells have been recently described as inflammatory cd4 + t cells that produce cytokines such as il-22 , il-26 , and il-13 of which il-22 is the most important functional cytokine .", "recent studies indicate that il-6 and tnf- , along with the help of plasmacytoid dendritic cells , can promote the th22 phenotype [ 14 , 16 ] . ", "the differentiation of main three t cell subsets involved in the pathogenesis of psoriasis , as well as th1 , th17 , and th22 cytokine production are illustrated in figure 1 . ", "the aim of the study was to assess the serum levels of some cytokines involved in the th17 and th22 responses in psoriatic patients .", " the study comprised 60 psoriatic patients , 50 males ( 83.33% ) and 10 females ( 16.67% ) , as well as 30 healthy controls .", "the studied patients ' age was between 18 and 69 years , 45.6 13.2 years on average .", "the history of the disease was from 1 to 45 years , 18.7 11.5 years on average .", "thirty - four of the studied patients ( 40% ) reported two psoriasis exacerbations a year , whereas 9 persons ( 15% ) observed as many as 4 exacerbations a year . ", "the skin lesions severity was assessed with the use of psoriasis area and severity index ( pasi ) , body surface area ( bsa ) , and physician global assessment ( pga ) scores .", "the pasi value in the studied group was from 4.8 to 64.2 and 15.7 9.7 on average .", "the bsa value was in the range of 9.096.9% and 31.4 18.2 on average .", "the pga score was 3 in 24 individuals ( 40% ) , 4 in another 24 individuals ( 40% ) , and 5 in 12 individuals ( 20% ) . ", "blood samples were collected from psoriatic patients and controls and were centrifuged for 15 minutes at 1000 g . then , serum samples were subdivided into small aliquots to be stored at 80c until tested for cytokine levels .", "elisa kits were used to determine il-6 , il-12 , il-17 , il-20 , il-22 , and il-23 ( r&d systems , minneapolis , mn , usa ) serum levels , according to the manufacturer 's instructions . ", "continuous variables were presented as mean standard deviation , while categorical variables were presented as absolute and relative frequencies .", "mann - whitney 's u test was used to compare continuous data between the studied and control groups .", " the statistical analyses of the conducted study results revealed significantly higher serum levels of il-6 , il-20 , and il-23 in psoriatic patients comparing to healthy controls ( table 1 and figure 2 ) . significant correlations between il-20 , il-22 , il-17 , and psoriasis severity were observed .", "no significant correlations were found between the concentrations of the studied cytokines and the sex , age , or psoriasis duration in the studied patients ( p > 0.05 ) .", "significantly higher il-6 values were found in the psoriatic patients in comparison to the control group ( p < 0.001 ) . ", "il-6 contributes to the th17 cell line 's involvement in numerous processes of inflammation and autoimmunity by preventing the proliferation of t regulatory cells .", "il-17 can induce fibroblasts to produce il-6 potentially activating a positive feedback loop that strengthens th17 inflammation . ", "analyzed 78 studies comparing the serum inflammatory markers , including il-6 , in psoriasis with healthy controls .", "the study showed that the standardized mean differences were higher in psoriatic patients compared to healthy controls for il-6 .", "elevated serum il-6 appears to be associated with greater psoriasis severity [ 18 , 21 , 24 ] .", "it was shown that il-6 might be the biomarker differentiating psoriasis arthritis from psoriasis without joint involvement [ 19 , 22 ] . ", "significantly higher il-20 values were found in the psoriatic patients in comparison to the control group ( p < 0.001 ) . a significant positive correlation between the", "il-20 concentrations and psoriasis severity measured by the pasi was detected ( p < 0.001 ; r = 0.698 ) . ", "il-20 is produced by keratinocytes in the presence of il-22 , tnf- , and il-17 but not ifn- or il-20 itself [ 15 , 25 ] .", "il-20 can play an important role in the later effector phase of psoriasis pathogenesis , in which it inhibits the terminal differentiation , increases antimicrobial competence , and production of chemokines for neutrophils in keratinocytes [ 25 , 29 ] . ", "there are not many studies concerning il-20 serum level , but increased levels of il-20 were noted in lesional skin as well as in the blood in psoriatic patients . as in our study , il-20 serum levels correlated with pasi scores . ", "a significantly higher increase in il-22 was observed in psoriatic patients in comparison with the healthy controls ( p < 0.001 ) .", "a significant positive correlation was found between the il-22 concentration and psoriasis severity measured by both the pasi and bsa score ; that is , p < 0.001 ; r = 0.557 and p < 0.001 ; r = 0.559 , respectively . ", "il-22 is a member of the il-10 cytokines family and is mainly produced by th17 , th22 , and mucosal nk cells [ 3032 ] .", "il-22 upregulates keratinocyte proliferation and migration , inhibits keratinocyte differentiation by downregulating a variety of genes as filaggrin and involucrin genes [ 33 , 34 ] , and augments the expression of inflammatory molecules by keratinocytes , which leads to an increase in skin thickness in vitro and in vivo [ 3537 ] .", "il-22 increased the expression of the hbd-2 and hbd-3 in human keratinocytes and mmp1 and mmp3 in the skin [ 3840 ] . in psoriasis", ", il-22 is overexpressed most probably as a result of upregulated il-23 and il-6 levels [ 12 , 41 , 42 ] .", "the treatment with tnf inhibitor ( etanercept ) reduced serum levels of il-17 and il-22 . ", "no significant differences were found in the il-12 and il-23 concentrations between the psoriatic patients and control group ( p > 0.05 ) . il-23", "together with il-12 belongs to the il-12 family and are both structurally related ; il-12 is formed by the p40 and p35 subunits ; il-23 consists of p40 and p19 subunits [ 44 , 45 ] . ", "although both il-12 and il-23 are present in psoriasis , studies support that il-23 , rather than il-12 , is crucial during the pathogenesis of psoriasis .", "il-23 is overexpressed in psoriasis lesional skin , as shown for example , by increased p40 and p19 mrna levels but not always p35 [ 4654 ] .", "il-23 is overproduced by dermal dendritic cells [ 48 , 53 ] and keratinocytes in lesional psoriatic skin . ", "most recent reports show no increased expression of the il-12 in psoriasis [ 3 , 49 , 52 , 53 ] .", "statistically significant differences in serum il-12 level have been found in psoriatic patients comparing to healthy controls [ 20 , 56 ] . however , there are no significant studies comparing the serum levels of il-23 in psoriatic patients and controls . ", "no significant differences were found in the serum il-17 concentrations between the psoriatic patients and control group ( p > 0.05 ) . a significant positive correlation between the", "il-17 concentrations and psoriasis severity measured by the pasi was detected ( p < 0.05 ; r = 0.277 ) . ", "il-17 ( il-17a ) is a member of a newly identified cytokine family comprising il-17a , il-17b , il-17c , il-17d , il-17e ( il-25 ) , and il-17f . il-17", "and il-17f have a proinflammatory activity inducing the expression of proinflammatory cytokines , colony - stimulating factors , and chemokines from dendritic cells , neutrophils , t cells , monocyte / macrophages , and epithelial cells [ 11 , 44 ] .", "il-17a and il-17f can mobilize , recruit , and activate neutrophils [ 44 , 57 ] . ", "il-17 is undetectable in normal skin , and biological therapy that inhibits th17 pathways results in reduced expression of il-17 and il-23 and improved disease outcomes [ 11 , 24 , 39 ] .", "th17 cells and the cytokines produced by these cells are found in increased levels within skin affected by psoriasis [ 31 , 4749 , 5860 ] .", "statistically significant differences in serum il-17a level have been found in psoriatic patients comparing to healthy controls .", "il-17 serum levels correlated with the psoriasis area and severity index ( pasi ) . ", "in addition , an analysis of mutual correlations between the concentrations of selected cytokines in the psoriatic patients was conducted .", "a significant positive correlation between the il-23 and il-17 values was found ( p < 0.05 ; r = 0.271 ) .", "an increase in the il-23 concentration was accompanied by an increase in the il-17 concentration . in previous studies", ", it was found that interaction of il-23 with its receptor on th17 cells stimulates the production of il-17 and other related proinflammatory cytokines activates nk cells and regulates antibody production [ 1 , 3 , 44 ] .", " we believe that the results of our study confirm involvement of th17 and th22 cytokines in psoriasis pathogenesis .", "elevated il-22 level without increase of il-17 level may suggest that th22 role is more significant in the inflammatory process of psoriasis .", "very high concentrations of il-22 in the patients ' serum can be connected with intensive il-6 stimulation . il-22 and il-20 itself induce production of il-20 , which is elevated in our study .", "it seems that il-6 , which initiates th17 and th22 pathways in psoriasis , may be helpful in the clinical practice as a soluble biomarker of the disease activity and its prognosis .", "the development of new therapeutical strategies targeting the initial step of cytokine network activation , for example , il-6 , may reduce the next events of inflammatory reactions and prevent the psoriasis exacerbation and systemic complications .", "furthermore , serum levels of il-20 , il-22 , and il-17 , which correlated with the clinical severity and activity of psoriasis , may be objective parameters of successful treatment and may be used in the followup ." ]

[ "<S> \n introduction . psoriasis is a t cell - mediated inflammatory disease in which pathogenesis t helper ( th ) lymphocytes ( th1 , th17 , and th22 ) play an important role . </S>", "<S> the aim of the study was to assess the serum levels of some cytokines involved in the th17 and th22 responses in psoriatic patients . </S>", "<S> material and methods . </S>", "<S> the study comprised 60 psoriatic patients and 30 healthy controls . in the serum collected from psoriatic patients and healthy controls , the concentrations of il-6 , il-12 , il-17 , il-20 , il-22 , and </S>", "<S> il-23 were examined with elisa kits . </S>", "<S> severity of psoriatic skin lesions was assessed by means of pasi , bsa , and pga scores . </S>", "<S> results . </S>", "<S> il-6 , il-20 , and il-22 concentrations were significantly higher in psoriatic patients in comparison with the control group . </S>", "<S> the positive correlations between the concentrations of il-22 and il-20 and severity of psoriasis assessed with pasi and bsa scores as well as il-17 and pasi score were found . </S>", "<S> there was also a positive correlation between il-23 and il-17 concentrations </S>", "<S> . conclusions </S>", "<S> . results of the conducted studies suggest that th22 response may contribute to the skin and systemic inflammatory disease in psoriasis . </S>", "<S> it seems that early identification of soluble biomarkers and initiation of well - matched treatment may prevent exacerbation and progression of psoriasis . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Conclusions" ]

[ [ "the knowledge about the role of cytokines in psoriasis has developed in the last several years .", "initially , only th1 cells and cytokines secreted by these cells , like tnf- , ifn- , and il-2 , have been associated with the development and maintenance of chronic inflammatory diseases , such as psoriasis .", "th1 cells differentiate from naive cd4 + cells in the presence of il-12 , il-18 , and ifn and . it is well known that th1 cytokines have strong inflammatory effects in activating macrophage , neutrophil , and cd8 + cytotoxic t cells . in the 1990s ,", "th17 cells were described as a new t - cell population that produces il-17 , il-6 , il-21 , il-22 , and tnf . transforming growth factor ( tgf)-1 , il-6 , il-23 , and il-15 stimulated initial th17 differentiation from naive t cells .", "tgf-1 is secreted by activated t cells and it initiates t cell and fibroblast activation , as well as angiogenesis and neovascularization [ 37 ] .", "il-6 , secreted by macrophages , endothelial cells , and epithelial cells , is responsible for augmentation of keratinocyte hyperplasia and invasion of macrophages and t cells [ 3 , 8 ] .", "il-15 , produced by monocytes , macrophages , dcs , and t cells , can appear to induce angiogenesis , immune cell recruitment , and activation of keratinocytes [ 4 , 9 , 10 ] .", "cytokines produced by th17 cells were found to initiate acanthosis , hyperkeratosis , and parakeratosis .", "th17 cells demonstrated involvement in neutrophil and monocyte chemotaxis , t - cell migration and activation , and neovascularization .", "th22 cells have been recently described as inflammatory cd4 + t cells that produce cytokines such as il-22 , il-26 , and il-13 of which il-22 is the most important functional cytokine .", "recent studies indicate that il-6 and tnf- , along with the help of plasmacytoid dendritic cells , can promote the th22 phenotype [ 14 , 16 ] . ", "the differentiation of main three t cell subsets involved in the pathogenesis of psoriasis , as well as th1 , th17 , and th22 cytokine production are illustrated in figure 1 . ", "the aim of the study was to assess the serum levels of some cytokines involved in the th17 and th22 responses in psoriatic patients ." ], [ " the study comprised 60 psoriatic patients , 50 males ( 83.33% ) and 10 females ( 16.67% ) , as well as 30 healthy controls .", "the studied patients ' age was between 18 and 69 years , 45.6 13.2 years on average .", "the history of the disease was from 1 to 45 years , 18.7 11.5 years on average .", "thirty - four of the studied patients ( 40% ) reported two psoriasis exacerbations a year , whereas 9 persons ( 15% ) observed as many as 4 exacerbations a year . ", "the skin lesions severity was assessed with the use of psoriasis area and severity index ( pasi ) , body surface area ( bsa ) , and physician global assessment ( pga ) scores .", "the pasi value in the studied group was from 4.8 to 64.2 and 15.7 9.7 on average .", "the bsa value was in the range of 9.096.9% and 31.4 18.2 on average .", "the pga score was 3 in 24 individuals ( 40% ) , 4 in another 24 individuals ( 40% ) , and 5 in 12 individuals ( 20% ) . ", "blood samples were collected from psoriatic patients and controls and were centrifuged for 15 minutes at 1000 g . then , serum samples were subdivided into small aliquots to be stored at 80c until tested for cytokine levels .", "elisa kits were used to determine il-6 , il-12 , il-17 , il-20 , il-22 , and il-23 ( r&d systems , minneapolis , mn , usa ) serum levels , according to the manufacturer 's instructions . ", "continuous variables were presented as mean standard deviation , while categorical variables were presented as absolute and relative frequencies .", "mann - whitney 's u test was used to compare continuous data between the studied and control groups ." ], [ " the statistical analyses of the conducted study results revealed significantly higher serum levels of il-6 , il-20 , and il-23 in psoriatic patients comparing to healthy controls ( table 1 and figure 2 ) . significant correlations between il-20 , il-22 , il-17 , and psoriasis severity were observed .", "no significant correlations were found between the concentrations of the studied cytokines and the sex , age , or psoriasis duration in the studied patients ( p > 0.05 ) .", "significantly higher il-6 values were found in the psoriatic patients in comparison to the control group ( p < 0.001 ) . ", "il-6 contributes to the th17 cell line 's involvement in numerous processes of inflammation and autoimmunity by preventing the proliferation of t regulatory cells .", "il-17 can induce fibroblasts to produce il-6 potentially activating a positive feedback loop that strengthens th17 inflammation . ", "analyzed 78 studies comparing the serum inflammatory markers , including il-6 , in psoriasis with healthy controls .", "the study showed that the standardized mean differences were higher in psoriatic patients compared to healthy controls for il-6 .", "elevated serum il-6 appears to be associated with greater psoriasis severity [ 18 , 21 , 24 ] .", "it was shown that il-6 might be the biomarker differentiating psoriasis arthritis from psoriasis without joint involvement [ 19 , 22 ] . ", "significantly higher il-20 values were found in the psoriatic patients in comparison to the control group ( p < 0.001 ) . a significant positive correlation between the", "il-20 concentrations and psoriasis severity measured by the pasi was detected ( p < 0.001 ; r = 0.698 ) . ", "il-20 is produced by keratinocytes in the presence of il-22 , tnf- , and il-17 but not ifn- or il-20 itself [ 15 , 25 ] .", "il-20 can play an important role in the later effector phase of psoriasis pathogenesis , in which it inhibits the terminal differentiation , increases antimicrobial competence , and production of chemokines for neutrophils in keratinocytes [ 25 , 29 ] . ", "there are not many studies concerning il-20 serum level , but increased levels of il-20 were noted in lesional skin as well as in the blood in psoriatic patients . as in our study , il-20 serum levels correlated with pasi scores . ", "a significantly higher increase in il-22 was observed in psoriatic patients in comparison with the healthy controls ( p < 0.001 ) .", "a significant positive correlation was found between the il-22 concentration and psoriasis severity measured by both the pasi and bsa score ; that is , p < 0.001 ; r = 0.557 and p < 0.001 ; r = 0.559 , respectively . ", "il-22 is a member of the il-10 cytokines family and is mainly produced by th17 , th22 , and mucosal nk cells [ 3032 ] .", "il-22 upregulates keratinocyte proliferation and migration , inhibits keratinocyte differentiation by downregulating a variety of genes as filaggrin and involucrin genes [ 33 , 34 ] , and augments the expression of inflammatory molecules by keratinocytes , which leads to an increase in skin thickness in vitro and in vivo [ 3537 ] .", "il-22 increased the expression of the hbd-2 and hbd-3 in human keratinocytes and mmp1 and mmp3 in the skin [ 3840 ] . in psoriasis", ", il-22 is overexpressed most probably as a result of upregulated il-23 and il-6 levels [ 12 , 41 , 42 ] .", "the treatment with tnf inhibitor ( etanercept ) reduced serum levels of il-17 and il-22 . ", "no significant differences were found in the il-12 and il-23 concentrations between the psoriatic patients and control group ( p > 0.05 ) . il-23", "together with il-12 belongs to the il-12 family and are both structurally related ; il-12 is formed by the p40 and p35 subunits ; il-23 consists of p40 and p19 subunits [ 44 , 45 ] . ", "although both il-12 and il-23 are present in psoriasis , studies support that il-23 , rather than il-12 , is crucial during the pathogenesis of psoriasis .", "il-23 is overexpressed in psoriasis lesional skin , as shown for example , by increased p40 and p19 mrna levels but not always p35 [ 4654 ] .", "il-23 is overproduced by dermal dendritic cells [ 48 , 53 ] and keratinocytes in lesional psoriatic skin . ", "most recent reports show no increased expression of the il-12 in psoriasis [ 3 , 49 , 52 , 53 ] .", "statistically significant differences in serum il-12 level have been found in psoriatic patients comparing to healthy controls [ 20 , 56 ] . however , there are no significant studies comparing the serum levels of il-23 in psoriatic patients and controls . ", "no significant differences were found in the serum il-17 concentrations between the psoriatic patients and control group ( p > 0.05 ) . a significant positive correlation between the", "il-17 concentrations and psoriasis severity measured by the pasi was detected ( p < 0.05 ; r = 0.277 ) . ", "il-17 ( il-17a ) is a member of a newly identified cytokine family comprising il-17a , il-17b , il-17c , il-17d , il-17e ( il-25 ) , and il-17f . il-17", "and il-17f have a proinflammatory activity inducing the expression of proinflammatory cytokines , colony - stimulating factors , and chemokines from dendritic cells , neutrophils , t cells , monocyte / macrophages , and epithelial cells [ 11 , 44 ] .", "il-17a and il-17f can mobilize , recruit , and activate neutrophils [ 44 , 57 ] . ", "il-17 is undetectable in normal skin , and biological therapy that inhibits th17 pathways results in reduced expression of il-17 and il-23 and improved disease outcomes [ 11 , 24 , 39 ] .", "th17 cells and the cytokines produced by these cells are found in increased levels within skin affected by psoriasis [ 31 , 4749 , 5860 ] .", "statistically significant differences in serum il-17a level have been found in psoriatic patients comparing to healthy controls .", "il-17 serum levels correlated with the psoriasis area and severity index ( pasi ) . ", "in addition , an analysis of mutual correlations between the concentrations of selected cytokines in the psoriatic patients was conducted .", "a significant positive correlation between the il-23 and il-17 values was found ( p < 0.05 ; r = 0.271 ) .", "an increase in the il-23 concentration was accompanied by an increase in the il-17 concentration . in previous studies", ", it was found that interaction of il-23 with its receptor on th17 cells stimulates the production of il-17 and other related proinflammatory cytokines activates nk cells and regulates antibody production [ 1 , 3 , 44 ] ." ], [ " we believe that the results of our study confirm involvement of th17 and th22 cytokines in psoriasis pathogenesis .", "elevated il-22 level without increase of il-17 level may suggest that th22 role is more significant in the inflammatory process of psoriasis .", "very high concentrations of il-22 in the patients ' serum can be connected with intensive il-6 stimulation . il-22 and il-20 itself induce production of il-20 , which is elevated in our study .", "it seems that il-6 , which initiates th17 and th22 pathways in psoriasis , may be helpful in the clinical practice as a soluble biomarker of the disease activity and its prognosis .", "the development of new therapeutical strategies targeting the initial step of cytokine network activation , for example , il-6 , may reduce the next events of inflammatory reactions and prevent the psoriasis exacerbation and systemic complications .", "furthermore , serum levels of il-20 , il-22 , and il-17 , which correlated with the clinical severity and activity of psoriasis , may be objective parameters of successful treatment and may be used in the followup ." ] ]

[ "chlamydia genital infection is the most common bacterial infection sexually transmitted in the world ( 1 ) . the highest age - specific rate was reported in females aged 15 - 35 years .", "the majority of the females are asymptomatic ( 2 ) , thus , providing a continuous reservoir for the infection ( 3 ) .", "it is estimated that 5 - 12% of sexually active adults in the age group of 16 - 34 years in the united kingdom are infected with chlamydia trachomatis ( 4 ) . according to the world health organization ( who )", ", 101.5 million people with chlamydia infection are detected globally each year ( 5 ) .", "the infection risk factors include being young or adolescent , having new or frequent sexual contacts , inconsistent use of barrier contraception , history of prior sexual transmitted infection , and low educational and socioeconomic levels ( 6 ) .", "thus , the prevalence of c. trachomatis may differ among racial groups because of differences in sexual risk behavior and cultural backgrounds ( 7 ) .", "untreated chlamydial infections may lead to pelvic inflammatory disease ( pid ) , ectopic pregnancy , premature delivery , spontaneous abortion , low birth weight , tubal infertility and subsequent scarring of the fallopian tubes ( 8) . during pregnancy ,", "chlamydial infection can cause miscarriage , premature rupture of membranes , preterm labor , low birth weight , infant mortality , neonatal chlamydial infection , and postpartum endometritis ( 9 ) .", "screening , early diagnosis and treatment is considered the main policy to prevent complications and further transmission of the infection ( 10 ) .", "conventional laboratory diagnosis of c. trachomatis infection is done by cell culture or antigen detection .", "commercially available naats using methods such as polymerase chain reaction ( pcr ) , and ligase chain reaction ( lcr ) , are now the gold standard tests for genital chlamydia infection ( 11 ) .", "pcr has good sensitivity ( 100% ) and specificity ( 99.3% ) for endocervical samples compared with urethral culture .", "it is the preferred test and the most commonly used method of diagnosis , which amplifies c. trachomatis dna sequences ( 12 ) .", "there are no accurate data regarding the prevalence of c. trachomatis infection in kashan and few studies from iran examined the prevalence of genital chlamydia infection based on age .", "the current study aimed to assess the prevalence of endocervical c. trachomatis infection in a population of females aged 17 - 35 years in kashan , iran .", "endocervical swab samples were collected by the gynecologists from 255 married females aged 17 - 35 years , with or without symptoms , referring to the obstetrics and gynecology clinics of kashan , iran , from december 2012 to july 2013 .", "the study was approved by the regional research ethics committee of kashan university of medical sciences ( p 2331 ) , and all participants signed informed consent forms prior to enrolment in the study .", "demographics , sexual behavior , method of contraception , previous history of sexually transmitted infections ( stis ) and symptoms were assessed by a questionnaire routinely used in the clinic .", "cervical samples were collected from the endocervical region after inserting a sterile speculum into the vagina .", "mucus was cleaned with a sterile cotton swab without causing any bleeding or using antiseptics .", "dacron swabs were used to collect endocervical specimens . the specimen placed into sterile collection tubes containing 2 ml of transport buffer ( nah2po4 20 mm , na2hpo4 20 mm , sucrose 20 mm", ", glutamin 5 mm , gentamycin 50 g / ml , vancomycin 100 g / ml , amphotericin b 25 g / ml supplemented with 10% of inactivated fetal calf serum ) and transported to the laboratory for further processing .", "pcr was set up using positive control dna from c. trachomatis serovar l2 type strain 434/bu ( atcc vr-902b ) . for the negative control ,", "the dna was isolated using a kiaspin pcr template purification kit , ( kiagen , iran ) according to the manufacturer s instructions .", "briefly , samples were vortexed and centrifuged for 30 minutes at 14000 g. then , dna purification was performed , 200 l of each specimen was transferred and mixed with 300 l of buffer lb and 2 l of dna / rna carrier and 5 l of proteinase k in a 1.5 ml microcentrifuge tube and incubated at 65c for 10 minutes .", "after incubation , 100 l of isopropanol was added to the tubes , mixed and centrifuged for 2 minutes at 5000 g. the supernatant was applied to the spin column and centrifuged at 12000 g for 1 minute , after which the filtrate was discarded and 500 l of buffers irb and 600 l of wb were added to each spin column and centrifuged again at 12000 g for 1 minute .", "chlamydia trachomatis was detected by a pcr designed in the laboratory to amplify a sequence in the cryptic plasmid generating a fragment of about 512 base pair .", "primers were hl1 forward , 5-tagagataggaaaccaactc-3and hl2 reverse , 5ctcgggttaatgttgcatga-3 ( 13 ) ( cinnagen , tehran , iran ) .", "amplification reactions were performed in a final volume of 25 l containing 5 l of sample , 12.5 l of the master mix pcr solution ( cinnagen , tehran , iran ) , and 1 l of each reverse and forward primers .", "the reaction mixture was incubated for 5 minutes at 94c , followed by 40 cycles of 1 minute at 94c , 1 minute at 45c , and 1 minute at 72c , and a final elongation step of 7 minutes at 72c .", "the pcr products were analyzed by electrophoresis in the 1% tbe buffer through 1.5% agarose gel , stained with ethidium bromide 0.5 g / ml and dna bands were visualized using a uv transilluminator ( figure 1 ) .", "lane m : marker ; lane 1 : negative control ; lane 2 : positive control ; lane 3 - 6 : positive samples ; 7 : negative sample .", "data were analyzed using spss version 16 and the outcome variables including prevalence rates and their 95% confidence intervals ( cis ) were calculated . the chi - square or fisher s exact test was used to analyze the categorical data .", "endocervical swab samples were collected by the gynecologists from 255 married females aged 17 - 35 years , with or without symptoms , referring to the obstetrics and gynecology clinics of kashan , iran , from december 2012 to july 2013 .", "the study was approved by the regional research ethics committee of kashan university of medical sciences ( p 2331 ) , and all participants signed informed consent forms prior to enrolment in the study .", "demographics , sexual behavior , method of contraception , previous history of sexually transmitted infections ( stis ) and symptoms were assessed by a questionnaire routinely used in the clinic .", "cervical samples were collected from the endocervical region after inserting a sterile speculum into the vagina .", "mucus was cleaned with a sterile cotton swab without causing any bleeding or using antiseptics .", "dacron swabs were used to collect endocervical specimens . the specimen placed into sterile collection tubes containing 2 ml of transport buffer ( nah2po4 20 mm , na2hpo4 20 mm , sucrose 20 mm", ", glutamin 5 mm , gentamycin 50 g / ml , vancomycin 100 g / ml , amphotericin b 25 g / ml supplemented with 10% of inactivated fetal calf serum ) and transported to the laboratory for further processing .", "pcr was set up using positive control dna from c. trachomatis serovar l2 type strain 434/bu ( atcc vr-902b ) .", "for the negative control , sterile deionized water was used as the pcr template instead of nucleic acid .", "the dna was isolated using a kiaspin pcr template purification kit , ( kiagen , iran ) according to the manufacturer s instructions .", "briefly , samples were vortexed and centrifuged for 30 minutes at 14000 g. then , dna purification was performed , 200 l of each specimen was transferred and mixed with 300 l of buffer lb and 2 l of dna / rna carrier and 5 l of proteinase k in a 1.5 ml microcentrifuge tube and incubated at 65c for 10 minutes .", "after incubation , 100 l of isopropanol was added to the tubes , mixed and centrifuged for 2 minutes at 5000 g. the supernatant was applied to the spin column and centrifuged at 12000 g for 1 minute , after which the filtrate was discarded and 500 l of buffers irb and 600 l of wb were added to each spin column and centrifuged again at 12000 g for 1 minute .", "chlamydia trachomatis was detected by a pcr designed in the laboratory to amplify a sequence in the cryptic plasmid generating a fragment of about 512 base pair .", "primers were hl1 forward , 5-tagagataggaaaccaactc-3and hl2 reverse , 5ctcgggttaatgttgcatga-3 ( 13 ) ( cinnagen , tehran , iran ) .", "amplification reactions were performed in a final volume of 25 l containing 5 l of sample , 12.5 l of the master mix pcr solution ( cinnagen , tehran , iran ) , and 1 l of each reverse and forward primers .", "the reaction mixture was incubated for 5 minutes at 94c , followed by 40 cycles of 1 minute at 94c , 1 minute at 45c , and 1 minute at 72c , and a final elongation step of 7 minutes at 72c .", "the pcr products were analyzed by electrophoresis in the 1% tbe buffer through 1.5% agarose gel , stained with ethidium bromide 0.5 g / ml and dna bands were visualized using a uv transilluminator ( figure 1 ) .", "lane m : marker ; lane 1 : negative control ; lane 2 : positive control ; lane 3 - 6 : positive samples ; 7 : negative sample .", "data were analyzed using spss version 16 and the outcome variables including prevalence rates and their 95% confidence intervals ( cis ) were calculated .", "the chi - square or fisher s exact test was used to analyze the categorical data", "the age of the 255 subjects ranged 17 - 35 years with a mean of 27.3 4.37 years .", "the majority of them were married or living with a partner ( 98% ) , had high school education or higher ( 74% ) .", "the prevalence of c. trachomatis was 2.4% ( 95% confidence interval [ ci ] 0.54 - 4.26% ) with a mean of 25.8 6.17 years ; 50% ( 3/6 ) of the subjects with a positive chlamydia test result were under the age of 25 .", "of the 92 subjects in the 25-year - old group , 3 ( 3.3% ) were positive for c. trachomatis compared to three out of 163 ( 1.8% ) in the > 25-year - group ; 83.3% ( 5/6 ) of the subjects with a positive chlamydia test result were older than 16 at first sexual encounter and all of the infected subjects lived in the city .", "the most general symptoms among c. trachomatis infected young subjects were vaginal discharge ( 66.6% ) and lumbar pain ( 50% ) .", "no significant relationships were found between c. trachomatis infection and the risk factors , probably reflecting the small number of those who were positive in the test .", "the prevalence of c. trachomatis genital infections among young females in kashan , iran was 2.4% .", "the previous published reports from iran showed various prevalence of 2% to 11% ( 14 ) .", "the study by chamani - tabriz et al . on the prevalence of chlamydia infection in females aged 15 - 42 attending obstetrics and gynecology clinics in tehran , from may 2003 to october 2003 showed that 12.6% were positive for chlamydia by pcr ( 15 ) . in another study on the females < 45 years attending gynecology clinics in babol , iran ,", "european centre for disease prevention and control ( ecdc ) described the prevalence ranging from 1.4% to 3.0% among females aged 15 - 40 years ( 17 ) .", "the prevalence of c. trachomatis infection and the risk factors in individuals < 35 years old attending sexual health clinics in barcelona province , spain , in 2006 was 3.8% and the prevalence was significantly higher in those under 25 years ( 5.8% ) compared with the ones aged 25 - 35 ( 1.6% ) ( 18 ) .", "another study in melbourne , australia , showed that the chlamydia prevalence was 3.7% among sexually active females aged 18 - 24 years compared with 0.2% among 25 - 35-year - old ones ( 19 ) .", "a study among the brazilian females referred to sexually transmitted diseases ( std ) clinic showed that 68% of the infected ones aged 15 - 25 year ( 13 ) . in the current study , the c. trachomatis prevalence in females", " 25 years ( 3.2% ) was similar to those of reported in australia 4.9% ( 20 ) , spain 4% ( 21 ) , france 3.6% ( 22 ) and the united kingdom 3% ( 23 ) .", "the current cdc guidelines recommend annual screening for chlamydia in sexually active females 25 years and the ones > 25 years old who are at increased risk ( 24 ) .", "screening is especially important in pregnant females who are at risk of transmission of their infection to the fetus ( 25 ) .", "chlamydia trachomatis infection is the main causative agent of abortion ( 26 ) . in the current study , abortions were reported by 16.7% of females with positive test results .", "early detection and appropriate treatment of c. trachomatis can prevent the development of the disease after abortion ( 27 ) .", "prevalence of c. trachomatis was found to be low among females in kashan , iran .", "the reason for c. trachomatis infection low prevalence in the population under study was the scarcity of pre - marriage sexual intercourse frequency in females , having stable sexual intercourse only after marriage .", "to date it was the first study in kashan to determine the prevalence of endocervical c. trachomatis infection in young females .", "there were several limitations to the study , which should be considered in the interpretation of the results .", "data on sexual behavior was not specifically collected for the current study . knowing a patient s number of sexual partners , sexual activity before and after sti diagnosis , and frequency of condom use may help identify the patients who are at increased infection risk .", "chlamydia prevalence was low in young sexually - active females , but there is a significant difference regarding chlamydia infection in females younger than 25 years .", "the new information provides evidence for the need to implement active opportunity to screen young sexually active females and the patients in all socioeconomic groups .", "all sexually active females should be offered a c. trachomatis test when attending a physician ." ]

[ "<S> background : chlamydia trachomatis is one of the major bacterial agents of the sexually transmitted diseases worldwide , especially among young females . </S>", "<S> there is no data regarding the prevalence of genital chlamydia infection among young females in kashan , iran.objectives:the current study aimed to determine the prevalence of endocervical c. trachomatis infection among females aged 17 - 35 years in kashan , iran.patients and methods : in the current descriptive study , 255 endocervical swab samples were collected from the obstetrics and gynecology clinics of kashan , iran from december 2012 to july 2013 . </S>", "<S> cervical swabs were placed in transport media and sent to the laboratory . to identify c. trachomatis in the samples polymerase chain reaction ( pcr ) </S>", "<S> was performed to amplify a sequence in the cryptic plasmid , generating a fragment of about 512base pair . </S>", "<S> demographic data was collected considering the relevant risk factors by a standard questionnaire.results:a total of 255 females were tested . the prevalence of genital c. trachomatis was 2.4% ( 95% confidence interval [ ci ] 0.54% - 4.26% ) ; 3.2% of the females in the 25-year - old group were positive versus 1.8% in the 26 - 35-year - old group . </S>", "<S> the most general presented symptoms of genital c. trachomatis infection were vaginal discharge ( 66.6% ) and lumbar pain ( 50% ) . </S>", "<S> no significant relationships were found between c. trachomatis infection and the risk factors.conclusions:to the authors knowledge this is the first study to describe endocervical c. trachomatis infection in this area . </S>", "<S> the obtained results also emphasized the importance of routine diagnosis of c. trachomatis to control of the infection . </S>" ]

[ "1. Background", "2. Objectives", "3. Patients and Methods", "3.1. Clinical Specimens", "3.2. Bacterial Strains", "3.3. DNA Extraction", "3.4. PCR Assay", "3.5. Statistical Analysis ", "4. Results", "5. Discussion" ]

[ [ "chlamydia genital infection is the most common bacterial infection sexually transmitted in the world ( 1 ) . the highest age - specific rate was reported in females aged 15 - 35 years .", "the majority of the females are asymptomatic ( 2 ) , thus , providing a continuous reservoir for the infection ( 3 ) .", "it is estimated that 5 - 12% of sexually active adults in the age group of 16 - 34 years in the united kingdom are infected with chlamydia trachomatis ( 4 ) . according to the world health organization ( who )", ", 101.5 million people with chlamydia infection are detected globally each year ( 5 ) .", "the infection risk factors include being young or adolescent , having new or frequent sexual contacts , inconsistent use of barrier contraception , history of prior sexual transmitted infection , and low educational and socioeconomic levels ( 6 ) .", "thus , the prevalence of c. trachomatis may differ among racial groups because of differences in sexual risk behavior and cultural backgrounds ( 7 ) .", "untreated chlamydial infections may lead to pelvic inflammatory disease ( pid ) , ectopic pregnancy , premature delivery , spontaneous abortion , low birth weight , tubal infertility and subsequent scarring of the fallopian tubes ( 8) . during pregnancy ,", "chlamydial infection can cause miscarriage , premature rupture of membranes , preterm labor , low birth weight , infant mortality , neonatal chlamydial infection , and postpartum endometritis ( 9 ) .", "screening , early diagnosis and treatment is considered the main policy to prevent complications and further transmission of the infection ( 10 ) .", "conventional laboratory diagnosis of c. trachomatis infection is done by cell culture or antigen detection .", "commercially available naats using methods such as polymerase chain reaction ( pcr ) , and ligase chain reaction ( lcr ) , are now the gold standard tests for genital chlamydia infection ( 11 ) .", "pcr has good sensitivity ( 100% ) and specificity ( 99.3% ) for endocervical samples compared with urethral culture .", "it is the preferred test and the most commonly used method of diagnosis , which amplifies c. trachomatis dna sequences ( 12 ) .", "there are no accurate data regarding the prevalence of c. trachomatis infection in kashan and few studies from iran examined the prevalence of genital chlamydia infection based on age ." ], [ "the current study aimed to assess the prevalence of endocervical c. trachomatis infection in a population of females aged 17 - 35 years in kashan , iran ." ], [ "endocervical swab samples were collected by the gynecologists from 255 married females aged 17 - 35 years , with or without symptoms , referring to the obstetrics and gynecology clinics of kashan , iran , from december 2012 to july 2013 .", "the study was approved by the regional research ethics committee of kashan university of medical sciences ( p 2331 ) , and all participants signed informed consent forms prior to enrolment in the study .", "demographics , sexual behavior , method of contraception , previous history of sexually transmitted infections ( stis ) and symptoms were assessed by a questionnaire routinely used in the clinic .", "cervical samples were collected from the endocervical region after inserting a sterile speculum into the vagina .", "mucus was cleaned with a sterile cotton swab without causing any bleeding or using antiseptics .", "dacron swabs were used to collect endocervical specimens . the specimen placed into sterile collection tubes containing 2 ml of transport buffer ( nah2po4 20 mm , na2hpo4 20 mm , sucrose 20 mm", ", glutamin 5 mm , gentamycin 50 g / ml , vancomycin 100 g / ml , amphotericin b 25 g / ml supplemented with 10% of inactivated fetal calf serum ) and transported to the laboratory for further processing .", "pcr was set up using positive control dna from c. trachomatis serovar l2 type strain 434/bu ( atcc vr-902b ) . for the negative control ,", "the dna was isolated using a kiaspin pcr template purification kit , ( kiagen , iran ) according to the manufacturer s instructions .", "briefly , samples were vortexed and centrifuged for 30 minutes at 14000 g. then , dna purification was performed , 200 l of each specimen was transferred and mixed with 300 l of buffer lb and 2 l of dna / rna carrier and 5 l of proteinase k in a 1.5 ml microcentrifuge tube and incubated at 65c for 10 minutes .", "after incubation , 100 l of isopropanol was added to the tubes , mixed and centrifuged for 2 minutes at 5000 g. the supernatant was applied to the spin column and centrifuged at 12000 g for 1 minute , after which the filtrate was discarded and 500 l of buffers irb and 600 l of wb were added to each spin column and centrifuged again at 12000 g for 1 minute .", "chlamydia trachomatis was detected by a pcr designed in the laboratory to amplify a sequence in the cryptic plasmid generating a fragment of about 512 base pair .", "primers were hl1 forward , 5-tagagataggaaaccaactc-3and hl2 reverse , 5ctcgggttaatgttgcatga-3 ( 13 ) ( cinnagen , tehran , iran ) .", "amplification reactions were performed in a final volume of 25 l containing 5 l of sample , 12.5 l of the master mix pcr solution ( cinnagen , tehran , iran ) , and 1 l of each reverse and forward primers .", "the reaction mixture was incubated for 5 minutes at 94c , followed by 40 cycles of 1 minute at 94c , 1 minute at 45c , and 1 minute at 72c , and a final elongation step of 7 minutes at 72c .", "the pcr products were analyzed by electrophoresis in the 1% tbe buffer through 1.5% agarose gel , stained with ethidium bromide 0.5 g / ml and dna bands were visualized using a uv transilluminator ( figure 1 ) .", "lane m : marker ; lane 1 : negative control ; lane 2 : positive control ; lane 3 - 6 : positive samples ; 7 : negative sample .", "data were analyzed using spss version 16 and the outcome variables including prevalence rates and their 95% confidence intervals ( cis ) were calculated . the chi - square or fisher s exact test was used to analyze the categorical data ." ], [ "endocervical swab samples were collected by the gynecologists from 255 married females aged 17 - 35 years , with or without symptoms , referring to the obstetrics and gynecology clinics of kashan , iran , from december 2012 to july 2013 .", "the study was approved by the regional research ethics committee of kashan university of medical sciences ( p 2331 ) , and all participants signed informed consent forms prior to enrolment in the study .", "demographics , sexual behavior , method of contraception , previous history of sexually transmitted infections ( stis ) and symptoms were assessed by a questionnaire routinely used in the clinic .", "cervical samples were collected from the endocervical region after inserting a sterile speculum into the vagina .", "mucus was cleaned with a sterile cotton swab without causing any bleeding or using antiseptics .", "dacron swabs were used to collect endocervical specimens . the specimen placed into sterile collection tubes containing 2 ml of transport buffer ( nah2po4 20 mm , na2hpo4 20 mm , sucrose 20 mm", ", glutamin 5 mm , gentamycin 50 g / ml , vancomycin 100 g / ml , amphotericin b 25 g / ml supplemented with 10% of inactivated fetal calf serum ) and transported to the laboratory for further processing ." ], [ "pcr was set up using positive control dna from c. trachomatis serovar l2 type strain 434/bu ( atcc vr-902b ) .", "for the negative control , sterile deionized water was used as the pcr template instead of nucleic acid ." ], [ "the dna was isolated using a kiaspin pcr template purification kit , ( kiagen , iran ) according to the manufacturer s instructions .", "briefly , samples were vortexed and centrifuged for 30 minutes at 14000 g. then , dna purification was performed , 200 l of each specimen was transferred and mixed with 300 l of buffer lb and 2 l of dna / rna carrier and 5 l of proteinase k in a 1.5 ml microcentrifuge tube and incubated at 65c for 10 minutes .", "after incubation , 100 l of isopropanol was added to the tubes , mixed and centrifuged for 2 minutes at 5000 g. the supernatant was applied to the spin column and centrifuged at 12000 g for 1 minute , after which the filtrate was discarded and 500 l of buffers irb and 600 l of wb were added to each spin column and centrifuged again at 12000 g for 1 minute ." ], [ "chlamydia trachomatis was detected by a pcr designed in the laboratory to amplify a sequence in the cryptic plasmid generating a fragment of about 512 base pair .", "primers were hl1 forward , 5-tagagataggaaaccaactc-3and hl2 reverse , 5ctcgggttaatgttgcatga-3 ( 13 ) ( cinnagen , tehran , iran ) .", "amplification reactions were performed in a final volume of 25 l containing 5 l of sample , 12.5 l of the master mix pcr solution ( cinnagen , tehran , iran ) , and 1 l of each reverse and forward primers .", "the reaction mixture was incubated for 5 minutes at 94c , followed by 40 cycles of 1 minute at 94c , 1 minute at 45c , and 1 minute at 72c , and a final elongation step of 7 minutes at 72c .", "the pcr products were analyzed by electrophoresis in the 1% tbe buffer through 1.5% agarose gel , stained with ethidium bromide 0.5 g / ml and dna bands were visualized using a uv transilluminator ( figure 1 ) .", "lane m : marker ; lane 1 : negative control ; lane 2 : positive control ; lane 3 - 6 : positive samples ; 7 : negative sample ." ], [ "data were analyzed using spss version 16 and the outcome variables including prevalence rates and their 95% confidence intervals ( cis ) were calculated .", "the chi - square or fisher s exact test was used to analyze the categorical data" ], [ "the age of the 255 subjects ranged 17 - 35 years with a mean of 27.3 4.37 years .", "the majority of them were married or living with a partner ( 98% ) , had high school education or higher ( 74% ) .", "the prevalence of c. trachomatis was 2.4% ( 95% confidence interval [ ci ] 0.54 - 4.26% ) with a mean of 25.8 6.17 years ; 50% ( 3/6 ) of the subjects with a positive chlamydia test result were under the age of 25 .", "of the 92 subjects in the 25-year - old group , 3 ( 3.3% ) were positive for c. trachomatis compared to three out of 163 ( 1.8% ) in the > 25-year - group ; 83.3% ( 5/6 ) of the subjects with a positive chlamydia test result were older than 16 at first sexual encounter and all of the infected subjects lived in the city .", "the most general symptoms among c. trachomatis infected young subjects were vaginal discharge ( 66.6% ) and lumbar pain ( 50% ) .", "no significant relationships were found between c. trachomatis infection and the risk factors , probably reflecting the small number of those who were positive in the test ." ], [ "the prevalence of c. trachomatis genital infections among young females in kashan , iran was 2.4% .", "the previous published reports from iran showed various prevalence of 2% to 11% ( 14 ) .", "the study by chamani - tabriz et al . on the prevalence of chlamydia infection in females aged 15 - 42 attending obstetrics and gynecology clinics in tehran , from may 2003 to october 2003 showed that 12.6% were positive for chlamydia by pcr ( 15 ) . in another study on the females < 45 years attending gynecology clinics in babol , iran ,", "european centre for disease prevention and control ( ecdc ) described the prevalence ranging from 1.4% to 3.0% among females aged 15 - 40 years ( 17 ) .", "the prevalence of c. trachomatis infection and the risk factors in individuals < 35 years old attending sexual health clinics in barcelona province , spain , in 2006 was 3.8% and the prevalence was significantly higher in those under 25 years ( 5.8% ) compared with the ones aged 25 - 35 ( 1.6% ) ( 18 ) .", "another study in melbourne , australia , showed that the chlamydia prevalence was 3.7% among sexually active females aged 18 - 24 years compared with 0.2% among 25 - 35-year - old ones ( 19 ) .", "a study among the brazilian females referred to sexually transmitted diseases ( std ) clinic showed that 68% of the infected ones aged 15 - 25 year ( 13 ) . in the current study , the c. trachomatis prevalence in females", " 25 years ( 3.2% ) was similar to those of reported in australia 4.9% ( 20 ) , spain 4% ( 21 ) , france 3.6% ( 22 ) and the united kingdom 3% ( 23 ) .", "the current cdc guidelines recommend annual screening for chlamydia in sexually active females 25 years and the ones > 25 years old who are at increased risk ( 24 ) .", "screening is especially important in pregnant females who are at risk of transmission of their infection to the fetus ( 25 ) .", "chlamydia trachomatis infection is the main causative agent of abortion ( 26 ) . in the current study , abortions were reported by 16.7% of females with positive test results .", "early detection and appropriate treatment of c. trachomatis can prevent the development of the disease after abortion ( 27 ) .", "prevalence of c. trachomatis was found to be low among females in kashan , iran .", "the reason for c. trachomatis infection low prevalence in the population under study was the scarcity of pre - marriage sexual intercourse frequency in females , having stable sexual intercourse only after marriage .", "to date it was the first study in kashan to determine the prevalence of endocervical c. trachomatis infection in young females .", "there were several limitations to the study , which should be considered in the interpretation of the results .", "data on sexual behavior was not specifically collected for the current study . knowing a patient s number of sexual partners , sexual activity before and after sti diagnosis , and frequency of condom use may help identify the patients who are at increased infection risk .", "chlamydia prevalence was low in young sexually - active females , but there is a significant difference regarding chlamydia infection in females younger than 25 years .", "the new information provides evidence for the need to implement active opportunity to screen young sexually active females and the patients in all socioeconomic groups .", "all sexually active females should be offered a c. trachomatis test when attending a physician ." ] ]

[ "rabies diagnosis is based on fluorescent antibody testing ( fat ) in brain smears and inoculation of brain suspension either in mouse neuroblastoma cell cultures or intracerebrally in mice as confirmatory assays with high sensitivity and specificity for postmortem diagnosis [ 13 ] .", "these techniques are well suited for rapid and reliable routine diagnosis , if brain material is available . for other diagnostic specimens sampled intra vitam in case of clinical suspicion , for example , saliva , cerebrospinal fluid , or skin biopsies , more suited molecular techniques with excellent sensitivity", "have been developed and validated , mostly targeting the conserved nucleoprotein gene of the lyssavirus genome , for example , [ 410 ] . among these , there are protocols both for classical rt - pcr and for real - time rt - pcr , which adds speed , efficiency , contamination safety , and reliability to the technique combined with the potential to quantify the viral load [ 11 , 12 ] .", "a major advantage offered by these molecular techniques is the characterisation of viral isolates by sequencing the given amplification products followed by phylogenetic or phylogeographic analysis [ 1315 ] .", "the greatest challenge for these powerful novel techniques is the ever growing spectrum of known lyssavirus species / genotypes ( gt ) , probably all of which having the potential to cause animal and human rabies fatalities [ 16 , 17 ] .", "so far , 12 lyssavirus species and three as yet unclassified species have been identified .", "the development of classical simple or ( hemi)nested rt - pcr methods with particular emphasis on the diagnosis of a broad spectrum of lyssaviruses species has been published in a number of studies [ 1823 ] . also several real - time rt - pcr protocols suited for the broad detection or differentiation of several genotypes have been developed [ 9 , 2428 ] .", "is of particular interest for the rabies epidemiology in europe concerning the european bat lyssaviruses types 1 and 2 [ 2932 ] apart from classical rabies [ 33 , 34 ] .", "this assay uses genotype ( gt ) specific probes and fluorophore signals for direct differentiation of gt1 , gt5 , and gt6 in a single - tube reaction .", "the goal of this work was , based on a comprehensive review of the rich literature on the theme and adapting thereof , to develop , establish , and validate classical and real - time rt - pcr protocols for ( intravitam ) diagnosis of rabies and molecular - epidemiological characterisation of viral strains with the main emphasis on the growing number of known species / genotypes .", "all operations with potentially infectious material apart from centrifugation in closed tubes were performed in a class ii biological safety cabinet .", "frozen cell culture supernatants of rabies virus strains propagated previously on bhk-21 cells ( baby hamster kidney cells , atcc , manassas , usa ) were thawed , centrifuged at 2,000 g for 10 min at 4c , and filtrated using the millex - ha 450 nm filter ( millipore , cork , ireland ) . approximately 1 g of cerebellum , medulla oblongata , and hippocampus from unfixed , freshly obtained brain material was homogenized to a 20% brain suspension using a mortar and pestle , after addition of approximately 1 g of quartz sand ( merck kgaa , darmstadt , germany ) and 5 ml of modified eagle medium with earle 's salts with 2.2 g / l nahco3 ( bioswisstec ag , schaffhausen , switzerland ) supplemented with 5% penicillin 10,000 iu / ml ( bioswisstec ag , schaffhausen , switzerland ) and 20% foetal calf serum ( fcs ; paa laboratories gmbh , pasching , austria ) . the suspension", "was then decanted into a 5 ml tube ( 5 ml polystyrene round - bottom tube ; bd biosciences , erembodegem , belgium ) to let it sediment for 1 hour at 4c and subsequently centrifuged at 1,400 g for 10 min and filtrated as above .", "frozen brain suspensions from former mouse inoculation tests were handled like frozen cell culture supernatant .", "500 l of rna storage solution ( ambion , foster city , usa ) was added to 100200 l of fresh or previously frozen saliva samples or swabs , which were subsequently vortexed ( vortex genie 2 , tewis laborbedarf ag , berne , switzerland ) for 1 min and centrifuged in a biofuge pico ( heraeus holding gmbh , hanau , germany ) at 5,000 rpm for 10 min .", "cerebrospinal fluid ( csf ) samples were diluted 1 : 2 to 1 : 4 with rna storage solution ( ambion , foster city , usa ) .", "skin biopsies were shaved , cut into small pieces with a sterile pair of scissors , and further processed as described for brain suspension .", "a panel of 31 lyssaviruses ( including 4 species ) was used to test the diagnostic performance of our pcr ( table 1 ) .", "stocks of viral strains were produced in neuroblastoma cells ( mna 42/13 ) as described for the rabies tissue culture infection test ( rtcit , described below ) .", "the maintenance medium used for the first passage was dulbecco 's mem supplemented with 0.5% neomycin 50 iu / ml ( bioswisstec ag , schaffhausen , switzerland ) , 5% tryptose phosphate ( bioconcept ltd .", "amimed , allschwil , switzerland ) , 1% nonessential amino acids ( bioswisstec ag , schaffhausen , switzerland ) , 3% foetal calf serum , and 1% l - glutamine 200 mm ( bioswisstec ag , schaffhausen , switzerland ) to which 1% diethylaminoethyl - dextran ( deae - dextran ; sigma - aldrich corporation , st .", "dulbecco 's mem with 10% foetal calf serum was used for the 3 consecutive passages .", "staining of the cells was performed after each passage using rabies dfa reagent ( millipore , livingston , uk ) as a conjugate .", "the standard fluorescent antibody test ( fat ) was performed with brain tissue samples as previously described", ". rabies tissue culture infection test ( rtcit ) using four consecutive passages on murine neuroblastoma cells [ 4143 ] was applied to clinical specimens like brain specimens , liquor , saliva , or skin biopsies .", "primers , probes , and synthetic dna were obtained from microsynth ( microsynth gmbh , balgach , switzerland ) . the location of suitable n - directed primers and probes for heminested rt - pcr and real - time rt - pcr , respectively , was chosen and evaluated based on published work [ 8 , 18 , 22 , 25 ] . using multiple sequence alignment ( clustalx 2.0.3 program [ 4446 ] ) of the n gene region of the available lyssavirus species rabv ( 33 sequences ) , lbv ( 4 sequences ) , mokv ( 5 sequences ) , duvv ( 4 sequences ) , eblv-1 ( 17 sequences ) , eblv-2 ( 14 sequences ) , ablv , aravan , khujand , irkut , and west caucasian bat ( each one ) from genbank ( national center for biotechnology information and national library of medicine , rockville pike , usa ) , variable positions of primers and probes were adjusted with wobble positions for a potentially broader match .", "several probes for real - time rt - pcr for broadening the spectrum of detectable species were designed in this work ( table 2 ) . as internal control for conventional rt - pcr , amplification of gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) was used . as internal inhibition control for real - time rt - pcr ,", "primers and probes for the amplification of sendai virus ( atcc , manassas , usa ) , which was added to the samples before rna isolation , were used ( table 2 ) .", "synthetic dna with a length of 125 bases encompassing positions 48172 according to the pasteur virus genome ( x03673 ) of the following lyssavirus species were obtained from microsynth : aravan virus ( arav ) ef614259 , khujand virus ( khuv ) ef614261 , bokeloh bat lyssavirus ( bblv ) jf311903 , australian bat lyssavirus ( ablv ) af418014 , irkut virus ( irkv ) ef614260 , lagos bat virus ( lbv ) eu293110 , mokola virus ( mokv ) 293117 , shimoni bat virus ( shiv ) gu170201 , and west caucasian bat virus ( wcbv ) ef614258 .", "rna was isolated from 140 l of sample supernatant using the qiaamp viral rna mini kit ( qiagen , germantown , usa ) according to the manufacturer 's instructions , except the usage of rna storage solution ( ambion , foster city , usa ) for elution .", "heminested rt - pcr was performed as previously described using the onestep rt - pcr kit . briefly , for the first round 3 l of extracted rna was amplified in the rt - pcr mix prepared according to manufacturer 's instructions , supplemented with 0.6 m of each primer ( jw12-f , jw6as1-r1 , and jw6as2-r1 ; table 2 ) and 5 u rnase inhibitor ( rnasin plus , 40 u/l ; promega , madison , usa ) , with the following cycling conditions : 50c for 30 min and 95 for 15 min for reverse transcription and subsequent activation of the polymerase , followed by 10 cycles of 95c for 20 s , 60c for 30 s ( 1c decrease / cycle ) , followed by 72c for 30 s and 35 cycles of 95c for 20 s , 52c for 30 s , followed by 72c for 30 s. for the second round , the taq dna polymerase kit ( qiagen , germantown , usa ) was used with 2 l of the first round product , 2.5 l 10x pcr buffer , 1.2 mm final dntp concentration ( invitrogen , foster city , usa ) , 0.4 m of heminested primers ( jw12-f , jw10as1-r2 , and jw10as2-r2 ; table 2 ) , 0.5 u of taq dna polymerase , and 19.35 l nuclease free water ( ambion , foster city , usa ) .", "cycling conditions were as follows : 95c for 5 min followed by 35 cycles of 94c for 20 s , 52c for 20 s , followed by 72c for 30 s. amplifications were performed in a 2720 thermal cycler .", "internal controls using gapdh primers , as described in the one - round pcr , were run in parallel in the first round .", "gel electrophoresis was performed using a 1.5% tae agarose gel ( agarose le , analytical grade ; promega , madison , usa ) stained with ethidium bromide ( eurobio , courtaboeuf , france ) . for sequencing , the desired band was excised under uv light .", "real - time rt - pcr ( ndwd ) adapted from nadin - davis et al . and wakeley et al . [ 8 , 25 ]", "was performed using the quantitect probe rt - pcr kit ( qiagen , germantown , usa ) .", "the 25 l reaction volume consisted of 9.5 l rnase - free water , or 9 l in the multiplex mix , 12.5 l 2x quantitect rt - pcr master mix , 0.25 l of the rabvd1 forward and reverse primer ( 0.4 m final concentration each ) , 0.25 l of the probes rabvd1 , lysgt5 , and lysgt6 together in the multiplex mix or 0.25 l of the probes ivvwcbv - p , ivvmok - p , ivvablv2n - p , ivvlbv - p , ivvkhujand - p , and ivvsblv2-p ( table 2 ) alone in a mix ( all 0.2 m final concentration ) , 0.25 l of quantitect rt mix , and 2 l of sample rna .", "additionally , for the internal sendai virus control , 5 l of sample rna was added to a mix containing 6.625 l of rnase - free water , 12.5 l 2x quantitect rt - pcr master mix , 0.625 l of sendai forward primer , reverse primer , and probe mix ( sendai - f , sendai - r , and sendai - p , 8 m ; table 2 ) , and 0.25 l of quantitect rt mix .", "the reactions were carried out in microamp fast optical 96-well reaction plates with barcode 0.1 ml ( applied biosystems , foster city , usa ) in a 7500 fast real - time pcr system v1.3.1 ( applied biosystems , foster city , usa ) using the following cycling conditions : 1 cycle of 50c for 30 min and 95c for 15 min followed by 45 cycles of 95c for 15 sec and 50c for 1 min .", "amplification curve analysis was performed using the 7500 software v2.0.6 ( applied biosystems , foster city , usa ) . for cloning ,", "a segment of viral genome covering a part of the n gene ( positions 1933 according to the pasteur virus genome accession number x03673 ) of cvs-11 was generated using primers designed for this purpose ( twclon - f and twclon - r , table 2 ) .", "amplification was performed using the onestep rt - pcr kit with the master mix consisting of 29.25 l of rnase - free water , 10 l 5x qiagen onestep rt - pcr buffer , 0.4 mm of each dntp , 0.6 m of the cloning primers , 10 u rnasin ( 40 u/l ) , and 2 l of qiagen onestep rt - pcr enzyme mixed with 5 l of extracted rna to a total volume of 50 l .", "amplifications were performed in a veriti 96-well thermal cycler ( applied biosystems , foster city , usa ) using the following cycling conditions : 30 min at 50c , followed by 15 min at 95c and 40 repetitive cycles of 1 min at 94c , 1 min with a temperature gradient from 56c to 46c in steps of 2c , and 1 min at 72c .", "after gel electrophoresis excised pcr fragments with a length of 933 bp were eluted with the qiaquick gel extraction kit ( qiagen , germantown , usa ) and cloned into the pcr - ii - topo vector using the topo ta cloning kit ( invitrogen , foster city , usa ) according to the manufacturer 's instructions .", "sequencing reactions were performed by microsynth ag , balgach , switzerland . for this purpose , reaction mixtures containing", "22.5 ng dna per 100 bp , 30 pmol of each primer used for the amplification ( table 2 ) , and depc treated water ( ambion , foster city , usa ) to a final volume of 15 l were prepared .", "sequences were edited using the seqman ii v5.01 software ( dnastar , madison , usa ) and subsequently evaluated using the clone manager 9 software ( scientific & educational software , cary , usa ) .", "the 543 bp nucleotide sequences of the nucleoprotein gene obtained from the heminested pcr reaction , together with additional sequences retrieved from genbank , were saved in fasta file format .", "the sequences were then aligned using the clustalx 2.0.3 software and the genedoc software v2.5.0 .", "thirty - one lyssaviruses ( 19x rabv , 7x eblv-1 , 4x eblv-2 , and 1x duvv ) were analysed in rtcit .", "all isolates were viable in murine neuroblastoma cells and could be visualized using rabies dfa reagent as a conjugate . for the comparison with infectious titres , rna of the classical rabies virus strain cvs-11", "was extracted from cell culture supernatants ( bhk-21 cells or neuroblastoma cells mna 42/13 ) and serially diluted from 10 to 10 .", "the infectious titre was determined according to the spearman - karber formula [ 49 , 50 ] in fluorescent focus forming doses 50% ( ffd50 ) . in the heminested rt - pcr ( hnrt - pcr ) ,", "the amplification product of 582 bp was visible up to a dilution of 10 corresponding to a detection limit of 0.4 ffd50 ( 10 ffd50 ; figure 1 ) . in the real - time rt - pcr ( ndwd )", "the limit of detection was defined as the last dilution at which at least 2 out of 3 replicates were positive . using this definition , cvs-11 was detectable up to a dilution of 10 ( once at a dilution of 10 ) .", "the amplification plot showed regular intervals of the curves with ct - values from 18.1 ( dilution 10 ) to 40.0 ( dilution 10 ) .", "relating to infectious titre real - time rt - pcr reached a detection limit of 0.003 ffd50 ( 10 ) for cvs-11 .", "the efficiency of the real - time rt - pcr ( ndwd ) was determined using linear regression of the ct - values and the titres of the samples ( figure 2 ) . on the basis of the slope coefficient", "the limit of detection in terms of dna copy numbers was determined using 10-fold serial dilutions of a cloned 933 bp segment of n of cvs-11 containing 10 to 10 dna copies / pcr reaction . in the hnpcr ,", "the limit of detection was 100 dna copies/3 l of starting material of the cvs-11 plasmid .", "in the real - time pcr ( ndwd ) , the 10-fold serial dilution of the cvs - plasmid was tested in triplicate .", "the limit of detection was 10 dna copies/2 l of the cvs-11 plasmid with ct - values between 38.6 and 47.5 .", "the efficiency was at 82.8% with a slope coefficient of 3.82 ( not shown ) .", "the intra - assay repeatability of the real - time pcr ( ndwd ) proved to be excellent with very low coefficients of variation up to 10 dna copies ( 0.181.37% ) with and outlier at 10 copies ( 12.05% ; table 3 ) .", "a total of 31 lyssaviruses ( 19 identified as classical rabv , 7 as eblv-1 , 4 as eblv-2 , and 1 as duvv ) were used to test the diagnostic performance of the pcr ( table 1 ) .", "all viruses used were detected with the expected band size in the hnrt - pcr technique ( table 4 ) .", "the specificity of the amplified product was confirmed as rabv , eblv-1 , eblv-2 , or duvv by sequencing in each case .", "host gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) was detected in all of the cell culture supernatants and mouse brain suspensions as internal control , indicating that sample material was present and rna isolation , reverse transcription , and amplification were not inhibited .", "all samples were also tested positive by the real - time rt - pcr assay ( table 4 ) .", "sendai virus as internal control for inhibition was run in parallel using the same cycling conditions . in the real - time rt - pcr ( ndwd ) with an annealing temperature of 50c , some samples were detected with more than one probe . with the exception of the sample derived from a raccoon dog from poland , the probe for the homologous genotype always yielded the lowest ct - value . in this exceptional case ,", "the probe for the detection of genotype 6 was slightly lower than the one for genotype 1 ( 15.0 versus 16.1 ; table 4 ) .", "comparison of these two probes with the sequence of the rabies variant in question showed 1 and 2 mismatches with the rabvd1-p and lysgt6-p probe , respectively ( figure 3 ) .", "duvenhage virus , also designated as genotype 4 , was detectable with the probe adapted for genotype 6 in spite of 3 mismatches ( lysgt6-p ) .", "the probe lysgt6-p was able to detect synthetic dna fragments of bblv , arav , and irkv , although containing one or two mismatches , respectively .", "the synthetic dna fragments of khuv , ablv , lbv , mokv , shibv , and wcbv , which exhibited from four ( ablv / khuv ) up to ten ( wcbv ) mismatches to the best fitting probe for gt1 , gt6 , or gt5 , respectively , were all detected with the corresponding species - specific new probe ( table 2 ) .", "10 copies of the synthetic dna were detected at ct - values of 27.933.5 for ablv , mokv , shibv , and wcbv ( not shown ) .", "the efficiency of detection of khuv , which showed an atypical amplification plot at the highest number of 10 copies of the synthetic dna , and that of lbv was lower with ct - values of 40.0 and 40.5 at a copy number of 10 , respectively ( not shown ) .", "multiplexing of combinations of these probes ( ivvlbv - p ( fam : 6-carboxyfluorescein ) , ivvkhujand - p ( yy : yakima yellow ) , and ivvsblv2-p ( cy5 : cyanine 5 ) , as well as ivvwcbv - p ( fam ) , ivvmok - p ( yy ) , and ivvablv2n - p ( cy5 ) ) as for the species 1 , 5 , and 6 was not successful .", "the 543 bp amplification products of the nucleoprotein gene obtained in the heminested pcr reactions were sequenced ( table 1 ) and analysed phylogenetically with additional sequences retrieved from genbank representing all known lyssaviruses .", "the similarity of the 543 bp nucleotide sequences of the nucleoprotein gene ( positions 74616 according to the pasteur virus genome ) among the lyssaviruses included ranged from 64.6% to 90.1% ( hamming distance ) .", "the resulting phylogenetic tree obtained by the neighbor - joining method implemented in the mega5 software is presented in figure 4 .", "clinical specimens like brain suspensions ( 1x human , 12x mouse ) , skin biopsies from the nape of the neck ( 3x human , 1x mouse ) , saliva ( 4x human ) , and cerebrospinal fluids ( 6x human ) were used for the establishment of the assays . both hnrt - pcr and real - time rt - pcr ( ndwd ) were shown to work properly on all these samples without significant inhibition using gapdh as internal control for conventional rt - pcr and sendai virus for real - time rt - pcr ( ndwd ) , which was mixed to the samples before rna isolation .", "skin biopsies taken from the neck and lip of a mouse ( white swiss mouse at an age of 3 weeks ) euthanized 2 weeks after intracerebral infection with sad bern virus 20 years ago were positive with the hnrt - pcr and real - time pcr ( ndwd ) .", "skin biopsies taken from other locations on the head were positive with the real - time pcr ( ndwd ) and weakly positive in hnrt - pcr whereas samples taken around the vibrissae were weakly positive in real - time pcr ( ndwd ) only .", "sequencing of amplification products excluded a contamination with the positive cvs-11 control ( not shown ) .", "brain suspension from an imported human rabies case in 2012 was already strongly positive after one round of the hnrt - pcr .", "phylogenetic analysis of the 543 bp nucleotide fragment revealed its close relationship to the classical rabies virus strains circulating in the insectivorous mexican free - tailed bat , tadarida brasiliensis , a species common in the southern united states and mexico .", "this allowed identification of the origin of the patient 's infection , who had travelled extensively and did not report any previous biting incident [ 51 , 52 ] .", "based on a large amount of published work , we were able to establish and quantitatively characterise rt - pcr protocols for the detection of lyssaviruses in clinical samples .", "well suited real - time protocols [ 8 , 25 ] for the detection of classical rabies virus and the genotypes 5 and 6 ( eblv-1 and eblv-2 ) were adapted and extended for the detection of at least 13 lyssavirus species . to this goal , 6 species - specific probes ( khuv , ablv , lbv , mokv , shibv , and wcbv )", "were designed and verified on synthetic dna fragments encompassing the targeted sequence of the lyssaviral nucleoprotein gene .", "multiplexing the probes in a single - tube reaction as described for the genotypes 1 , 5 , and 6 was not possible with the newly designed probes , probably due to false priming and/or interference within the mix of reagents and synthetic target sequence .", "as far as evaluated with the multiplexed probes for the genotypes 1 , 5 , and 6 using 31 viral isolates , which were all detectable with high sensitivity , direct differentiation of targeted genotypes on the base of the quantitative reaction ( ct - values ) was mostly possible .", "furthermore , a single probe for genotype 6 ( species eblv-2 ) was able to detect up to 7 genotypes / species .", "we consider this type of cross hybridization as an advantage of the technique using degenerate primers and probes in terms of a broad detectability of lyssavirus rather than a lack of accuracy of discrimination . in an approach using sybr green qpcr with similarly degenerate primers spanning the same part of n as in this work , the detection of all lyssavirus species known at that time was achieved .", "using several genotype - specific probes in a taqman real - time protocol , we were able to add more intrinsic confidence to the specificity of the analysis .", "quantitative characterisation of the assay using the probe for genotypes 1 , 5 , and 6 on cvs-11 showed excellent sensitivity and repeatability with a detection limit of as low as an infectious dose of 0.003 ffd50 combined with an analytical sensitivity of 10 dna copies at efficiencies of 94.5% and 82.8% , respectively . considering the amount of degeneration in both primers and probes , the efficiencies determined must be considered satisfactory .", "as far as the absolute detection limit in terms of target copies is concerned , the usage of plasmid dna rather than rna must be kept in mind . since reverse transcription can not be assumed as 100% efficient and reproducible , the limit for rna might be somewhat lower . for further characterisation of samples with a positive reaction in real - time rt - pcr , excellent simple and ( hemi)nested rt - pcr protocols are available . with the protocol used for this work ,", "all 31 viral strains used could be amplified and sequenced , confirming the diagnostic broadness of the assay .", "phylogenetic analysis of these partial nucleotide sequences belonging to genotypes 1 , 4 , 5 , and 6 along with known sequences confirmed the suitability of this relatively conserved genomic region for molecular - epidemiological characterisation of lyssaviruses circulating worldwide [ 5658 ] .", "this was also confirmed in the context of a human rabies case imported to switzerland in 2012 , which could be attributed unequivocally to an exposure to a bat of the species tadarida brasiliensis in california , usa .", "in this work we could show and validate the suitability of an adapted and further developed real - time rt - pcr protocol for the rapid , efficient , and highly sensitive intravitam diagnosis of a wide spectrum of lyssavirus species followed by rapid molecular - epidemiological characterisation of viral isolates respective to origin of the virus and source of exposure , using heminested rt - pcr .", "this new tool is also particularly promising for active surveillance of european bat lyssaviruses using oral swabs in live - captured bats ." ]

[ "<S> the causative agents of rabies are single - stranded , negative - sense rna viruses in the genus lyssavirus of rhabdoviridae , consisting of twelve classified and three as yet unclassified species including classical rabies virus ( rabv ) . </S>", "<S> highly neurotropic rabv causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome . </S>", "<S> rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health . due to growing variety of </S>", "<S> the genus lyssavirus observed , the development of suitable molecular assays for diagnosis and differentiation is challenging . </S>", "<S> this work focused on the establishment of a suitable real - time rt - pcr technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation . </S>", "<S> the real - time rt - pcr was adapted with the goal to detect the whole spectrum of lyssavirus species , for nine of which synthesized dna fragments were used . for the detection of species , seven probes were developed . </S>", "<S> serial dilutions of the rabies virus strain cvs-11 showed a 100-fold higher sensitivity of real - time pcr compared to heminested rt - pcr . using a panel of thirty - one lyssaviruses representing four species , </S>", "<S> the suitability of the protocol could be shown . </S>", "<S> phylogenetic analysis of the sequences obtained by heminested pcr allowed correct classification of all viruses used . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion" ]

[ [ "rabies diagnosis is based on fluorescent antibody testing ( fat ) in brain smears and inoculation of brain suspension either in mouse neuroblastoma cell cultures or intracerebrally in mice as confirmatory assays with high sensitivity and specificity for postmortem diagnosis [ 13 ] .", "these techniques are well suited for rapid and reliable routine diagnosis , if brain material is available . for other diagnostic specimens sampled intra vitam in case of clinical suspicion , for example , saliva , cerebrospinal fluid , or skin biopsies , more suited molecular techniques with excellent sensitivity", "have been developed and validated , mostly targeting the conserved nucleoprotein gene of the lyssavirus genome , for example , [ 410 ] . among these , there are protocols both for classical rt - pcr and for real - time rt - pcr , which adds speed , efficiency , contamination safety , and reliability to the technique combined with the potential to quantify the viral load [ 11 , 12 ] .", "a major advantage offered by these molecular techniques is the characterisation of viral isolates by sequencing the given amplification products followed by phylogenetic or phylogeographic analysis [ 1315 ] .", "the greatest challenge for these powerful novel techniques is the ever growing spectrum of known lyssavirus species / genotypes ( gt ) , probably all of which having the potential to cause animal and human rabies fatalities [ 16 , 17 ] .", "so far , 12 lyssavirus species and three as yet unclassified species have been identified .", "the development of classical simple or ( hemi)nested rt - pcr methods with particular emphasis on the diagnosis of a broad spectrum of lyssaviruses species has been published in a number of studies [ 1823 ] . also several real - time rt - pcr protocols suited for the broad detection or differentiation of several genotypes have been developed [ 9 , 2428 ] .", "is of particular interest for the rabies epidemiology in europe concerning the european bat lyssaviruses types 1 and 2 [ 2932 ] apart from classical rabies [ 33 , 34 ] .", "this assay uses genotype ( gt ) specific probes and fluorophore signals for direct differentiation of gt1 , gt5 , and gt6 in a single - tube reaction .", "the goal of this work was , based on a comprehensive review of the rich literature on the theme and adapting thereof , to develop , establish , and validate classical and real - time rt - pcr protocols for ( intravitam ) diagnosis of rabies and molecular - epidemiological characterisation of viral strains with the main emphasis on the growing number of known species / genotypes ." ], [ "all operations with potentially infectious material apart from centrifugation in closed tubes were performed in a class ii biological safety cabinet .", "frozen cell culture supernatants of rabies virus strains propagated previously on bhk-21 cells ( baby hamster kidney cells , atcc , manassas , usa ) were thawed , centrifuged at 2,000 g for 10 min at 4c , and filtrated using the millex - ha 450 nm filter ( millipore , cork , ireland ) . approximately 1 g of cerebellum , medulla oblongata , and hippocampus from unfixed , freshly obtained brain material was homogenized to a 20% brain suspension using a mortar and pestle , after addition of approximately 1 g of quartz sand ( merck kgaa , darmstadt , germany ) and 5 ml of modified eagle medium with earle 's salts with 2.2 g / l nahco3 ( bioswisstec ag , schaffhausen , switzerland ) supplemented with 5% penicillin 10,000 iu / ml ( bioswisstec ag , schaffhausen , switzerland ) and 20% foetal calf serum ( fcs ; paa laboratories gmbh , pasching , austria ) . the suspension", "was then decanted into a 5 ml tube ( 5 ml polystyrene round - bottom tube ; bd biosciences , erembodegem , belgium ) to let it sediment for 1 hour at 4c and subsequently centrifuged at 1,400 g for 10 min and filtrated as above .", "frozen brain suspensions from former mouse inoculation tests were handled like frozen cell culture supernatant .", "500 l of rna storage solution ( ambion , foster city , usa ) was added to 100200 l of fresh or previously frozen saliva samples or swabs , which were subsequently vortexed ( vortex genie 2 , tewis laborbedarf ag , berne , switzerland ) for 1 min and centrifuged in a biofuge pico ( heraeus holding gmbh , hanau , germany ) at 5,000 rpm for 10 min .", "cerebrospinal fluid ( csf ) samples were diluted 1 : 2 to 1 : 4 with rna storage solution ( ambion , foster city , usa ) .", "skin biopsies were shaved , cut into small pieces with a sterile pair of scissors , and further processed as described for brain suspension .", "a panel of 31 lyssaviruses ( including 4 species ) was used to test the diagnostic performance of our pcr ( table 1 ) .", "stocks of viral strains were produced in neuroblastoma cells ( mna 42/13 ) as described for the rabies tissue culture infection test ( rtcit , described below ) .", "the maintenance medium used for the first passage was dulbecco 's mem supplemented with 0.5% neomycin 50 iu / ml ( bioswisstec ag , schaffhausen , switzerland ) , 5% tryptose phosphate ( bioconcept ltd .", "amimed , allschwil , switzerland ) , 1% nonessential amino acids ( bioswisstec ag , schaffhausen , switzerland ) , 3% foetal calf serum , and 1% l - glutamine 200 mm ( bioswisstec ag , schaffhausen , switzerland ) to which 1% diethylaminoethyl - dextran ( deae - dextran ; sigma - aldrich corporation , st .", "dulbecco 's mem with 10% foetal calf serum was used for the 3 consecutive passages .", "staining of the cells was performed after each passage using rabies dfa reagent ( millipore , livingston , uk ) as a conjugate .", "the standard fluorescent antibody test ( fat ) was performed with brain tissue samples as previously described", ". rabies tissue culture infection test ( rtcit ) using four consecutive passages on murine neuroblastoma cells [ 4143 ] was applied to clinical specimens like brain specimens , liquor , saliva , or skin biopsies .", "primers , probes , and synthetic dna were obtained from microsynth ( microsynth gmbh , balgach , switzerland ) . the location of suitable n - directed primers and probes for heminested rt - pcr and real - time rt - pcr , respectively , was chosen and evaluated based on published work [ 8 , 18 , 22 , 25 ] . using multiple sequence alignment ( clustalx 2.0.3 program [ 4446 ] ) of the n gene region of the available lyssavirus species rabv ( 33 sequences ) , lbv ( 4 sequences ) , mokv ( 5 sequences ) , duvv ( 4 sequences ) , eblv-1 ( 17 sequences ) , eblv-2 ( 14 sequences ) , ablv , aravan , khujand , irkut , and west caucasian bat ( each one ) from genbank ( national center for biotechnology information and national library of medicine , rockville pike , usa ) , variable positions of primers and probes were adjusted with wobble positions for a potentially broader match .", "several probes for real - time rt - pcr for broadening the spectrum of detectable species were designed in this work ( table 2 ) . as internal control for conventional rt - pcr , amplification of gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) was used . as internal inhibition control for real - time rt - pcr ,", "primers and probes for the amplification of sendai virus ( atcc , manassas , usa ) , which was added to the samples before rna isolation , were used ( table 2 ) .", "synthetic dna with a length of 125 bases encompassing positions 48172 according to the pasteur virus genome ( x03673 ) of the following lyssavirus species were obtained from microsynth : aravan virus ( arav ) ef614259 , khujand virus ( khuv ) ef614261 , bokeloh bat lyssavirus ( bblv ) jf311903 , australian bat lyssavirus ( ablv ) af418014 , irkut virus ( irkv ) ef614260 , lagos bat virus ( lbv ) eu293110 , mokola virus ( mokv ) 293117 , shimoni bat virus ( shiv ) gu170201 , and west caucasian bat virus ( wcbv ) ef614258 .", "rna was isolated from 140 l of sample supernatant using the qiaamp viral rna mini kit ( qiagen , germantown , usa ) according to the manufacturer 's instructions , except the usage of rna storage solution ( ambion , foster city , usa ) for elution .", "heminested rt - pcr was performed as previously described using the onestep rt - pcr kit . briefly , for the first round 3 l of extracted rna was amplified in the rt - pcr mix prepared according to manufacturer 's instructions , supplemented with 0.6 m of each primer ( jw12-f , jw6as1-r1 , and jw6as2-r1 ; table 2 ) and 5 u rnase inhibitor ( rnasin plus , 40 u/l ; promega , madison , usa ) , with the following cycling conditions : 50c for 30 min and 95 for 15 min for reverse transcription and subsequent activation of the polymerase , followed by 10 cycles of 95c for 20 s , 60c for 30 s ( 1c decrease / cycle ) , followed by 72c for 30 s and 35 cycles of 95c for 20 s , 52c for 30 s , followed by 72c for 30 s. for the second round , the taq dna polymerase kit ( qiagen , germantown , usa ) was used with 2 l of the first round product , 2.5 l 10x pcr buffer , 1.2 mm final dntp concentration ( invitrogen , foster city , usa ) , 0.4 m of heminested primers ( jw12-f , jw10as1-r2 , and jw10as2-r2 ; table 2 ) , 0.5 u of taq dna polymerase , and 19.35 l nuclease free water ( ambion , foster city , usa ) .", "cycling conditions were as follows : 95c for 5 min followed by 35 cycles of 94c for 20 s , 52c for 20 s , followed by 72c for 30 s. amplifications were performed in a 2720 thermal cycler .", "internal controls using gapdh primers , as described in the one - round pcr , were run in parallel in the first round .", "gel electrophoresis was performed using a 1.5% tae agarose gel ( agarose le , analytical grade ; promega , madison , usa ) stained with ethidium bromide ( eurobio , courtaboeuf , france ) . for sequencing , the desired band was excised under uv light .", "real - time rt - pcr ( ndwd ) adapted from nadin - davis et al . and wakeley et al . [ 8 , 25 ]", "was performed using the quantitect probe rt - pcr kit ( qiagen , germantown , usa ) .", "the 25 l reaction volume consisted of 9.5 l rnase - free water , or 9 l in the multiplex mix , 12.5 l 2x quantitect rt - pcr master mix , 0.25 l of the rabvd1 forward and reverse primer ( 0.4 m final concentration each ) , 0.25 l of the probes rabvd1 , lysgt5 , and lysgt6 together in the multiplex mix or 0.25 l of the probes ivvwcbv - p , ivvmok - p , ivvablv2n - p , ivvlbv - p , ivvkhujand - p , and ivvsblv2-p ( table 2 ) alone in a mix ( all 0.2 m final concentration ) , 0.25 l of quantitect rt mix , and 2 l of sample rna .", "additionally , for the internal sendai virus control , 5 l of sample rna was added to a mix containing 6.625 l of rnase - free water , 12.5 l 2x quantitect rt - pcr master mix , 0.625 l of sendai forward primer , reverse primer , and probe mix ( sendai - f , sendai - r , and sendai - p , 8 m ; table 2 ) , and 0.25 l of quantitect rt mix .", "the reactions were carried out in microamp fast optical 96-well reaction plates with barcode 0.1 ml ( applied biosystems , foster city , usa ) in a 7500 fast real - time pcr system v1.3.1 ( applied biosystems , foster city , usa ) using the following cycling conditions : 1 cycle of 50c for 30 min and 95c for 15 min followed by 45 cycles of 95c for 15 sec and 50c for 1 min .", "amplification curve analysis was performed using the 7500 software v2.0.6 ( applied biosystems , foster city , usa ) . for cloning ,", "a segment of viral genome covering a part of the n gene ( positions 1933 according to the pasteur virus genome accession number x03673 ) of cvs-11 was generated using primers designed for this purpose ( twclon - f and twclon - r , table 2 ) .", "amplification was performed using the onestep rt - pcr kit with the master mix consisting of 29.25 l of rnase - free water , 10 l 5x qiagen onestep rt - pcr buffer , 0.4 mm of each dntp , 0.6 m of the cloning primers , 10 u rnasin ( 40 u/l ) , and 2 l of qiagen onestep rt - pcr enzyme mixed with 5 l of extracted rna to a total volume of 50 l .", "amplifications were performed in a veriti 96-well thermal cycler ( applied biosystems , foster city , usa ) using the following cycling conditions : 30 min at 50c , followed by 15 min at 95c and 40 repetitive cycles of 1 min at 94c , 1 min with a temperature gradient from 56c to 46c in steps of 2c , and 1 min at 72c .", "after gel electrophoresis excised pcr fragments with a length of 933 bp were eluted with the qiaquick gel extraction kit ( qiagen , germantown , usa ) and cloned into the pcr - ii - topo vector using the topo ta cloning kit ( invitrogen , foster city , usa ) according to the manufacturer 's instructions .", "sequencing reactions were performed by microsynth ag , balgach , switzerland . for this purpose , reaction mixtures containing", "22.5 ng dna per 100 bp , 30 pmol of each primer used for the amplification ( table 2 ) , and depc treated water ( ambion , foster city , usa ) to a final volume of 15 l were prepared .", "sequences were edited using the seqman ii v5.01 software ( dnastar , madison , usa ) and subsequently evaluated using the clone manager 9 software ( scientific & educational software , cary , usa ) .", "the 543 bp nucleotide sequences of the nucleoprotein gene obtained from the heminested pcr reaction , together with additional sequences retrieved from genbank , were saved in fasta file format .", "the sequences were then aligned using the clustalx 2.0.3 software and the genedoc software v2.5.0 ." ], [ "thirty - one lyssaviruses ( 19x rabv , 7x eblv-1 , 4x eblv-2 , and 1x duvv ) were analysed in rtcit .", "all isolates were viable in murine neuroblastoma cells and could be visualized using rabies dfa reagent as a conjugate . for the comparison with infectious titres , rna of the classical rabies virus strain cvs-11", "was extracted from cell culture supernatants ( bhk-21 cells or neuroblastoma cells mna 42/13 ) and serially diluted from 10 to 10 .", "the infectious titre was determined according to the spearman - karber formula [ 49 , 50 ] in fluorescent focus forming doses 50% ( ffd50 ) . in the heminested rt - pcr ( hnrt - pcr ) ,", "the amplification product of 582 bp was visible up to a dilution of 10 corresponding to a detection limit of 0.4 ffd50 ( 10 ffd50 ; figure 1 ) . in the real - time rt - pcr ( ndwd )", "the limit of detection was defined as the last dilution at which at least 2 out of 3 replicates were positive . using this definition , cvs-11 was detectable up to a dilution of 10 ( once at a dilution of 10 ) .", "the amplification plot showed regular intervals of the curves with ct - values from 18.1 ( dilution 10 ) to 40.0 ( dilution 10 ) .", "relating to infectious titre real - time rt - pcr reached a detection limit of 0.003 ffd50 ( 10 ) for cvs-11 .", "the efficiency of the real - time rt - pcr ( ndwd ) was determined using linear regression of the ct - values and the titres of the samples ( figure 2 ) . on the basis of the slope coefficient", "the limit of detection in terms of dna copy numbers was determined using 10-fold serial dilutions of a cloned 933 bp segment of n of cvs-11 containing 10 to 10 dna copies / pcr reaction . in the hnpcr ,", "the limit of detection was 100 dna copies/3 l of starting material of the cvs-11 plasmid .", "in the real - time pcr ( ndwd ) , the 10-fold serial dilution of the cvs - plasmid was tested in triplicate .", "the limit of detection was 10 dna copies/2 l of the cvs-11 plasmid with ct - values between 38.6 and 47.5 .", "the efficiency was at 82.8% with a slope coefficient of 3.82 ( not shown ) .", "the intra - assay repeatability of the real - time pcr ( ndwd ) proved to be excellent with very low coefficients of variation up to 10 dna copies ( 0.181.37% ) with and outlier at 10 copies ( 12.05% ; table 3 ) .", "a total of 31 lyssaviruses ( 19 identified as classical rabv , 7 as eblv-1 , 4 as eblv-2 , and 1 as duvv ) were used to test the diagnostic performance of the pcr ( table 1 ) .", "all viruses used were detected with the expected band size in the hnrt - pcr technique ( table 4 ) .", "the specificity of the amplified product was confirmed as rabv , eblv-1 , eblv-2 , or duvv by sequencing in each case .", "host gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) was detected in all of the cell culture supernatants and mouse brain suspensions as internal control , indicating that sample material was present and rna isolation , reverse transcription , and amplification were not inhibited .", "all samples were also tested positive by the real - time rt - pcr assay ( table 4 ) .", "sendai virus as internal control for inhibition was run in parallel using the same cycling conditions . in the real - time rt - pcr ( ndwd ) with an annealing temperature of 50c , some samples were detected with more than one probe . with the exception of the sample derived from a raccoon dog from poland , the probe for the homologous genotype always yielded the lowest ct - value . in this exceptional case ,", "the probe for the detection of genotype 6 was slightly lower than the one for genotype 1 ( 15.0 versus 16.1 ; table 4 ) .", "comparison of these two probes with the sequence of the rabies variant in question showed 1 and 2 mismatches with the rabvd1-p and lysgt6-p probe , respectively ( figure 3 ) .", "duvenhage virus , also designated as genotype 4 , was detectable with the probe adapted for genotype 6 in spite of 3 mismatches ( lysgt6-p ) .", "the probe lysgt6-p was able to detect synthetic dna fragments of bblv , arav , and irkv , although containing one or two mismatches , respectively .", "the synthetic dna fragments of khuv , ablv , lbv , mokv , shibv , and wcbv , which exhibited from four ( ablv / khuv ) up to ten ( wcbv ) mismatches to the best fitting probe for gt1 , gt6 , or gt5 , respectively , were all detected with the corresponding species - specific new probe ( table 2 ) .", "10 copies of the synthetic dna were detected at ct - values of 27.933.5 for ablv , mokv , shibv , and wcbv ( not shown ) .", "the efficiency of detection of khuv , which showed an atypical amplification plot at the highest number of 10 copies of the synthetic dna , and that of lbv was lower with ct - values of 40.0 and 40.5 at a copy number of 10 , respectively ( not shown ) .", "multiplexing of combinations of these probes ( ivvlbv - p ( fam : 6-carboxyfluorescein ) , ivvkhujand - p ( yy : yakima yellow ) , and ivvsblv2-p ( cy5 : cyanine 5 ) , as well as ivvwcbv - p ( fam ) , ivvmok - p ( yy ) , and ivvablv2n - p ( cy5 ) ) as for the species 1 , 5 , and 6 was not successful .", "the 543 bp amplification products of the nucleoprotein gene obtained in the heminested pcr reactions were sequenced ( table 1 ) and analysed phylogenetically with additional sequences retrieved from genbank representing all known lyssaviruses .", "the similarity of the 543 bp nucleotide sequences of the nucleoprotein gene ( positions 74616 according to the pasteur virus genome ) among the lyssaviruses included ranged from 64.6% to 90.1% ( hamming distance ) .", "the resulting phylogenetic tree obtained by the neighbor - joining method implemented in the mega5 software is presented in figure 4 .", "clinical specimens like brain suspensions ( 1x human , 12x mouse ) , skin biopsies from the nape of the neck ( 3x human , 1x mouse ) , saliva ( 4x human ) , and cerebrospinal fluids ( 6x human ) were used for the establishment of the assays . both hnrt - pcr and real - time rt - pcr ( ndwd ) were shown to work properly on all these samples without significant inhibition using gapdh as internal control for conventional rt - pcr and sendai virus for real - time rt - pcr ( ndwd ) , which was mixed to the samples before rna isolation .", "skin biopsies taken from the neck and lip of a mouse ( white swiss mouse at an age of 3 weeks ) euthanized 2 weeks after intracerebral infection with sad bern virus 20 years ago were positive with the hnrt - pcr and real - time pcr ( ndwd ) .", "skin biopsies taken from other locations on the head were positive with the real - time pcr ( ndwd ) and weakly positive in hnrt - pcr whereas samples taken around the vibrissae were weakly positive in real - time pcr ( ndwd ) only .", "sequencing of amplification products excluded a contamination with the positive cvs-11 control ( not shown ) .", "brain suspension from an imported human rabies case in 2012 was already strongly positive after one round of the hnrt - pcr .", "phylogenetic analysis of the 543 bp nucleotide fragment revealed its close relationship to the classical rabies virus strains circulating in the insectivorous mexican free - tailed bat , tadarida brasiliensis , a species common in the southern united states and mexico .", "this allowed identification of the origin of the patient 's infection , who had travelled extensively and did not report any previous biting incident [ 51 , 52 ] ." ], [ "based on a large amount of published work , we were able to establish and quantitatively characterise rt - pcr protocols for the detection of lyssaviruses in clinical samples .", "well suited real - time protocols [ 8 , 25 ] for the detection of classical rabies virus and the genotypes 5 and 6 ( eblv-1 and eblv-2 ) were adapted and extended for the detection of at least 13 lyssavirus species . to this goal , 6 species - specific probes ( khuv , ablv , lbv , mokv , shibv , and wcbv )", "were designed and verified on synthetic dna fragments encompassing the targeted sequence of the lyssaviral nucleoprotein gene .", "multiplexing the probes in a single - tube reaction as described for the genotypes 1 , 5 , and 6 was not possible with the newly designed probes , probably due to false priming and/or interference within the mix of reagents and synthetic target sequence .", "as far as evaluated with the multiplexed probes for the genotypes 1 , 5 , and 6 using 31 viral isolates , which were all detectable with high sensitivity , direct differentiation of targeted genotypes on the base of the quantitative reaction ( ct - values ) was mostly possible .", "furthermore , a single probe for genotype 6 ( species eblv-2 ) was able to detect up to 7 genotypes / species .", "we consider this type of cross hybridization as an advantage of the technique using degenerate primers and probes in terms of a broad detectability of lyssavirus rather than a lack of accuracy of discrimination . in an approach using sybr green qpcr with similarly degenerate primers spanning the same part of n as in this work , the detection of all lyssavirus species known at that time was achieved .", "using several genotype - specific probes in a taqman real - time protocol , we were able to add more intrinsic confidence to the specificity of the analysis .", "quantitative characterisation of the assay using the probe for genotypes 1 , 5 , and 6 on cvs-11 showed excellent sensitivity and repeatability with a detection limit of as low as an infectious dose of 0.003 ffd50 combined with an analytical sensitivity of 10 dna copies at efficiencies of 94.5% and 82.8% , respectively . considering the amount of degeneration in both primers and probes , the efficiencies determined must be considered satisfactory .", "as far as the absolute detection limit in terms of target copies is concerned , the usage of plasmid dna rather than rna must be kept in mind . since reverse transcription can not be assumed as 100% efficient and reproducible , the limit for rna might be somewhat lower . for further characterisation of samples with a positive reaction in real - time rt - pcr , excellent simple and ( hemi)nested rt - pcr protocols are available . with the protocol used for this work ,", "all 31 viral strains used could be amplified and sequenced , confirming the diagnostic broadness of the assay .", "phylogenetic analysis of these partial nucleotide sequences belonging to genotypes 1 , 4 , 5 , and 6 along with known sequences confirmed the suitability of this relatively conserved genomic region for molecular - epidemiological characterisation of lyssaviruses circulating worldwide [ 5658 ] .", "this was also confirmed in the context of a human rabies case imported to switzerland in 2012 , which could be attributed unequivocally to an exposure to a bat of the species tadarida brasiliensis in california , usa ." ], [ "in this work we could show and validate the suitability of an adapted and further developed real - time rt - pcr protocol for the rapid , efficient , and highly sensitive intravitam diagnosis of a wide spectrum of lyssavirus species followed by rapid molecular - epidemiological characterisation of viral isolates respective to origin of the virus and source of exposure , using heminested rt - pcr .", "this new tool is also particularly promising for active surveillance of european bat lyssaviruses using oral swabs in live - captured bats ." ] ]

[ "poly implant prothse ( pip ) company is known to manufacture breast implants using unapproved industrial grade silicone and their products have been banned after numerous studies reporting an increased risk of complications such as implant rupture and gel leakage.[14 ] over 400,000 pip implants have been utilized for breast augmentation worldwide , and approximately 10,000 have been sold in ukraine .", "overall , various complications have been reported in patients with ruptured breast implants or after free biomaterial injections , which also include , but not limited to neoplasms as breast carcinoma , mesenchymal tumors , and anaplastic large cell lymphoma ( alcl).[510 ] however , little is known about possible biological effect of industrial grade silicone in individuals with ruptured pip implants .", "xanthoma is a benign lesion that is frequently diagnosed in skin , kidney or gallbladder , but rare in breast .", "xanthoma is rarely diagnosed in patients with breast implants and is mainly described in case reports .", "we report a clinical case of a patient previously operated on for breast augmentation , who opted for implant replacement and was diagnosed with a unilateral breast xanthoma associated with pip implant rupture .", "a 30-year - old female was seen by the senior plastic surgeon ( vk ) in january 2015 after bilateral aesthetic breast augmentation in 2007 .", "the patient was concerned about her pip breast implants and knowing possible risks of the devices opted for their removal and replacement with other authorized breast implants .", "preoperative clinical assessment revealed subglandular breast implants with no signs of capsular contracture or other implant - related concerns .", "the woman was otherwise healthy ; non - obese ( body mass index 20 kg / m ) and non - smoker .", "we started with the right side where no noteworthy changes have been seen , i.e. no paraprosthetic fluid , implant was intact with no irregularities , and the capsule looked normal . during the removal of the left implant ,", "further , some visible air bubbles within the implant and small fatty smears on the gloves were discovered , indicating possible subtle ruptures of the implant s shell . to test this hypothesis ,", "this maneuver resulted in rupture of the left implant , whereas the right one remained intact ( figure 1 ) .", "( a ) a yellow implant without signs of rupture and with visible air bubbles inside indicating possible microruptures of the implant shell .", "( b ) ruptured pip device with leakage of the silicone gel after manual compression further revision of the left implant pocket revealed a yellowish well - delimited mass 4 5 cm in the central lower part of the breast over the fascia of the pectoral major muscle .", "a malignant neoplasm was suspected and an excisional biopsy with histopathological and immunohistochemical ( ihc ) analyses were performed .", "we did not proceed with concurrent breast implantation and prioritized rapid tumor verification , evaluation and treatment .", "the histopathology of the specimen revealed xanthoma with no evidence of malignant neoplasm ( figure 2 ) .", "ihc was performed to discriminate the case from soft tissue sarcomas by using antibodies against proteins cd68 ( clone kp1 ) and s100 ( clone 4c4.9 ) ; proliferation was evaluated by analyses of ki-67 ( clone mib-1 ) expression .", "analyses of ihc for histiocytic marker cd68 revealed a strong positive expression in 100% of xanthoma cells , a weak staining of s100 in < 10% of xanthoma cells ( considered as negative ) .", "several positively stained cells with mib-1 indicated the absence of sarcoma and low proliferation , which supported the histopathological diagnosis of benign lesion , i.e. xanthoma ( figure 2 ) .", "( a ) histopathological features of h&e stained xanthoma tissue showing foamy histiocytes with multinucleate giant cells ( magnification 200 ) .", "( b ) negative expression of protein s100 by ihc in xanthoma ( magnification 400 ) .", "( c ) illustration of several xanthoma cells with positive nuclear expression of mib-1 indicating low proliferation ( magnification 400 ) .", "( d ) strong cytoplasmatic expression of cd68 in xanthoma ( magnification 400 ) both breasts were evaluated by ultrasonography , showing some signs of remaining xanthoma tissue but no evidence of other pathological findings postoperatively ( figure 3 ) .", "considering the clinical findings , and to perform a secondary reconstruction with the new breast implants creating new submuscular pockets ( figure 4 ) .", "the remnant xanthoma tissue is visualized as 1 mm hypoechogenic mass ( marked by white crosses ) surgical findings .", "( a ) remnant xanthoma tissue was identified as 2 2 cm yellowish neoplasm in the lower part of the left breast .", "blood biochemistry was performed to evaluate the post - operative status of certain compounds relating to lipid methabolism .", "the analyses of clinical biochemistry revealed that majority of studied parameters were within the normal ranges : triglycerides 0.74 mmol / l ( normal range : < 1.7 ) , cholesterol 4.24 mmol / l ( normal range : < 5 ) , high - density lipoprotein cholesterol 2.04 mmol / l ( normal range : > 1.2 ) , low - density lipoprotein cholesterol 1.9 mmol / l ( normal range : < 3 ) , very low - density lipoprotein cholesterol 0.34 mmol / l ( normal range : 0.261.04 ) , apolipoprotein a1 1.33 g / l ( normal range : 0.762.1 ) , apolipoprotein b 0.58 g / l ( normal range : 0.461.42 ) , lipoprotein ( a ) 9.59 mg / dl ( normal range : < 30 ) . considering the findings and patient s desire to reconstruct the breast , we proceeded with the excision of the remained xanthoma tissue and the delayed secondary breast augmentation was scheduled ( figure 4 ) .", "thus , a total capsulectomy was performed and new implants ( 450 ml , round shaped , polytech health & aesthetics , germany ) were placed in the submuscular position .", "the 6-months follow up revealed good results with no post - operative complications clinically or on ultrasound . as part of the routine assessment , health - related quality of life ( hrqol )", "was evaluated before surgery , after implant removal and 6 months after secondary breast augmentation .", "an eq-5d-5l instrument with a visual analogue scale ( vas ) was used to assess self - rated overall health status ( http://www.euroqol.org/euroqol group , netherlands ) according to the previously published protocol .", "the patient reported no problems related to physical activity , but a problem in the anxiety / depression domain was detected after implants removal ; this returned to normal after the breast implantation .", "self - rated overall health status revealed a decrease from 50 vas units preoperatively to 30 units after implants removal .", "however , 90 vas units were reported 6 months after the placement of the new implants .", "in this study , we reported an unusual case of a ruptured pip implant associated with breast xanthoma .", "this neoplasm is a rare lesion of the breast and an uncommon complication of pip implant rupture . to the best of our knowledge ,", "xanthoma is not included in the list of clinical findings in the current opinion of the scientific committee on emerging and newly identified health risks of european commission ( scenihr , february , 2014 ) . according to scenihr and other studies ,", "commonly reported signs of the pip implant rupture include breast pain , breast and axillary lymphadenopathy , and palpable breast mass associating with migration of silicone gel . in the current case", ", we did not observe any of these signs , which might indicate a different clinical course for patients with xanthoma and micro ruptured pip , also supported by the findings from other studies .", "another differential diagnosis to consider in patients with a breast mass following breast augmentation is anaplastic large cell lymphoma ( alcl ) .", "although alcl is a rare breast malignancy , it is reported as an implant - associated malignant neoplasm developing due to a chronic periprosthetic inflammation.[(7 ] however , no clear association between pip implants and alcl has been yet confirmed according to scenihr or other investigations .", "it is also worth to mention , that before the surgery we were unaware of any seroma in the breast and did not perform any fluid aspiration and cytological evaluation .", "however , an excisional biopsy of the tumor with histhopatological assessment had been performed with following immunohistochemistry , which was considered sufficient to describe the morphological patterns of the tumor and rule out alcl .", "apart from alcl , various mesenchymal tumors ( i.e. fibromatosis , lipomatous tumors or sarcomas ) are known to be associated with breast silicone implants .", "these neoplasms are infrequent and differential diagnosis could be established using histopathological analyses along with ihc staining for s100 protein .", "although malignant mesenchymal tumors were not reported among patients with ruptured pip implants , we performed ihc analyses of s100 expression in our patient , which is in agreement with other studies .", "previously , xanthogranulomatous inflammatory processes in breast were demonstrated in patients with implant rupture , breast cancer patient , or individuals without history of breast lesions . similarly to hernanz et al .", "we did not observe any specific symptoms of xanthoma , unlike hussain et al . who reported an abnormal mass in a breast cancer patient , possibly indicating different mechanisms of xanthoma pathogenesis .", "an association between xanthoma and foreign body reaction is a rare observation and was previously demonstrated in several case reports . according to the data from these studies xanthoma was associated with : oophoritis as a reaction to talcum powder , with cystitis secondary to sutures , as well as with cholecystitis as a reaction to gallstones .", "literature search revealed no evidence of association between breast xanthoma and foreign body reaction . in our study , the microrupture of the pip device was suspected only after removal of the left implant .", "the risk of rupture is known and it was previously shown in a study on elastomeric properties of pip implants that detected a random reduction of thickness and defects of microscopical structures of the shell . moreover ,", "the hypothesis of microscopical structural defects is supported by the presence of the yellow color of the ruptured implant in our case .", "this may arise due to absorption of the fat - soluble compounds due to a high permeability of the pip shell . similarly to other studies we observed some fluid effusion during implant removal as a result of synovial metaplasia associating with implant rupture .", "finally , we also identified a negative impact of implants removal and neoplasm identification on hrqol ; levels of anxiety and depression increased postoperatively , but improved 6 months after the secondary breast augmentation and tumor related reassurance . to summarize , we report a case of breast xanthoma following breast augmentation associated with an asymptomatic microrupture of the pip implant .", "current study supports findings of other papers investigating larger cohorts of patients with pip implants ; it also raises awareness of possible risks among individuals without clinical symptoms of implant rupture and leakage of the silicone gel . in asymptomatic patients who would like to keep their pip implants we recommend more intensive clinical surveillance with the breast ultrasound screening ." ]

[ "<S> abstracta suspicious breast mass was intra - operatively found in a female patient previously operated for breast augmentation with the poly implant prothse ( pip ) implants . </S>", "<S> the neoplasm was verified as xanthoma and an asymptomatic microrupture of pip was also detected . </S>", "<S> this report discusses possible association of breast xanthoma with pip implant rupture . </S>" ]

[ "Introduction", "Case report", "Discussion", "Disclosure statement" ]

[ [ "poly implant prothse ( pip ) company is known to manufacture breast implants using unapproved industrial grade silicone and their products have been banned after numerous studies reporting an increased risk of complications such as implant rupture and gel leakage.[14 ] over 400,000 pip implants have been utilized for breast augmentation worldwide , and approximately 10,000 have been sold in ukraine .", "overall , various complications have been reported in patients with ruptured breast implants or after free biomaterial injections , which also include , but not limited to neoplasms as breast carcinoma , mesenchymal tumors , and anaplastic large cell lymphoma ( alcl).[510 ] however , little is known about possible biological effect of industrial grade silicone in individuals with ruptured pip implants .", "xanthoma is a benign lesion that is frequently diagnosed in skin , kidney or gallbladder , but rare in breast .", "xanthoma is rarely diagnosed in patients with breast implants and is mainly described in case reports .", "we report a clinical case of a patient previously operated on for breast augmentation , who opted for implant replacement and was diagnosed with a unilateral breast xanthoma associated with pip implant rupture ." ], [ "a 30-year - old female was seen by the senior plastic surgeon ( vk ) in january 2015 after bilateral aesthetic breast augmentation in 2007 .", "the patient was concerned about her pip breast implants and knowing possible risks of the devices opted for their removal and replacement with other authorized breast implants .", "preoperative clinical assessment revealed subglandular breast implants with no signs of capsular contracture or other implant - related concerns .", "the woman was otherwise healthy ; non - obese ( body mass index 20 kg / m ) and non - smoker .", "we started with the right side where no noteworthy changes have been seen , i.e. no paraprosthetic fluid , implant was intact with no irregularities , and the capsule looked normal . during the removal of the left implant ,", "further , some visible air bubbles within the implant and small fatty smears on the gloves were discovered , indicating possible subtle ruptures of the implant s shell . to test this hypothesis ,", "this maneuver resulted in rupture of the left implant , whereas the right one remained intact ( figure 1 ) .", "( a ) a yellow implant without signs of rupture and with visible air bubbles inside indicating possible microruptures of the implant shell .", "( b ) ruptured pip device with leakage of the silicone gel after manual compression further revision of the left implant pocket revealed a yellowish well - delimited mass 4 5 cm in the central lower part of the breast over the fascia of the pectoral major muscle .", "a malignant neoplasm was suspected and an excisional biopsy with histopathological and immunohistochemical ( ihc ) analyses were performed .", "we did not proceed with concurrent breast implantation and prioritized rapid tumor verification , evaluation and treatment .", "the histopathology of the specimen revealed xanthoma with no evidence of malignant neoplasm ( figure 2 ) .", "ihc was performed to discriminate the case from soft tissue sarcomas by using antibodies against proteins cd68 ( clone kp1 ) and s100 ( clone 4c4.9 ) ; proliferation was evaluated by analyses of ki-67 ( clone mib-1 ) expression .", "analyses of ihc for histiocytic marker cd68 revealed a strong positive expression in 100% of xanthoma cells , a weak staining of s100 in < 10% of xanthoma cells ( considered as negative ) .", "several positively stained cells with mib-1 indicated the absence of sarcoma and low proliferation , which supported the histopathological diagnosis of benign lesion , i.e. xanthoma ( figure 2 ) .", "( a ) histopathological features of h&e stained xanthoma tissue showing foamy histiocytes with multinucleate giant cells ( magnification 200 ) .", "( b ) negative expression of protein s100 by ihc in xanthoma ( magnification 400 ) .", "( c ) illustration of several xanthoma cells with positive nuclear expression of mib-1 indicating low proliferation ( magnification 400 ) .", "( d ) strong cytoplasmatic expression of cd68 in xanthoma ( magnification 400 ) both breasts were evaluated by ultrasonography , showing some signs of remaining xanthoma tissue but no evidence of other pathological findings postoperatively ( figure 3 ) .", "considering the clinical findings , and to perform a secondary reconstruction with the new breast implants creating new submuscular pockets ( figure 4 ) .", "the remnant xanthoma tissue is visualized as 1 mm hypoechogenic mass ( marked by white crosses ) surgical findings .", "( a ) remnant xanthoma tissue was identified as 2 2 cm yellowish neoplasm in the lower part of the left breast .", "blood biochemistry was performed to evaluate the post - operative status of certain compounds relating to lipid methabolism .", "the analyses of clinical biochemistry revealed that majority of studied parameters were within the normal ranges : triglycerides 0.74 mmol / l ( normal range : < 1.7 ) , cholesterol 4.24 mmol / l ( normal range : < 5 ) , high - density lipoprotein cholesterol 2.04 mmol / l ( normal range : > 1.2 ) , low - density lipoprotein cholesterol 1.9 mmol / l ( normal range : < 3 ) , very low - density lipoprotein cholesterol 0.34 mmol / l ( normal range : 0.261.04 ) , apolipoprotein a1 1.33 g / l ( normal range : 0.762.1 ) , apolipoprotein b 0.58 g / l ( normal range : 0.461.42 ) , lipoprotein ( a ) 9.59 mg / dl ( normal range : < 30 ) . considering the findings and patient s desire to reconstruct the breast , we proceeded with the excision of the remained xanthoma tissue and the delayed secondary breast augmentation was scheduled ( figure 4 ) .", "thus , a total capsulectomy was performed and new implants ( 450 ml , round shaped , polytech health & aesthetics , germany ) were placed in the submuscular position .", "the 6-months follow up revealed good results with no post - operative complications clinically or on ultrasound . as part of the routine assessment , health - related quality of life ( hrqol )", "was evaluated before surgery , after implant removal and 6 months after secondary breast augmentation .", "an eq-5d-5l instrument with a visual analogue scale ( vas ) was used to assess self - rated overall health status ( http://www.euroqol.org/euroqol group , netherlands ) according to the previously published protocol .", "the patient reported no problems related to physical activity , but a problem in the anxiety / depression domain was detected after implants removal ; this returned to normal after the breast implantation .", "self - rated overall health status revealed a decrease from 50 vas units preoperatively to 30 units after implants removal .", "however , 90 vas units were reported 6 months after the placement of the new implants ." ], [ "in this study , we reported an unusual case of a ruptured pip implant associated with breast xanthoma .", "this neoplasm is a rare lesion of the breast and an uncommon complication of pip implant rupture . to the best of our knowledge ,", "xanthoma is not included in the list of clinical findings in the current opinion of the scientific committee on emerging and newly identified health risks of european commission ( scenihr , february , 2014 ) . according to scenihr and other studies ,", "commonly reported signs of the pip implant rupture include breast pain , breast and axillary lymphadenopathy , and palpable breast mass associating with migration of silicone gel . in the current case", ", we did not observe any of these signs , which might indicate a different clinical course for patients with xanthoma and micro ruptured pip , also supported by the findings from other studies .", "another differential diagnosis to consider in patients with a breast mass following breast augmentation is anaplastic large cell lymphoma ( alcl ) .", "although alcl is a rare breast malignancy , it is reported as an implant - associated malignant neoplasm developing due to a chronic periprosthetic inflammation.[(7 ] however , no clear association between pip implants and alcl has been yet confirmed according to scenihr or other investigations .", "it is also worth to mention , that before the surgery we were unaware of any seroma in the breast and did not perform any fluid aspiration and cytological evaluation .", "however , an excisional biopsy of the tumor with histhopatological assessment had been performed with following immunohistochemistry , which was considered sufficient to describe the morphological patterns of the tumor and rule out alcl .", "apart from alcl , various mesenchymal tumors ( i.e. fibromatosis , lipomatous tumors or sarcomas ) are known to be associated with breast silicone implants .", "these neoplasms are infrequent and differential diagnosis could be established using histopathological analyses along with ihc staining for s100 protein .", "although malignant mesenchymal tumors were not reported among patients with ruptured pip implants , we performed ihc analyses of s100 expression in our patient , which is in agreement with other studies .", "previously , xanthogranulomatous inflammatory processes in breast were demonstrated in patients with implant rupture , breast cancer patient , or individuals without history of breast lesions . similarly to hernanz et al .", "we did not observe any specific symptoms of xanthoma , unlike hussain et al . who reported an abnormal mass in a breast cancer patient , possibly indicating different mechanisms of xanthoma pathogenesis .", "an association between xanthoma and foreign body reaction is a rare observation and was previously demonstrated in several case reports . according to the data from these studies xanthoma was associated with : oophoritis as a reaction to talcum powder , with cystitis secondary to sutures , as well as with cholecystitis as a reaction to gallstones .", "literature search revealed no evidence of association between breast xanthoma and foreign body reaction . in our study , the microrupture of the pip device was suspected only after removal of the left implant .", "the risk of rupture is known and it was previously shown in a study on elastomeric properties of pip implants that detected a random reduction of thickness and defects of microscopical structures of the shell . moreover ,", "the hypothesis of microscopical structural defects is supported by the presence of the yellow color of the ruptured implant in our case .", "this may arise due to absorption of the fat - soluble compounds due to a high permeability of the pip shell . similarly to other studies we observed some fluid effusion during implant removal as a result of synovial metaplasia associating with implant rupture .", "finally , we also identified a negative impact of implants removal and neoplasm identification on hrqol ; levels of anxiety and depression increased postoperatively , but improved 6 months after the secondary breast augmentation and tumor related reassurance . to summarize , we report a case of breast xanthoma following breast augmentation associated with an asymptomatic microrupture of the pip implant .", "current study supports findings of other papers investigating larger cohorts of patients with pip implants ; it also raises awareness of possible risks among individuals without clinical symptoms of implant rupture and leakage of the silicone gel . in asymptomatic patients who would like to keep their pip implants we recommend more intensive clinical surveillance with the breast ultrasound screening ." ], [ "" ] ]

[ "invasive meningococcal disease ( imd ) is due to infection with the gram - negative bacterium neisseria meningitidis ( n. meningitidis ) .", "although fulminant septicemia is less common , it is highly fatal , even if treated .", "approximately 500 000 cases occur worldwide annually , resulting in approximately 50 000 deaths and severe long - term complications in up to 20% of survivors .", "deafness , intellectual and learning impairment , epilepsy , and other neurological deficits are frequent sequelae that lead to long - term reduction in quality of life . according to the world health organisation ( who ) estimates , the overall notification rate of imd in europe ranges from 0.214 cases per 100 000 in the general population .", "although the rates of imd can be low in european and other industrialized countries , the disease is potentially devastating , with a case mortality rate that has remained at about 510% despite treatment . in europe ,", "the highest numbers of confirmed imd cases are reported in children younger than 5 y of age ( 7.37 per 100 000 ) , followed by children aged 1524 y ( 1.44 per 100 000 ) .", "most of these cases of imd occur sporadically , although small local outbreaks are not uncommon .", "while the majority of imd infections are caused by n. meningitidis serogroups a , b , c , x , w-135 , or y , their relative prevalence has varied considerably over time and by geographical location , making trends in disease epidemiology unpredictable . in europe , the majority of imd cases ( 85% ) are caused by serogroups b and c , while serogroup y is the third most frequently reported cause ( 3% in 2008 , and 4% in 2009 ) .", "overall , the number of cases of imd attributable to serogroup y increased by 42% across european countries between 2007 and 2009 . however , the incidence of n. meningitidis infection due to serogroup c has declined since the introduction of the meningococcal group c conjugate vaccine ( mcc ) in some european countries while the incidence of imd across europe has also changed .", "active surveillance is crucial to assess the epidemiology and burden of imd , as well as the impact of vaccination . in european countries , in which adequate surveillance systems have been in place for > 10 y", ", long - term surveillance has detected large reductions in meningococcal serogroup c disease following the introduction of the mcc .", "however , the scarcity of comprehensive country - specific data on the current burden of meningococcal disease in turkey arises from the lack of homogenous surveillance system , making it difficult to understand and to predict the dynamic changes in serogroup - specific epidemiology .", "meningococcal conjugate acwy vaccine is used for both individual protection ( e.g. , target groups such as hajj pilgrims , military recruits , or teenagers in the united states ) and mass campaign vaccinations .", "it is established that mcv has a great impact in reducing meningitis if used at large scale , such as the use of menc conjugated vaccine in the uk . while commonly used meningococcal vaccines in europe cover serogroups a , c , y , and w-135 , an effective vaccine against serogroup b has not been available until recently . a quadrivalent protein - based serogroup b vaccine developed by novartis vaccines and diagnostics has been licensed in the eu for infant vaccination . although its use has not been implemented in large - scale vaccination programmes , phase iii studies have demonstrated that the vaccine has the potential to protect against 78% of all imd - causative serogroup b strains .", "lack of systemic surveillance data on serogroup - specific meningococcal epidemiology complicates the decision to be made by health authorities with regard to implementation and choice of available meningococcal vaccines .", "the purpose of this review is to provide up - to - date information on the epidemiological situation regarding the burden of imd in turkey .", "in turkey , data available on the burden of imd at the national level are based on records collected by the turkish statistical institute ( tsi ) and by the ministry of health .", "according to ministry of health data , between 1989 and 1999 , the incidence of imd varied between 1 and 35 cases per 100 000 and the mortality rate varied between 0.71 and 2.62 per million cases .", "the tsi reports that meningococcal infections were responsible for 10.2% ( 1336 ) of all deaths in children under 5 y of age in year 2008 .", "tsi reports 21 , 14 , and 15 deaths from meningococcal infections in all age groups in 2010 , 2011 , and 2012 , respectively based on icd-10 coding system .", "these contradictory data might be due to either the notification procedure ( report of death due to imd or report of newly identified imd cases ) or the identification of causes .", "the current situation on meningococcal epidemiology may be better reflected by the prospective multicenter sentinel surveillance for childhood bacterial meningitis conducted by 2 groups since year 2005 .", "one of the investigator - initiated sentinel surveillance studies was conducted by ceyhan et al . between february 16 , 2005 , through february 15 , 2006 , for acute bacterial meningitis among children admitted to 12 participating hospitals from 7 geographic areas and 12 health centers in 9 cities .", "this study reports that n. meningitidis was the leading cause of bacterial meningitis in children aged < 1 y ( 56.5% ) followed by those aged 14 y. among the samples that were positive for n. meningitidis following pcr analysis , serogroup w-135 was the cause of most infections59 ( 42.7% ) cases were due to serogroup w-135 , 43 ( 31.1% ) were due to serogroup b , 3 ( 2.2% ) were due to serogroup y , and 1 ( 0.7% ) was due to serogroup a. there were no cases with a positive result for serogroup c. in this study , serogroup w-135 was also more common in children aged 416 y , while serogroup b was slightly more common in children aged <3 y. n. meningitidis serogroup w-135 was more prominent than the other meningococcal serogroups in the southeast anatolia while n. meningitidis serogroup b was much more common in the marmara ( northwestern turkey ) and the central region . during ongoing surveillance by ceyhan et al . between 20072009 , while the overall imd incidence was stable , the prevalence of imd due to serogroup b was reported to increase to 35% and the prevalence of serogroup w-135 decreased .", "interestingly , the same group states that during the first 9 mo of 2011 , serogroup w135 was the predominant serogroup , followed by serogroup a , and serogroup b was observed in only 2.5% of cases .", "another nationwide prospective rt - pcr - based surveillance for childhood bacterial meningitis was conducted between july 2006 and january 2009 and the turkish ministry of health with participation of 37 referral hospitals located in 23 cities in 7 geographic regions of the country .", "rt - pcr detected bacterial dna in 246 ( 29% ) of 841 csf specimens from children with suspected bacterial meningitis , of which 131 ( 53% ) were due to streptococcus pneumoniae , 47 ( 19% ) were due to neisseria meningitidis , and 40 ( 16% ) were due to hemophilus influenzae type b. in this study , 29 ( 62% ) of 47 n. meningitidis isolates were determined as serogroup b , 2 ( 4% ) serogroup c , 1 ( 2% ) serogroup x , 1 ( 2% ) serogroup a , 1 ( 2% ) serogroup w135 , and 13 ( 28% ) other serogroups ( undetermined ) .", "sixteen ( 32% ) of n. meningitidis were detected from the marmara ( north - western turkey ) region and only 5 ( 11% ) from the southeast anatolia .", "to prevent imd among young infants , protection needs to be achieved early in life . since the introduction of the first meningococcal conjugate vaccines in 1999", ", remarkable progress has been made in reducing the morbidity and mortality caused by meningococcal disease .", "studies have demonstrated that meningococcal c conjugate ( mcc ) vaccination confers protection in infancy ( 012 mo ) from the first dose but this protection is short - term .", "however , administration of a booster dose early in the second year of life results in longer protection .", "the burden of imd is the highest among infants aged < 1 y ( 2.6 cases per 100 000 persons ) in the us . among infants younger than 1 y of age , more than 50% of cases", "it was recently reported that bacterial meningitis was most frequently observed in the first 7 mo of life , regardless of the etiologic agent .", "all meningococcal meningitis cases occurred in those < 6 mo of age and mostly in those <3 mo of age .", "all cases in the youngest infants were due to meningococcal serogroup b and w135 in turkey .", "if meningococcal vaccines against men b and serogroups a , c , w135 , y with early infant indications may also be registered , we would have an opportunity to protect young infants starting from 2 mo of age in turkey .", "the growing - complexity of national immunization programme ( nip ) in turkey has resulted in as many as 4 required injections during a single healthy baby visit .", "the number of injections in a well - baby visit in the us is up to 7 .", "protecting against vaccine preventable meningococcal serogroups will result in an increase in the number of injections up to 6 in turkey , but this is acceptable when we compare with developed countries ( table 1 ) current nip schedule .", "contrasting data obtained from different surveillance studies may be related to regional differences in the weight of subjects enrolled from different regions of the country or methods used .", "argues that higher rates of serogroup w-135 since 2005 in their earlier study is due to 100 000 turkish hajj pilgrims traveling to saudi arabia every year , which was also seen in malaysia and singapore .", "the results reported by bakir et al . showing the predominance of serogroup b may be explained by the millions of the turkish diaspora living in europe , where serogroup b is predominant , who travel back and forth to the country many times annually .", "this is a more likely explanation of the existing sero - epidemiology because other explanations of the sharp decrease of serogroup b imd from 35% down to 2.5% in only a few years in the absence of serogroup - specific vaccination are unlikely .", "this variation may also reflect the introduction and implementation of detection techniques such as the pcr .", "sero - epidemiology data on imd in 2 different surveillance studies in turkey at the same time periods had different results .", "this complicates the decision to be made by health authorities on the implementation and the type of meningococcal vaccine to be chosen . in the light of available surveillance data in turkey , a vaccine effective against meningococcus serogroup b as well as serogroup", "systematic continuous surveillance is warranted to measure the urgency of need and effectiveness of a vaccination program for imd . the who recommends the introduction of appropriate large - scale meningococcal vaccination programmes in countries with high ( > 10 cases/100 000 population / year ) or intermediate ( 210 cases/100 000", "population / year ) imd incidence rates , and in countries with frequent epidemics . in countries with lower imd incidence rates ( < 2 cases/100 000 population / year ) , vaccination is recommended for defined risk groups , including children and young adults residing in close communities , laboratory workers at risk of exposure , travelers to high - endemic areas and immunodeficient individuals .", "overall , each country should choose the appropriate vaccination program depending on the locally prevalent serogroup of n. meningitidis .", "conjugate vaccines are preferred over polysaccharide vaccines due to their potential for herd protection , immunologic memory and their increased immunogenicity , particularly in children < 2 y of age ." ]

[ "<S> each country chooses the appropriate vaccine against imd depending on the locally prevalent serogroups of n. meningitides . </S>", "<S> frequency of each meningococcal serogroup varies considerably over time and by geographical location . despite the majority of imd cases ( 85% ) </S>", "<S> are caused by serogroups b and c in europe , true prevalence of serogroup b imd cases in turkey is unclear . in one of the recent studies , the sharp decrease of serogroup b imd from 35% down to 2.5% in a few years despite the absence of vaccination is confusing . </S>", "<S> millions of european turkish people travels from europe to turkey every year who could most probably carry serogroup b. it is obvious that a nationwide active surveillance is crucial to assess the the true epidemiology and burden of imd which has a major impact on the choice of vaccine type and age interval the vaccination to be implemented . </S>" ]

[ "Introduction", "Available Data on the Burden of IMD in Turkey", "Discussion" ]

[ [ "invasive meningococcal disease ( imd ) is due to infection with the gram - negative bacterium neisseria meningitidis ( n. meningitidis ) .", "although fulminant septicemia is less common , it is highly fatal , even if treated .", "approximately 500 000 cases occur worldwide annually , resulting in approximately 50 000 deaths and severe long - term complications in up to 20% of survivors .", "deafness , intellectual and learning impairment , epilepsy , and other neurological deficits are frequent sequelae that lead to long - term reduction in quality of life . according to the world health organisation ( who ) estimates , the overall notification rate of imd in europe ranges from 0.214 cases per 100 000 in the general population .", "although the rates of imd can be low in european and other industrialized countries , the disease is potentially devastating , with a case mortality rate that has remained at about 510% despite treatment . in europe ,", "the highest numbers of confirmed imd cases are reported in children younger than 5 y of age ( 7.37 per 100 000 ) , followed by children aged 1524 y ( 1.44 per 100 000 ) .", "most of these cases of imd occur sporadically , although small local outbreaks are not uncommon .", "while the majority of imd infections are caused by n. meningitidis serogroups a , b , c , x , w-135 , or y , their relative prevalence has varied considerably over time and by geographical location , making trends in disease epidemiology unpredictable . in europe , the majority of imd cases ( 85% ) are caused by serogroups b and c , while serogroup y is the third most frequently reported cause ( 3% in 2008 , and 4% in 2009 ) .", "overall , the number of cases of imd attributable to serogroup y increased by 42% across european countries between 2007 and 2009 . however , the incidence of n. meningitidis infection due to serogroup c has declined since the introduction of the meningococcal group c conjugate vaccine ( mcc ) in some european countries while the incidence of imd across europe has also changed .", "active surveillance is crucial to assess the epidemiology and burden of imd , as well as the impact of vaccination . in european countries , in which adequate surveillance systems have been in place for > 10 y", ", long - term surveillance has detected large reductions in meningococcal serogroup c disease following the introduction of the mcc .", "however , the scarcity of comprehensive country - specific data on the current burden of meningococcal disease in turkey arises from the lack of homogenous surveillance system , making it difficult to understand and to predict the dynamic changes in serogroup - specific epidemiology .", "meningococcal conjugate acwy vaccine is used for both individual protection ( e.g. , target groups such as hajj pilgrims , military recruits , or teenagers in the united states ) and mass campaign vaccinations .", "it is established that mcv has a great impact in reducing meningitis if used at large scale , such as the use of menc conjugated vaccine in the uk . while commonly used meningococcal vaccines in europe cover serogroups a , c , y , and w-135 , an effective vaccine against serogroup b has not been available until recently . a quadrivalent protein - based serogroup b vaccine developed by novartis vaccines and diagnostics has been licensed in the eu for infant vaccination . although its use has not been implemented in large - scale vaccination programmes , phase iii studies have demonstrated that the vaccine has the potential to protect against 78% of all imd - causative serogroup b strains .", "lack of systemic surveillance data on serogroup - specific meningococcal epidemiology complicates the decision to be made by health authorities with regard to implementation and choice of available meningococcal vaccines .", "the purpose of this review is to provide up - to - date information on the epidemiological situation regarding the burden of imd in turkey ." ], [ "in turkey , data available on the burden of imd at the national level are based on records collected by the turkish statistical institute ( tsi ) and by the ministry of health .", "according to ministry of health data , between 1989 and 1999 , the incidence of imd varied between 1 and 35 cases per 100 000 and the mortality rate varied between 0.71 and 2.62 per million cases .", "the tsi reports that meningococcal infections were responsible for 10.2% ( 1336 ) of all deaths in children under 5 y of age in year 2008 .", "tsi reports 21 , 14 , and 15 deaths from meningococcal infections in all age groups in 2010 , 2011 , and 2012 , respectively based on icd-10 coding system .", "these contradictory data might be due to either the notification procedure ( report of death due to imd or report of newly identified imd cases ) or the identification of causes .", "the current situation on meningococcal epidemiology may be better reflected by the prospective multicenter sentinel surveillance for childhood bacterial meningitis conducted by 2 groups since year 2005 .", "one of the investigator - initiated sentinel surveillance studies was conducted by ceyhan et al . between february 16 , 2005 , through february 15 , 2006 , for acute bacterial meningitis among children admitted to 12 participating hospitals from 7 geographic areas and 12 health centers in 9 cities .", "this study reports that n. meningitidis was the leading cause of bacterial meningitis in children aged < 1 y ( 56.5% ) followed by those aged 14 y. among the samples that were positive for n. meningitidis following pcr analysis , serogroup w-135 was the cause of most infections59 ( 42.7% ) cases were due to serogroup w-135 , 43 ( 31.1% ) were due to serogroup b , 3 ( 2.2% ) were due to serogroup y , and 1 ( 0.7% ) was due to serogroup a. there were no cases with a positive result for serogroup c. in this study , serogroup w-135 was also more common in children aged 416 y , while serogroup b was slightly more common in children aged <3 y. n. meningitidis serogroup w-135 was more prominent than the other meningococcal serogroups in the southeast anatolia while n. meningitidis serogroup b was much more common in the marmara ( northwestern turkey ) and the central region . during ongoing surveillance by ceyhan et al . between 20072009 , while the overall imd incidence was stable , the prevalence of imd due to serogroup b was reported to increase to 35% and the prevalence of serogroup w-135 decreased .", "interestingly , the same group states that during the first 9 mo of 2011 , serogroup w135 was the predominant serogroup , followed by serogroup a , and serogroup b was observed in only 2.5% of cases .", "another nationwide prospective rt - pcr - based surveillance for childhood bacterial meningitis was conducted between july 2006 and january 2009 and the turkish ministry of health with participation of 37 referral hospitals located in 23 cities in 7 geographic regions of the country .", "rt - pcr detected bacterial dna in 246 ( 29% ) of 841 csf specimens from children with suspected bacterial meningitis , of which 131 ( 53% ) were due to streptococcus pneumoniae , 47 ( 19% ) were due to neisseria meningitidis , and 40 ( 16% ) were due to hemophilus influenzae type b. in this study , 29 ( 62% ) of 47 n. meningitidis isolates were determined as serogroup b , 2 ( 4% ) serogroup c , 1 ( 2% ) serogroup x , 1 ( 2% ) serogroup a , 1 ( 2% ) serogroup w135 , and 13 ( 28% ) other serogroups ( undetermined ) .", "sixteen ( 32% ) of n. meningitidis were detected from the marmara ( north - western turkey ) region and only 5 ( 11% ) from the southeast anatolia ." ], [ "to prevent imd among young infants , protection needs to be achieved early in life . since the introduction of the first meningococcal conjugate vaccines in 1999", ", remarkable progress has been made in reducing the morbidity and mortality caused by meningococcal disease .", "studies have demonstrated that meningococcal c conjugate ( mcc ) vaccination confers protection in infancy ( 012 mo ) from the first dose but this protection is short - term .", "however , administration of a booster dose early in the second year of life results in longer protection .", "the burden of imd is the highest among infants aged < 1 y ( 2.6 cases per 100 000 persons ) in the us . among infants younger than 1 y of age , more than 50% of cases", "it was recently reported that bacterial meningitis was most frequently observed in the first 7 mo of life , regardless of the etiologic agent .", "all meningococcal meningitis cases occurred in those < 6 mo of age and mostly in those <3 mo of age .", "all cases in the youngest infants were due to meningococcal serogroup b and w135 in turkey .", "if meningococcal vaccines against men b and serogroups a , c , w135 , y with early infant indications may also be registered , we would have an opportunity to protect young infants starting from 2 mo of age in turkey .", "the growing - complexity of national immunization programme ( nip ) in turkey has resulted in as many as 4 required injections during a single healthy baby visit .", "the number of injections in a well - baby visit in the us is up to 7 .", "protecting against vaccine preventable meningococcal serogroups will result in an increase in the number of injections up to 6 in turkey , but this is acceptable when we compare with developed countries ( table 1 ) current nip schedule .", "contrasting data obtained from different surveillance studies may be related to regional differences in the weight of subjects enrolled from different regions of the country or methods used .", "argues that higher rates of serogroup w-135 since 2005 in their earlier study is due to 100 000 turkish hajj pilgrims traveling to saudi arabia every year , which was also seen in malaysia and singapore .", "the results reported by bakir et al . showing the predominance of serogroup b may be explained by the millions of the turkish diaspora living in europe , where serogroup b is predominant , who travel back and forth to the country many times annually .", "this is a more likely explanation of the existing sero - epidemiology because other explanations of the sharp decrease of serogroup b imd from 35% down to 2.5% in only a few years in the absence of serogroup - specific vaccination are unlikely .", "this variation may also reflect the introduction and implementation of detection techniques such as the pcr .", "sero - epidemiology data on imd in 2 different surveillance studies in turkey at the same time periods had different results .", "this complicates the decision to be made by health authorities on the implementation and the type of meningococcal vaccine to be chosen . in the light of available surveillance data in turkey , a vaccine effective against meningococcus serogroup b as well as serogroup", "systematic continuous surveillance is warranted to measure the urgency of need and effectiveness of a vaccination program for imd . the who recommends the introduction of appropriate large - scale meningococcal vaccination programmes in countries with high ( > 10 cases/100 000 population / year ) or intermediate ( 210 cases/100 000", "population / year ) imd incidence rates , and in countries with frequent epidemics . in countries with lower imd incidence rates ( < 2 cases/100 000 population / year ) , vaccination is recommended for defined risk groups , including children and young adults residing in close communities , laboratory workers at risk of exposure , travelers to high - endemic areas and immunodeficient individuals .", "overall , each country should choose the appropriate vaccination program depending on the locally prevalent serogroup of n. meningitidis .", "conjugate vaccines are preferred over polysaccharide vaccines due to their potential for herd protection , immunologic memory and their increased immunogenicity , particularly in children < 2 y of age ." ] ]

[ "correct estimation of protein concentration in aqueous biological samples is an essential step in biochemical research and the pharmaceutical industry impacting downstream applications ranging from biomarker studies to quality control in the production of biotherapeutics .", "determination of protein concentration in most popular assays is accomplished via comparison to a sequence - based extinction coefficient ( uv measurements ) or in relation to a standard ( traditional dye - based absorbance assays such as bca , lowry , and bradford ) [ 13 ] .", "however , most recent reviews point out the fact that , due to assay - specific limitations , there is no single method dominating protein quantification [ 1 , 3 ] . while uv based quantification is reliant upon absorbance of tryptophan , tyrosine , and cysteine at 280 nm [ 4 , 5 ] , a protein 's extinction coefficient can vary widely with sequence .", "in fact , a greater than two - fold difference is observed between extinction coefficients calculated for albumin and immunoglobulin g. also , mixtures of unknown composition ( most notably , biologically relevant samples ) can not be confidently quantified based on absorption at 280 nm .", "moreover , amino acid analysis ( aaa ) is capable of delivering possibly the most accurate protein quantitation , but the method is expensive with lengthy turnaround times if samples are sent to a third party for analysis . performed in - house , aaa requires time - consuming sample manipulation and specialized equipment .", "infrared ( ir ) spectroscopy is a powerful and growing analytical tool for the detection and analysis of biomedically relevant compounds such as proteins , lipids , carbohydrates , and nucleic acids [ 69 ] .", "midinfrared ( mir ) spectroscopy is based on the absorption of radiation in the approximate range 4000400 cm and is currently considered among the most promising spectroscopic techniques for application in biomedical research and diagnostics [ 1012 ] .", "also , mir spectroscopy has been recognized as a viable method for lipid analysis [ 1316 ] and is one of the oldest and well established experimental techniques for the analysis of protein and carbohydrate structure [ 1721 ] .", "attenuated total reflection ( atr ) spectroscopy and transmission flow - through cells used in combination with complex chemometric data analysis have recently enabled fast quantitative protein analysis directly from aqueous samples [ 2229 ] . however , while flow - through cells for protein quantification allow for automated sample analysis , these devices have a propensity for clogging , requiring frequent , time - consuming cleaning procedures . while atr cells are more robust , the required sample volumes ( 1025 l ) may be considered significant , particularly in situations where biological samples , limited by both volume and repeat access , are to be analyzed .", "however , due to enhanced sensitivity , the multivariate approach ( e.g. , partial least - squares analysis ( pls ) ) is usually applied to data analysis [ 14 , 2229 ] reducing attractiveness of the method for routine application in biological laboratories that usually lack the time and expertise required for method development and validation . to our knowledge , an easy , fast , and robust method utilizing atr and univariate data analysis for accurate and reproducible protein quantitation from complex biological samples in a dried format has not yet been reported .", "several amide bands have been identified in mir spectroscopy allowing for characterization and quantification of proteins . among these ,", "amide i ( 16001690 cm ) and amide ii ( 14801575 cm ) are recognized as the most representative of all vibration modes [ 17 , 18 ] .", "the amide i absorption consists predominantly of c = o stretching vibration ( about 80% ) with a minor contribution from the c ", "n stretching vibration ( 20% ) , while the amide ii band is more equally split between n h bending ( 60% ) and c ", "until recently , analysis of the amide i and ii absorption regions has been severely hampered in aqueous samples due to spectral interference of a strong water absorption band at 15001700 cm . while the advent of atr and flow - through cells has circumvented the water interference issues , their utility is limited due to practical drawbacks which include instrumentation cost , expertise , and time required for method development and accurate data analysis . a simple univariate ( beer - lambert ) analysis ,", "applied in the method reported here , relies on the integration of amide i band and uses directly searchable absorptions on the spectrum curve .", "reported protein quantification by mir , while still based on a curve - fitting technique , presents substantial advantages over other current methodologies like uv absorbance or colorimetric assays .", "first , unlike uv absorbance at 280 nm , mir - based protein quantitation is much less dependent upon amino acid composition . also", ", amide bond quantitation by mir is not subject to signal interference from many common biological buffer components such as detergents , reducing agents , and chelators , demonstrating superiority over standard colorimetric assays .", "moreover , the mir - based method enables fast and accurate peptide quantitation providing researchers with a robust substitute for time - consuming amino acid analysis .", "however , when compared to uv spectroscopy , ir instruments are more costly and require technical expertise as well as time - consuming method development preventing widespread applicability of mir for general protein quantification .", "thus , an instrument or method , allowing for simple and more cost - effective analysis of samples , while at the same time combining the advantages of atr and flow - through based systems , would be of unique value .", "additionally , in contrast to uv or any other known protein quantitation method , simple , mir - based analysis can also be employed for simultaneous analysis of lipids or detergents [ 14 , 3036 ] . due to their complex and varied chemical composition ,", "characteristic lipid bands , such as the aliphatic group stretching ( 30002800 cm ) , ester c = o stretching ( around 1740 cm ) , or phosphate stretching ( around 1235 cm ) permit qualitative and quantitative analysis of lipid content [ 14 , 31 , 32 ] . due to similarities in composition , detergents possess mir absorption spectra that closely resemble lipids present in the cell membrane and can be analyzed along with lipids .", "this commonality offers researchers a means for monitoring the efficiency of residual detergent removal from lysate - derived samples prior to downstream application .", "the method reported in this paper uses a hydrophilic polytetrafluoroethylene ( ptfe ) membrane engineered for sample application and retention .", "the membrane is transparent in the mir regions used for protein and lipid / detergent analysis .", "the size of the sample application spot was further designed to be slightly smaller than the ir beam , ensuring probing of the entire sample .", "this constraining feature is important in enabling simple , univariate , quantitative measurements . in comparison to currently available techniques and instruments ( e.g. , hts - xt system by bruker optics ) , the method described here is technically less complex ; thus , it is more cost - effective and especially well suited for routine analysis of small sample numbers .", "also , given the minimal volume ( 2 l ) required for measurement , this method can be successfully applied for the analysis of precious material available in limited quantities .", "the presented paper provides a detailed description of method development along with a comparison to other protein quantification techniques routinely used in biological laboratories , with respect to the required sample volume , time - consumption , labor - intensity , accuracy , and robustness .", "in addition to general protein quantification , the ability for simultaneous lipid analysis was also investigated .", "well characterized solutions of several purified proteins , protein mixtures , and examples of lipids and detergents were used to assess quantification limits , dynamic range , linearity , accuracy , precision , and robustness of the reported method .", "further applicability of the method for the analysis of biological samples was demonstrated using cell lysates and tissue homogenates .", "in order to avoid cleaning steps in between series of successive measurements , a disposable device for sample application was designed . from several investigated options ,", "a membrane - based system emerged as the most cost - efficient and the easiest to use , predominantly because it allowed rapid water removal and drying of the sample .", "various membranes were tested ( data not shown ) ; ultimately a hydrophilic ptfe - based membrane was selected due to its transparency within the relevant mir regions , allowing for the quantification of proteins and lipids . as shown in figure 1(a ) , the membrane spectrum contains a strong signal between 1100 and 1300 cm but is transparent in the mir region above 1300 cm used for the analysis of biological samples reported here .", "the hydrophilic ptfe membrane ( 30 m thick ) is mounted on cardboard frame to allow easier handling and provide a place to record sample names , while also assuring consistent presentation for interrogation by the mir beam .", "the card configuration contains four analysis areas designed for easy application and containment of the entire sample within the focused ir beam .", "the hydrophilic spot for sample application ( 4.5 mm diameter ; see figure 1(b ) and supplementary figure s1a , the supplementary material will be available online at http://dx.doi.org/10.1155/2014/657079 . ) is surrounded by a hydrophobic ring generated by mechanical removal of hydrophilic surface ; the ring prevents analyte dispersal promoting precise presentation of the entire sample to the mir beam ( diameter 6.5 mm ) [ 37 , 38 ] .", "the reported method permits two ways of drying aqueous sample spotted onto ptfe membrane . on average", ", 2 l of sample placed on the membrane spot requires around 10 to 15 minutes to dry completely at room temperature .", "the sample can also be dried within around 30 seconds , on average , by the heater and fan located in the sample carrier chamber of the dedicated ftir spectrometer .", "4-spot card was selected for the final configuration to allow rapid analysis of an individual sample in triplicate or three separate samples in comparison to an appropriate background buffer spot .", " measurements were performed using the direct detect spectrometer ( emd millipore ) , a fourier transform ( ft)-ir system analyzing the spectral range 5006000 cm .", "a classical univariate quantification method , based on the fact that mir spectroscopy of nonscattering samples obeys beer 's law , was applied to data analysis . for each protein and lipid / detergent measurement ,", "a buffer subtraction step was performed only on regions of the spectra used for amide i and/or aliphatic symmetric stretching signal analysis .", "proteins are quantified using the amide i region ( 17021602 cm ) only ; thus , all other regions of the mir spectrum are not critical for the analysis .", "consequently , for accurate protein quantification , it was sufficient to subtract buffer in the region ( 18501350 cm ) surrounding amide i area .", "the same procedure was applied to aliphatic stretching region used for lipid / detergent analysis .", "buffer subtraction is performed between 3100 and 2600 cm ; the rest of mir spectra is not being utilized .", "amide i band integration is performed by anchoring the baseline at fixed points between 1702 and 1602 cm and determining the amide i signal value at the highest point between these wavenumbers .", "two additional integration methods ( not discussed here ) accounting for possible buffer interference have also been developed .", "aliphatic symmetric stretching band integration is performed by anchoring the baseline between 2990 and 2810 cm and determining the signal strength at the highest point between 2868 and 2838 cm . to compare the theoretical variability of various quantitation methods ,", "a protein database was examined . for example , the theoretical extinction coefficient at 280 nm is based on the number of tryptophan , tyrosine , and cysteine residues in the protein .", "the mir - based analysis is dependent on the number of amide bonds , which not only link the amino acids together but are also present in the side chains of asparagine and glutamine . to automate the examination of the database , a visual basic for applications ( vba ) program was written for excel ( microsoft ) to check all 20233 proteins in the human protein database ( uniprot release 2012_10 , ) for ( 1 ) total count of amino acids , ( 2 ) number of glutamines , ( 3 ) number of asparagines , ( 4 ) number of tryptophans , ( 5 ) total molecular weight , and ( 6 ) theoretical molar extinction coefficient at 280 nm .", "the theoretical molar extinction coefficient was estimated by summing the number of tryptophans in the protein multiplied by 5690 , the number of tyrosines by 1280 , and the number of cysteines by 120 .", "this molar extinction coefficient was converted to a mass coefficient by dividing by the molecular weight of the protein to derive an extinction coefficient with units of au ml mg cm .", "the average mass per residue was estimated by dividing the molecular weight by the number of amino acids in the protein .", "the number of amides was determined by adding the number of asparagines and glutamines to the total number of amino acids and subtracting one .", "the average mass per amide was determined by dividing the molecular weight by the number of amides .", "sample protein concentrations were determined with reference to a calibration curve ; the method requires prior generation of a reusable standard curve derived from serial dilution of a reference protein . for all reported experiments ,", "the system was calibrated using bovine serum albumin ( bsa ) from the national institute of standards and technology ( nist ) diluted in phosphate - buffered saline ( pbs ) .", "a series of ten concentration points ( 0.1255 mg / ml ) was used to generate the protein calibration curve .", "all reported measurements were performed in triplicate using 2 l of sample solution per membrane position . from pilot experiments ( not discussed here ) , 2 l was chosen as a trade - off between minimum sample volume required and acceptable quantitation results .", "although smaller quantities could be applied to the membrane , human error in the pipetting of such low volumes would introduce additional variability and thus was not considered further .", "performance of the method , within the standard curve - defined dynamic range , was assessed using pure protein solutions as well as protein mixtures .", "pure protein solutions were prepared with lysozyme solubilized in milli - q water and protein a in pbs .", "protein mixtures consisted of bsa , cytochrome c , alcohol dehydrogenase , human transferrin , concanavalin a , lysozyme , -globulins from rabbit , and protein a in pbs , used at two distinct concentrations . for reference purposes ,", "the concentration of all examined protein solutions was determined by amino acid analysis ( aaa ) . to obtain a 1 mg / ml solution analyzed by mir - based quantitation", ", a lysozyme sample ( aaa determination at 68 mg / ml ) was diluted with pbs at 1/68 ratio . protein", "a ( aaa determination at 52 mg / ml ) was diluted , also with pbs , 1/13 to obtain 4 mg / ml sample .", "the protein mixture ( aaa determination at 1.98 mg / ml ) was used at the aaa estimated concentration as well as a 1/8 dilution .", "potential interference from detergents and reducing agents , which are known to impact bradford and lowry protein determination assays , was investigated using known concentrations of bsa in pbs ( from 0.25 to 2 mg / ml ) spiked with increasing amount of investigated additive .", "influence of tween 20 and triton x-100 on protein quantitation was tested in the range of 0.15% detergent .", "-mercaptoethanol ( bme ) and dl - dithiothreitol ( dtt ) were analyzed at up to 150 mm and 50 mm , respectively .", "possible obstruction of protein quantification due to the presence of sodium dodecyl sulfate ( sds ) was analyzed using buffers containing up to 1% of the detergent .", "while all investigated detergents and reducing agents produced mir spectra , none was absorbing within the region used for protein quantification ( 17001600 cm ) .", "empirical sample concentration values were determined by interpolation from calibration curves developed for each lipid or detergent analyzed .", "the reported method relied on strength of bands produced by vibrations of aliphatic groups ( 30002800 cm ) .", "for the experiments reported here , the system was calibrated using tetracosanoic acid in chloroform and 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) in pbs .", "a series of seven concentrations ( 0.251.75 mg / ml ) was used to generate a calibration curve for tetracosanoic acid . for chaps , the calibration curve was also derived from seven concentration points ( 0.254% ) .", "to investigate whether protein and lipid can be quantified simultaneously in complex biological samples , tissue lysates , originating from breast cancer tissues , were analyzed .", "surgical frozen tissue , derived from a human breast ductal carcinoma , was obtained from analytical biological services inc . and divided into 2 equal samples ( 115 mg each ) .", "tissue was covered with 2 ml 1x ripa buffer ( emd millipore ; final composition : 50 mm tris - hcl ph 7.4 , 150 mm nacl , 0.25% deoxycholic acid , 1% np-40 , and 1 mm edta ) or cytobuster protein extraction reagent ( emd millipore ; composition not available ) , both supplemented with an inhibitor cocktail and disrupted with a glass tissue homogenizer .", "liquid fractions from homogenized and lysed tissue were transferred to separate tubes ( fraction 1 , supplementary figure s2 ) while the remaining pieces of tissue were covered with fresh volume of corresponding lysis buffer and homogenized again ( fraction 2 , supplementary figure s2 ) .", "protein containing fractions ( bottom of the supernatant layers ( p1a and p2a ) ) were separated and analyzed , using mir - based method , for protein and lipid content .", "the top layers were saved for future lipid analysis . to remove substantial amounts of lipids detected in both protein fractions , an additional centrifugation step ( 15,000 g for 10 minutes )", "resulting layers , top lipid fraction ( l1b and l2b ) , and bottom protein fractions ( p1b and p2b ) were analyzed , using mir - based method , for protein and lipid content .", "lysates were prepared from mcf-7 ( atcc htb-22 ) breast cancer cells ( 1.4 10 cells per sample ) in 1 ml of either 1x ripa buffer or cytobuster protein extraction reagent , both supplemented with protease inhibitor cocktail , by homogenization for few seconds with a handheld homogenizer followed by centrifugation at 15,000 g for 10 minutes .", "the mir - based method was used to measure both protein and lipid content of the supernatant .", "mir - based protein data was compared to the results obtained using bca protein assay kit ( thermo scientific ) .", "mir - based analysis of biological samples was achieved by the application of 2 l aqueous samples onto hydrophilic ptfe membrane and the presentation of dried sample to the mir beam . while znse and calcium fluoride are commonly used in mir spectroscopy , they are cost - prohibitive for single - use applications . also , the intention of the presented method was to avoid cleaning of the deposition window after each use and ptfe - based single - use card permitted such application .", "the ptfe membrane displays strong signal between 1100 and 1300 cm ( figure 1(a ) ) ; however , it is relatively inert in the remaining mir spectrum , including the amide i region used for protein quantitation and the aliphatic group stretching region used for lipid and detergent measurement . during development", ", it became clear that precise overlap between the site of sample application and the mir beam is critical for quantitative accuracy . in order to achieve highly reproducible sample presentation to the mir beam", ", a hydrophobic ring was introduced through mechanical membrane crushing and removal of the hydrophilic surface .", "introduction of this ring allowed for precise confinement of applied sample within the designated spot .", "spot was surrounded by a hydrophobic embossment ( figure 1(b ) ) preventing dispersion of the aqueous sample during application and drying .", "design significantly improved the overlap between the mir beam intensity profile , being the strongest in the center of the spotting area , and the dried sample area , thereby promoting higher assay accuracy .", "because the comparative measurements are performed on dried samples , and therefore volume - dependent , reproducible and precise deposition of the employed 2 l onto the membrane is critical .", "all results reported here were obtained using manual pipette ( rainin , pipet - plus r2 ) with a latch trigger mechanism and aspiration rate controller , features that improve precision from sample to sample and from operator to operator . in order to enable a fast , cost - effective , and simple analysis method ,", "although multivariate quantification procedures can provide better sensitivity when applied to the analysis of complex samples , they require ir expertise . as the method described here is intended for general protein quantification that is rapid and straightforward to non - ir specialists , the simpler univariate approach was pursued .", "the influence of protein secondary structure on amide i extinction coefficient ( exact location of amide i band ) has been well documented [ 17 , 18 , 40 ] .", "different spectral regions within the amide i area were analyzed and the best results for general protein quantification were obtained for the region between 1702 and 1602 cm . additionally , to account for possible buffer interference , a buffer signal ( e.g. , originating from buffer salts deposited and dried on the membrane ) was subtracted from the protein signal .", "investigation of various spectral regions considered for buffer signal subtraction delivered the most promising results for the region between 1850 and 1350 cm . following subtraction ,", "the strength of the remaining amide i signal was used to interpolate the estimated protein concentration from a known standard curve . under standard conditions , aqueous samples spotted on hydrophilic materials , including ptfe membrane , dry forming a coffee ring effect where the majority of sample is preferentially deposited around the edges of a spot .", "distribution of the coffee ring is strongly influenced by the nature of the sample buffer .", "effect is displayed in water ( supplementary figure s1b ) while use of buffers , such as pbs , reduces water tension allowing more uniform sample distribution across the membrane surface ( supplementary figure s1c ) . in the presence of detergent ,", "the buffer - dependent differences in dried sample distribution are highly reproducible but require generation of a reusable calibration curve and confirmation of linearity for each matrix to ensure the most accurate results .", "one major limitation of uv / vis - based protein quantitation is its strict dependence on amino acid composition , in particular the presence of tryptophan and tyrosine .", "comparative analysis between amide i - derived signal and uv / vis absorbance at 280 nm was performed for a range of concentrations of bsa , rabbit igg , and protein a ( figure 2 ) . unlike uv / vis ,", "mir - based analysis was unaffected by the proteins ' vast differences in amino acid composition or size . in reviewing the human protein database ( table 1 )", ", it is clear that many protein properties , such as protein length and mass , the number of specific amino acids , and even the predicted extinction coefficients at 280 nm , vary widely from protein to protein .", "however , the average mass per residue tends to be surprisingly consistent considering that individual residue contributions can range from 57 da for glycine to 186 da for tryptophan .", "further , since the number of amide bonds in a protein or peptide is only one less than the number of residues , plus the number of asparagines and glutamines , the mass per amide bond is also very consistent , even for short proteins and peptides . one could therefore postulate that mir absorbance ( on a mass basis , not a molar basis ) would be very consistent and that a single protein could serve as a reliable reference for just about any other protein or peptide , assuming that its average residue mass did not deviate significantly from the typical 110 da per residue ( e.g. , polyglycine or polytryptophan would be expected to deviate ) . in order to test the mir - based protein quantitation method ,", "a series of ten dilutions ( in triplicate ) , spanning the range 0.1255 mg / ml , was used to prepare a calibration curve .", "amide i signal strength delivered by each concentration point was fitted to a regression line that was ultimately used to determine protein concentration in the analyzed samples ( figure 3 ) .", "accuracy of mir - based concentration estimation was determined using single - protein solutions as well as a protein mixture .", "concentration values were derived by interpolation from a bsa reference curve and compared to amino acid analysis ( aaa ) , a method recognized currently as the gold standard for estimating protein concentration .", "first , a 1 mg / ml lysozyme sample in pbs , prepared by 1/68 dilution of a 68 mg / ml sample ( concentration determined by aaa ) was found by the mir method to be 0.922 0.061 mg / ml .", "next , protein a ( 52 mg / ml by aaa ) was diluted 1/13 with pbs to obtain a 4 mg / ml solution . when analyzed by mir , a concentration of 4.047 0.184 mg / ml was determined .", "lastly , a mixture of proteins ( 1.98 mg / ml by aaa ) was quantified at 1.944 0.028 mg / ml ; a 1/8 dilution of the same sample ( expected 0.25 mg / ml ) delivered a concentration of 0.273 0.028 mg / ml when analyzed by the mir - based method .", "overall , the method showed very good accuracy and linearity in response to samples between 0.25 mg / ml and 5 mg / ml . measurement accuracy for samples below 0.25 mg / ml decreased significantly ; thus , a limit of 0.25 mg / ml was selected for this method . in regard to the upper detection limits , a small set of samples up to 100 mg / ml was successfully measured ( data not shown ) .", "however , as the intended application of this method was for the analysis of precious samples , such high sample concentrations were not analyzed within the context of this paper .", "sample cards prepared using protein mixtures , at 0.25 and 1.98 mg / ml ( each in triplicate ; total of 9 spots per sample ) , were analyzed multiple times to determine measurement reproducibility .", "each card ( 3 protein spots + 1 control spot ) was measured four times and the concentrations obtained for each position as well as for an entire card were compared . assuring correct and complete deposition of the samples onto the membrane ,", "the average concentrations were 0.277 mg / ml ( 4.9% cv ) and 1.942 mg / ml ( 1.5% cv ) , respectively .", "the precision at each individual card position was measured , with cvs of 1.3 and 0.3% , respectively , at position 2 .", "position 3 delivered data with cvs of 1.2 and 0.1% , while cvs at position 4 were at 2.3 and 0.1% , respectively .", "protein quantitation method reported here relies on mir - based evaluation of biological samples ( from original buffers ) spotted and dried onto a membrane .", "therefore , measurements in the presence of buffer salts containing amide bonds , such as urea , can potentially affect the accuracy of the results . in the majority of cases ,", "interference from buffer salts is accounted for in the buffer subtraction step ; however , since the method relies on a simple univariate approach , high concentrations of interfering components can still overwhelm the protein signal and preclude proper measurement . reducing agents and detergents", "are known to interfere with colorimetric protein quantitation methods . to elucidate whether these agents would also interfere with mir - based measurements ,", "the results reveal that accurate mir - based protein quantitation can be achieved in the presence of up to 50 mm dithiothreitol ( dtt ) ( figure 4(a ) ) and up to 150 mm of -mercaptoethanol ( spectra not shown ) .", "in addition , the infrared absorption pattern of sodium dodecyl sulfate ( sds ; analyzed up to 1% ) did not overlap with the protein region allowing unbiased protein quantification in the presence of this detergent ( figure 4(b ) ) .", "method performance was also unaltered by the presence of up to 5% tween 20 ( data not shown ) or triton x-100 ( figure 4(c ) ) .", "at the same time , the presence of amide containing detergent , like , for example , chaps , might interfere with or prevent accurate protein quantitation . also , since detergents are known to bind to proteins with varying affinities , subtracting the buffer contribution might not always be sufficient for accurate analysis .", "for example , in cases when analyzed proteins bind a significant amount of the detergent , the bound detergent will most likely not be accounted for by the blank influencing the accuracy of concentration determination . by virtue of its ability to detect the spectral absorbance bands for many structural entities ,", "the mir - based method is not limited to the analysis of protein species . given the large number of lipid - associated absorption bands , application of the described method for the quantification of lipid biomolecules", "h stretching region ( 28502870 cm ) provides an ideal candidate for the analysis of lipids and detergents . while a single - protein standard can be used to quantify a wide range of protein and peptide samples , due to the vast complexity and variability among lipids and detergents , it was speculated that each quantitation would require the generation of a standard curve using the specific species in question", ". experiments using various lipids , including fatty acids ( supplementary figure s3a ) , phospholipids , triglycerides , liposaccharides , and many detergents , demonstrated a high degree of variability in the detection limits and slope of the employed calibration curves , thus confirming requirement for individualized calibrations for each of the analyzed lipids and detergents . in order to validate the compatibility of mir - based quantitation method for lipid and detergent analysis ,", "the ftir spectrometer was initially calibrated using either tetracosanoic acid ( in chloroform ) or chaps ( in pbs ) .", "two concentration ranges ( performed in triplicate ) , spanning 0.251.75 mg / ml ( tetracosanoic acid ) and 0.254% ( chaps ) , were used to derive lipid and detergent calibration curves ( supplementary figure s3b ) .", "the strength of symmetric c h vibration for each concentration was fitted to a regression line .", "the accuracy of concentration estimation within the established dynamic ranges for tetracosanoic acid and chaps was assessed using 0.8 mg / ml tetracosanoic acid and 1.8% chaps .", "the results showed that , for a well defined calibration range , the method was capable of estimating lipid and detergent concentration with low error . assuring precise pipetting when applying the samples , mir - based quantitation of the tetracosanoic acid sample returned 0.853 0.14 mg / ml ( 2.4% cv ) and chaps sample was quantified as 1.8 0.004% ( 2.3% cv ) .", "the experiments using several formulations of lysis buffers , like ripa and cytobuster protein extraction reagent , spiked with known concentration of bsa and measured alone , as well as in the presence of phospholipids ( data not shown ) , demonstrated that the reported method allows accurate protein quantification from lysis buffers . to determine", "if the reported method can be further applied to more complex biological samples , protein content in variously prepared breast cancer cell line ( mcf-7 ) lysates was measured and compared to values derived from a bca protein assay .", "total protein content in the sample obtained using cytobuster protein extraction reagent was estimated at 2.4 mg / ml ( bca assay ) or 2.7 mg / ml ( mir - based method ) .", "protein concentration in samples lysed with ripa buffer was estimated at 3.4 mg / ml ( bca assay ) or 4.6 mg / ml ( mir - based method ) .", "the results obtained using bca assay were consistently lower ( 10% and 26% , resp . ) than the mir - based method .", "for the two methods , the differences in estimated protein concentration were most likely caused by a combination of the following factors : presence of various detergents ( documented to influence the accuracy of bca assay ) in lysis buffers , documented inaccuracy of bca in the analysis of native proteins , an error associated with use of bsa as a standard in colorimetric assays , and the fact that accurate quantification of peptides by the bca assay could not be achieved without additional sample manipulations that are incompatible with biological material .", "the possibility that mir - based quantification is inflated by signal from nucleic acids present in cell lysates was excluded because the amount of nucleic acids generally present in such mixtures is significantly below the detection level of the reported method .", "the findings reported here are consistent with a previous report where the mir - based method was used to quantify protein content in crude human skin carcinoma cell lysates or to adjust protein concentration prior to mass spectrometry and nmr analysis [ 47 , 48 ] .", "the method described here was able to detect and examine protein and lipid content of all analyzed samples simultaneously and selectively ( figure 5(a ) ) , in contrast with conventional analysis techniques such as uv spectroscopy .", "this dual functionality could enable the monitoring of changes in total lipid content and protein liberation yield , thereby simplifying and improving the analytical process .", "the efficacy of mir - based analysis for use in downstream sample qualification was evaluated during a short study of breast cancer cell lysate fractionation and biomarker detection .", "breast tissue was chosen because it is documented to have a high fat content ( 69.9 22.9% ) . in the reported study", ", breast tissue lysates were prepared using ripa buffer and cytobuster protein extraction reagent . following tissue homogenization ,", "the efficiency of fat removal and total protein liberation during centrifugal extractions was monitored using the mir - based method ( see supplementary figure s2 for separation flow chart ) .", "the collected spectral data were used to determine protein recovery across the centrifugal fractions ( figure 5(b ) ) and to examine the efficiency of gradual fat removal from these samples ( figure 5(c ) ) .", "information delivered by mir - based analysis was utilized in further sample manipulations facilitating faster and more consistent immunodetection of the investigated breast cancer biomarkers ( study in progress ) .", "fast and accurate quantification of proteins from complex aqueous biological samples such as plasma , cerebrospinal fluid , or cell / tissue lysates remains a challenge .", "critical understanding of results from downstream applications is often dependent on proper preparation and accurate qualification of the applied samples .", "classical methods for protein quantification , like uv and colorimetric assays , permit precise estimation of purified protein concentration but are less reliable when applied to the analysis of complex mixtures . while mir is a promising technique allowing accurate quantification in complex samples ,", "existing techniques for mir - based protein quantification require regular time - consuming cleaning procedures ( flow - through cells ) , larger sample volume ( atr cells ) , or expertise in method development ( atr of dried samples ) , thus precluding their suitability for fast analysis in a typical research laboratory .", "although high - throughput mir sampling and analysis techniques are already available on the market , they are more suited for larger pharmaceutical laboratories that require recurring analysis of repetitive sample sets and possess the requisite analytical skills for effective chemometrics - based method development .", "currently available instrumentation is often too costly or requires certain ir expertise to be easily adapted for general protein quantification by non - ir savvy personnel in small - scale laboratories or academia .", "the method developed and described herein relies on a simple , cost - effective approach permitting accurate quantification results based on a univariate sample analysis technique that does not require advanced ir expertise .", "the system relies on a membrane , transparent in most of the mir region , which permits robust analysis of aqueous samples in a dried format .", "the reported method is also compatible with organic solvents commonly used in peptide and lipid research . to achieve accurate quantitative results , strict adherence to a consistent loading volume between samples and controls", "is required . while a general calibration curve can be employed , the method is more accurate when a specific calibration curve is prepared for each buffer to be utilized .", "based on the experimental data , a general lower quantification range was set to 0.25 mg / ml ; for some samples , a detection limit as low as 0.1 mg / ml can be achieved . because the developed method focuses solely on small volumes and biologically relevant samples , the upper detection limit was not investigated thoroughly .", "however , from the narrow set of experimental results , such limits can exceed 100 mg / ml . although some buffer salts might not be compatible with the reported technique , in pbs , the method allows development of a general linear calibration between 0.25 mg / ml and 5 mg / ml . accuracy and precision of the reported method , within the linear range , are comparable with results obtained by amino acid analysis , providing researchers with a fast and cost - effective alternative to other protein quantitation tools available today . due", "to chemical and structural variation observed within lipids and detergents , mir signal strengths display considerable variability ; for this reason , a universal standard for all lipids and detergents could not be identified .", "therefore , quantitation of lipids and detergents requires development of separate standard curves for each of the analyzed compounds .", "in addition , qualitative analysis of mixtures or unknown compounds can be performed using a relative absorbance ", "mode where the method delivers information about strength of mir signal without any comparison to a known standard . in summary , the mir - based method reported here enables simultaneous measurement of total protein recovery and monitoring of fat removal from lysed samples .", "this technique was also successfully applied to complex sample analysis during a small - scale investigation of surgical breast tissue processing and fractionation .", "mir - based analysis facilitates more in - depth sample characterization and offers higher quality control over the sample preparation process .", "given its unbiased biomolecular detection capabilities and amenability to liquid samples , many more applications are easily envisioned for this method ." ]

[ "<S> biological samples present a range of complexities from homogeneous purified protein to multicomponent mixtures . accurate qualification of such samples is paramount to downstream applications . </S>", "<S> we describe the development of an mir spectroscopy - based analytical method offering simultaneous protein quantitation ( 0.255 mg / ml ) and analysis of total lipid or detergent species , as well as the identification of other biomolecules present in biological samples . </S>", "<S> the method utilizes a hydrophilic ptfe membrane engineered for presentation of aqueous samples in a dried format compatible with fast infrared analysis . unlike classical quantification techniques , the reported method is amino acid sequence independent and thus applicable to complex samples of unknown composition . by comparison to existing platforms , </S>", "<S> this mir - based method enables direct quantification using minimal sample volume ( 2 l ) ; it is well - suited where repeat access and limited sample size are critical parameters . </S>", "<S> further , accurate results can be derived without specialized training or knowledge of ir spectroscopy . </S>", "<S> overall , the simplified application and analysis system provides a more cost - effective alternative to high - throughput ir systems for research laboratories with minimal throughput demands . in summary </S>", "<S> , the mir - based system provides a viable alternative to current protein quantitation methods ; it also uniquely offers simultaneous qualification of other components , notably lipids and detergents . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion" ]

[ [ "correct estimation of protein concentration in aqueous biological samples is an essential step in biochemical research and the pharmaceutical industry impacting downstream applications ranging from biomarker studies to quality control in the production of biotherapeutics .", "determination of protein concentration in most popular assays is accomplished via comparison to a sequence - based extinction coefficient ( uv measurements ) or in relation to a standard ( traditional dye - based absorbance assays such as bca , lowry , and bradford ) [ 13 ] .", "however , most recent reviews point out the fact that , due to assay - specific limitations , there is no single method dominating protein quantification [ 1 , 3 ] . while uv based quantification is reliant upon absorbance of tryptophan , tyrosine , and cysteine at 280 nm [ 4 , 5 ] , a protein 's extinction coefficient can vary widely with sequence .", "in fact , a greater than two - fold difference is observed between extinction coefficients calculated for albumin and immunoglobulin g. also , mixtures of unknown composition ( most notably , biologically relevant samples ) can not be confidently quantified based on absorption at 280 nm .", "moreover , amino acid analysis ( aaa ) is capable of delivering possibly the most accurate protein quantitation , but the method is expensive with lengthy turnaround times if samples are sent to a third party for analysis . performed in - house , aaa requires time - consuming sample manipulation and specialized equipment .", "infrared ( ir ) spectroscopy is a powerful and growing analytical tool for the detection and analysis of biomedically relevant compounds such as proteins , lipids , carbohydrates , and nucleic acids [ 69 ] .", "midinfrared ( mir ) spectroscopy is based on the absorption of radiation in the approximate range 4000400 cm and is currently considered among the most promising spectroscopic techniques for application in biomedical research and diagnostics [ 1012 ] .", "also , mir spectroscopy has been recognized as a viable method for lipid analysis [ 1316 ] and is one of the oldest and well established experimental techniques for the analysis of protein and carbohydrate structure [ 1721 ] .", "attenuated total reflection ( atr ) spectroscopy and transmission flow - through cells used in combination with complex chemometric data analysis have recently enabled fast quantitative protein analysis directly from aqueous samples [ 2229 ] . however , while flow - through cells for protein quantification allow for automated sample analysis , these devices have a propensity for clogging , requiring frequent , time - consuming cleaning procedures . while atr cells are more robust , the required sample volumes ( 1025 l ) may be considered significant , particularly in situations where biological samples , limited by both volume and repeat access , are to be analyzed .", "however , due to enhanced sensitivity , the multivariate approach ( e.g. , partial least - squares analysis ( pls ) ) is usually applied to data analysis [ 14 , 2229 ] reducing attractiveness of the method for routine application in biological laboratories that usually lack the time and expertise required for method development and validation . to our knowledge , an easy , fast , and robust method utilizing atr and univariate data analysis for accurate and reproducible protein quantitation from complex biological samples in a dried format has not yet been reported .", "several amide bands have been identified in mir spectroscopy allowing for characterization and quantification of proteins . among these ,", "amide i ( 16001690 cm ) and amide ii ( 14801575 cm ) are recognized as the most representative of all vibration modes [ 17 , 18 ] .", "the amide i absorption consists predominantly of c = o stretching vibration ( about 80% ) with a minor contribution from the c ", "n stretching vibration ( 20% ) , while the amide ii band is more equally split between n h bending ( 60% ) and c ", "until recently , analysis of the amide i and ii absorption regions has been severely hampered in aqueous samples due to spectral interference of a strong water absorption band at 15001700 cm . while the advent of atr and flow - through cells has circumvented the water interference issues , their utility is limited due to practical drawbacks which include instrumentation cost , expertise , and time required for method development and accurate data analysis . a simple univariate ( beer - lambert ) analysis ,", "applied in the method reported here , relies on the integration of amide i band and uses directly searchable absorptions on the spectrum curve .", "reported protein quantification by mir , while still based on a curve - fitting technique , presents substantial advantages over other current methodologies like uv absorbance or colorimetric assays .", "first , unlike uv absorbance at 280 nm , mir - based protein quantitation is much less dependent upon amino acid composition . also", ", amide bond quantitation by mir is not subject to signal interference from many common biological buffer components such as detergents , reducing agents , and chelators , demonstrating superiority over standard colorimetric assays .", "moreover , the mir - based method enables fast and accurate peptide quantitation providing researchers with a robust substitute for time - consuming amino acid analysis .", "however , when compared to uv spectroscopy , ir instruments are more costly and require technical expertise as well as time - consuming method development preventing widespread applicability of mir for general protein quantification .", "thus , an instrument or method , allowing for simple and more cost - effective analysis of samples , while at the same time combining the advantages of atr and flow - through based systems , would be of unique value .", "additionally , in contrast to uv or any other known protein quantitation method , simple , mir - based analysis can also be employed for simultaneous analysis of lipids or detergents [ 14 , 3036 ] . due to their complex and varied chemical composition ,", "characteristic lipid bands , such as the aliphatic group stretching ( 30002800 cm ) , ester c = o stretching ( around 1740 cm ) , or phosphate stretching ( around 1235 cm ) permit qualitative and quantitative analysis of lipid content [ 14 , 31 , 32 ] . due to similarities in composition , detergents possess mir absorption spectra that closely resemble lipids present in the cell membrane and can be analyzed along with lipids .", "this commonality offers researchers a means for monitoring the efficiency of residual detergent removal from lysate - derived samples prior to downstream application .", "the method reported in this paper uses a hydrophilic polytetrafluoroethylene ( ptfe ) membrane engineered for sample application and retention .", "the membrane is transparent in the mir regions used for protein and lipid / detergent analysis .", "the size of the sample application spot was further designed to be slightly smaller than the ir beam , ensuring probing of the entire sample .", "this constraining feature is important in enabling simple , univariate , quantitative measurements . in comparison to currently available techniques and instruments ( e.g. , hts - xt system by bruker optics ) , the method described here is technically less complex ; thus , it is more cost - effective and especially well suited for routine analysis of small sample numbers .", "also , given the minimal volume ( 2 l ) required for measurement , this method can be successfully applied for the analysis of precious material available in limited quantities .", "the presented paper provides a detailed description of method development along with a comparison to other protein quantification techniques routinely used in biological laboratories , with respect to the required sample volume , time - consumption , labor - intensity , accuracy , and robustness .", "in addition to general protein quantification , the ability for simultaneous lipid analysis was also investigated .", "well characterized solutions of several purified proteins , protein mixtures , and examples of lipids and detergents were used to assess quantification limits , dynamic range , linearity , accuracy , precision , and robustness of the reported method .", "further applicability of the method for the analysis of biological samples was demonstrated using cell lysates and tissue homogenates ." ], [ "in order to avoid cleaning steps in between series of successive measurements , a disposable device for sample application was designed . from several investigated options ,", "a membrane - based system emerged as the most cost - efficient and the easiest to use , predominantly because it allowed rapid water removal and drying of the sample .", "various membranes were tested ( data not shown ) ; ultimately a hydrophilic ptfe - based membrane was selected due to its transparency within the relevant mir regions , allowing for the quantification of proteins and lipids . as shown in figure 1(a ) , the membrane spectrum contains a strong signal between 1100 and 1300 cm but is transparent in the mir region above 1300 cm used for the analysis of biological samples reported here .", "the hydrophilic ptfe membrane ( 30 m thick ) is mounted on cardboard frame to allow easier handling and provide a place to record sample names , while also assuring consistent presentation for interrogation by the mir beam .", "the card configuration contains four analysis areas designed for easy application and containment of the entire sample within the focused ir beam .", "the hydrophilic spot for sample application ( 4.5 mm diameter ; see figure 1(b ) and supplementary figure s1a , the supplementary material will be available online at http://dx.doi.org/10.1155/2014/657079 . ) is surrounded by a hydrophobic ring generated by mechanical removal of hydrophilic surface ; the ring prevents analyte dispersal promoting precise presentation of the entire sample to the mir beam ( diameter 6.5 mm ) [ 37 , 38 ] .", "the reported method permits two ways of drying aqueous sample spotted onto ptfe membrane . on average", ", 2 l of sample placed on the membrane spot requires around 10 to 15 minutes to dry completely at room temperature .", "the sample can also be dried within around 30 seconds , on average , by the heater and fan located in the sample carrier chamber of the dedicated ftir spectrometer .", "4-spot card was selected for the final configuration to allow rapid analysis of an individual sample in triplicate or three separate samples in comparison to an appropriate background buffer spot .", " measurements were performed using the direct detect spectrometer ( emd millipore ) , a fourier transform ( ft)-ir system analyzing the spectral range 5006000 cm .", "a classical univariate quantification method , based on the fact that mir spectroscopy of nonscattering samples obeys beer 's law , was applied to data analysis . for each protein and lipid / detergent measurement ,", "a buffer subtraction step was performed only on regions of the spectra used for amide i and/or aliphatic symmetric stretching signal analysis .", "proteins are quantified using the amide i region ( 17021602 cm ) only ; thus , all other regions of the mir spectrum are not critical for the analysis .", "consequently , for accurate protein quantification , it was sufficient to subtract buffer in the region ( 18501350 cm ) surrounding amide i area .", "the same procedure was applied to aliphatic stretching region used for lipid / detergent analysis .", "buffer subtraction is performed between 3100 and 2600 cm ; the rest of mir spectra is not being utilized .", "amide i band integration is performed by anchoring the baseline at fixed points between 1702 and 1602 cm and determining the amide i signal value at the highest point between these wavenumbers .", "two additional integration methods ( not discussed here ) accounting for possible buffer interference have also been developed .", "aliphatic symmetric stretching band integration is performed by anchoring the baseline between 2990 and 2810 cm and determining the signal strength at the highest point between 2868 and 2838 cm . to compare the theoretical variability of various quantitation methods ,", "a protein database was examined . for example , the theoretical extinction coefficient at 280 nm is based on the number of tryptophan , tyrosine , and cysteine residues in the protein .", "the mir - based analysis is dependent on the number of amide bonds , which not only link the amino acids together but are also present in the side chains of asparagine and glutamine . to automate the examination of the database , a visual basic for applications ( vba ) program was written for excel ( microsoft ) to check all 20233 proteins in the human protein database ( uniprot release 2012_10 , ) for ( 1 ) total count of amino acids , ( 2 ) number of glutamines , ( 3 ) number of asparagines , ( 4 ) number of tryptophans , ( 5 ) total molecular weight , and ( 6 ) theoretical molar extinction coefficient at 280 nm .", "the theoretical molar extinction coefficient was estimated by summing the number of tryptophans in the protein multiplied by 5690 , the number of tyrosines by 1280 , and the number of cysteines by 120 .", "this molar extinction coefficient was converted to a mass coefficient by dividing by the molecular weight of the protein to derive an extinction coefficient with units of au ml mg cm .", "the average mass per residue was estimated by dividing the molecular weight by the number of amino acids in the protein .", "the number of amides was determined by adding the number of asparagines and glutamines to the total number of amino acids and subtracting one .", "the average mass per amide was determined by dividing the molecular weight by the number of amides .", "sample protein concentrations were determined with reference to a calibration curve ; the method requires prior generation of a reusable standard curve derived from serial dilution of a reference protein . for all reported experiments ,", "the system was calibrated using bovine serum albumin ( bsa ) from the national institute of standards and technology ( nist ) diluted in phosphate - buffered saline ( pbs ) .", "a series of ten concentration points ( 0.1255 mg / ml ) was used to generate the protein calibration curve .", "all reported measurements were performed in triplicate using 2 l of sample solution per membrane position . from pilot experiments ( not discussed here ) , 2 l was chosen as a trade - off between minimum sample volume required and acceptable quantitation results .", "although smaller quantities could be applied to the membrane , human error in the pipetting of such low volumes would introduce additional variability and thus was not considered further .", "performance of the method , within the standard curve - defined dynamic range , was assessed using pure protein solutions as well as protein mixtures .", "pure protein solutions were prepared with lysozyme solubilized in milli - q water and protein a in pbs .", "protein mixtures consisted of bsa , cytochrome c , alcohol dehydrogenase , human transferrin , concanavalin a , lysozyme , -globulins from rabbit , and protein a in pbs , used at two distinct concentrations . for reference purposes ,", "the concentration of all examined protein solutions was determined by amino acid analysis ( aaa ) . to obtain a 1 mg / ml solution analyzed by mir - based quantitation", ", a lysozyme sample ( aaa determination at 68 mg / ml ) was diluted with pbs at 1/68 ratio . protein", "a ( aaa determination at 52 mg / ml ) was diluted , also with pbs , 1/13 to obtain 4 mg / ml sample .", "the protein mixture ( aaa determination at 1.98 mg / ml ) was used at the aaa estimated concentration as well as a 1/8 dilution .", "potential interference from detergents and reducing agents , which are known to impact bradford and lowry protein determination assays , was investigated using known concentrations of bsa in pbs ( from 0.25 to 2 mg / ml ) spiked with increasing amount of investigated additive .", "influence of tween 20 and triton x-100 on protein quantitation was tested in the range of 0.15% detergent .", "-mercaptoethanol ( bme ) and dl - dithiothreitol ( dtt ) were analyzed at up to 150 mm and 50 mm , respectively .", "possible obstruction of protein quantification due to the presence of sodium dodecyl sulfate ( sds ) was analyzed using buffers containing up to 1% of the detergent .", "while all investigated detergents and reducing agents produced mir spectra , none was absorbing within the region used for protein quantification ( 17001600 cm ) .", "empirical sample concentration values were determined by interpolation from calibration curves developed for each lipid or detergent analyzed .", "the reported method relied on strength of bands produced by vibrations of aliphatic groups ( 30002800 cm ) .", "for the experiments reported here , the system was calibrated using tetracosanoic acid in chloroform and 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) in pbs .", "a series of seven concentrations ( 0.251.75 mg / ml ) was used to generate a calibration curve for tetracosanoic acid . for chaps , the calibration curve was also derived from seven concentration points ( 0.254% ) .", "to investigate whether protein and lipid can be quantified simultaneously in complex biological samples , tissue lysates , originating from breast cancer tissues , were analyzed .", "surgical frozen tissue , derived from a human breast ductal carcinoma , was obtained from analytical biological services inc . and divided into 2 equal samples ( 115 mg each ) .", "tissue was covered with 2 ml 1x ripa buffer ( emd millipore ; final composition : 50 mm tris - hcl ph 7.4 , 150 mm nacl , 0.25% deoxycholic acid , 1% np-40 , and 1 mm edta ) or cytobuster protein extraction reagent ( emd millipore ; composition not available ) , both supplemented with an inhibitor cocktail and disrupted with a glass tissue homogenizer .", "liquid fractions from homogenized and lysed tissue were transferred to separate tubes ( fraction 1 , supplementary figure s2 ) while the remaining pieces of tissue were covered with fresh volume of corresponding lysis buffer and homogenized again ( fraction 2 , supplementary figure s2 ) .", "protein containing fractions ( bottom of the supernatant layers ( p1a and p2a ) ) were separated and analyzed , using mir - based method , for protein and lipid content .", "the top layers were saved for future lipid analysis . to remove substantial amounts of lipids detected in both protein fractions , an additional centrifugation step ( 15,000 g for 10 minutes )", "resulting layers , top lipid fraction ( l1b and l2b ) , and bottom protein fractions ( p1b and p2b ) were analyzed , using mir - based method , for protein and lipid content .", "lysates were prepared from mcf-7 ( atcc htb-22 ) breast cancer cells ( 1.4 10 cells per sample ) in 1 ml of either 1x ripa buffer or cytobuster protein extraction reagent , both supplemented with protease inhibitor cocktail , by homogenization for few seconds with a handheld homogenizer followed by centrifugation at 15,000 g for 10 minutes .", "the mir - based method was used to measure both protein and lipid content of the supernatant .", "mir - based protein data was compared to the results obtained using bca protein assay kit ( thermo scientific ) ." ], [ "mir - based analysis of biological samples was achieved by the application of 2 l aqueous samples onto hydrophilic ptfe membrane and the presentation of dried sample to the mir beam . while znse and calcium fluoride are commonly used in mir spectroscopy , they are cost - prohibitive for single - use applications . also , the intention of the presented method was to avoid cleaning of the deposition window after each use and ptfe - based single - use card permitted such application .", "the ptfe membrane displays strong signal between 1100 and 1300 cm ( figure 1(a ) ) ; however , it is relatively inert in the remaining mir spectrum , including the amide i region used for protein quantitation and the aliphatic group stretching region used for lipid and detergent measurement . during development", ", it became clear that precise overlap between the site of sample application and the mir beam is critical for quantitative accuracy . in order to achieve highly reproducible sample presentation to the mir beam", ", a hydrophobic ring was introduced through mechanical membrane crushing and removal of the hydrophilic surface .", "introduction of this ring allowed for precise confinement of applied sample within the designated spot .", "spot was surrounded by a hydrophobic embossment ( figure 1(b ) ) preventing dispersion of the aqueous sample during application and drying .", "design significantly improved the overlap between the mir beam intensity profile , being the strongest in the center of the spotting area , and the dried sample area , thereby promoting higher assay accuracy .", "because the comparative measurements are performed on dried samples , and therefore volume - dependent , reproducible and precise deposition of the employed 2 l onto the membrane is critical .", "all results reported here were obtained using manual pipette ( rainin , pipet - plus r2 ) with a latch trigger mechanism and aspiration rate controller , features that improve precision from sample to sample and from operator to operator . in order to enable a fast , cost - effective , and simple analysis method ,", "although multivariate quantification procedures can provide better sensitivity when applied to the analysis of complex samples , they require ir expertise . as the method described here is intended for general protein quantification that is rapid and straightforward to non - ir specialists , the simpler univariate approach was pursued .", "the influence of protein secondary structure on amide i extinction coefficient ( exact location of amide i band ) has been well documented [ 17 , 18 , 40 ] .", "different spectral regions within the amide i area were analyzed and the best results for general protein quantification were obtained for the region between 1702 and 1602 cm . additionally , to account for possible buffer interference , a buffer signal ( e.g. , originating from buffer salts deposited and dried on the membrane ) was subtracted from the protein signal .", "investigation of various spectral regions considered for buffer signal subtraction delivered the most promising results for the region between 1850 and 1350 cm . following subtraction ,", "the strength of the remaining amide i signal was used to interpolate the estimated protein concentration from a known standard curve . under standard conditions , aqueous samples spotted on hydrophilic materials , including ptfe membrane , dry forming a coffee ring effect where the majority of sample is preferentially deposited around the edges of a spot .", "distribution of the coffee ring is strongly influenced by the nature of the sample buffer .", "effect is displayed in water ( supplementary figure s1b ) while use of buffers , such as pbs , reduces water tension allowing more uniform sample distribution across the membrane surface ( supplementary figure s1c ) . in the presence of detergent ,", "the buffer - dependent differences in dried sample distribution are highly reproducible but require generation of a reusable calibration curve and confirmation of linearity for each matrix to ensure the most accurate results .", "one major limitation of uv / vis - based protein quantitation is its strict dependence on amino acid composition , in particular the presence of tryptophan and tyrosine .", "comparative analysis between amide i - derived signal and uv / vis absorbance at 280 nm was performed for a range of concentrations of bsa , rabbit igg , and protein a ( figure 2 ) . unlike uv / vis ,", "mir - based analysis was unaffected by the proteins ' vast differences in amino acid composition or size . in reviewing the human protein database ( table 1 )", ", it is clear that many protein properties , such as protein length and mass , the number of specific amino acids , and even the predicted extinction coefficients at 280 nm , vary widely from protein to protein .", "however , the average mass per residue tends to be surprisingly consistent considering that individual residue contributions can range from 57 da for glycine to 186 da for tryptophan .", "further , since the number of amide bonds in a protein or peptide is only one less than the number of residues , plus the number of asparagines and glutamines , the mass per amide bond is also very consistent , even for short proteins and peptides . one could therefore postulate that mir absorbance ( on a mass basis , not a molar basis ) would be very consistent and that a single protein could serve as a reliable reference for just about any other protein or peptide , assuming that its average residue mass did not deviate significantly from the typical 110 da per residue ( e.g. , polyglycine or polytryptophan would be expected to deviate ) . in order to test the mir - based protein quantitation method ,", "a series of ten dilutions ( in triplicate ) , spanning the range 0.1255 mg / ml , was used to prepare a calibration curve .", "amide i signal strength delivered by each concentration point was fitted to a regression line that was ultimately used to determine protein concentration in the analyzed samples ( figure 3 ) .", "accuracy of mir - based concentration estimation was determined using single - protein solutions as well as a protein mixture .", "concentration values were derived by interpolation from a bsa reference curve and compared to amino acid analysis ( aaa ) , a method recognized currently as the gold standard for estimating protein concentration .", "first , a 1 mg / ml lysozyme sample in pbs , prepared by 1/68 dilution of a 68 mg / ml sample ( concentration determined by aaa ) was found by the mir method to be 0.922 0.061 mg / ml .", "next , protein a ( 52 mg / ml by aaa ) was diluted 1/13 with pbs to obtain a 4 mg / ml solution . when analyzed by mir , a concentration of 4.047 0.184 mg / ml was determined .", "lastly , a mixture of proteins ( 1.98 mg / ml by aaa ) was quantified at 1.944 0.028 mg / ml ; a 1/8 dilution of the same sample ( expected 0.25 mg / ml ) delivered a concentration of 0.273 0.028 mg / ml when analyzed by the mir - based method .", "overall , the method showed very good accuracy and linearity in response to samples between 0.25 mg / ml and 5 mg / ml . measurement accuracy for samples below 0.25 mg / ml decreased significantly ; thus , a limit of 0.25 mg / ml was selected for this method . in regard to the upper detection limits , a small set of samples up to 100 mg / ml was successfully measured ( data not shown ) .", "however , as the intended application of this method was for the analysis of precious samples , such high sample concentrations were not analyzed within the context of this paper .", "sample cards prepared using protein mixtures , at 0.25 and 1.98 mg / ml ( each in triplicate ; total of 9 spots per sample ) , were analyzed multiple times to determine measurement reproducibility .", "each card ( 3 protein spots + 1 control spot ) was measured four times and the concentrations obtained for each position as well as for an entire card were compared . assuring correct and complete deposition of the samples onto the membrane ,", "the average concentrations were 0.277 mg / ml ( 4.9% cv ) and 1.942 mg / ml ( 1.5% cv ) , respectively .", "the precision at each individual card position was measured , with cvs of 1.3 and 0.3% , respectively , at position 2 .", "position 3 delivered data with cvs of 1.2 and 0.1% , while cvs at position 4 were at 2.3 and 0.1% , respectively .", "protein quantitation method reported here relies on mir - based evaluation of biological samples ( from original buffers ) spotted and dried onto a membrane .", "therefore , measurements in the presence of buffer salts containing amide bonds , such as urea , can potentially affect the accuracy of the results . in the majority of cases ,", "interference from buffer salts is accounted for in the buffer subtraction step ; however , since the method relies on a simple univariate approach , high concentrations of interfering components can still overwhelm the protein signal and preclude proper measurement . reducing agents and detergents", "are known to interfere with colorimetric protein quantitation methods . to elucidate whether these agents would also interfere with mir - based measurements ,", "the results reveal that accurate mir - based protein quantitation can be achieved in the presence of up to 50 mm dithiothreitol ( dtt ) ( figure 4(a ) ) and up to 150 mm of -mercaptoethanol ( spectra not shown ) .", "in addition , the infrared absorption pattern of sodium dodecyl sulfate ( sds ; analyzed up to 1% ) did not overlap with the protein region allowing unbiased protein quantification in the presence of this detergent ( figure 4(b ) ) .", "method performance was also unaltered by the presence of up to 5% tween 20 ( data not shown ) or triton x-100 ( figure 4(c ) ) .", "at the same time , the presence of amide containing detergent , like , for example , chaps , might interfere with or prevent accurate protein quantitation . also , since detergents are known to bind to proteins with varying affinities , subtracting the buffer contribution might not always be sufficient for accurate analysis .", "for example , in cases when analyzed proteins bind a significant amount of the detergent , the bound detergent will most likely not be accounted for by the blank influencing the accuracy of concentration determination . by virtue of its ability to detect the spectral absorbance bands for many structural entities ,", "the mir - based method is not limited to the analysis of protein species . given the large number of lipid - associated absorption bands , application of the described method for the quantification of lipid biomolecules", "h stretching region ( 28502870 cm ) provides an ideal candidate for the analysis of lipids and detergents . while a single - protein standard can be used to quantify a wide range of protein and peptide samples , due to the vast complexity and variability among lipids and detergents , it was speculated that each quantitation would require the generation of a standard curve using the specific species in question", ". experiments using various lipids , including fatty acids ( supplementary figure s3a ) , phospholipids , triglycerides , liposaccharides , and many detergents , demonstrated a high degree of variability in the detection limits and slope of the employed calibration curves , thus confirming requirement for individualized calibrations for each of the analyzed lipids and detergents . in order to validate the compatibility of mir - based quantitation method for lipid and detergent analysis ,", "the ftir spectrometer was initially calibrated using either tetracosanoic acid ( in chloroform ) or chaps ( in pbs ) .", "two concentration ranges ( performed in triplicate ) , spanning 0.251.75 mg / ml ( tetracosanoic acid ) and 0.254% ( chaps ) , were used to derive lipid and detergent calibration curves ( supplementary figure s3b ) .", "the strength of symmetric c h vibration for each concentration was fitted to a regression line .", "the accuracy of concentration estimation within the established dynamic ranges for tetracosanoic acid and chaps was assessed using 0.8 mg / ml tetracosanoic acid and 1.8% chaps .", "the results showed that , for a well defined calibration range , the method was capable of estimating lipid and detergent concentration with low error . assuring precise pipetting when applying the samples , mir - based quantitation of the tetracosanoic acid sample returned 0.853 0.14 mg / ml ( 2.4% cv ) and chaps sample was quantified as 1.8 0.004% ( 2.3% cv ) .", "the experiments using several formulations of lysis buffers , like ripa and cytobuster protein extraction reagent , spiked with known concentration of bsa and measured alone , as well as in the presence of phospholipids ( data not shown ) , demonstrated that the reported method allows accurate protein quantification from lysis buffers . to determine", "if the reported method can be further applied to more complex biological samples , protein content in variously prepared breast cancer cell line ( mcf-7 ) lysates was measured and compared to values derived from a bca protein assay .", "total protein content in the sample obtained using cytobuster protein extraction reagent was estimated at 2.4 mg / ml ( bca assay ) or 2.7 mg / ml ( mir - based method ) .", "protein concentration in samples lysed with ripa buffer was estimated at 3.4 mg / ml ( bca assay ) or 4.6 mg / ml ( mir - based method ) .", "the results obtained using bca assay were consistently lower ( 10% and 26% , resp . ) than the mir - based method .", "for the two methods , the differences in estimated protein concentration were most likely caused by a combination of the following factors : presence of various detergents ( documented to influence the accuracy of bca assay ) in lysis buffers , documented inaccuracy of bca in the analysis of native proteins , an error associated with use of bsa as a standard in colorimetric assays , and the fact that accurate quantification of peptides by the bca assay could not be achieved without additional sample manipulations that are incompatible with biological material .", "the possibility that mir - based quantification is inflated by signal from nucleic acids present in cell lysates was excluded because the amount of nucleic acids generally present in such mixtures is significantly below the detection level of the reported method .", "the findings reported here are consistent with a previous report where the mir - based method was used to quantify protein content in crude human skin carcinoma cell lysates or to adjust protein concentration prior to mass spectrometry and nmr analysis [ 47 , 48 ] .", "the method described here was able to detect and examine protein and lipid content of all analyzed samples simultaneously and selectively ( figure 5(a ) ) , in contrast with conventional analysis techniques such as uv spectroscopy .", "this dual functionality could enable the monitoring of changes in total lipid content and protein liberation yield , thereby simplifying and improving the analytical process .", "the efficacy of mir - based analysis for use in downstream sample qualification was evaluated during a short study of breast cancer cell lysate fractionation and biomarker detection .", "breast tissue was chosen because it is documented to have a high fat content ( 69.9 22.9% ) . in the reported study", ", breast tissue lysates were prepared using ripa buffer and cytobuster protein extraction reagent . following tissue homogenization ,", "the efficiency of fat removal and total protein liberation during centrifugal extractions was monitored using the mir - based method ( see supplementary figure s2 for separation flow chart ) .", "the collected spectral data were used to determine protein recovery across the centrifugal fractions ( figure 5(b ) ) and to examine the efficiency of gradual fat removal from these samples ( figure 5(c ) ) .", "information delivered by mir - based analysis was utilized in further sample manipulations facilitating faster and more consistent immunodetection of the investigated breast cancer biomarkers ( study in progress ) ." ], [ "fast and accurate quantification of proteins from complex aqueous biological samples such as plasma , cerebrospinal fluid , or cell / tissue lysates remains a challenge .", "critical understanding of results from downstream applications is often dependent on proper preparation and accurate qualification of the applied samples .", "classical methods for protein quantification , like uv and colorimetric assays , permit precise estimation of purified protein concentration but are less reliable when applied to the analysis of complex mixtures . while mir is a promising technique allowing accurate quantification in complex samples ,", "existing techniques for mir - based protein quantification require regular time - consuming cleaning procedures ( flow - through cells ) , larger sample volume ( atr cells ) , or expertise in method development ( atr of dried samples ) , thus precluding their suitability for fast analysis in a typical research laboratory .", "although high - throughput mir sampling and analysis techniques are already available on the market , they are more suited for larger pharmaceutical laboratories that require recurring analysis of repetitive sample sets and possess the requisite analytical skills for effective chemometrics - based method development .", "currently available instrumentation is often too costly or requires certain ir expertise to be easily adapted for general protein quantification by non - ir savvy personnel in small - scale laboratories or academia .", "the method developed and described herein relies on a simple , cost - effective approach permitting accurate quantification results based on a univariate sample analysis technique that does not require advanced ir expertise .", "the system relies on a membrane , transparent in most of the mir region , which permits robust analysis of aqueous samples in a dried format .", "the reported method is also compatible with organic solvents commonly used in peptide and lipid research . to achieve accurate quantitative results , strict adherence to a consistent loading volume between samples and controls", "is required . while a general calibration curve can be employed , the method is more accurate when a specific calibration curve is prepared for each buffer to be utilized .", "based on the experimental data , a general lower quantification range was set to 0.25 mg / ml ; for some samples , a detection limit as low as 0.1 mg / ml can be achieved . because the developed method focuses solely on small volumes and biologically relevant samples , the upper detection limit was not investigated thoroughly .", "however , from the narrow set of experimental results , such limits can exceed 100 mg / ml . although some buffer salts might not be compatible with the reported technique , in pbs , the method allows development of a general linear calibration between 0.25 mg / ml and 5 mg / ml . accuracy and precision of the reported method , within the linear range , are comparable with results obtained by amino acid analysis , providing researchers with a fast and cost - effective alternative to other protein quantitation tools available today . due", "to chemical and structural variation observed within lipids and detergents , mir signal strengths display considerable variability ; for this reason , a universal standard for all lipids and detergents could not be identified .", "therefore , quantitation of lipids and detergents requires development of separate standard curves for each of the analyzed compounds .", "in addition , qualitative analysis of mixtures or unknown compounds can be performed using a relative absorbance ", "mode where the method delivers information about strength of mir signal without any comparison to a known standard . in summary , the mir - based method reported here enables simultaneous measurement of total protein recovery and monitoring of fat removal from lysed samples .", "this technique was also successfully applied to complex sample analysis during a small - scale investigation of surgical breast tissue processing and fractionation .", "mir - based analysis facilitates more in - depth sample characterization and offers higher quality control over the sample preparation process .", "given its unbiased biomolecular detection capabilities and amenability to liquid samples , many more applications are easily envisioned for this method ." ] ]

[ "the northern house mosquito , culex pipiens l. ( diptera : culicidae ) , is a member of a species complex that includes vectors of lymphatic filariasis and several arboviral diseases , such as st .", "louis encephalitis ( sle ) , rift valley fever ( rfv ) , and west nile virus ( wnv ) . in the northeastern quadrant of the united states , c. pipiens recently has become important because of its role in the transmission of wnv .", "the virus was introduced into new york city in 1999 and is currently the most widespread arboviral disease in the u.s .", "( cdc , 2010 ) . the blood - feeding pattern of c. pipiens ( shifting from avian to human feeding within the transmission season ) ( edman and taylor , 1968 ; kilpatrick et al . , 2006 ) , coupled with its high vector competence ( turell et al . , 2005 ) and", "ability to carry wnv through the winter , has allowed the virus to spread from its point of origin and maintain transmission throughout the mosquito s range .", "thus , c. pipiens is one of the major targets for mosquito vector control and surveillance in the u.s .", "current methods of monitoring by abatement districts and health departments include centers for disease control ( cdc ) light traps and co2-baited traps , which mainly trap blood - seeking females , along with gravid traps , which target gravid females .", "plant - derived attractants have the potential to act as surveillance - trap lures and , when combined with poison - laced sugar solutions ( mller et al . ,", "mosquitoes are attracted to various plant species , from which they obtain nectar and other juices .", "sugar feeding by mosquitoes has a significant influence on dispersal ( hocking , 1953 ; magnarelli , 1977 ) and vectorial capacity ( gary and foster , 2001 ; gu et al . , 2011 ) , and , contrary to long - held conjecture , is required by both males and females throughout the adult stage ( downes , 1958 ; yuval , 1992 ; foster , 1995 ) .", "both sexes typically first visit plants soon after emergence . males then require sugar at frequent intervals to maintain their energy reserves in order to join nightly mating swarms ( yuval et al . , 1992 ) .", "females take sugar between blood meals , when they are digesting blood , or when they are gravid ( clements , 1999 ; foster , 2008 ) .", "furthermore , females of most temperate - climate culex and anopheles species enter reproductive diapause in late summer , after which they no longer are attracted to blood hosts , but engage heavily in plant - sugar feeding ( bowen , 1992a ) .", "the potential for plant volatiles to lure mosquitoes has been demonstrated in several laboratory behavioral assays that have used stimuli such as natural plant extracts ( vargo and foster , 1982 ; jepson and healy , 1988 ; mauer and rowley , 1999 ) or single floral compounds ( jhumur et al . , 2006 ) .", "in field studies , bates ( 1949 ) reported the successful trapping of anopheline mosquitoes with plant and fruit baits ; fruit also proved to be an effective attractant in cdc traps for c. tarsalis ( reisen et al . , 1986 ) .", "sandholm and price ( 1962 ) observed that mosquitoes in the field were attracted to light - colored flowers with distinct fragrances ; odor appears to be primarily responsible for long - range attraction , with visual cues playing a role at shorter range ( thorsteinson and brust , 1962 ; healy and jepson , 1988 ; jepson and healy , 1988 ) . orientation to commercially obtained floral extracts and honey has been demonstrated for various mosquito species ( thorsteinson and brust , 1962 ; hancock and foster , 1997 ; foster and takken , 2004 ) . schlein and mller ( 2008 ) and mller et al .", "( 2008 , 2010 ) reported dramatic population reductions of c. pipiens and other mosquito species by spraying a fruit - based sugar bait containing insecticide on vegetation surrounding larval habitats .", "light - less cdc traps baited with the blossoms of tamarix jordanis were highly effective in trapping c. pipiens , and populations were reduced where these blossoms were treated with spinosad insecticide ( schlein and mller , 2008 ) .", "flower species that elicit the highest attraction probably vary by region . during mid - summer in the u.s .", ", mosquitoes have been observed probing blossoms of common milkweed , asclepias syriaca , at rates disproportionate to their abundance relative to other flowering plants , with one study reporting 54 species occurring in the study area that did not serve as nectar sources ( grimstad and defoliart , 1974 ) .", "another study collected 25 aedes vexans from one milkweed cluster in just 15 min ( sandholm and price , 1962 ) .", "the observed preference of insects for milkweed flowers , both day and night , possibly is due to its strong and distinctive fragrance , lighter - colored flowers , and greater nectar production ( sandholm and price , 1962 ) .", "common milkweed is indigenous to eastern and midwestern north america , where its principal native pollinators are mainly large hymenoptera and lepidoptera ( e.g. , jennersten and morse , 1991 ) .", "mosquitoes are incapable of transferring milkweed pollinia and therefore function as nectar thieves , appearing to co - opt the attractive properties used by the pollinators to locate the flowers ( foster , 1995 ) .", "solvent extracts of common milkweed flowers also are attractive to mosquitoes ( vargo and foster , 1982 ; mauer and rowley , 1999 ) , confirming that volatile chemicals are at least partly responsible for that attraction .", "mauer and rowley ( 1999 ) determined the headspace profile of common milkweed flower to be predominantly 2-phenylethanol and benzyl alcohol ; however , they found that a synthetic blend of these two compounds was not attractive to c. pipiens . due to the multiple demonstrations of c. pipiens attraction to milkweed , both in the laboratory and the field , we used a solvent extract of the flower as a potential model for synthetic mosquito lures .", "experiments were conducted with c. pipiens from a colony established in 2009 from larvae collected near columbus , oh , usa . larvae were identified at l4 by siphonal hair tufts ( vinogradova , 2000 ) .", "adults were maintained in 41-l clear acrylic cages on a diet of 10 % sucrose , water , and weekly blood meals from the legs of a rooster ( ilacuc permit no .", "oviposition water was prepared by soaking grass clippings in aged tap water and allowing fermentation over a 3-d period .", "three days after each blood meal , oviposition cups were placed with caged adults , and eggs were collected the following day .", "two hundred first - instar larvae were placed into 22.8 33.0 cm aluminum pans with 450 ml of aged tap water .", "the larvae were provided finely ground tetramin flakes , increasing the quantity daily from 50 mg for first instars to 500 mg for final instars until pupae appeared on the 8th and 9th day post - hatching .", "pupae then were counted and transferred to plastic cups and placed in a 41-l cage supplied with water wicks .", "emerging adults were given ad libitum access to water , but were deprived of sugar .", "the mosquito rearing and maintenance conditions were 27 1 c , 85 5 % rh , and 16:8 ( l : d ) , with 30-min gradual crepuscular transitions between photophase and scotophase .", "phenylacetaldehyde ( > 90 % ) , benzaldehyde ( 99.5 % , purified by redistillation ) , nonanal ( 95 % ) , ( e)-2-nonenal ( 97 % ) , and an alkane - standards mixture ( c8 - c20 ) were purchased from sigma - aldrich ( st . louis , mo , usa ) .", "( phillipsburg , nj , usa ) , and -ocimene [ ( 75 % ( e)--ocimene ) ] was synthesized by chemos gmbh ( regenstauf , germany ) .", "synthetics were diluted with hplc - grade n - pentane ( fisher scientific , pittsburgh , pa , usa ) .", "flowers of common milkweed , a. syriaca , were collected in early summer from the ohio state university campus in columbus ( 400018.95 \" n , 830247.11 \" w ) , placed in an ice cooler , and transported 10-min to the laboratory .", "single florets were plucked from the main milkweed umbel and separated from the calyx and other green parts of the flower , weighed , and placed into a 500 ml narrow - mouth glass erlenmeyer flask .", "milkweed florets were submerged in hplc - grade n - pentane ( fisher scientific ) in a 1:8 ratio ( w / v ) for a total solvent volume of 480 ml and held for 24 h at room temperature , at which time the extract was decanted into 21-ml borosilicate glass vials , capped with teflon - lined screw caps and stored at -20 c .", "the volatile profile of milkweed flower was characterized by placing a single floret into a 21-ml borosilicate glass vial with a teflon - lined rubber septum .", "volatiles were allowed to equilibrate in the vial headspace at 30 c for 10 min before collections were made .", "volatiles were collected by using a divinylbenzene / carboxen / polydimethylsiloxane ( dvb / car / pdms ) ( 50/30 m ) solid phase micro - extraction ( spme ) fiber ( supelco , bellefonte , pa , usa ) .", "this mixed - chemistry fiber provides affinity for chemicals with a far broader range of polarity and volatility than pdms alone .", "the fiber was introduced to the vial through the septum and exposed for 30 min .", "the collected volatiles were analyzed immediately by using an agilent technologies 6890 series gas chromatograph interfaced to an agilent technologies 5973 quadrupole mass selective detector ( gc - ms ) .", "the spme fiber was desorbed at 275 c for 3 min with a splitless injection onto a zebron zb-1 ms column ( 30 m 0.25 mm , 0.25 m film thickness ) ( phenomenex , torrance , ca , usa ) .", "the oven temperature was held at 25 c for 3 min , then ramped at 15 c / min to 250 c , where it was held for 2 min .", "the carrier gas was helium at a flow rate of 1 ml / min .", "the mass selective detector was operated in ei mode at 70 ev , scanning 19 - 350 m / z with a quadrupole temperature of 180 c and source temperature of 240 c . to guide the construction of a synthetic floral blend , pentane extracts of milkweed florets", "were analyzed by gc - ms on a zebron zb-50 column ( 15 m 0.25 mm , 0.25 m film thickness ) ( phenomenex ) , with a 1-l splitless injection at 210 c .", "the temperature program began at 25 c for 3 min and increased at 13 c / min to 200 c , followed by a 25 c / min increase to 280 c to remove high - boiling lipids .", "peak identities were validated by re - injection on a finnegan trace gc / ms ( thermo fisher scientific inc . , waltham , ma , usa ) , with a 1:10 split at 180 c and a rtx-5ms column ( 30 m 0.25 mm , 0.25 m film thickness ) ( restek , bellefonte , pa , usa ) .", "the helium flow was 1 ml / min , and temperature program was 40 c for 1 min , ramped at 15 c / min to 275 c , with a 2-min final hold time .", "retention indices ( ri ) of compounds were determined on both columns relative to n - alkanes ( c8c20 ) .", "components of floral headspace and extract were identified by matching their mass spectra to nist / epa / nih mass spectral library 2005 and comparison of retention indices to published values ( adams , 1995 ) .", "identity of major components was confirmed by comparison of spectra and retention times to authentic standards .", "peak purity was determined by using amdis deconvolution software ( nist , gaithersburg , md , usa ) to assure the absence of any co - eluting components .", "a synthetic mimic of the extract was constructed by blending authentic standards of the major components identified in the extract .", "quantities of each component were individually adjusted until the overall concentration and component proportions closely approximated those of the floral extract ( fig . ", "1total ion chromatograms ( a ) of florets of milkweed , asclepias syriaca , pentane extract ( broken line ) , and a synthetic blend ( solid line ) used to assay upwind attraction of culex pipiens ( peak # 1 .", "phenylethanol ; column = zebron zb-50 ) and total ion chromatogram ( b ) of the headspace profile of a single a. syraica floret captured on a divinylbenzene / carboxen / polydimethylsiloxane spme fiber ( peak # 1 .", "( e , e)-alloocimene ; column = zebron zb-1 ) total ion chromatograms ( a ) of florets of milkweed , asclepias syriaca , pentane extract ( broken line ) , and a synthetic blend ( solid line ) used to assay upwind attraction of culex pipiens ( peak # 1 .", "phenylethanol ; column = zebron zb-50 ) and total ion chromatogram ( b ) of the headspace profile of a single a. syraica floret captured on a divinylbenzene / carboxen / polydimethylsiloxane spme fiber ( peak # 1 .", "( e , e)-alloocimene ; column = zebron zb-1 ) all behavioral assays were conducted in a clear acrylic dual - port flight olfactometer , modified from hancock and foster ( 1993 ) , with three main parts : an introduction / release chamber , flight chamber , and trapping ports . the introduction", "/ release chamber was located at the downwind end , measuring 30 40 cm wide and 30 cm long .", "a sliding gate separated the release chamber from the main flight chamber ( 30 40 cm wide , 90 cm long ) , which had two cylindrical glass jar trapping ports ( 15 cm long by 7 cm diameter ) on its upwind end .", "ports were fitted with borosilicate glass funnels , with the wide end opening into the flight chamber and the narrower ( 3 mm diam ) end pointing into a glass jar .", "thus , mosquitoes entering the wide end of the funnel from the flight chamber were channeled into the jar , where they were retained .", "trapping ports were located 11 cm above the flight chamber floor and were separated by 21 cm . the funnel and glass jar were held together by parafilm sealing film ( pechiney plastic company , menasha , wi , usa ) , which was replaced between treatments .", "we recognized the pitfalls associated with the use of parafilm in solvent - based behavioral assays ( millar and haynes , 1998 ) , and we ensured that this sealant would not come in contact with any chemicals in the olfactometer .", "a 7.5-mm diam hole in the upwind end of each jar allowed the introduction of purified / humidified air after it had passed from an oil - free air pump through an activated carbon canister , and then through a water column .", "air flow was maintained at 50 ml / s into each port , providing a velocity of 72 mm / s in the center of the flight chamber . a black cotton cloth , covering a dampened layer of cotton wool , covered the entire floor of the flight chamber , to maintain a level of humidity similar to that within the choice ports .", "data loggers ( hobo , onset computer corporation , bourne , ma , usa ) recorded temperature and humidity in the olfactometer , which was 25.0 - 27.5 c and 75 - 95 % rh . an exhaust duct , connected at the downwind end of the release chamber , directed the effluent air out of the building through an exhaust hood . to test for positional bias of the ports , mosquitoes were released into the chamber after either baiting both ports with honey or leaving both empty .", "mosquitoes were divided equally between left and right ports when baited with honey and were found rarely in ports when both were empty .", "response to whole milkweed flowers was determined in the flight olfactometer with 2 g of florets placed in an aluminum weighing boat in one of the trapping jars , and compared with an empty control jar .", "extracts and synthetic chemicals were applied to 15-cm long cotton wicks ( tidi products , neenah , wi , usa ) and compared to a pentane control .", "the positions of the treatment and control ports were alternated between bioassays as a further safeguard against positional bias .", "the olfactometer parts were cleaned with 70 % ethanol followed by water after each experiment ; gloves were used at all times to avoid contamination with human - related kairomones .", "the 16:8 ( l : d ) light cycle used for mosquito rearing was maintained in the bioassay room . at 2 h", "prior to scotophase , approximately 200 - 300 mosquitoes of both sexes in similar numbers were released through a sleeve connection , directly from an acrylic plastic cage ( 30 30 30 cm ) into the holding / release chamber , where they were held for 15 min to acclimatize before the release gate was opened .", "after 12 h , the numbers of mosquitoes in the treatment port , the control port , and remaining in the flight chamber were recorded .", "use of a 12-h period captured the bimodal nocturnal - crepuscular sugar - feeding rhythm of c. pipiens , which includes some sugar feeding throughout the night ( yee and foster , 1992 ) .", "subtractive bioassays were conducted on the synthetic blend to determine the relative activity of the identified components .", "the full six - component blend was presented in one of the paired olfactometer ports alongside a reducted five - compound blend , in which one of the components was removed .", "after establishing the significance of each chemical on mosquito attraction , we compared the minimal attractive blend against the full six - component blend .", "this was followed by a dose response study of the minimal blend to determine the optimal concentration for response .", "doses were presented in a randomized block design ( n = 5 ) , with percentage response calculated as the number in the synthetic blend port minus the number in the control port and expressed relative to the number of mosquitoes released .", "mosquito flight behavior was recorded by using three infrared rca closed - circuit video cameras ( lancaster , pa , usa ) onto which was mounted a tc1824b wide - angle es 25 mm 1:1.4 lens for flight - chamber recordings or a tc1874c es 75 mm 1:1.8 lens for each of the choice ports .", "( lancaster , pa , usa ) eight - channel switcher , with video output to two rca tc1109 video monitors .", "video output was recorded with an emerson ewv404 vcr ( parsippany , nj , usa ) onto vhs tape in time - lapse mode , 20 s each min .", "mosquito response in the flight olfactometer was analyzed by comparing numbers caught in the two ports by a goodness - of - fit chi - squared test with spss v. 17 ( spss inc , chicago , il , usa ) .", "response experiment , a regression model of mosquito response to log - transformed blend dose was determined by using the fitted line plot module of minitab v. 16 ( minitab inc .", "six compounds comprised > 90 % of the relative abundance of the milkweed floret components in the pentane extract : ( e)--ocimene , benzaldehyde , nonanal , benzyl alcohol , phenylacetaldehyde , and ( e)-2-nonenal ( fig . 1a , table 1 ) .", "the volatile profile collected by dvb / car / pdms spme directly from fresh florets was dominated by three of these compounds : ( e)--ocimene , benzaldehyde , and phenylacetaldehyde ( > 75 % of the total ) , but there were also substantial differences compared to the pentane extract ( fig . ", "most notably , ( e , z)- and ( e , e)-alloocimene were collected from the flower headspace , but were almost completely absent from the extract , whereas nonanal , a major constituent of floret extract , was missing in the headspace sample .", "these disparities can be explained partly by variation in volatility among the components , but also likely reflect differences between the chemical composition in flower tissue and what is released .", "the extract also contained higher - boiling hydrocarbons and fatty acids , but due to their low volatility , they were not included in behavioral studies . based on the quantities of synthetic standards", "used to mimic the extract gc profile ( table 1 ) , the total floral concentration of the six major volatiles was estimated at 32 g / g floret fresh weight.table 1major volatile constituents of a pentane extract of the common milkweed , asclepias syriaca , as determined by gas chromatography ", "mass spectrometry , and quantities used in a synthetic mimiccompoundrelative retention indexdiagnostic ei - ms fragment ions ( % intensity)synthetic blend ( g / ml)(e)--ocimene112477(36 ) , 79(42 ) , 91(53 ) , 93(100)6.14benzaldehyde115551(54 ) , 77(100 ) , 105(88 ) , 106(88)3.27nonanal122029(66 ) , 41(100 ) , 56(61 ) , 57(94)4.14benzyl alcohol123677(85 ) , 79(100 ) , 107(59 ) , 108(75)4.58phenylacetaldehyde125765(22 ) , 91(100 ) , 92(24 ) , 120(16)11.25(e)-2-nonenal130429(56 ) , 41(100 ) , 55(80 ) , 70(58)2.12identity established by comparison to authentic standard of each compoundgc retention times relative to n - alkane standards on a phenomenex zb-50 column phasesynthetic -ocimene contained a 3:1 ( e : z ) isomer mixture major volatile constituents of a pentane extract of the common milkweed , asclepias syriaca , as determined by gas chromatography ", "mass spectrometry , and quantities used in a synthetic mimic identity established by comparison to authentic standard of each compound gc retention times relative to n - alkane standards on a phenomenex zb-50 column phase synthetic -ocimene contained a 3:1 ( e : z ) isomer mixture infrared video recordings showed c. pipiens in the flight chamber engaged in a zigzagging flight that was directed upwind towards ports baited with florets , extracts , or synthetic blends .", "the angle of the turns progressively decreased as the mosquitoes approached the target . in most cases , mosquitoes flew into the ports , but in a few cases they landed on the entrance of the port and walked in .", "there was no observed oriented flight towards the control port . in the first behavioral assay , mosquitoes showed a significant response to whole milkweed florets , where 67 % of released mosquitoes were captured in the floret - baited port , compared to only 6 % in the control port ( fig . ", "2a ; \n = 515.02 df = 1 , p < 0.001 ) . despite the chemical differences measured between the headspace and extract profiles of milkweed florets , mosquitoes showed a similarly strong response to a pentane extract of milkweed florets ( 52 % ) compared to pentane alone ( 11 % ) ( fig . ", "2b ; = 61.44 , df = 1 , p < 0.001 ) .", "moreover , mosquitoes were observed in video recordings to probe the extract - treated cotton wick during the early scotophase and early photophase , suggesting that the extract also stimulated a feeding response .", "2percentage of culex pipiens flying upwind in a dual - port flight olfactometer to : a whole milkweed , asclepias syriaca , florets ; b pentane extract of a. syriaca florets ; and ( c ) synthetic a. syriaca floret blend compared against a water a or solvent ( b , c ) control . * * * p < 0.001 by chi - squared test percentage of culex pipiens flying upwind in a dual - port flight olfactometer to : a whole milkweed , asclepias syriaca , florets ; b pentane extract of a. syriaca florets ; and ( c ) synthetic a. syriaca floret blend compared against a water a or solvent ( b , c ) control . * * * p < 0.001 by chi - squared test based on the positive response to the milkweed floret extract , a six - component synthetic blend was formulated to simulate the concentrations and relative proportions of major constituents of the pentane extract ( fig . ", "one discrepancy in the gc profiles was caused by the presence of the ( z)-isomer in synthetic ( e)--ocimene , resulting in a synthetic blend that exceeded the levels of this isomer in the floret extract . in the olfactometer ,", "the synthetic floret blend performed similarly to the natural extract , with 48 % of released mosquitoes trapped , compared to 16 % for the control ( fig . 2c ; \n = 120.6 , df = 1 , p < 0.001 ) .", "again , video - recordings showed a directed upwind flight response to ports containing the synthetic blend , accompanied by vigorous probing of the treated wicks , but not to the controls .", "males and females responded to milkweed floret odors in similar numbers in all three experiments as the sex ratio of mosquitoes captured in either the treatment or control ports did not deviate significantly from 1:1 .", "the subtractive bioassay of the synthetic blend indicated a significant role for three compounds in c. pipiens response to milkweed : benzaldehyde ( \n = 13.22 , df = 1 , p < 0.001 ) , phenylacetaldehyde ( \n = 8.25 , df = 1 , p = 0.004 ) , and ( e)-2-nonenal ( \n = 3.62 , df = 1 , p = 0.05 ) ( fig . 3 ) .", "when any of these compounds was removed , mosquitoes showed a significant preference for the full blend over the reducted blend .", "a nearly significant preference was seen for blends missing either -ocimene ( p = 0.078 ) or benzyl alcohol ( p = 0.082 ) compared to the full blend .", "the activity of benzaldehyde , phenylacetaldehyde , and ( e)-2-nonenal was confirmed when the three were combined : the three - component blend captured 31 % of mosquitoes compared to only 7 % for the control ( fig . ", "4a ; \n = 196.56 , df = 1 , p < 0.001 ) and was as active as the full blend ( fig . 4b , 4 ; = 3.02 , df = 1 , p = 0.082).fig .", "3choice response of culex pipiens in a dual - port flight olfactometer to a six - component ( full ) synthetic blend of milkweed , asclepias syriaca , floret volatiles compared to blends in which each component has been removed individually ( reducted ) .", "total response indicates the percentage of c. pipiens released that were captured by one of the blends ( no significant differences by 1-way anova ) .", "relative response shows the proportional response to the reducted ( gray bars ) and full ( black bars ) blends .", "numbers in bars indicate the mean number of c. pipiens responding ( n = 46 ) .", "asterisks denote significant differences in mean response to reducted versus full blend by chi - squared test ( * p < 0.05 , * * p < 0.005)fig", ". 4a percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to a three - component blend , consisting of benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ; * * * p < 0.001 by chi - squared test .", "b percentage of c. pipiens flying upwind in response to the full ( six - component ) and minimal ( three - component ) synthetic blends of a. syriaca floral odor .", "ns = no significant difference choice response of culex pipiens in a dual - port flight olfactometer to a six - component ( full ) synthetic blend of milkweed , asclepias syriaca , floret volatiles compared to blends in which each component has been removed individually ( reducted ) .", "total response indicates the percentage of c. pipiens released that were captured by one of the blends ( no significant differences by 1-way anova ) .", "relative response shows the proportional response to the reducted ( gray bars ) and full ( black bars ) blends .", "numbers in bars indicate the mean number of c. pipiens responding ( n = 46 ) .", "asterisks denote significant differences in mean response to reducted versus full blend by chi - squared test ( * p < 0.05 , * * p < ", "0.005 ) a percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to a three - component blend , consisting of benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ; * * * p < 0.001 by chi - squared test .", "b percentage of c. pipiens flying upwind in response to the full ( six - component ) and minimal ( three - component ) synthetic blends of a. syriaca floral odor .", "ns = no significant difference because quantity as well as quality can determine the intensity of chemically mediated behavior , c. pipiens response was measured in the flight olfactometer to different doses of the three - component blend compared to a solvent control .", "there was a significant positive quadratic response [ log(dose ) ( y = 20.527x + 73.476x43.709 ; r = 57.6 % ] , with a response maximum predicted at 62 g ( fig . 5 ) .", "the response flattened out at both the lowest and highest doses ; removal of the two most extreme doses produced a stronger - fitting regression model ( r = 93.3 % ) , but with a similar predicted optimal dose of 65 g.fig .", "5percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to different concentrations of a three - component kairomone blend ( benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ) after subtracting the response to a paired solvent ( pentane ) control percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to different concentrations of a three - component kairomone blend ( benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ) after subtracting the response to a paired solvent ( pentane ) control", "a positive upwind orientation by c. pipiens to floret volatiles of a. syriaca was demonstrated in this study , suggesting a new potential source of attractants for use in the field as lures .", "males and females showed significant response to milkweed florets , a pentane extract , and a synthetic blend of the extract s six major constituents : benzaldehyde , ( e)--ocimene , benzyl alcohol , phenylacetaldehyde , nonanal , and ( e)-2-nonenal .", "the first three of these compounds are among the most common floral odor constituents in plants ( knudsen et al . , 2006 ) .", "subsequent subtractive bioassays of the synthetic blend demonstrated that only three of its components contributed to attraction : benzaldehyde , phenylacetaldehyde , and ( e)-2-nonenal . the attraction of mosquitoes to plant odors has been demonstrated in numerous field and laboratory studies , but few studies have shown significant attraction to synthetics .", "mauer and rowley ( 1999 ) observed orientation of c. pipiens to a methylene chloride extract of a. syriaca in a dual port olfactometer .", "however , a synthetic blend of the two dominant compounds in the extract headspace , benzyl alcohol and 2-phenylethanol , failed to attract the mosquitoes .", "we did not test 2-phenylethanol , but benzyl alcohol was not active in our flight olfactometer bioassays .", "( 2007 ) also found benzyl alcohol to not be very attractive to c. pipiens var .", "flowers of tanacetum vulgare also attract culex species in the field ( andersson and jaenson , 1987 ) , and bowen ( 1992b ) discovered a high proportion of both broadly- and narrowly - tuned antennal receptor neurons of c. pipiens sensitive to thujone , the primary constituent of t. vulgare fragrance", ". however , thujone only elicited a close - range dose - dependent probing response in c. pipiens ; it did not stimulate upwind flight in a wind - tunnel olfactometer ( bowen , 1992b ) .", "the only previous flower - based synthetic blend causing mosquito orientation was developed from silene otites , whose flowers attract mosquitoes in the field ( brantjes and leemans , 1976 ) .", "the synthetic blend mimic consisted of phenylacetaldehyde , veratrole , and 2-methoxyphenol , the first of which elicited the strongest attraction in c. pipiens var .", "phenylacetaldehyde also was a prominent component of our a. syriaca headspace and solvent extract , and it was essential for maximum attraction to the synthetic blend .", "although nonanal was a major component of a. syriaca floret extract , we saw no evidence that it played a role in attraction in our bioassays .", "in contrast , from single - cell recordings , syed and leal ( 2009 ) determined that ca .", "40 % of all antennal olfactory receptors of c. quinquefasciatus were acutely sensitive to nonanal , and it produced a synergistic effect on trap catch when presented with co2 .", "nonanal is a major skin odorant of birds and mammals , including humans , resulting from the oxidation of sebaceous fatty acids ( haze et al . , 2001 )", "this fact , along with the significant behavioral interaction with co2 , suggests that nonanal may play an important role in mediating culex host - finding , but does not attract sugar - seeking mosquitoes .", "we found notable differences between profiles of the pentane extract and the spme - collected headspace of a. syriaca florets .", "pentane extracts were dominated by phenylacetaldehyde , ( e)--ocimene , and nonanal , whereas the headspace profile contained primarily benzaldehyde , ocimene isomers , and phenylacetaldehyde .", "disparities between extracts and chemicals collected in the headspace might be explained by differences in the physico - chemical properties of the constituents , deep penetration by solvents to extract compounds that are not normally released by the tissue , and/or selective trapping of chemicals by spme . in this study ,", "benzaldehyde , -ocimene , and alloocimene have the highest vapor pressures of all the chemicals identified from milkweed , and they make up most of the volatile profile .", "the vapor pressure of benzaldehyde is almost twice that of nonanal , almost four times that of ( e)-2-nonenal , and more than six times that of benzyl alcohol .", "the latter three compounds were either absent or found in very low levels in the headspace analysis .", "phenylacetaldehyde is intermediate in its volatility , but was also the component found in the highest levels in the pentane extract .", "volatile collection analyses can produce misleading results , as they may not accurately reflect the actual proportionality of constituents in the headspace .", "we do not believe that this was a major factor for explaining the differences in the headspace and extract profiles of milkweed due to our choice of spme phase .", "although pdms has been the most widely used spme phase , it is actually a poor choice for characterizing plant volatile profiles that contain constituents with a range of volatilities and functional groups . in preliminary studies ( not shown ) , we found a broader array of volatile compounds was trapped from various flowers by dvb / car / pdms compared to pdms alone .", "this mixed - bed fiber not only employs a broader range of phase polarity , but also incorporates both adsorption and partitioning as mechanisms of collection ( koziel and novak , 2002 ) .", "a number of recent studies have demonstrated the higher recovery efficiency and linearity by dvb / car / pdms compared to other spme phases for all of the compound classes that we identified from milkweed ( cui et al . , 2009 ; ferreira et al . , 2009 ; zhang et al . , 2009 ) .", "2009 ) found that volatile profiles of longan fruit extracted by a 50/30 m dvb / car / pdms fiber were 3x higher in terpenes , 5x higher in alcohols , and 14x higher in esters than those produced by a 100 m pdms fiber , and revealed volatile carbonyls and acids , which were completely absent from pdms profiles .", "this study points to floral odors as a potential new model for chemical lures useful for mosquito sampling or control . if similar attraction can be demonstrated at the field level , as has already been demonstrated with human kairomone blends ( mukabana et al .", ", 2012 ) , synthetic floral blends could potentially be used in trapping devices to sample adult populations .", "relative to animal - derived odors ( mukabana et al . , 2012 ) , floral odors have the advantage of attracting both sexes of mosquitoes in proportional numbers , and females in all gonotrophic states and in reproductive diapause . given that c. pipiens visits a variety of flowers for nectar - feeding , the three compounds identified here are likely not the only floral components attractive to them , and more effective blends may remain to be discovered .", "future research should seek additional attractants and determine optimal blend release rates , delivery systems , and trap designs for maximizing capture in the field ." ]

[ "<S> a pentane extract of flowers of common milkweed , asclepias syriaca ( asclepiadaceae ) , elicited significant orientation from both male and female culex pipiens in a dual - port flight olfactometer . </S>", "<S> analysis of the extract by gas chromatography </S>", "<S> mass spectrometry revealed six major constituents in order of relative abundance : benzaldehyde , ( e)--ocimene , phenylacetaldehyde , benzyl alcohol , nonanal , and ( e)-2-nonenal . </S>", "<S> although not all were collected from the headspace profile of live flowers , a synthetic blend of these six compounds , when presented to mosquitoes in the same levels and proportions that occur in the extract , elicited a response comparable to the extract . </S>", "<S> subtractive behavioral bioassays demonstrated that a three - component blend consisting of benzaldehyde , phenylacetaldehyde , and ( e)-2-nonenal was as attractive as the full blend . </S>", "<S> these findings suggest the potential use of synthetic floral - odor blends for monitoring or control of both male and female disease - vectoring mosquitoes . </S>" ]

[ "Introduction", "Methods and Materials", "Results", "Discussion" ]

[ [ "the northern house mosquito , culex pipiens l. ( diptera : culicidae ) , is a member of a species complex that includes vectors of lymphatic filariasis and several arboviral diseases , such as st .", "louis encephalitis ( sle ) , rift valley fever ( rfv ) , and west nile virus ( wnv ) . in the northeastern quadrant of the united states , c. pipiens recently has become important because of its role in the transmission of wnv .", "the virus was introduced into new york city in 1999 and is currently the most widespread arboviral disease in the u.s .", "( cdc , 2010 ) . the blood - feeding pattern of c. pipiens ( shifting from avian to human feeding within the transmission season ) ( edman and taylor , 1968 ; kilpatrick et al . , 2006 ) , coupled with its high vector competence ( turell et al . , 2005 ) and", "ability to carry wnv through the winter , has allowed the virus to spread from its point of origin and maintain transmission throughout the mosquito s range .", "thus , c. pipiens is one of the major targets for mosquito vector control and surveillance in the u.s .", "current methods of monitoring by abatement districts and health departments include centers for disease control ( cdc ) light traps and co2-baited traps , which mainly trap blood - seeking females , along with gravid traps , which target gravid females .", "plant - derived attractants have the potential to act as surveillance - trap lures and , when combined with poison - laced sugar solutions ( mller et al . ,", "mosquitoes are attracted to various plant species , from which they obtain nectar and other juices .", "sugar feeding by mosquitoes has a significant influence on dispersal ( hocking , 1953 ; magnarelli , 1977 ) and vectorial capacity ( gary and foster , 2001 ; gu et al . , 2011 ) , and , contrary to long - held conjecture , is required by both males and females throughout the adult stage ( downes , 1958 ; yuval , 1992 ; foster , 1995 ) .", "both sexes typically first visit plants soon after emergence . males then require sugar at frequent intervals to maintain their energy reserves in order to join nightly mating swarms ( yuval et al . , 1992 ) .", "females take sugar between blood meals , when they are digesting blood , or when they are gravid ( clements , 1999 ; foster , 2008 ) .", "furthermore , females of most temperate - climate culex and anopheles species enter reproductive diapause in late summer , after which they no longer are attracted to blood hosts , but engage heavily in plant - sugar feeding ( bowen , 1992a ) .", "the potential for plant volatiles to lure mosquitoes has been demonstrated in several laboratory behavioral assays that have used stimuli such as natural plant extracts ( vargo and foster , 1982 ; jepson and healy , 1988 ; mauer and rowley , 1999 ) or single floral compounds ( jhumur et al . , 2006 ) .", "in field studies , bates ( 1949 ) reported the successful trapping of anopheline mosquitoes with plant and fruit baits ; fruit also proved to be an effective attractant in cdc traps for c. tarsalis ( reisen et al . , 1986 ) .", "sandholm and price ( 1962 ) observed that mosquitoes in the field were attracted to light - colored flowers with distinct fragrances ; odor appears to be primarily responsible for long - range attraction , with visual cues playing a role at shorter range ( thorsteinson and brust , 1962 ; healy and jepson , 1988 ; jepson and healy , 1988 ) . orientation to commercially obtained floral extracts and honey has been demonstrated for various mosquito species ( thorsteinson and brust , 1962 ; hancock and foster , 1997 ; foster and takken , 2004 ) . schlein and mller ( 2008 ) and mller et al .", "( 2008 , 2010 ) reported dramatic population reductions of c. pipiens and other mosquito species by spraying a fruit - based sugar bait containing insecticide on vegetation surrounding larval habitats .", "light - less cdc traps baited with the blossoms of tamarix jordanis were highly effective in trapping c. pipiens , and populations were reduced where these blossoms were treated with spinosad insecticide ( schlein and mller , 2008 ) .", "flower species that elicit the highest attraction probably vary by region . during mid - summer in the u.s .", ", mosquitoes have been observed probing blossoms of common milkweed , asclepias syriaca , at rates disproportionate to their abundance relative to other flowering plants , with one study reporting 54 species occurring in the study area that did not serve as nectar sources ( grimstad and defoliart , 1974 ) .", "another study collected 25 aedes vexans from one milkweed cluster in just 15 min ( sandholm and price , 1962 ) .", "the observed preference of insects for milkweed flowers , both day and night , possibly is due to its strong and distinctive fragrance , lighter - colored flowers , and greater nectar production ( sandholm and price , 1962 ) .", "common milkweed is indigenous to eastern and midwestern north america , where its principal native pollinators are mainly large hymenoptera and lepidoptera ( e.g. , jennersten and morse , 1991 ) .", "mosquitoes are incapable of transferring milkweed pollinia and therefore function as nectar thieves , appearing to co - opt the attractive properties used by the pollinators to locate the flowers ( foster , 1995 ) .", "solvent extracts of common milkweed flowers also are attractive to mosquitoes ( vargo and foster , 1982 ; mauer and rowley , 1999 ) , confirming that volatile chemicals are at least partly responsible for that attraction .", "mauer and rowley ( 1999 ) determined the headspace profile of common milkweed flower to be predominantly 2-phenylethanol and benzyl alcohol ; however , they found that a synthetic blend of these two compounds was not attractive to c. pipiens . due to the multiple demonstrations of c. pipiens attraction to milkweed , both in the laboratory and the field , we used a solvent extract of the flower as a potential model for synthetic mosquito lures ." ], [ "experiments were conducted with c. pipiens from a colony established in 2009 from larvae collected near columbus , oh , usa . larvae were identified at l4 by siphonal hair tufts ( vinogradova , 2000 ) .", "adults were maintained in 41-l clear acrylic cages on a diet of 10 % sucrose , water , and weekly blood meals from the legs of a rooster ( ilacuc permit no .", "oviposition water was prepared by soaking grass clippings in aged tap water and allowing fermentation over a 3-d period .", "three days after each blood meal , oviposition cups were placed with caged adults , and eggs were collected the following day .", "two hundred first - instar larvae were placed into 22.8 33.0 cm aluminum pans with 450 ml of aged tap water .", "the larvae were provided finely ground tetramin flakes , increasing the quantity daily from 50 mg for first instars to 500 mg for final instars until pupae appeared on the 8th and 9th day post - hatching .", "pupae then were counted and transferred to plastic cups and placed in a 41-l cage supplied with water wicks .", "emerging adults were given ad libitum access to water , but were deprived of sugar .", "the mosquito rearing and maintenance conditions were 27 1 c , 85 5 % rh , and 16:8 ( l : d ) , with 30-min gradual crepuscular transitions between photophase and scotophase .", "phenylacetaldehyde ( > 90 % ) , benzaldehyde ( 99.5 % , purified by redistillation ) , nonanal ( 95 % ) , ( e)-2-nonenal ( 97 % ) , and an alkane - standards mixture ( c8 - c20 ) were purchased from sigma - aldrich ( st . louis , mo , usa ) .", "( phillipsburg , nj , usa ) , and -ocimene [ ( 75 % ( e)--ocimene ) ] was synthesized by chemos gmbh ( regenstauf , germany ) .", "synthetics were diluted with hplc - grade n - pentane ( fisher scientific , pittsburgh , pa , usa ) .", "flowers of common milkweed , a. syriaca , were collected in early summer from the ohio state university campus in columbus ( 400018.95 \" n , 830247.11 \" w ) , placed in an ice cooler , and transported 10-min to the laboratory .", "single florets were plucked from the main milkweed umbel and separated from the calyx and other green parts of the flower , weighed , and placed into a 500 ml narrow - mouth glass erlenmeyer flask .", "milkweed florets were submerged in hplc - grade n - pentane ( fisher scientific ) in a 1:8 ratio ( w / v ) for a total solvent volume of 480 ml and held for 24 h at room temperature , at which time the extract was decanted into 21-ml borosilicate glass vials , capped with teflon - lined screw caps and stored at -20 c .", "the volatile profile of milkweed flower was characterized by placing a single floret into a 21-ml borosilicate glass vial with a teflon - lined rubber septum .", "volatiles were allowed to equilibrate in the vial headspace at 30 c for 10 min before collections were made .", "volatiles were collected by using a divinylbenzene / carboxen / polydimethylsiloxane ( dvb / car / pdms ) ( 50/30 m ) solid phase micro - extraction ( spme ) fiber ( supelco , bellefonte , pa , usa ) .", "this mixed - chemistry fiber provides affinity for chemicals with a far broader range of polarity and volatility than pdms alone .", "the fiber was introduced to the vial through the septum and exposed for 30 min .", "the collected volatiles were analyzed immediately by using an agilent technologies 6890 series gas chromatograph interfaced to an agilent technologies 5973 quadrupole mass selective detector ( gc - ms ) .", "the spme fiber was desorbed at 275 c for 3 min with a splitless injection onto a zebron zb-1 ms column ( 30 m 0.25 mm , 0.25 m film thickness ) ( phenomenex , torrance , ca , usa ) .", "the oven temperature was held at 25 c for 3 min , then ramped at 15 c / min to 250 c , where it was held for 2 min .", "the carrier gas was helium at a flow rate of 1 ml / min .", "the mass selective detector was operated in ei mode at 70 ev , scanning 19 - 350 m / z with a quadrupole temperature of 180 c and source temperature of 240 c . to guide the construction of a synthetic floral blend , pentane extracts of milkweed florets", "were analyzed by gc - ms on a zebron zb-50 column ( 15 m 0.25 mm , 0.25 m film thickness ) ( phenomenex ) , with a 1-l splitless injection at 210 c .", "the temperature program began at 25 c for 3 min and increased at 13 c / min to 200 c , followed by a 25 c / min increase to 280 c to remove high - boiling lipids .", "peak identities were validated by re - injection on a finnegan trace gc / ms ( thermo fisher scientific inc . , waltham , ma , usa ) , with a 1:10 split at 180 c and a rtx-5ms column ( 30 m 0.25 mm , 0.25 m film thickness ) ( restek , bellefonte , pa , usa ) .", "the helium flow was 1 ml / min , and temperature program was 40 c for 1 min , ramped at 15 c / min to 275 c , with a 2-min final hold time .", "retention indices ( ri ) of compounds were determined on both columns relative to n - alkanes ( c8c20 ) .", "components of floral headspace and extract were identified by matching their mass spectra to nist / epa / nih mass spectral library 2005 and comparison of retention indices to published values ( adams , 1995 ) .", "identity of major components was confirmed by comparison of spectra and retention times to authentic standards .", "peak purity was determined by using amdis deconvolution software ( nist , gaithersburg , md , usa ) to assure the absence of any co - eluting components .", "a synthetic mimic of the extract was constructed by blending authentic standards of the major components identified in the extract .", "quantities of each component were individually adjusted until the overall concentration and component proportions closely approximated those of the floral extract ( fig . ", "1total ion chromatograms ( a ) of florets of milkweed , asclepias syriaca , pentane extract ( broken line ) , and a synthetic blend ( solid line ) used to assay upwind attraction of culex pipiens ( peak # 1 .", "phenylethanol ; column = zebron zb-50 ) and total ion chromatogram ( b ) of the headspace profile of a single a. syraica floret captured on a divinylbenzene / carboxen / polydimethylsiloxane spme fiber ( peak # 1 .", "( e , e)-alloocimene ; column = zebron zb-1 ) total ion chromatograms ( a ) of florets of milkweed , asclepias syriaca , pentane extract ( broken line ) , and a synthetic blend ( solid line ) used to assay upwind attraction of culex pipiens ( peak # 1 .", "phenylethanol ; column = zebron zb-50 ) and total ion chromatogram ( b ) of the headspace profile of a single a. syraica floret captured on a divinylbenzene / carboxen / polydimethylsiloxane spme fiber ( peak # 1 .", "( e , e)-alloocimene ; column = zebron zb-1 ) all behavioral assays were conducted in a clear acrylic dual - port flight olfactometer , modified from hancock and foster ( 1993 ) , with three main parts : an introduction / release chamber , flight chamber , and trapping ports . the introduction", "/ release chamber was located at the downwind end , measuring 30 40 cm wide and 30 cm long .", "a sliding gate separated the release chamber from the main flight chamber ( 30 40 cm wide , 90 cm long ) , which had two cylindrical glass jar trapping ports ( 15 cm long by 7 cm diameter ) on its upwind end .", "ports were fitted with borosilicate glass funnels , with the wide end opening into the flight chamber and the narrower ( 3 mm diam ) end pointing into a glass jar .", "thus , mosquitoes entering the wide end of the funnel from the flight chamber were channeled into the jar , where they were retained .", "trapping ports were located 11 cm above the flight chamber floor and were separated by 21 cm . the funnel and glass jar were held together by parafilm sealing film ( pechiney plastic company , menasha , wi , usa ) , which was replaced between treatments .", "we recognized the pitfalls associated with the use of parafilm in solvent - based behavioral assays ( millar and haynes , 1998 ) , and we ensured that this sealant would not come in contact with any chemicals in the olfactometer .", "a 7.5-mm diam hole in the upwind end of each jar allowed the introduction of purified / humidified air after it had passed from an oil - free air pump through an activated carbon canister , and then through a water column .", "air flow was maintained at 50 ml / s into each port , providing a velocity of 72 mm / s in the center of the flight chamber . a black cotton cloth , covering a dampened layer of cotton wool , covered the entire floor of the flight chamber , to maintain a level of humidity similar to that within the choice ports .", "data loggers ( hobo , onset computer corporation , bourne , ma , usa ) recorded temperature and humidity in the olfactometer , which was 25.0 - 27.5 c and 75 - 95 % rh . an exhaust duct , connected at the downwind end of the release chamber , directed the effluent air out of the building through an exhaust hood . to test for positional bias of the ports , mosquitoes were released into the chamber after either baiting both ports with honey or leaving both empty .", "mosquitoes were divided equally between left and right ports when baited with honey and were found rarely in ports when both were empty .", "response to whole milkweed flowers was determined in the flight olfactometer with 2 g of florets placed in an aluminum weighing boat in one of the trapping jars , and compared with an empty control jar .", "extracts and synthetic chemicals were applied to 15-cm long cotton wicks ( tidi products , neenah , wi , usa ) and compared to a pentane control .", "the positions of the treatment and control ports were alternated between bioassays as a further safeguard against positional bias .", "the olfactometer parts were cleaned with 70 % ethanol followed by water after each experiment ; gloves were used at all times to avoid contamination with human - related kairomones .", "the 16:8 ( l : d ) light cycle used for mosquito rearing was maintained in the bioassay room . at 2 h", "prior to scotophase , approximately 200 - 300 mosquitoes of both sexes in similar numbers were released through a sleeve connection , directly from an acrylic plastic cage ( 30 30 30 cm ) into the holding / release chamber , where they were held for 15 min to acclimatize before the release gate was opened .", "after 12 h , the numbers of mosquitoes in the treatment port , the control port , and remaining in the flight chamber were recorded .", "use of a 12-h period captured the bimodal nocturnal - crepuscular sugar - feeding rhythm of c. pipiens , which includes some sugar feeding throughout the night ( yee and foster , 1992 ) .", "subtractive bioassays were conducted on the synthetic blend to determine the relative activity of the identified components .", "the full six - component blend was presented in one of the paired olfactometer ports alongside a reducted five - compound blend , in which one of the components was removed .", "after establishing the significance of each chemical on mosquito attraction , we compared the minimal attractive blend against the full six - component blend .", "this was followed by a dose response study of the minimal blend to determine the optimal concentration for response .", "doses were presented in a randomized block design ( n = 5 ) , with percentage response calculated as the number in the synthetic blend port minus the number in the control port and expressed relative to the number of mosquitoes released .", "mosquito flight behavior was recorded by using three infrared rca closed - circuit video cameras ( lancaster , pa , usa ) onto which was mounted a tc1824b wide - angle es 25 mm 1:1.4 lens for flight - chamber recordings or a tc1874c es 75 mm 1:1.8 lens for each of the choice ports .", "( lancaster , pa , usa ) eight - channel switcher , with video output to two rca tc1109 video monitors .", "video output was recorded with an emerson ewv404 vcr ( parsippany , nj , usa ) onto vhs tape in time - lapse mode , 20 s each min .", "mosquito response in the flight olfactometer was analyzed by comparing numbers caught in the two ports by a goodness - of - fit chi - squared test with spss v. 17 ( spss inc , chicago , il , usa ) .", "response experiment , a regression model of mosquito response to log - transformed blend dose was determined by using the fitted line plot module of minitab v. 16 ( minitab inc ." ], [ "six compounds comprised > 90 % of the relative abundance of the milkweed floret components in the pentane extract : ( e)--ocimene , benzaldehyde , nonanal , benzyl alcohol , phenylacetaldehyde , and ( e)-2-nonenal ( fig . 1a , table 1 ) .", "the volatile profile collected by dvb / car / pdms spme directly from fresh florets was dominated by three of these compounds : ( e)--ocimene , benzaldehyde , and phenylacetaldehyde ( > 75 % of the total ) , but there were also substantial differences compared to the pentane extract ( fig . ", "most notably , ( e , z)- and ( e , e)-alloocimene were collected from the flower headspace , but were almost completely absent from the extract , whereas nonanal , a major constituent of floret extract , was missing in the headspace sample .", "these disparities can be explained partly by variation in volatility among the components , but also likely reflect differences between the chemical composition in flower tissue and what is released .", "the extract also contained higher - boiling hydrocarbons and fatty acids , but due to their low volatility , they were not included in behavioral studies . based on the quantities of synthetic standards", "used to mimic the extract gc profile ( table 1 ) , the total floral concentration of the six major volatiles was estimated at 32 g / g floret fresh weight.table 1major volatile constituents of a pentane extract of the common milkweed , asclepias syriaca , as determined by gas chromatography ", "mass spectrometry , and quantities used in a synthetic mimiccompoundrelative retention indexdiagnostic ei - ms fragment ions ( % intensity)synthetic blend ( g / ml)(e)--ocimene112477(36 ) , 79(42 ) , 91(53 ) , 93(100)6.14benzaldehyde115551(54 ) , 77(100 ) , 105(88 ) , 106(88)3.27nonanal122029(66 ) , 41(100 ) , 56(61 ) , 57(94)4.14benzyl alcohol123677(85 ) , 79(100 ) , 107(59 ) , 108(75)4.58phenylacetaldehyde125765(22 ) , 91(100 ) , 92(24 ) , 120(16)11.25(e)-2-nonenal130429(56 ) , 41(100 ) , 55(80 ) , 70(58)2.12identity established by comparison to authentic standard of each compoundgc retention times relative to n - alkane standards on a phenomenex zb-50 column phasesynthetic -ocimene contained a 3:1 ( e : z ) isomer mixture major volatile constituents of a pentane extract of the common milkweed , asclepias syriaca , as determined by gas chromatography ", "mass spectrometry , and quantities used in a synthetic mimic identity established by comparison to authentic standard of each compound gc retention times relative to n - alkane standards on a phenomenex zb-50 column phase synthetic -ocimene contained a 3:1 ( e : z ) isomer mixture infrared video recordings showed c. pipiens in the flight chamber engaged in a zigzagging flight that was directed upwind towards ports baited with florets , extracts , or synthetic blends .", "the angle of the turns progressively decreased as the mosquitoes approached the target . in most cases , mosquitoes flew into the ports , but in a few cases they landed on the entrance of the port and walked in .", "there was no observed oriented flight towards the control port . in the first behavioral assay , mosquitoes showed a significant response to whole milkweed florets , where 67 % of released mosquitoes were captured in the floret - baited port , compared to only 6 % in the control port ( fig . ", "2a ; \n = 515.02 df = 1 , p < 0.001 ) . despite the chemical differences measured between the headspace and extract profiles of milkweed florets , mosquitoes showed a similarly strong response to a pentane extract of milkweed florets ( 52 % ) compared to pentane alone ( 11 % ) ( fig . ", "2b ; = 61.44 , df = 1 , p < 0.001 ) .", "moreover , mosquitoes were observed in video recordings to probe the extract - treated cotton wick during the early scotophase and early photophase , suggesting that the extract also stimulated a feeding response .", "2percentage of culex pipiens flying upwind in a dual - port flight olfactometer to : a whole milkweed , asclepias syriaca , florets ; b pentane extract of a. syriaca florets ; and ( c ) synthetic a. syriaca floret blend compared against a water a or solvent ( b , c ) control . * * * p < 0.001 by chi - squared test percentage of culex pipiens flying upwind in a dual - port flight olfactometer to : a whole milkweed , asclepias syriaca , florets ; b pentane extract of a. syriaca florets ; and ( c ) synthetic a. syriaca floret blend compared against a water a or solvent ( b , c ) control . * * * p < 0.001 by chi - squared test based on the positive response to the milkweed floret extract , a six - component synthetic blend was formulated to simulate the concentrations and relative proportions of major constituents of the pentane extract ( fig . ", "one discrepancy in the gc profiles was caused by the presence of the ( z)-isomer in synthetic ( e)--ocimene , resulting in a synthetic blend that exceeded the levels of this isomer in the floret extract . in the olfactometer ,", "the synthetic floret blend performed similarly to the natural extract , with 48 % of released mosquitoes trapped , compared to 16 % for the control ( fig . 2c ; \n = 120.6 , df = 1 , p < 0.001 ) .", "again , video - recordings showed a directed upwind flight response to ports containing the synthetic blend , accompanied by vigorous probing of the treated wicks , but not to the controls .", "males and females responded to milkweed floret odors in similar numbers in all three experiments as the sex ratio of mosquitoes captured in either the treatment or control ports did not deviate significantly from 1:1 .", "the subtractive bioassay of the synthetic blend indicated a significant role for three compounds in c. pipiens response to milkweed : benzaldehyde ( \n = 13.22 , df = 1 , p < 0.001 ) , phenylacetaldehyde ( \n = 8.25 , df = 1 , p = 0.004 ) , and ( e)-2-nonenal ( \n = 3.62 , df = 1 , p = 0.05 ) ( fig . 3 ) .", "when any of these compounds was removed , mosquitoes showed a significant preference for the full blend over the reducted blend .", "a nearly significant preference was seen for blends missing either -ocimene ( p = 0.078 ) or benzyl alcohol ( p = 0.082 ) compared to the full blend .", "the activity of benzaldehyde , phenylacetaldehyde , and ( e)-2-nonenal was confirmed when the three were combined : the three - component blend captured 31 % of mosquitoes compared to only 7 % for the control ( fig . ", "4a ; \n = 196.56 , df = 1 , p < 0.001 ) and was as active as the full blend ( fig . 4b , 4 ; = 3.02 , df = 1 , p = 0.082).fig .", "3choice response of culex pipiens in a dual - port flight olfactometer to a six - component ( full ) synthetic blend of milkweed , asclepias syriaca , floret volatiles compared to blends in which each component has been removed individually ( reducted ) .", "total response indicates the percentage of c. pipiens released that were captured by one of the blends ( no significant differences by 1-way anova ) .", "relative response shows the proportional response to the reducted ( gray bars ) and full ( black bars ) blends .", "numbers in bars indicate the mean number of c. pipiens responding ( n = 46 ) .", "asterisks denote significant differences in mean response to reducted versus full blend by chi - squared test ( * p < 0.05 , * * p < 0.005)fig", ". 4a percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to a three - component blend , consisting of benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ; * * * p < 0.001 by chi - squared test .", "b percentage of c. pipiens flying upwind in response to the full ( six - component ) and minimal ( three - component ) synthetic blends of a. syriaca floral odor .", "ns = no significant difference choice response of culex pipiens in a dual - port flight olfactometer to a six - component ( full ) synthetic blend of milkweed , asclepias syriaca , floret volatiles compared to blends in which each component has been removed individually ( reducted ) .", "total response indicates the percentage of c. pipiens released that were captured by one of the blends ( no significant differences by 1-way anova ) .", "relative response shows the proportional response to the reducted ( gray bars ) and full ( black bars ) blends .", "numbers in bars indicate the mean number of c. pipiens responding ( n = 46 ) .", "asterisks denote significant differences in mean response to reducted versus full blend by chi - squared test ( * p < 0.05 , * * p < ", "0.005 ) a percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to a three - component blend , consisting of benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ; * * * p < 0.001 by chi - squared test .", "b percentage of c. pipiens flying upwind in response to the full ( six - component ) and minimal ( three - component ) synthetic blends of a. syriaca floral odor .", "ns = no significant difference because quantity as well as quality can determine the intensity of chemically mediated behavior , c. pipiens response was measured in the flight olfactometer to different doses of the three - component blend compared to a solvent control .", "there was a significant positive quadratic response [ log(dose ) ( y = 20.527x + 73.476x43.709 ; r = 57.6 % ] , with a response maximum predicted at 62 g ( fig . 5 ) .", "the response flattened out at both the lowest and highest doses ; removal of the two most extreme doses produced a stronger - fitting regression model ( r = 93.3 % ) , but with a similar predicted optimal dose of 65 g.fig .", "5percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to different concentrations of a three - component kairomone blend ( benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ) after subtracting the response to a paired solvent ( pentane ) control percentage of culex pipiens flying upwind in a dual - choice flight olfactometer in response to different concentrations of a three - component kairomone blend ( benzaldehyde , ( e)-2-nonenal , and phenylacetaldehyde ) after subtracting the response to a paired solvent ( pentane ) control" ], [ "a positive upwind orientation by c. pipiens to floret volatiles of a. syriaca was demonstrated in this study , suggesting a new potential source of attractants for use in the field as lures .", "males and females showed significant response to milkweed florets , a pentane extract , and a synthetic blend of the extract s six major constituents : benzaldehyde , ( e)--ocimene , benzyl alcohol , phenylacetaldehyde , nonanal , and ( e)-2-nonenal .", "the first three of these compounds are among the most common floral odor constituents in plants ( knudsen et al . , 2006 ) .", "subsequent subtractive bioassays of the synthetic blend demonstrated that only three of its components contributed to attraction : benzaldehyde , phenylacetaldehyde , and ( e)-2-nonenal . the attraction of mosquitoes to plant odors has been demonstrated in numerous field and laboratory studies , but few studies have shown significant attraction to synthetics .", "mauer and rowley ( 1999 ) observed orientation of c. pipiens to a methylene chloride extract of a. syriaca in a dual port olfactometer .", "however , a synthetic blend of the two dominant compounds in the extract headspace , benzyl alcohol and 2-phenylethanol , failed to attract the mosquitoes .", "we did not test 2-phenylethanol , but benzyl alcohol was not active in our flight olfactometer bioassays .", "( 2007 ) also found benzyl alcohol to not be very attractive to c. pipiens var .", "flowers of tanacetum vulgare also attract culex species in the field ( andersson and jaenson , 1987 ) , and bowen ( 1992b ) discovered a high proportion of both broadly- and narrowly - tuned antennal receptor neurons of c. pipiens sensitive to thujone , the primary constituent of t. vulgare fragrance", ". however , thujone only elicited a close - range dose - dependent probing response in c. pipiens ; it did not stimulate upwind flight in a wind - tunnel olfactometer ( bowen , 1992b ) .", "the only previous flower - based synthetic blend causing mosquito orientation was developed from silene otites , whose flowers attract mosquitoes in the field ( brantjes and leemans , 1976 ) .", "the synthetic blend mimic consisted of phenylacetaldehyde , veratrole , and 2-methoxyphenol , the first of which elicited the strongest attraction in c. pipiens var .", "phenylacetaldehyde also was a prominent component of our a. syriaca headspace and solvent extract , and it was essential for maximum attraction to the synthetic blend .", "although nonanal was a major component of a. syriaca floret extract , we saw no evidence that it played a role in attraction in our bioassays .", "in contrast , from single - cell recordings , syed and leal ( 2009 ) determined that ca .", "40 % of all antennal olfactory receptors of c. quinquefasciatus were acutely sensitive to nonanal , and it produced a synergistic effect on trap catch when presented with co2 .", "nonanal is a major skin odorant of birds and mammals , including humans , resulting from the oxidation of sebaceous fatty acids ( haze et al . , 2001 )", "this fact , along with the significant behavioral interaction with co2 , suggests that nonanal may play an important role in mediating culex host - finding , but does not attract sugar - seeking mosquitoes .", "we found notable differences between profiles of the pentane extract and the spme - collected headspace of a. syriaca florets .", "pentane extracts were dominated by phenylacetaldehyde , ( e)--ocimene , and nonanal , whereas the headspace profile contained primarily benzaldehyde , ocimene isomers , and phenylacetaldehyde .", "disparities between extracts and chemicals collected in the headspace might be explained by differences in the physico - chemical properties of the constituents , deep penetration by solvents to extract compounds that are not normally released by the tissue , and/or selective trapping of chemicals by spme . in this study ,", "benzaldehyde , -ocimene , and alloocimene have the highest vapor pressures of all the chemicals identified from milkweed , and they make up most of the volatile profile .", "the vapor pressure of benzaldehyde is almost twice that of nonanal , almost four times that of ( e)-2-nonenal , and more than six times that of benzyl alcohol .", "the latter three compounds were either absent or found in very low levels in the headspace analysis .", "phenylacetaldehyde is intermediate in its volatility , but was also the component found in the highest levels in the pentane extract .", "volatile collection analyses can produce misleading results , as they may not accurately reflect the actual proportionality of constituents in the headspace .", "we do not believe that this was a major factor for explaining the differences in the headspace and extract profiles of milkweed due to our choice of spme phase .", "although pdms has been the most widely used spme phase , it is actually a poor choice for characterizing plant volatile profiles that contain constituents with a range of volatilities and functional groups . in preliminary studies ( not shown ) , we found a broader array of volatile compounds was trapped from various flowers by dvb / car / pdms compared to pdms alone .", "this mixed - bed fiber not only employs a broader range of phase polarity , but also incorporates both adsorption and partitioning as mechanisms of collection ( koziel and novak , 2002 ) .", "a number of recent studies have demonstrated the higher recovery efficiency and linearity by dvb / car / pdms compared to other spme phases for all of the compound classes that we identified from milkweed ( cui et al . , 2009 ; ferreira et al . , 2009 ; zhang et al . , 2009 ) .", "2009 ) found that volatile profiles of longan fruit extracted by a 50/30 m dvb / car / pdms fiber were 3x higher in terpenes , 5x higher in alcohols , and 14x higher in esters than those produced by a 100 m pdms fiber , and revealed volatile carbonyls and acids , which were completely absent from pdms profiles .", "this study points to floral odors as a potential new model for chemical lures useful for mosquito sampling or control . if similar attraction can be demonstrated at the field level , as has already been demonstrated with human kairomone blends ( mukabana et al .", ", 2012 ) , synthetic floral blends could potentially be used in trapping devices to sample adult populations .", "relative to animal - derived odors ( mukabana et al . , 2012 ) , floral odors have the advantage of attracting both sexes of mosquitoes in proportional numbers , and females in all gonotrophic states and in reproductive diapause . given that c. pipiens visits a variety of flowers for nectar - feeding , the three compounds identified here are likely not the only floral components attractive to them , and more effective blends may remain to be discovered .", "future research should seek additional attractants and determine optimal blend release rates , delivery systems , and trap designs for maximizing capture in the field ." ] ]

[ "two male patients with type 2 diabetes ( t2d ) and coronary heart disease had plasma levels of brain natriuretic peptide ( bnp ) and n - terminal probrain natriuretic peptide ( nt - probnp ) measured as part of routine evaluation .", "their glomerular filtration rate was normal ( > 75 ml / min per 1.73 m ) , hba1c was < 7% with metformin , and c - reactive protein ( crp ) was < 1 mg / dl in both .", "patient a was 71 years old and had suffered an anterior myocardial infarction three years ago with st - segment elevation , which was initially treated with fibrinolytics and 24 h later using percutaneous coronary intervention with a drug eluting stent on the anterior coronary descending artery .", "he presented with a > 70% stenosis on a diagonal artery , which was not treated and showed no significant stenosis in the remaining coronary arteries .", "his bmi was 34.8 kg / m , waist circumference 116 cm and was stable without chest pain in nyha class i. the echocardiogram showed apical and lateral hypokinesia with left ventricular ejection fraction ( lvef ) of 58% .", "plasma bnp and nt - probnp were 72 and 100 pg / ml , respectively ( reference values for age and sex : bnp median 28 pg / ml ( percentile 2575th , 1058 pg / ml ) ; nt - probnp median 45 pg / ml ( percentile 595th , 14140 pg / ml ) ) .", "patient b , 58 years of age , had suffered a non - st - elevation myocardial infarction two years earlier , and had received standard treatment .", "his bmi was 44.3 kg / m and his waist circumference 137 cm .", "the echocardiogram showed mild hypokinesia of the basal segment of the inferior wall and mild dilation of left ventricle with eccentric hypertrophy .", "lvef was 66% and plasma bnp and nt - probnp were 10 and < 20 pg / ml , respectively ( reference values for age and sex : bnp median 31 pg / ml , percentile 2575th , 14 - -49 pg / ml ; nt - probnp median 25 pg / ml , percentile 595th , 588 pg / ml ) .", "these findings posed the question as to why it is that in some patients the plasma levels of cardiac natriuretic peptides ( cnp ) are unexpectedly low , and what is their value in the prognosis for these patients ?", "the cnp act as a basic functional link between cardiovascular system homeostasis , inflammation and certain metabolic functions ( fig .", "1 ) . increased secretion of cnp is a result of both mechanical and neurohumoral stimuli ( 1 , 2 ) . the main mechanical stimulus for the secretion of cnp is the increased pressure in the cardiac chambers leading to stretching of the myocardial fibers .", "neurohumoral stimuli include endothelin-1 , angiotensin ii , adrenergic agonists and various cytokines ( 4 ) . increased left ventricular end - diastolic wall stress and left ventricular end - systolic wall stress correlate with an increase in plasma cnp in heart failure ( 5 , 6 ) .", "however , studies on cardiac transplant patients have shown that plasma cnp levels remain high even after intra - cardiac pressures normalize following transplantation ( 7 ) . during an acute cardiac allograft rejection episode , bnp but not atrial natriuretic peptide ( anp ) ", "plasma levels increase significantly above pre - rejection values independently of the surgical technique used ( 8) .", "plasma bnp levels during acute rejection episodes do not correlate with hemodynamic variables but correlate with the levels of regulated on activation , normal t expressed and secreted , insulin growth factor binding protein-1 and neutrophil activating protein-2 ( 4 ) . in non - rejecting transplanted patients , despite the normalization of endothelin-1 plasma levels and diastolic and systolic functions , bnp remains high by comparison with control subjects ( 9 ) .", "measuring cnp is useful as a diagnostic and prognostic tool ( 10 , 11 , 12 ) .", "high plasma levels of cnp ( e.g. bnp > 63pg / ml or nt - probnp > 206 pg / ml ) can quite precisely predict which patients are at risk for events such as hospitalization due to systolic heart failure or cardiovascular death ( 13 , 14 , 15 , 16 , 17 , 18 ) .", "obese patients may have strikingly low levels of plasma cnp ( 19 , 20 , 21 , 22 ) due to multiple mechanisms that might include increased neprilysin - neutral endopeptidase activity resulting in increased degradation of circulating cnp and increased adipose tissue expression of the np clearance receptor c ( nprc ) .", "it has been proposed that suppression of probnp1 - 108 prohormone processing due to o - glycosylation at its cleavage site where furine or corin convertases act , prevents the formation of bnp77 - 108 ( functionally active ) and nt - probnp1 - 76 ( inactive ) fragments , an event that is purportedly more frequent in diabetics and in insulin - resistant states ( 23 , 24 , 25 ) . in heart failure patients ,", "the precursor pro - hormone probnp1 - 108 is found circulating at lower than normal levels ( 26 , 27 ) .", "recently , the paradigm - hf trial showed that angiotensin receptor - neprilysin inhibition using the compound lcz-696 , 200 mg twice daily , compared with enalapril at 10 mg twice daily significantly reduced the risk of cardiovascular death and hospitalization in patients with heart failure with an lvef 40% .", "however , death rates from cardiovascular causes were similar among diabetics compared with non - diabetics ( 28 ) .", "although this interesting finding could be an accidental one , other heart failure trials found that the benefit of this therapy in diabetics seems to be less than that in non - diabetics ( 29 , 30 , 31 ) .", "this finding merits further research , since diabetics comprise 2535% of the subjects in most studies ( 29 , 30 , 31 , 32 ) .", "the obese are differentiated from the metabolically healthy obese by an unhealthy metabolic profile ( 33 ) . the latter is defined by increased waist circumference ( > 94 cm in men and > 80 cm in women ) or bmi 30 kg / m accompanied by two or more of the following : hypertriglyceridemia ( 1.7 mmol / l ) , low hdl cholesterol ( < 1.03 mmol / l in men and < 1.29 mmol / l in women ) , hyperglycemia ( > 11.1 mmol / l or a diagnosis of diabetes ) , hypertension ( 130/85 mmhg ) or medication for high blood pressure ( 34 , 35 ) . in a 12-year follow - up of 61 299 subjects free from cardiovascular disease at baseline mrkedal et al . ( 36 ) observed that systolic heart failure development was similar among ", "metabolically healthy compared to non - metabolically healthy obese subjects , meaning that obesity per se creates a higher risk for developing systolic heart failure , especially if it is long - lasting and severe ( bmi > 40 kg / m ) .", "recently , it was reported that subjects with high plasma levels of nt - probnp are at a greater risk of developing systolic heart failure whether they are obese or non - obese . however , unlike the non - obese who show a direct linear relationship between levels of nt - probnp and systolic heart failure , the obese show a u - shaped relationship ; i.e. those who have the lowest plasma levels are at just as much risk of developing systolic heart failure as those with the highest plasma levels ( 37 ) .", "in contrast , the pathological weight loss caused by anorexia nervosa seems to be related to high plasma levels of anp ( 38 ) . in severe obesity ,", "bariatric surgery reduces the bmi and improves control of diabetes but it is not yet known if it reduces the risk of cardiovascular events ( 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ) . in patients with severe obesity ( bmi > 40 kg / m ) before gastric bypass surgery , the median level of nt - probnp was 54 pg / ml increasing by 125% after 1 year with no apparent relation to weight loss or glucometabolic parameters .", "it was speculated that an improvement in secretory cardiac function following surgery was responsible for the increase in circulating nt - probnp ( 46 ) .", "myocardial ischemia and cardiomyocyte stretch trigger the immediate release of cnp ( 47 , 48 ) even when the lvef is normal ( 49 , 50 ) . in a 9.2 year follow - up study of diabetic ( about 7% of the cohort ) and non - diabetic patients with chronic stable myocardial ischemia , high plasma levels of nt - probnp were significantly associated with mortality regardless of left ventricular function ( 51 ) .", "recently , it was shown that in patients with stable coronary heart disease bnp and nt - probnp were strong predictors of long - term ( 6.53.3 years ) cardiovascular events and notably , when nt - probnp was added to the clinical predictors , it performed better than bnp in risk classification for adverse cardiovascular events ( 52 ) . serial measurement data for nt - probnp in patients with chronic stable angina have shown a wide intra - individual variation .", "an increase by > 42% or a decrease by > 30% relative to baseline values is required to indicate a significant change ( 53 , 54 ) . in patients presenting with acute chest pain ,", "cnp plasma levels help the diagnosis and prognosis when used together with a non - diagnostic ecg and a negative troponin .", "cnp measured in these patients at rest predict future cardiac events at 30 and 180 days and at 1 year ( 55 , 56 , 57 , 58 ) .", "if myocardial ischemia is evident during the stress test , an elevation of the plasma levels of nt - probnp detects ischemia and predicts cardiovascular events ( 59 ) .", "likewise , in non - st - elevation acute coronary syndromes , cnp add prognostic information to the clinical indicators ( 60 , 61 , 62 , 63 ) ( table 1 ) . in a recent investigation ( 62 ) , bnp was shown to be an independent predictor of mortality on entering the study . at 1 year of", "follow - up bnp plasma levels provided added value over the timi and grace scores .", "cnp are stronger predictors of mortality than troponin in patients that will eventually develop myocardial infarction . in st - segment - elevation", "acute coronary syndromes cnp plasma levels also add prognostic information independently of the lvef ( 50 , 64 , 65 ) and in addition , predict which patients with primary percutaneous coronary intervention ( pci ) may be discharged earlier thus saving health resources ( 66 ) .", "it has been shown experimentally that anp is associated with salt - sensitive high blood pressure ( 67 ) . in humans", "alleles associated with the highest levels of np were also associated with a lower risk of high blood pressure ( 68 , 69 ) . in keeping with these findings", ", another study showed a relative deficiency of cnp at all the different stages of high blood pressure in humans consisting of low levels of probnp1 - 108 and anp99 - 126 .", "low plasma levels of bnp76 - 108 and nt - probnp1 - 76 were observed in the first hypertensive stages together with a reduction of nt - anp1 - 98 in stage i ( 70 ) .", "a possible therapeutic modality to supplement cnp in those cases in which circulating cnp are deemed deficient ( like in hypertension or heart failure ) is suggested by the development of a recombinant human serum albumin - atrial natriuretic factor ( anf ) ( 71 ) molecule .", "this compound , when injected into mice increased circulating cgmp and decreased blood pressure demonstrated a half - life of > 80 min , which is considerably longer than the 510 min half - life for native anf ( 71 , 72 , 73 ) . from the preceding , it may be surmised that elevated cnp levels are strong markers of future cardiovascular events , especially cardiovascular death . on the other hand , cnp plasma levels lower than expected in obese and diabetic patients", "the two patients discussed above had similar metabolic profiles , lvef > 50% but a different bmi and cnp profile .", "patient a was not severely obese and had slight residual myocardial ischemia with bnp and nt - probnp within low to moderate values expected for systolic heart failure and cardiovascular mortality on follow - up .", "patient b suffered from severe obesity , with very low plasma levels of cnp with a prognosis related to his bmi of > 40 ", "kg / m and probably also to a deficit of cnp , placing him in the high - risk category for developing systolic heart failure and eventual cardiovascular death .", "our patient a has markedly higher bnp and nt - probnp levels compared to patient b. this can largely be explained by the better ejection fraction of patient b , but there might also be other contributing factors .", "for example , recent studies suggest an inverse relationship between circulating cnp levels and bodyweight as well as with insulin resistance and t2d ( 19 , 74 , 75 , 76 , 77 , 78 ) ( table 1 ) . this correlation can also be observed in patients with congestive heart failure , despite increased cnp levels , due to cardiac wall stress ( 79 ) .", "cnp are cleared and degraded by neutral endopeptidase neprilysin and natriuretic peptide receptor c ( nprc ) ( 80 , 81 ) .", "natriuretic peptide receptor a ( npra ) and nprc have been identified in human adipose tissue in abundance , implying that adipose tissue sustains a regulatory function on the np system ( 82 , 83 , 84 ) .", "interestingly , compared to non - obese and normotensive individuals , nprc is increased in adipose tissue of obese hypertensive patients ( 85 ) .", "insulin has been observed to induce nprc expression in human adipocytes ( 84 ) and monocytes ( 83 ) , and might , hence , link conditions associated with hyperinsulinemia ( e.g. obesity and insulin resistance ) , to a relative np deficit", ". additionally , neprilysin , the np degrading endopeptidase , is expressed at increased levels in obesity ( 86 ) .", "these data argue for obesity and insulin resistance being conditions in which cnp are degraded at an accelerated pace .", "second , very recent experimental data also suggest that myocardial bnp expression is markedly decreased in mice fed a high fat diet ( 87 ) , an observation that warrants clinical confirmation .", "a cnp deficit in patients with components of the metabolic syndrome might be of clinical relevance .", "obese individuals have a higher prevalence of arterial hypertension compared to lean subjects ( 88 ) .", "although obesity - related arterial hypertension has been intensively studied , not all mechanisms are well understood ( 89 , 90 ) . by reduced vasodilatory and sodium - excretion activity , as well as", "aldosterone - system ( raas ) , a cnp deficit might contribute to obesity related hypertension .", "interestingly , in lean subjects , application of a sodium load induces myocardial cnp secretion and stimulates natriuresis ( 91 , 92 , 93 , 94 ) , a response which is blunted in patients with obesity ( 95 ) .", "together , these data argue that obesity promotes hypertension partly through reduced vascular and renal np responses as well as through impaired np - mediated raas inhibition .", "second , cnp also have beneficial actions on cardiac remodelling in essential hypertension , reducing left ventricular hypertrophy ( 96 ) .", "conversely , conditions with a cnp deficit are associated with cardiac hypertrophy in hypertensive patients . in this", "regard , hypertensive patients with the metabolic syndrome present with lower anp and nt - probnp levels and increased left ventricular mass compared to hypertensive patients without the metabolic syndrome and insulin resistance ( 97 ) .", "finally , the cnp deficit in patients with the metabolic syndrome might be part of a vicious circle which maintains metabolic disease .", "for instance , cnp exert lipolytic properties mediated by a cgmp - dependent protein kinase g activating pathway .", "cgmp - activated protein kinase g ( gk - i ) activates perilipin a and hormone sensitive lipase mediated triglyceride hydrolysis ( 98 , 99 , 100 , 101 ) . interestingly , these effects seem not to interact with the lipolytic effects of catecholamines ( 99 , 102 ) and the effect seems to be independent of the regulation of insulin ( 103 ) . besides a direct activating effect on lipolysis and lipid oxidation , cnp also control secretion of the insulin sensitizing adipokine adiponectin . in humans , anp acutely increases systemic levels of total and high molecular weight adiponectin ( 104 ) .", "these findings are in line with some observational studies showing positive associations between systemic cnp and adiponectin concentrations , as for example in heart failure patients ( 105 , 106 ) .", "this finding might also explain the adiponectin paradox in patients with congestive heart failure .", "cnp also exert other relevant metabolic actions on adipose tissue . treating a human derived adipose cell line with anp results in the uncoupling of cellular respiration ( 107 ) as well as ", "the response seems to be mediated by p38 map kinase , which increases uncoupling protein 1 ( ucp1 ) transcription ( 107 ) . upon cold exposure , systemic np concentrations increase and nprc expression in", "similarly , forced pkg expression in primary adipocytes leads to an increase in ucp1 expression and activates a thermogenic program .", "administration of anp acutely increases lipid oxidation ( 102 , 109 ) and postprandial energy expenditure in healthy individuals ( 110 ) . circulating -hydroxybutyrate increases , indicating enhanced hepatic lipid oxidation .", "apart from acute effects on lipid oxidation ( 110 ) , anp and bnp induce skeletal muscle mitochondrial biogenesis , respiration and lipid oxidation in human cells and in rodents , in vitro and in vivo \n ( 111 , 112 ) .", "chronic overexpression of bnp and gk - i each led to increased muscle mitochondrial content , oxidative capacity and lipid oxidation in rodents ( 111 ) .", "enhanced oxidative metabolism is associated with protection from diet induced obesity and insulin resistance ( 113 ) .", "heterozygous npra knockout mice are prone to gaining weight and become insulin resistant ( 111 ) .", "the mechanism linking np signalling to mitochondrial biogenesis and lipid oxidation in skeletal muscle includes activation of the co - transcriptional activator peroxisome proliferator activated receptor coactivator ( pgc)-1 and peroxisome proliferator activated receptor- , both of which are important factors of mitochondrial biogenesis in skeletal muscle ( 111 ) . in human myotubes , we observed that cnp stimulate pgc-1 , maximal mitochondrial respiratory capacity and lipid oxidation ( 112 ) .", "moreover , npra expression positively correlates with pgc-1 expression in the skeletal muscle of individuals after a chronic physical training program .", "similarly , cell culture studies show that cgmp restores glucose and insulin induced mitochondrial dysfunction in cultured c2c12 myotubes ( 114 ) , nitric oxide signalling via cgmp mediates activation of pgc-1 , and mitochondrial biogenesis in various murine tissues ( 115 ) .", "hepatic and skeletal muscle lipid content has been associated with insulin resistance ( 113 ) . in liver and skeletal muscle ,", "insulin resistance develops when bioactive lipid species accumulate , such as intracellular diacylglycerol ( 113 ) . in obesity and the metabolic syndrome , this lipid accumulation is primarily due to excessive caloric intake that exceeds the capacity of hepatocytes and myocytes to metabolize or export fatty acids , while refining mitochondrial respiration and enhancing lipid oxidation have been proved to improve lipid utilization and insulin sensitivity ( 116 , 117 , 118 ) . so far , there is no evidence that cnp directly interact with the insulin signalling cascade ( 109 , 119 ) .", "it is tempting to speculate that cnp could ameliorate lipid - induced insulin resistance through improvements in hepatic ( 110 ) and muscular ( 112 ) lipid oxidation .", "in line with this notion , cnp preserves mitochondrial function and insulin sensitivity in high fat diet mice ( 111 ) . cross sectional studies support the hypothesis that cnp protect from the development of obesity and t2d in patients ( 78 , 120 , 121,122 ) .", "conversely , data from the framingham heart study and the malm diet and cancer study show that reduced cnp concentrations correlate with a higher probability of insulin resistance in lean and obese individuals ( 76 ) .", "moreover , in the latter study low cnp concentrations are predictive of new - onset diabetes ( 123 ) .", "together , these data suggest that a cnp deficit , as observed in obese patients with the metabolic syndrome , contributes and aggravates metabolic vascular disease , and thus , prognosis .", "life style interventions as well as pharmacological approaches might be of benefit in this regard ( 28 , 124 ) .", "while physical activity increases cnp levels acutely and augments npra expression in skeletal muscle cells ( 112 ) , physical activity coupled with a low calorie diet seems to increase cnp levels chronically ( 125 ) ; an observation that seems to depend on the amount of weight loss ( 126 , 127 ) .", "experimental data further suggests that the incretin glucagon like peptide 1 ( glp1 ) might be of relevance in the regulation of np secretion .", "the glp1 receptor agonist liraglutide , which is widely used for the treatment of t2d , has been shown to induce cardiac anp release in mice , leading to enhanced natriuresis and vasodilatation ( 128 ) without inducing congestive heart failure .", "so far 25 clinical studies failed to demonstrate a similar effect of liraglutide when administered acutely ( 129 ) or sub - chronically to patients ( 130 ) .", "chronic liraglutide treatment , accompanied by weight loss , however , seems to increase circulating anp as well as bnp concentrations in diabetic patients ( 131 ) .", "clearly , future studies need to address the important question how a cnp deficit in obesity and t2d can be reversed most efficiently ( 132 ) .", "the authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review .", "this work was supported by a grant from the secretara de ciencia y tecnologa de la universidad nacional de crdoba ( grant number 69/08 ) to h r ramos , a grant from the german research foundation ( grant number bi1292/4 - 2 ) to a l birkenfeld and by funds from the university of ottawa to a j de bold ." ]

[ "<S> since their discovery in 1981 , the cardiac natriuretic peptides ( cnp ) atrial natriuretic peptide ( also referred to as atrial natriuretic factor ) and brain natriuretic peptide have been well characterised in terms of their renal and cardiovascular actions . </S>", "<S> in addition , it has been shown that cnp plasma levels are strong predictors of cardiovascular events and mortality in populations with no apparent heart disease as well as in patients with established cardiac pathology . </S>", "<S> cnp secretion from the heart is increased by humoral and mechanical stimuli . </S>", "<S> the clinical significance of cnp plasma levels has been shown to differ in obese and non - obese subjects . </S>", "<S> recent lines of evidence suggest important metabolic effects of the cnp system , which has been shown to activate lipolysis , enhance lipid oxidation and mitochondrial respiration . </S>", "<S> clinically , these properties lead to browning of white adipose tissue and to increased muscular oxidative capacity . in human association studies in patients without heart disease </S>", "<S> higher cnp concentrations were observed in lean , insulin - sensitive subjects . </S>", "<S> highly elevated cnp levels are generally observed in patients with systolic heart failure or high blood pressure , while obese and type-2 diabetics display reduced cnp levels . </S>", "<S> together , these observations suggest that the cnp system plays a role in the pathophysiology of metabolic vascular disease . understanding </S>", "<S> this role should help define novel principles in the treatment of cardiometabolic disease . </S>" ]

[ "Introduction", "The cardiologist's perspective", "The endocrinologist's perspective", "Declaration of interest", "Funding" ]

[ [ "two male patients with type 2 diabetes ( t2d ) and coronary heart disease had plasma levels of brain natriuretic peptide ( bnp ) and n - terminal probrain natriuretic peptide ( nt - probnp ) measured as part of routine evaluation .", "their glomerular filtration rate was normal ( > 75 ml / min per 1.73 m ) , hba1c was < 7% with metformin , and c - reactive protein ( crp ) was < 1 mg / dl in both .", "patient a was 71 years old and had suffered an anterior myocardial infarction three years ago with st - segment elevation , which was initially treated with fibrinolytics and 24 h later using percutaneous coronary intervention with a drug eluting stent on the anterior coronary descending artery .", "he presented with a > 70% stenosis on a diagonal artery , which was not treated and showed no significant stenosis in the remaining coronary arteries .", "his bmi was 34.8 kg / m , waist circumference 116 cm and was stable without chest pain in nyha class i. the echocardiogram showed apical and lateral hypokinesia with left ventricular ejection fraction ( lvef ) of 58% .", "plasma bnp and nt - probnp were 72 and 100 pg / ml , respectively ( reference values for age and sex : bnp median 28 pg / ml ( percentile 2575th , 1058 pg / ml ) ; nt - probnp median 45 pg / ml ( percentile 595th , 14140 pg / ml ) ) .", "patient b , 58 years of age , had suffered a non - st - elevation myocardial infarction two years earlier , and had received standard treatment .", "his bmi was 44.3 kg / m and his waist circumference 137 cm .", "the echocardiogram showed mild hypokinesia of the basal segment of the inferior wall and mild dilation of left ventricle with eccentric hypertrophy .", "lvef was 66% and plasma bnp and nt - probnp were 10 and < 20 pg / ml , respectively ( reference values for age and sex : bnp median 31 pg / ml , percentile 2575th , 14 - -49 pg / ml ; nt - probnp median 25 pg / ml , percentile 595th , 588 pg / ml ) .", "these findings posed the question as to why it is that in some patients the plasma levels of cardiac natriuretic peptides ( cnp ) are unexpectedly low , and what is their value in the prognosis for these patients ?" ], [ "the cnp act as a basic functional link between cardiovascular system homeostasis , inflammation and certain metabolic functions ( fig .", "1 ) . increased secretion of cnp is a result of both mechanical and neurohumoral stimuli ( 1 , 2 ) . the main mechanical stimulus for the secretion of cnp is the increased pressure in the cardiac chambers leading to stretching of the myocardial fibers .", "neurohumoral stimuli include endothelin-1 , angiotensin ii , adrenergic agonists and various cytokines ( 4 ) . increased left ventricular end - diastolic wall stress and left ventricular end - systolic wall stress correlate with an increase in plasma cnp in heart failure ( 5 , 6 ) .", "however , studies on cardiac transplant patients have shown that plasma cnp levels remain high even after intra - cardiac pressures normalize following transplantation ( 7 ) . during an acute cardiac allograft rejection episode , bnp but not atrial natriuretic peptide ( anp ) ", "plasma levels increase significantly above pre - rejection values independently of the surgical technique used ( 8) .", "plasma bnp levels during acute rejection episodes do not correlate with hemodynamic variables but correlate with the levels of regulated on activation , normal t expressed and secreted , insulin growth factor binding protein-1 and neutrophil activating protein-2 ( 4 ) . in non - rejecting transplanted patients , despite the normalization of endothelin-1 plasma levels and diastolic and systolic functions , bnp remains high by comparison with control subjects ( 9 ) .", "measuring cnp is useful as a diagnostic and prognostic tool ( 10 , 11 , 12 ) .", "high plasma levels of cnp ( e.g. bnp > 63pg / ml or nt - probnp > 206 pg / ml ) can quite precisely predict which patients are at risk for events such as hospitalization due to systolic heart failure or cardiovascular death ( 13 , 14 , 15 , 16 , 17 , 18 ) .", "obese patients may have strikingly low levels of plasma cnp ( 19 , 20 , 21 , 22 ) due to multiple mechanisms that might include increased neprilysin - neutral endopeptidase activity resulting in increased degradation of circulating cnp and increased adipose tissue expression of the np clearance receptor c ( nprc ) .", "it has been proposed that suppression of probnp1 - 108 prohormone processing due to o - glycosylation at its cleavage site where furine or corin convertases act , prevents the formation of bnp77 - 108 ( functionally active ) and nt - probnp1 - 76 ( inactive ) fragments , an event that is purportedly more frequent in diabetics and in insulin - resistant states ( 23 , 24 , 25 ) . in heart failure patients ,", "the precursor pro - hormone probnp1 - 108 is found circulating at lower than normal levels ( 26 , 27 ) .", "recently , the paradigm - hf trial showed that angiotensin receptor - neprilysin inhibition using the compound lcz-696 , 200 mg twice daily , compared with enalapril at 10 mg twice daily significantly reduced the risk of cardiovascular death and hospitalization in patients with heart failure with an lvef 40% .", "however , death rates from cardiovascular causes were similar among diabetics compared with non - diabetics ( 28 ) .", "although this interesting finding could be an accidental one , other heart failure trials found that the benefit of this therapy in diabetics seems to be less than that in non - diabetics ( 29 , 30 , 31 ) .", "this finding merits further research , since diabetics comprise 2535% of the subjects in most studies ( 29 , 30 , 31 , 32 ) .", "the obese are differentiated from the metabolically healthy obese by an unhealthy metabolic profile ( 33 ) . the latter is defined by increased waist circumference ( > 94 cm in men and > 80 cm in women ) or bmi 30 kg / m accompanied by two or more of the following : hypertriglyceridemia ( 1.7 mmol / l ) , low hdl cholesterol ( < 1.03 mmol / l in men and < 1.29 mmol / l in women ) , hyperglycemia ( > 11.1 mmol / l or a diagnosis of diabetes ) , hypertension ( 130/85 mmhg ) or medication for high blood pressure ( 34 , 35 ) . in a 12-year follow - up of 61 299 subjects free from cardiovascular disease at baseline mrkedal et al . ( 36 ) observed that systolic heart failure development was similar among ", "metabolically healthy compared to non - metabolically healthy obese subjects , meaning that obesity per se creates a higher risk for developing systolic heart failure , especially if it is long - lasting and severe ( bmi > 40 kg / m ) .", "recently , it was reported that subjects with high plasma levels of nt - probnp are at a greater risk of developing systolic heart failure whether they are obese or non - obese . however , unlike the non - obese who show a direct linear relationship between levels of nt - probnp and systolic heart failure , the obese show a u - shaped relationship ; i.e. those who have the lowest plasma levels are at just as much risk of developing systolic heart failure as those with the highest plasma levels ( 37 ) .", "in contrast , the pathological weight loss caused by anorexia nervosa seems to be related to high plasma levels of anp ( 38 ) . in severe obesity ,", "bariatric surgery reduces the bmi and improves control of diabetes but it is not yet known if it reduces the risk of cardiovascular events ( 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ) . in patients with severe obesity ( bmi > 40 kg / m ) before gastric bypass surgery , the median level of nt - probnp was 54 pg / ml increasing by 125% after 1 year with no apparent relation to weight loss or glucometabolic parameters .", "it was speculated that an improvement in secretory cardiac function following surgery was responsible for the increase in circulating nt - probnp ( 46 ) .", "myocardial ischemia and cardiomyocyte stretch trigger the immediate release of cnp ( 47 , 48 ) even when the lvef is normal ( 49 , 50 ) . in a 9.2 year follow - up study of diabetic ( about 7% of the cohort ) and non - diabetic patients with chronic stable myocardial ischemia , high plasma levels of nt - probnp were significantly associated with mortality regardless of left ventricular function ( 51 ) .", "recently , it was shown that in patients with stable coronary heart disease bnp and nt - probnp were strong predictors of long - term ( 6.53.3 years ) cardiovascular events and notably , when nt - probnp was added to the clinical predictors , it performed better than bnp in risk classification for adverse cardiovascular events ( 52 ) . serial measurement data for nt - probnp in patients with chronic stable angina have shown a wide intra - individual variation .", "an increase by > 42% or a decrease by > 30% relative to baseline values is required to indicate a significant change ( 53 , 54 ) . in patients presenting with acute chest pain ,", "cnp plasma levels help the diagnosis and prognosis when used together with a non - diagnostic ecg and a negative troponin .", "cnp measured in these patients at rest predict future cardiac events at 30 and 180 days and at 1 year ( 55 , 56 , 57 , 58 ) .", "if myocardial ischemia is evident during the stress test , an elevation of the plasma levels of nt - probnp detects ischemia and predicts cardiovascular events ( 59 ) .", "likewise , in non - st - elevation acute coronary syndromes , cnp add prognostic information to the clinical indicators ( 60 , 61 , 62 , 63 ) ( table 1 ) . in a recent investigation ( 62 ) , bnp was shown to be an independent predictor of mortality on entering the study . at 1 year of", "follow - up bnp plasma levels provided added value over the timi and grace scores .", "cnp are stronger predictors of mortality than troponin in patients that will eventually develop myocardial infarction . in st - segment - elevation", "acute coronary syndromes cnp plasma levels also add prognostic information independently of the lvef ( 50 , 64 , 65 ) and in addition , predict which patients with primary percutaneous coronary intervention ( pci ) may be discharged earlier thus saving health resources ( 66 ) .", "it has been shown experimentally that anp is associated with salt - sensitive high blood pressure ( 67 ) . in humans", "alleles associated with the highest levels of np were also associated with a lower risk of high blood pressure ( 68 , 69 ) . in keeping with these findings", ", another study showed a relative deficiency of cnp at all the different stages of high blood pressure in humans consisting of low levels of probnp1 - 108 and anp99 - 126 .", "low plasma levels of bnp76 - 108 and nt - probnp1 - 76 were observed in the first hypertensive stages together with a reduction of nt - anp1 - 98 in stage i ( 70 ) .", "a possible therapeutic modality to supplement cnp in those cases in which circulating cnp are deemed deficient ( like in hypertension or heart failure ) is suggested by the development of a recombinant human serum albumin - atrial natriuretic factor ( anf ) ( 71 ) molecule .", "this compound , when injected into mice increased circulating cgmp and decreased blood pressure demonstrated a half - life of > 80 min , which is considerably longer than the 510 min half - life for native anf ( 71 , 72 , 73 ) . from the preceding , it may be surmised that elevated cnp levels are strong markers of future cardiovascular events , especially cardiovascular death . on the other hand , cnp plasma levels lower than expected in obese and diabetic patients", "the two patients discussed above had similar metabolic profiles , lvef > 50% but a different bmi and cnp profile .", "patient a was not severely obese and had slight residual myocardial ischemia with bnp and nt - probnp within low to moderate values expected for systolic heart failure and cardiovascular mortality on follow - up .", "patient b suffered from severe obesity , with very low plasma levels of cnp with a prognosis related to his bmi of > 40 ", "kg / m and probably also to a deficit of cnp , placing him in the high - risk category for developing systolic heart failure and eventual cardiovascular death ." ], [ "our patient a has markedly higher bnp and nt - probnp levels compared to patient b. this can largely be explained by the better ejection fraction of patient b , but there might also be other contributing factors .", "for example , recent studies suggest an inverse relationship between circulating cnp levels and bodyweight as well as with insulin resistance and t2d ( 19 , 74 , 75 , 76 , 77 , 78 ) ( table 1 ) . this correlation can also be observed in patients with congestive heart failure , despite increased cnp levels , due to cardiac wall stress ( 79 ) .", "cnp are cleared and degraded by neutral endopeptidase neprilysin and natriuretic peptide receptor c ( nprc ) ( 80 , 81 ) .", "natriuretic peptide receptor a ( npra ) and nprc have been identified in human adipose tissue in abundance , implying that adipose tissue sustains a regulatory function on the np system ( 82 , 83 , 84 ) .", "interestingly , compared to non - obese and normotensive individuals , nprc is increased in adipose tissue of obese hypertensive patients ( 85 ) .", "insulin has been observed to induce nprc expression in human adipocytes ( 84 ) and monocytes ( 83 ) , and might , hence , link conditions associated with hyperinsulinemia ( e.g. obesity and insulin resistance ) , to a relative np deficit", ". additionally , neprilysin , the np degrading endopeptidase , is expressed at increased levels in obesity ( 86 ) .", "these data argue for obesity and insulin resistance being conditions in which cnp are degraded at an accelerated pace .", "second , very recent experimental data also suggest that myocardial bnp expression is markedly decreased in mice fed a high fat diet ( 87 ) , an observation that warrants clinical confirmation .", "a cnp deficit in patients with components of the metabolic syndrome might be of clinical relevance .", "obese individuals have a higher prevalence of arterial hypertension compared to lean subjects ( 88 ) .", "although obesity - related arterial hypertension has been intensively studied , not all mechanisms are well understood ( 89 , 90 ) . by reduced vasodilatory and sodium - excretion activity , as well as", "aldosterone - system ( raas ) , a cnp deficit might contribute to obesity related hypertension .", "interestingly , in lean subjects , application of a sodium load induces myocardial cnp secretion and stimulates natriuresis ( 91 , 92 , 93 , 94 ) , a response which is blunted in patients with obesity ( 95 ) .", "together , these data argue that obesity promotes hypertension partly through reduced vascular and renal np responses as well as through impaired np - mediated raas inhibition .", "second , cnp also have beneficial actions on cardiac remodelling in essential hypertension , reducing left ventricular hypertrophy ( 96 ) .", "conversely , conditions with a cnp deficit are associated with cardiac hypertrophy in hypertensive patients . in this", "regard , hypertensive patients with the metabolic syndrome present with lower anp and nt - probnp levels and increased left ventricular mass compared to hypertensive patients without the metabolic syndrome and insulin resistance ( 97 ) .", "finally , the cnp deficit in patients with the metabolic syndrome might be part of a vicious circle which maintains metabolic disease .", "for instance , cnp exert lipolytic properties mediated by a cgmp - dependent protein kinase g activating pathway .", "cgmp - activated protein kinase g ( gk - i ) activates perilipin a and hormone sensitive lipase mediated triglyceride hydrolysis ( 98 , 99 , 100 , 101 ) . interestingly , these effects seem not to interact with the lipolytic effects of catecholamines ( 99 , 102 ) and the effect seems to be independent of the regulation of insulin ( 103 ) . besides a direct activating effect on lipolysis and lipid oxidation , cnp also control secretion of the insulin sensitizing adipokine adiponectin . in humans , anp acutely increases systemic levels of total and high molecular weight adiponectin ( 104 ) .", "these findings are in line with some observational studies showing positive associations between systemic cnp and adiponectin concentrations , as for example in heart failure patients ( 105 , 106 ) .", "this finding might also explain the adiponectin paradox in patients with congestive heart failure .", "cnp also exert other relevant metabolic actions on adipose tissue . treating a human derived adipose cell line with anp results in the uncoupling of cellular respiration ( 107 ) as well as ", "the response seems to be mediated by p38 map kinase , which increases uncoupling protein 1 ( ucp1 ) transcription ( 107 ) . upon cold exposure , systemic np concentrations increase and nprc expression in", "similarly , forced pkg expression in primary adipocytes leads to an increase in ucp1 expression and activates a thermogenic program .", "administration of anp acutely increases lipid oxidation ( 102 , 109 ) and postprandial energy expenditure in healthy individuals ( 110 ) . circulating -hydroxybutyrate increases , indicating enhanced hepatic lipid oxidation .", "apart from acute effects on lipid oxidation ( 110 ) , anp and bnp induce skeletal muscle mitochondrial biogenesis , respiration and lipid oxidation in human cells and in rodents , in vitro and in vivo \n ( 111 , 112 ) .", "chronic overexpression of bnp and gk - i each led to increased muscle mitochondrial content , oxidative capacity and lipid oxidation in rodents ( 111 ) .", "enhanced oxidative metabolism is associated with protection from diet induced obesity and insulin resistance ( 113 ) .", "heterozygous npra knockout mice are prone to gaining weight and become insulin resistant ( 111 ) .", "the mechanism linking np signalling to mitochondrial biogenesis and lipid oxidation in skeletal muscle includes activation of the co - transcriptional activator peroxisome proliferator activated receptor coactivator ( pgc)-1 and peroxisome proliferator activated receptor- , both of which are important factors of mitochondrial biogenesis in skeletal muscle ( 111 ) . in human myotubes , we observed that cnp stimulate pgc-1 , maximal mitochondrial respiratory capacity and lipid oxidation ( 112 ) .", "moreover , npra expression positively correlates with pgc-1 expression in the skeletal muscle of individuals after a chronic physical training program .", "similarly , cell culture studies show that cgmp restores glucose and insulin induced mitochondrial dysfunction in cultured c2c12 myotubes ( 114 ) , nitric oxide signalling via cgmp mediates activation of pgc-1 , and mitochondrial biogenesis in various murine tissues ( 115 ) .", "hepatic and skeletal muscle lipid content has been associated with insulin resistance ( 113 ) . in liver and skeletal muscle ,", "insulin resistance develops when bioactive lipid species accumulate , such as intracellular diacylglycerol ( 113 ) . in obesity and the metabolic syndrome , this lipid accumulation is primarily due to excessive caloric intake that exceeds the capacity of hepatocytes and myocytes to metabolize or export fatty acids , while refining mitochondrial respiration and enhancing lipid oxidation have been proved to improve lipid utilization and insulin sensitivity ( 116 , 117 , 118 ) . so far , there is no evidence that cnp directly interact with the insulin signalling cascade ( 109 , 119 ) .", "it is tempting to speculate that cnp could ameliorate lipid - induced insulin resistance through improvements in hepatic ( 110 ) and muscular ( 112 ) lipid oxidation .", "in line with this notion , cnp preserves mitochondrial function and insulin sensitivity in high fat diet mice ( 111 ) . cross sectional studies support the hypothesis that cnp protect from the development of obesity and t2d in patients ( 78 , 120 , 121,122 ) .", "conversely , data from the framingham heart study and the malm diet and cancer study show that reduced cnp concentrations correlate with a higher probability of insulin resistance in lean and obese individuals ( 76 ) .", "moreover , in the latter study low cnp concentrations are predictive of new - onset diabetes ( 123 ) .", "together , these data suggest that a cnp deficit , as observed in obese patients with the metabolic syndrome , contributes and aggravates metabolic vascular disease , and thus , prognosis .", "life style interventions as well as pharmacological approaches might be of benefit in this regard ( 28 , 124 ) .", "while physical activity increases cnp levels acutely and augments npra expression in skeletal muscle cells ( 112 ) , physical activity coupled with a low calorie diet seems to increase cnp levels chronically ( 125 ) ; an observation that seems to depend on the amount of weight loss ( 126 , 127 ) .", "experimental data further suggests that the incretin glucagon like peptide 1 ( glp1 ) might be of relevance in the regulation of np secretion .", "the glp1 receptor agonist liraglutide , which is widely used for the treatment of t2d , has been shown to induce cardiac anp release in mice , leading to enhanced natriuresis and vasodilatation ( 128 ) without inducing congestive heart failure .", "so far 25 clinical studies failed to demonstrate a similar effect of liraglutide when administered acutely ( 129 ) or sub - chronically to patients ( 130 ) .", "chronic liraglutide treatment , accompanied by weight loss , however , seems to increase circulating anp as well as bnp concentrations in diabetic patients ( 131 ) .", "clearly , future studies need to address the important question how a cnp deficit in obesity and t2d can be reversed most efficiently ( 132 ) ." ], [ "the authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review ." ], [ "this work was supported by a grant from the secretara de ciencia y tecnologa de la universidad nacional de crdoba ( grant number 69/08 ) to h r ramos , a grant from the german research foundation ( grant number bi1292/4 - 2 ) to a l birkenfeld and by funds from the university of ottawa to a j de bold ." ] ]

[ "isovaleric acidemia ( iva ) is an inherited autosomal recessive metabolic disease caused by a defect in the metabolism of amino acid leucine .", "the disease may present as metabolically severe form in the initial 2 weeks of life with acute onset vomiting and lethargy , progressing to coma . the other manifestation may be a metabolically mild form of failure to thrive and/or delayed development / mental retardation .", "these patients as well as those who survive acute illness may develop acute intercurrent episodes of decompensation with stressors like infection , immunization , or fasting .", "diagnosis is made by identifying elevated levels of isovaleric acid and its derivatives and their glycine and carnitine conjugates in plasma and urine of patients .", "free isovaleric acid may increase several hundredfold in blood during acute decompensation , but is not readily seen in blood and urine as it is conjugated rapidly .", "elevated levels of isovalerylcarnitine in blood and isovalerylglycine in urine are the hallmarks of this disease regardless of the metabolic condition .", "other methods of diagnosis based on isovaleryl coa dehydrogenase ( ivd ) enzyme activity in the body cells and identifying dna changes in the gene encoding ivd can confirm the disease .", "we report the magnetic resonance imaging ( mri ) findings in a case of iva seen as hyperintense signal intensity in basal ganglia on t1- and t2-weighted images .", "a breastfed 4-month - old female presented to our emergency room with decreased feeding , a generalized clonic seizure , lethargy , and acidotic breathing for 3 h. a product of third - degree consanguinity , she was born by term normal delivery with no significant perinatal event .", ", she had a vacant stare with a glasgow coma score ( gcs ) of 7 .", "her heart rate was 120/min , respiratory rate 66/min , capillary refill time 2 s , and temperature 97.8f .", "on anthropometric assessment , she was found to have failure to thrive with a weight of 3.8 kg , length 59 cm , upper segment / lower segment ratio 1.7:1 , and head circumference 40 cm .", "arterial blood gases were consistent with high anion gap severe metabolic acidosis ( ph = 6.8 , hco3 = 3 meq / l , pco2 = 16 mmhg , anion gap = 26 ) .", "serum chemistry showed a calcium level of 8.5 meq / l , phosphorus 3.2 mg / dl , sodium 137 mmol / l , and potassium level of 4.5 mmol / l .", "patient was dilantinised and started on maintenance intravenous fluids containing dextrose 10% and antibiotics pending cultures which turned out to be sterile .", "examination of cns showed rigidity of all limbs with decreased movement of lower limbs , which improved over a period of time in the hospital .", "mri of head was performed with 1.5 t ( siemens , erlangen germany ) mr imager using inversion recovery ( ir ) t1-weighted ( t1w ) and t2-weighted ( t2-w ) sequences in various orthogonal planes .", "diffusion - weighted imaging ( dwi ) , susceptibility - weighted imaging ( swi ) , and magnetic resonance spectroscopy ( mrs ) were also performed .", "mri brain revealed altered signal intensity in bilateral basal ganglia involving lentiform nuclei symmetrically with predominant involvement of putamen than globus pallidus . on t1w images , bilateral putamen and globus pallidi appeared to be involved with a peripheral hyperintense signal intensity rim and central low heterogeneous signal compared to white matter [ figure 1a and b ] .", "corresponding lentiform lesions showed predominant hyperintense signal intensity on t2w images with small , slightly hypointense signal in the center [ figure 2a and b ] which was more clearly seen in axial images .", "dwi showed hyperintense signal intensity in bilateral lentiform nuclei on high diffusion - weighted ( b value of 1000 s / mm ) images [ figure 3a ] and hypointense signal on apparent diffusion coefficient adc [ figure 3b ] compatible with restricted diffusion .", "adc maps revealed low adc value in the lentiform nuclei ( 0.9 10 mm / s ) than thalami ( 1.110 mm / s ) . restricted", "diffusivity area was slightly larger than the corresponding t2w - hyperintense signal intensity lesion and was distinctly more extensive than the hyperintense ring seen on t1w images in the bilateral lentiform nuclei .", "no convincing blooming was noticed on swi ( t2 * * ) images in the lentiform nuclei in the corresponding t1w- and t2w - hyperintense signal intensity lesions [ figure 4 ] .", "mrs ( at te = 135 ms ) of the lentiform lesions revealed a non - specific appearance of reduced n - acetylaspartate naa peak and elevated choline peak with slightly elevated lactate peak [ figure 5 ] .", "there was no other significant abnormality of cerebellum , brainstem , or cerebral cortex on mri brain . t1w inversion recovery images at the basal ganglia level in axial ( a ) and sagittal ( b ) planes showing peripheral hyperintense signal intensity ring ( downward arrow ) in bilateral lentiform nuclei around heterogeneous low signal center ( upward / backward arrow ) t2w images at the level of basal ganglia in axial ( a ) and coronal ( b ) planes showing symmetric hyperintense signal intensity in bilateral lentiform nuclei ( upward arrow ) with small hypointense signal area toward the center ( downward arrow ) axial diffusion - weighted mri at b value of 1000 s / mm ( a ) at the level of basal ganglia shows hyperintense signal intensity in bilateral lentiform nuclei ( arrow ) .", "adc map ( b ) at the same level shows reduced adc value in bilateral lentiform nuclei seen as hypointense signal ( arrow ) susceptibility - weighted axial mri ( t2 * * ) at the level of basal ganglia shows indistinct loss of signal / blooming ( arrow ) in the region of lentiform nuclei which is seen as hyperintense on t1w and t2w images mrs at te 135 ms of lentiform lesion showing elevated choline peak , reduced n - acetyl aspartate peak , and slight raised lactate level biochemical analysis of urine was done for possible inborn error of metabolism quantitative measurement of urinary organic acids by gas chromatography ", "mass spectrometry revealed 3-hydroxyisovaleric acid = 5.95 , isovaleroglycine = 96.10 , isovalerylglycine-(2 ) = 0.40 ( all in the units of relative peak area to internal standard ) with elevation factors of 7.44 , 961 , and 0.40 respectively .", "a diagnosis of iva was made on the basis of biochemical analysis and clinical presentation of the child .", "patient was put on low - protein , high - calorie diet after initial stabilization and is being followed for physical and mental development milestones .", "the enzyme catalyzes an early reaction in the metabolism of branched chain amino acid , namely leucine .", "consequent to ivd deficiency , isovaleric acid and its other derivatives accumulate in the body . these metabolites may impart a peculiar sweaty feet smell to the body , which may aid in the diagnosis .", "iva may present acutely in neonates with poor feeding , vomiting , decreased level of consciousness , and seizures .", "characteristic smell of dirty socks may be noticed in the body sweat or cerumen from ear during an acute crisis .", "intermittent iva patients beyond newborn period , also called catabolic episodes , may be triggered by infection or other stress and may be mistaken as diabetic ketoacidosis .", "although genetic mutations and biochemical changes with gas chromatography and mass / tandem spectrometry have been extensively described in iva , imaging with computed tomography ( ct ) and mri of brain is rarely reported .", "we came across a single report describing ct and mri findings in a case of iva which documented altered signal intensity on mri in globus pallidus and brainstem .", "changes in globus pallidi have been described as t1 and t2 relaxation time prolongation seen as symmetric t1w - hypointense and t2w - hyperintense signal intensity in the bilateral globus pallidus .", "we report signal intensity changes on mri in the bilateral putamen and globus pallidi in a case of chronic ", "mri brain revealed t1w - hyperintense signal ( t1 shortening ) and t2w- hyperintense signal ( t2 prolongation ) in bilateral lentiform nuclei , with indistinct blooming / signal loss on swi and concomitant evidence of restricted diffusion .", "involvement of putamen besides globus pallidus and t1w - hyperintense ring - shaped signal change in the lentiform nuclei differentiated basal ganglia lesions in our case from the one described in literature .", "the underlying cause of mri signal alteration in basal ganglia can be presumed to be the metabolites accumulating due to ivd deficiency .", "putamen and globus pallidus are highly vulnerable to generalized metabolic abnormalities due to their richness of mitochondria , blood supply , and neurotransmitters and their increased utilization of glucose and oxygen .", "oxidative stress and reduced na+ k+-atpase activity could be caused by metabolites accumulating in iva , including isovaleric acid , which can result in t2 prolongation and restricted diffusion due to cytotoxic edema .", "acute hyperammonemia also is known to cause t2 prolongation and restricted diffusion in basal ganglia .", "we postulate oxidative stress due to accumulated metabolites to be responsible for reduced diffusivity seen in lentiform nuclei in our case .", "oxidative stress and energy depletion could be substantiated by the presence of lactate in the mrs of lentiform lesion .", "t1 relaxation time shortening of basal ganglia seen as hyperintense signal intensity on t1w mri has been proposed to result from deposition of proteins , myelin breakdown products , blood , calcium or other minerals .", "we hypothesize that t1w shortening of lentiform nuclei in our case may possibly be related to protein and/or myelin breakdown product deposition resulting due to passage of isovaleric acid across the blood ", "brain barrier in acute crisis , as calcium , iron , and hemosiderin deposition was excluded by susceptibility - weighted mri .", "protein hydration layer within the cytoplasm of reactive astrocytes may result in t1 relaxation time shortening and consequent t1w - hyperintense signal in the basal ganglia .", "reactive astrocytes ( gemistocytes ) have been described to accumulate after acute insult and result in t1w - hyperintense signal in the basal ganglia .", "differential diagnoses of t1w - hyperintense signal intensity in bilateral basal ganglia include manganese deposition in acute hepatic decompensation of chronic liver disease , other causes of hyperammonemia like inborn errors of metabolism , manganese toxicity , and total parenteral nutrition .", "hypoxic ischemic injury may involve basal ganglia and result in t1 relaxation time shortening in children .", "neurofibromatosis 1 ( the most common phakomatosis ) may manifest as t1w- and t2w - hyperintense signal intensity lesions in bilateral globus pallidus .", "relevant medical history and laboratory investigations ( wide anion gap metabolic acidosis , raised glycine and carnitine conjugates of isovaleryl coa ) can help in making the correct diagnosis of iva in a case of bilateral t1w - hyperintense signal intensity lesions involving lentiform nuclei .", "early diagnosis is the key to treatment in the form of restricted protein ( specifically leucine ) intake , high - calorie diet , and glycine and carnitine supplementation ." ]

[ "<S> isovaleric acidemia ( iva ) is an inborn error of branched chain amino acid metabolism that may manifest as acute neonatal metabolic acidosis or as chronic intermittent form with developmental delay or recurrent episodes of acute metabolic acidosis . </S>", "<S> early diagnosis is the key to prevent morbidity and mortality . </S>", "<S> brain imaging abnormalities are rarely described in iva . </S>", "<S> we report a case of chronic intermittent iva with acute presentation in a 4-month - old infant who presented with acute metabolic acidosis . </S>", "<S> brain magnetic resonance imaging ( mri ) revealed symmetric signal intensity changes in bilateral lentiform nuclei with an unreported t1-weighted ( t1w ) symmetric hyperintense ring - like appearance in bilateral putamen . </S>" ]

[ "Introduction", "Case Report", "Discussion", "Conclusion" ]

[ [ "isovaleric acidemia ( iva ) is an inherited autosomal recessive metabolic disease caused by a defect in the metabolism of amino acid leucine .", "the disease may present as metabolically severe form in the initial 2 weeks of life with acute onset vomiting and lethargy , progressing to coma . the other manifestation may be a metabolically mild form of failure to thrive and/or delayed development / mental retardation .", "these patients as well as those who survive acute illness may develop acute intercurrent episodes of decompensation with stressors like infection , immunization , or fasting .", "diagnosis is made by identifying elevated levels of isovaleric acid and its derivatives and their glycine and carnitine conjugates in plasma and urine of patients .", "free isovaleric acid may increase several hundredfold in blood during acute decompensation , but is not readily seen in blood and urine as it is conjugated rapidly .", "elevated levels of isovalerylcarnitine in blood and isovalerylglycine in urine are the hallmarks of this disease regardless of the metabolic condition .", "other methods of diagnosis based on isovaleryl coa dehydrogenase ( ivd ) enzyme activity in the body cells and identifying dna changes in the gene encoding ivd can confirm the disease .", "we report the magnetic resonance imaging ( mri ) findings in a case of iva seen as hyperintense signal intensity in basal ganglia on t1- and t2-weighted images ." ], [ "a breastfed 4-month - old female presented to our emergency room with decreased feeding , a generalized clonic seizure , lethargy , and acidotic breathing for 3 h. a product of third - degree consanguinity , she was born by term normal delivery with no significant perinatal event .", ", she had a vacant stare with a glasgow coma score ( gcs ) of 7 .", "her heart rate was 120/min , respiratory rate 66/min , capillary refill time 2 s , and temperature 97.8f .", "on anthropometric assessment , she was found to have failure to thrive with a weight of 3.8 kg , length 59 cm , upper segment / lower segment ratio 1.7:1 , and head circumference 40 cm .", "arterial blood gases were consistent with high anion gap severe metabolic acidosis ( ph = 6.8 , hco3 = 3 meq / l , pco2 = 16 mmhg , anion gap = 26 ) .", "serum chemistry showed a calcium level of 8.5 meq / l , phosphorus 3.2 mg / dl , sodium 137 mmol / l , and potassium level of 4.5 mmol / l .", "patient was dilantinised and started on maintenance intravenous fluids containing dextrose 10% and antibiotics pending cultures which turned out to be sterile .", "examination of cns showed rigidity of all limbs with decreased movement of lower limbs , which improved over a period of time in the hospital .", "mri of head was performed with 1.5 t ( siemens , erlangen germany ) mr imager using inversion recovery ( ir ) t1-weighted ( t1w ) and t2-weighted ( t2-w ) sequences in various orthogonal planes .", "diffusion - weighted imaging ( dwi ) , susceptibility - weighted imaging ( swi ) , and magnetic resonance spectroscopy ( mrs ) were also performed .", "mri brain revealed altered signal intensity in bilateral basal ganglia involving lentiform nuclei symmetrically with predominant involvement of putamen than globus pallidus . on t1w images , bilateral putamen and globus pallidi appeared to be involved with a peripheral hyperintense signal intensity rim and central low heterogeneous signal compared to white matter [ figure 1a and b ] .", "corresponding lentiform lesions showed predominant hyperintense signal intensity on t2w images with small , slightly hypointense signal in the center [ figure 2a and b ] which was more clearly seen in axial images .", "dwi showed hyperintense signal intensity in bilateral lentiform nuclei on high diffusion - weighted ( b value of 1000 s / mm ) images [ figure 3a ] and hypointense signal on apparent diffusion coefficient adc [ figure 3b ] compatible with restricted diffusion .", "adc maps revealed low adc value in the lentiform nuclei ( 0.9 10 mm / s ) than thalami ( 1.110 mm / s ) . restricted", "diffusivity area was slightly larger than the corresponding t2w - hyperintense signal intensity lesion and was distinctly more extensive than the hyperintense ring seen on t1w images in the bilateral lentiform nuclei .", "no convincing blooming was noticed on swi ( t2 * * ) images in the lentiform nuclei in the corresponding t1w- and t2w - hyperintense signal intensity lesions [ figure 4 ] .", "mrs ( at te = 135 ms ) of the lentiform lesions revealed a non - specific appearance of reduced n - acetylaspartate naa peak and elevated choline peak with slightly elevated lactate peak [ figure 5 ] .", "there was no other significant abnormality of cerebellum , brainstem , or cerebral cortex on mri brain . t1w inversion recovery images at the basal ganglia level in axial ( a ) and sagittal ( b ) planes showing peripheral hyperintense signal intensity ring ( downward arrow ) in bilateral lentiform nuclei around heterogeneous low signal center ( upward / backward arrow ) t2w images at the level of basal ganglia in axial ( a ) and coronal ( b ) planes showing symmetric hyperintense signal intensity in bilateral lentiform nuclei ( upward arrow ) with small hypointense signal area toward the center ( downward arrow ) axial diffusion - weighted mri at b value of 1000 s / mm ( a ) at the level of basal ganglia shows hyperintense signal intensity in bilateral lentiform nuclei ( arrow ) .", "adc map ( b ) at the same level shows reduced adc value in bilateral lentiform nuclei seen as hypointense signal ( arrow ) susceptibility - weighted axial mri ( t2 * * ) at the level of basal ganglia shows indistinct loss of signal / blooming ( arrow ) in the region of lentiform nuclei which is seen as hyperintense on t1w and t2w images mrs at te 135 ms of lentiform lesion showing elevated choline peak , reduced n - acetyl aspartate peak , and slight raised lactate level biochemical analysis of urine was done for possible inborn error of metabolism quantitative measurement of urinary organic acids by gas chromatography ", "mass spectrometry revealed 3-hydroxyisovaleric acid = 5.95 , isovaleroglycine = 96.10 , isovalerylglycine-(2 ) = 0.40 ( all in the units of relative peak area to internal standard ) with elevation factors of 7.44 , 961 , and 0.40 respectively .", "a diagnosis of iva was made on the basis of biochemical analysis and clinical presentation of the child .", "patient was put on low - protein , high - calorie diet after initial stabilization and is being followed for physical and mental development milestones ." ], [ "the enzyme catalyzes an early reaction in the metabolism of branched chain amino acid , namely leucine .", "consequent to ivd deficiency , isovaleric acid and its other derivatives accumulate in the body . these metabolites may impart a peculiar sweaty feet smell to the body , which may aid in the diagnosis .", "iva may present acutely in neonates with poor feeding , vomiting , decreased level of consciousness , and seizures .", "characteristic smell of dirty socks may be noticed in the body sweat or cerumen from ear during an acute crisis .", "intermittent iva patients beyond newborn period , also called catabolic episodes , may be triggered by infection or other stress and may be mistaken as diabetic ketoacidosis .", "although genetic mutations and biochemical changes with gas chromatography and mass / tandem spectrometry have been extensively described in iva , imaging with computed tomography ( ct ) and mri of brain is rarely reported .", "we came across a single report describing ct and mri findings in a case of iva which documented altered signal intensity on mri in globus pallidus and brainstem .", "changes in globus pallidi have been described as t1 and t2 relaxation time prolongation seen as symmetric t1w - hypointense and t2w - hyperintense signal intensity in the bilateral globus pallidus .", "we report signal intensity changes on mri in the bilateral putamen and globus pallidi in a case of chronic ", "mri brain revealed t1w - hyperintense signal ( t1 shortening ) and t2w- hyperintense signal ( t2 prolongation ) in bilateral lentiform nuclei , with indistinct blooming / signal loss on swi and concomitant evidence of restricted diffusion .", "involvement of putamen besides globus pallidus and t1w - hyperintense ring - shaped signal change in the lentiform nuclei differentiated basal ganglia lesions in our case from the one described in literature .", "the underlying cause of mri signal alteration in basal ganglia can be presumed to be the metabolites accumulating due to ivd deficiency .", "putamen and globus pallidus are highly vulnerable to generalized metabolic abnormalities due to their richness of mitochondria , blood supply , and neurotransmitters and their increased utilization of glucose and oxygen .", "oxidative stress and reduced na+ k+-atpase activity could be caused by metabolites accumulating in iva , including isovaleric acid , which can result in t2 prolongation and restricted diffusion due to cytotoxic edema .", "acute hyperammonemia also is known to cause t2 prolongation and restricted diffusion in basal ganglia .", "we postulate oxidative stress due to accumulated metabolites to be responsible for reduced diffusivity seen in lentiform nuclei in our case .", "oxidative stress and energy depletion could be substantiated by the presence of lactate in the mrs of lentiform lesion .", "t1 relaxation time shortening of basal ganglia seen as hyperintense signal intensity on t1w mri has been proposed to result from deposition of proteins , myelin breakdown products , blood , calcium or other minerals .", "we hypothesize that t1w shortening of lentiform nuclei in our case may possibly be related to protein and/or myelin breakdown product deposition resulting due to passage of isovaleric acid across the blood ", "brain barrier in acute crisis , as calcium , iron , and hemosiderin deposition was excluded by susceptibility - weighted mri .", "protein hydration layer within the cytoplasm of reactive astrocytes may result in t1 relaxation time shortening and consequent t1w - hyperintense signal in the basal ganglia .", "reactive astrocytes ( gemistocytes ) have been described to accumulate after acute insult and result in t1w - hyperintense signal in the basal ganglia .", "differential diagnoses of t1w - hyperintense signal intensity in bilateral basal ganglia include manganese deposition in acute hepatic decompensation of chronic liver disease , other causes of hyperammonemia like inborn errors of metabolism , manganese toxicity , and total parenteral nutrition .", "hypoxic ischemic injury may involve basal ganglia and result in t1 relaxation time shortening in children .", "neurofibromatosis 1 ( the most common phakomatosis ) may manifest as t1w- and t2w - hyperintense signal intensity lesions in bilateral globus pallidus ." ], [ "relevant medical history and laboratory investigations ( wide anion gap metabolic acidosis , raised glycine and carnitine conjugates of isovaleryl coa ) can help in making the correct diagnosis of iva in a case of bilateral t1w - hyperintense signal intensity lesions involving lentiform nuclei .", "early diagnosis is the key to treatment in the form of restricted protein ( specifically leucine ) intake , high - calorie diet , and glycine and carnitine supplementation ." ] ]

[ "dr . coyne - beasley receives research funding from merck & co. none of these funds were used in the conduction or completion of the research contained in this manuscript .", "this article was supported by grants from the national institutes of health 1r01ai113305 - 01 ( cates pi ) , and by the north carolina translational and clinical sciences institute , through support from the national center for advancing translational sciences ( ncats ) , national institutes of health , grant award number 1ul1tr001111 .", "the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih ." ]

[ "<S> a significant barrier to the delivery of hpv vaccine is reluctance by both healthcare providers and parents to vaccinate at age 11 or 12 , which may be considered a young age . </S>", "<S> this barrier has been called vaccine hesitancy in recent research . in this commentary </S>", "<S> , we suggest using social marketing strategies to promote hpv vaccination at the recommended preteen ages . </S>", "<S> we emphasize a critical public health message of a sexually transmitted infection ( sti ) as preventable and vaccination against hpv as a way to protect against its consequences . </S>", "<S> the message tackles the issue of vaccine hesitancy head on , by saying that most people are at risk for hpv and there is a way to prevent hpv 's serious consequences of cancer . </S>", "<S> our approach to this conversation in the clinical setting is also to engage the preteen in a dialog with the parent and provider . </S>", "<S> we expect our emphasis on the risk of sti infection will not only lead to increased hpv vaccination at preteen ages but also lay important groundwork for clinical adoption of other sti vaccines in development ( hiv , hsv , chlamydia , and gonorrhea ) as well as begin conversations to promote sexual health . </S>" ]

[ "Disclosure of Potential Conflicts of Interest", "Funding" ]

[ [ "dr . coyne - beasley receives research funding from merck & co. none of these funds were used in the conduction or completion of the research contained in this manuscript ." ], [ "this article was supported by grants from the national institutes of health 1r01ai113305 - 01 ( cates pi ) , and by the north carolina translational and clinical sciences institute , through support from the national center for advancing translational sciences ( ncats ) , national institutes of health , grant award number 1ul1tr001111 .", "the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih ." ] ]

[ "in 1994 , waldinger et al . introduced the intravaginal ejaculation latency time ( ielt ) as a measure for the ejaculation time of heterosexual intercourse .", "the ielt is defined as the time from the moment of vaginal penetration until the moment of intravaginal ejaculation . in case of ejaculation outside the vagina", "the most accurate way to measure the ielt is the use of a stopwatch handled by the female partner . in a stopwatch study in a cohort of dutch men with lifelong premature ejaculation", ", it was shown that the ielt had a skewed distribution and that 85% of men ejaculated within 1 minute after penetration .", "in addition , in a meta - analysis of all drug treatment studies of premature ejaculation from 1943 to 2003 , it was shown that prospective real - time stopwatch measurement of the ielt during selective serotonin reuptake inhibitors ( ssri ) treatment led to a smaller , and therefore more valid , confidence interval of the ielt compared to retrospective questionnaire studies of the ielt . in 2005 ,", "a prospective stopwatch study of 491 men of the general male population in 5 countries ( the netherlands , united kingdom , spain , turkey , and the united states ) also showed a skewed distribution to the right with a median ielt of 5.4 minutes ( range , 0.5544.1 minutes ) . using a blinded timer device ,", "a second study was performed in 2009 in a new group of 474 men of the general population in the same countries .", "this study also showed a remarkable similar skewed distribution to the right , with a median ielt of 6.0 minutes ( range , 0.152.7 minutes ) .", "the precise and similar method of ielt measurement by a stopwatch on various intercourse events in the 3 aforementioned studies facilitates comparison of their ielt distributions .", "however , a prerequisite for further research of the ejaculation time , is a better knowledge of the type of distribution that is formed by the ielt in different populations of men .", "the purpose of the current study , was to investigate which type of well - known mathematical distribution fitted to the ielt distribution of men with lifelong pe and of men in the general male population .", "by application of the statistical program easy fit professional ver . 5.6 ( mathwave technologies inc . ) , we analysed the ielt data of 2 previous stopwatch studies of the ielt in the general male population ( fig .", "1a , b ) and of the ielt data of a previous stopwatch study in a clinical cohort of men with lifelong premature ejaculation ( fig .", "in addition , we investigated which of the wellknown mathematical probability distributions , fitted most accurately to the curves of the aforementioned ielt distributions . this fitness of the distribution is expressed by the goodness of fit ( gof ) , which is calculated by the kolmogorov - smirnov test ( ks test ) the gof therefore is a measure for the accuracy of the fitness .", "the ks test is a non parametric statistical test of the equality of continuous , one - dimensional probability distributions . in the current study", "it was used to compare the ielt distributions with various well - known theoretical reference probability distributions ( one - sample ks test ) .", "the difference between the actual measured ielt data ( as previously published in the three stopwatch studies ) and the theoretical mathematical distribution models is measured with the kolmogorov smirnov test . in other words , the ks test measures the gof .", "the smaller the difference between the theoretical distribution and the ielt distribution , the more accurate is the fitness of the theoretical distributions on the ielt distributions .", "gof , measured by the ks test showed that the ielt distributions in the 2 studies of the ielt in the general male population ( the 2005 study and the 2009 study ) and in the ielt distribution of dutch caucasian men with lifelong pe were a gamma - distribution , which is characterized by ( 1 ) a boundary on the left at zero , and therefore excluding negative ielt values , ( 2 ) a positively skewed shape , including high ielt values , and ( 3 ) a significant flexibility in the shape , allowing the gamma distribution any number of ielts , with reasonable accuracy .", "of the multiple operationalized gamma - distributions , the lognormal distribution fitted most well on the gamma - ielt distribution in the general male population .", "the combined lognormal distribution of men in the general male population ( combination of ielt study 2005 and 2009 ) is shown in fig .", "the form of this lognormal distribution is mirrored by the mathematical formula , shown in fig .", "3 . in contrast , the gumbel max distribution fitted most well on the gamma - distribution of the ielt in the cohort of men with lifelong premature ejaculation ( fig .", "the form of this gumbel max distribution is mirrored by the mathematical formula , shown in fig .", "according to kolmogorov smirnov the gof of the lognormal distribution for the 2005 study was 0.11708 , and for the 2009 study the gof was 0.06284 .", "the gof of the combined lognormal distribution of both the 2005 and 2009 study was 0.057 . according to kolmogorov smirnov", "the gof of the gumbel max distribution of the ielt distribution in the 1998 study of men with lifelong pe was 0.179 . by using the lognormal distribution , the various ielt values of all men in the general male population of the 2005 study ,", "the 2009 study and their combination were calculated ( table 1 ) . by using the gumbel max distribution ,", "the various ielt values of all men in the 1998 study of dutch caucasian men with lifelong pe were calculated ( table 2 ) .", "table 3 shows that in the general male population 1.08% of men ejaculates within 0 to 60 seconds after vaginal penetration ( in 2005 study , 1.23% ; in 2009 study , 0.99% ; in combined study , 1.08% ) .", "in contrast , in the 1998 study of men with lifelong pe , 85.4% ejaculates within 1 minute , and 55.1% ejaculates within 30 seconds .", "notably , in the general male population 7.1% of men ejaculates between 1 and 2 minutes .", "in contrast in men with lifelong pe , 14% of men ejaculates within 1 and 2 minutes .", "2 and 4 show that the ielt distribution of the cohort of men with lifelong pe is not represented in the ielt distribution of the men in the general male population .", "this is also shown by the ielt data , as represented in table 3 , which shows that 85.4% of men ejaculates within 1 minute in men with lpe , whereas only 1.08% of men in the general male population ejaculates within 1 minute .", "by investigating which theoretical mathematical distribution fitted most accurately on the ielt distributions of 3 previously published ielt distributions , it appeared that the 3 ielt distributions were gamma distributions , but that the type of gamma distribution of men in the general male population does not fit in the gamma distribution of men with lifelong pe .", "therefore this study showed that men with lifelong pe not only differ from men in the general population in their ielt values , e.g. , men with lifelong pe have ielts of less than 1 minute , but also in the mathematical type , e.g. , curve of their ielt distribution . in order to investigate the ielt distributions , we investigated which of the well - known mathematically described distributions fitted most accurately to the ielt distribution of men in the general male population and of men with lifelong pe .", "for that purpose we summarized the data of 2 prospective real - time stopwatch studies of caucasian men in the general population , performed in the same five countries ( the netherlands , united kingdom , spain , turkey , and the united states ) , to one set of ielt data , including 965 males , and compared this ielt distribution with the ielt distribution of a cohort of 117 dutch caucasian men with lifelong pe . in the current study , it was found by application of the ks test on various mathematical distributions that the ielt distribution of the males in the general male population most accurately fitted the lognormal distribution , which belongs to the family of gamma distributions .", "however , the ielt distribution of males with lifelong pe did not fit to the lognormal distribution .", "importantly , the ielt distribution of the males with lifelong pe most accurately fitted to the gumbel max distribution , which , on its turn , inaccurately fits to the ielt distribution of men in the general male population .", "for example , whereas only a neglectable minority of men in the general male population ejaculates within 1 minute ( e.g. , 1.08% ) and within 30 seconds after vaginal penetration ( e.g. , 0.08% ) , the current study shows that the opposite occurs in men with lifelong pe . in these men , the majority ejaculates within 30 seconds ( e.g. , 55.1% ) and within 30 to 60 seconds ( e.g. , 30.3% ) . in other words , there are significantly more men with lifelong pe who ejaculate within 30 seconds than can be extrapolated from the probability density curve of the ielt distribution of men in the general population .", "an advantage of the formula for the lognormal distribution , is that it enables the calculation of the number and percentage of men in a specific population with a specific ielt value .", "similarly , based on the gumbel max distribution , the number and percentage of men with a specific ielt value can be calculated in a cohort of men with lifelong pe .", "our calculations by means of the formula of the lognormal distribution ( table 1 ) showed a striking similarity of the percentages of males with a specific ielt value in the 2 separate studies of men in the general population , supporting the reliability and validity of stopwatch measurement of the ielt in an epidemiological study of the ielt .", "notably , as the 2 general population ielt stopwatch studies , e.g. , the 2005 and 2009 studies , have been conducted in caucasian men , a limitation of the current study is that the formula may only be valid for caucasian men in western countries , whereas for african , middle east , and far east asian countries our formula remains to be investigated on the condition that similar ielt stopwatch studies in random samples of their general populations have been conducted .", "one may argue that another limitation of the current study is the number of men with lifelong pe .", "however , a simple calculation shows that in order to discover the gumbel max ielt distribution of men with lifelong pe in the lognormal ielt distribution in the general male population , one has to multiply the number of men with lifelong pe by 100 .", "1c could be extracted out of a general male population of 11.000 men ( 1% = 1/100 of the population ) . as it is impossible to perform a stopwatch study of the ielt in 11.000 males", "still , with a few hundreds of men with lifelong pe who have measured their ielt prospectively with a stopwatch it is argued that the current formula of the gumbel max ielt distribution can be become more accurate for application in a cohort of men with lifelong pe .", "interestingly , based on the formula of the lognormal distribution that most accurately describes the ielt data of men in the general male population , it is possible to answer certain questions regarding the ielt of men in the general male population without the necessity of conducting realtime stopwatch measurements in very large samples of men .", "for example , according to the statistics netherlands ( centraal bureau voor de statistiek or cbs ) , the netherlands currently has 16.982.433 inhibitants and 8.372.858 of them are males . of these males , 5.912.984 are aged between 18 and 70 years .", "application of our lognormal formula shows that in the netherlands there are 70.305 men ( 1.19% ) who ejaculate within 1 minute , and 48.546 ( 0.82% ) men who ejaculate within 30 seconds .", "in addition , using this formula , 502.012 ( 8.5% ) men ejaculate between 0 and 2 minutes , 2.419.002 men ( 40.9% ) who ejaculate between 1 and 5 minutes , and 1.971.330 ( 33.3% ) men who ejaculate between 5 and 10 minutes .", "in addition , there are 1.112.291 men ( 18.8% ) who ejaculate between 10 and 20 minutes .", "notably , after 20 minutes , the number and percentages decrease : 233.208 men ( 3.9% ) ejaculate between 20 and 30 minutes and 66.225 men ( 1.12% ) ejaculate between 30 and 40 minutes .", "a minority of 40.633 ( 0.687% ) ejaculates with an ielt of more than 40 minutes .", "the application of the formula , which describes the probability density of the ielt , shows that it may provide answers to questions that can not be measured by a stopwatch , due to the extremely large number of men that otherwise ought to be included in an epidemiological study , or just can not be measured as the ielt duration is too long and potentially painful for the female partner of a subject participating in a scientific study .", "notably , it is questioned why the ielt distribution of the men with lifelong pe is so completely different from the ielt distribution of men in the general male population .", "further research into this question is warranted and is presumably associated with genetic and epi - genetic factors . according to the recent classification by waldinger and schweitzer , there are 4 pe subtypes , e.g. , lifelong pe , acquired pe , subjective pe , and variable pe .", "their prevalence in the general population differs significantly , as has been shown by serefoglu et al . and gao et al . , with the lowest prevalence of lifelong pe .", "notably , lifelong pe is characterized by a hypertonic state , consisting of premature ejaculation ( ejaculatio praecox ) , a facilitated erection ( erectio praecox ) and a facilitated penile detumescence ( detumescentia praecox ) , when the male is becoming involved in an erotic situation .", "the current study shows that lifelong pe not only differs from the 3 other pe subtypes with regard to this triad of symptoms , but that lifelong pe has a different ielt and a different ielt distribution than males in the general population , of which men with variable and subjective pe are part of .", "the separate state of lifelong pe among the 3 other pe subtypes with regard to the ielt and its ielt distribution has consequences for the methodology and design of drug treatment studies . as has recently been argued by waldinger , the method and design for drug treatment studies of men with lifelong pe who ejaculate within seconds , are not required for drug treatment studies of men with variable and subjective pe with normal ielt values .", "the ielt distribution of men in the general population is most accurately fitted by the lognormal distribution , whereas the ielt distribution of men with lifelong pe is most accurately fitted by the gumbel max distribution .", "as there are significantly more men with lifelong pe who ejaculate within 30 and 60 seconds than can be extrapolated from the probability density curve of the ielt distribution of men in the general population it is concluded that men with lifelong pe have a distinct ielt distribution that can only be retrieved from the general male population ielt distribution when thousands of men would participate in a stopwatch study of the ielt .", "the cause of the difference in ielt distribution should be further investigated and may be due to genetic and epigenetic factors .", "application of the mathematical formula of the lognormal ielt distribution is useful for epidemiological research of the ielt in large populations of men ." ]

[ "<S> purposeto find the most accurate mathematical description of the intravaginal ejaculation latency time ( ielt ) distribution in the general male population.materials and methodswe compared the fitness of various well - known mathematical distributions with the ielt distribution of two previously published stopwatch studies of the caucasian general male population and a stopwatch study of dutch caucasian men with lifelong premature ejaculation ( pe ) . </S>", "<S> the accuracy of fitness is expressed by the goodness of fit ( gof ) . </S>", "<S> the smaller the gof , the more accurate is the fitness.resultsthe 3 ielt distributions are gamma distributions , but the ielt distribution of lifelong pe is another gamma distribution than the ielt distribution of men in the general male population . </S>", "<S> the lognormal distribution of the gamma distributions most accurately fits the ielt distribution of 965 men in the general population , with a gof of 0.057 . </S>", "<S> the gumbel max distribution most accurately fits the ielt distribution of 110 men with lifelong pe with a gof of 0.179 . </S>", "<S> there are more men with lifelong pe ejaculating within 30 and 60 seconds than can be extrapolated from the probability density curve of the lognormal ielt distribution of men in the general population.conclusionsmen with lifelong pe have a distinct ielt distribution , e.g. , a gumbel max ielt distribution , that can only be retrieved from the general male population lognormal ielt distribution when thousands of men would participate in a ielt stopwatch study . the mathematical formula of the lognormal ielt distribution is useful for epidemiological research of the ielt . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS" ]

[ [ "in 1994 , waldinger et al . introduced the intravaginal ejaculation latency time ( ielt ) as a measure for the ejaculation time of heterosexual intercourse .", "the ielt is defined as the time from the moment of vaginal penetration until the moment of intravaginal ejaculation . in case of ejaculation outside the vagina", "the most accurate way to measure the ielt is the use of a stopwatch handled by the female partner . in a stopwatch study in a cohort of dutch men with lifelong premature ejaculation", ", it was shown that the ielt had a skewed distribution and that 85% of men ejaculated within 1 minute after penetration .", "in addition , in a meta - analysis of all drug treatment studies of premature ejaculation from 1943 to 2003 , it was shown that prospective real - time stopwatch measurement of the ielt during selective serotonin reuptake inhibitors ( ssri ) treatment led to a smaller , and therefore more valid , confidence interval of the ielt compared to retrospective questionnaire studies of the ielt . in 2005 ,", "a prospective stopwatch study of 491 men of the general male population in 5 countries ( the netherlands , united kingdom , spain , turkey , and the united states ) also showed a skewed distribution to the right with a median ielt of 5.4 minutes ( range , 0.5544.1 minutes ) . using a blinded timer device ,", "a second study was performed in 2009 in a new group of 474 men of the general population in the same countries .", "this study also showed a remarkable similar skewed distribution to the right , with a median ielt of 6.0 minutes ( range , 0.152.7 minutes ) .", "the precise and similar method of ielt measurement by a stopwatch on various intercourse events in the 3 aforementioned studies facilitates comparison of their ielt distributions .", "however , a prerequisite for further research of the ejaculation time , is a better knowledge of the type of distribution that is formed by the ielt in different populations of men .", "the purpose of the current study , was to investigate which type of well - known mathematical distribution fitted to the ielt distribution of men with lifelong pe and of men in the general male population ." ], [ "by application of the statistical program easy fit professional ver . 5.6 ( mathwave technologies inc . ) , we analysed the ielt data of 2 previous stopwatch studies of the ielt in the general male population ( fig .", "1a , b ) and of the ielt data of a previous stopwatch study in a clinical cohort of men with lifelong premature ejaculation ( fig .", "in addition , we investigated which of the wellknown mathematical probability distributions , fitted most accurately to the curves of the aforementioned ielt distributions . this fitness of the distribution is expressed by the goodness of fit ( gof ) , which is calculated by the kolmogorov - smirnov test ( ks test ) the gof therefore is a measure for the accuracy of the fitness .", "the ks test is a non parametric statistical test of the equality of continuous , one - dimensional probability distributions . in the current study", "it was used to compare the ielt distributions with various well - known theoretical reference probability distributions ( one - sample ks test ) .", "the difference between the actual measured ielt data ( as previously published in the three stopwatch studies ) and the theoretical mathematical distribution models is measured with the kolmogorov smirnov test . in other words , the ks test measures the gof .", "the smaller the difference between the theoretical distribution and the ielt distribution , the more accurate is the fitness of the theoretical distributions on the ielt distributions ." ], [ "gof , measured by the ks test showed that the ielt distributions in the 2 studies of the ielt in the general male population ( the 2005 study and the 2009 study ) and in the ielt distribution of dutch caucasian men with lifelong pe were a gamma - distribution , which is characterized by ( 1 ) a boundary on the left at zero , and therefore excluding negative ielt values , ( 2 ) a positively skewed shape , including high ielt values , and ( 3 ) a significant flexibility in the shape , allowing the gamma distribution any number of ielts , with reasonable accuracy .", "of the multiple operationalized gamma - distributions , the lognormal distribution fitted most well on the gamma - ielt distribution in the general male population .", "the combined lognormal distribution of men in the general male population ( combination of ielt study 2005 and 2009 ) is shown in fig .", "the form of this lognormal distribution is mirrored by the mathematical formula , shown in fig .", "3 . in contrast , the gumbel max distribution fitted most well on the gamma - distribution of the ielt in the cohort of men with lifelong premature ejaculation ( fig .", "the form of this gumbel max distribution is mirrored by the mathematical formula , shown in fig .", "according to kolmogorov smirnov the gof of the lognormal distribution for the 2005 study was 0.11708 , and for the 2009 study the gof was 0.06284 .", "the gof of the combined lognormal distribution of both the 2005 and 2009 study was 0.057 . according to kolmogorov smirnov", "the gof of the gumbel max distribution of the ielt distribution in the 1998 study of men with lifelong pe was 0.179 . by using the lognormal distribution , the various ielt values of all men in the general male population of the 2005 study ,", "the 2009 study and their combination were calculated ( table 1 ) . by using the gumbel max distribution ,", "the various ielt values of all men in the 1998 study of dutch caucasian men with lifelong pe were calculated ( table 2 ) .", "table 3 shows that in the general male population 1.08% of men ejaculates within 0 to 60 seconds after vaginal penetration ( in 2005 study , 1.23% ; in 2009 study , 0.99% ; in combined study , 1.08% ) .", "in contrast , in the 1998 study of men with lifelong pe , 85.4% ejaculates within 1 minute , and 55.1% ejaculates within 30 seconds .", "notably , in the general male population 7.1% of men ejaculates between 1 and 2 minutes .", "in contrast in men with lifelong pe , 14% of men ejaculates within 1 and 2 minutes .", "2 and 4 show that the ielt distribution of the cohort of men with lifelong pe is not represented in the ielt distribution of the men in the general male population .", "this is also shown by the ielt data , as represented in table 3 , which shows that 85.4% of men ejaculates within 1 minute in men with lpe , whereas only 1.08% of men in the general male population ejaculates within 1 minute ." ], [ "by investigating which theoretical mathematical distribution fitted most accurately on the ielt distributions of 3 previously published ielt distributions , it appeared that the 3 ielt distributions were gamma distributions , but that the type of gamma distribution of men in the general male population does not fit in the gamma distribution of men with lifelong pe .", "therefore this study showed that men with lifelong pe not only differ from men in the general population in their ielt values , e.g. , men with lifelong pe have ielts of less than 1 minute , but also in the mathematical type , e.g. , curve of their ielt distribution . in order to investigate the ielt distributions , we investigated which of the well - known mathematically described distributions fitted most accurately to the ielt distribution of men in the general male population and of men with lifelong pe .", "for that purpose we summarized the data of 2 prospective real - time stopwatch studies of caucasian men in the general population , performed in the same five countries ( the netherlands , united kingdom , spain , turkey , and the united states ) , to one set of ielt data , including 965 males , and compared this ielt distribution with the ielt distribution of a cohort of 117 dutch caucasian men with lifelong pe . in the current study , it was found by application of the ks test on various mathematical distributions that the ielt distribution of the males in the general male population most accurately fitted the lognormal distribution , which belongs to the family of gamma distributions .", "however , the ielt distribution of males with lifelong pe did not fit to the lognormal distribution .", "importantly , the ielt distribution of the males with lifelong pe most accurately fitted to the gumbel max distribution , which , on its turn , inaccurately fits to the ielt distribution of men in the general male population .", "for example , whereas only a neglectable minority of men in the general male population ejaculates within 1 minute ( e.g. , 1.08% ) and within 30 seconds after vaginal penetration ( e.g. , 0.08% ) , the current study shows that the opposite occurs in men with lifelong pe . in these men , the majority ejaculates within 30 seconds ( e.g. , 55.1% ) and within 30 to 60 seconds ( e.g. , 30.3% ) . in other words , there are significantly more men with lifelong pe who ejaculate within 30 seconds than can be extrapolated from the probability density curve of the ielt distribution of men in the general population .", "an advantage of the formula for the lognormal distribution , is that it enables the calculation of the number and percentage of men in a specific population with a specific ielt value .", "similarly , based on the gumbel max distribution , the number and percentage of men with a specific ielt value can be calculated in a cohort of men with lifelong pe .", "our calculations by means of the formula of the lognormal distribution ( table 1 ) showed a striking similarity of the percentages of males with a specific ielt value in the 2 separate studies of men in the general population , supporting the reliability and validity of stopwatch measurement of the ielt in an epidemiological study of the ielt .", "notably , as the 2 general population ielt stopwatch studies , e.g. , the 2005 and 2009 studies , have been conducted in caucasian men , a limitation of the current study is that the formula may only be valid for caucasian men in western countries , whereas for african , middle east , and far east asian countries our formula remains to be investigated on the condition that similar ielt stopwatch studies in random samples of their general populations have been conducted .", "one may argue that another limitation of the current study is the number of men with lifelong pe .", "however , a simple calculation shows that in order to discover the gumbel max ielt distribution of men with lifelong pe in the lognormal ielt distribution in the general male population , one has to multiply the number of men with lifelong pe by 100 .", "1c could be extracted out of a general male population of 11.000 men ( 1% = 1/100 of the population ) . as it is impossible to perform a stopwatch study of the ielt in 11.000 males", "still , with a few hundreds of men with lifelong pe who have measured their ielt prospectively with a stopwatch it is argued that the current formula of the gumbel max ielt distribution can be become more accurate for application in a cohort of men with lifelong pe .", "interestingly , based on the formula of the lognormal distribution that most accurately describes the ielt data of men in the general male population , it is possible to answer certain questions regarding the ielt of men in the general male population without the necessity of conducting realtime stopwatch measurements in very large samples of men .", "for example , according to the statistics netherlands ( centraal bureau voor de statistiek or cbs ) , the netherlands currently has 16.982.433 inhibitants and 8.372.858 of them are males . of these males , 5.912.984 are aged between 18 and 70 years .", "application of our lognormal formula shows that in the netherlands there are 70.305 men ( 1.19% ) who ejaculate within 1 minute , and 48.546 ( 0.82% ) men who ejaculate within 30 seconds .", "in addition , using this formula , 502.012 ( 8.5% ) men ejaculate between 0 and 2 minutes , 2.419.002 men ( 40.9% ) who ejaculate between 1 and 5 minutes , and 1.971.330 ( 33.3% ) men who ejaculate between 5 and 10 minutes .", "in addition , there are 1.112.291 men ( 18.8% ) who ejaculate between 10 and 20 minutes .", "notably , after 20 minutes , the number and percentages decrease : 233.208 men ( 3.9% ) ejaculate between 20 and 30 minutes and 66.225 men ( 1.12% ) ejaculate between 30 and 40 minutes .", "a minority of 40.633 ( 0.687% ) ejaculates with an ielt of more than 40 minutes .", "the application of the formula , which describes the probability density of the ielt , shows that it may provide answers to questions that can not be measured by a stopwatch , due to the extremely large number of men that otherwise ought to be included in an epidemiological study , or just can not be measured as the ielt duration is too long and potentially painful for the female partner of a subject participating in a scientific study .", "notably , it is questioned why the ielt distribution of the men with lifelong pe is so completely different from the ielt distribution of men in the general male population .", "further research into this question is warranted and is presumably associated with genetic and epi - genetic factors . according to the recent classification by waldinger and schweitzer , there are 4 pe subtypes , e.g. , lifelong pe , acquired pe , subjective pe , and variable pe .", "their prevalence in the general population differs significantly , as has been shown by serefoglu et al . and gao et al . , with the lowest prevalence of lifelong pe .", "notably , lifelong pe is characterized by a hypertonic state , consisting of premature ejaculation ( ejaculatio praecox ) , a facilitated erection ( erectio praecox ) and a facilitated penile detumescence ( detumescentia praecox ) , when the male is becoming involved in an erotic situation .", "the current study shows that lifelong pe not only differs from the 3 other pe subtypes with regard to this triad of symptoms , but that lifelong pe has a different ielt and a different ielt distribution than males in the general population , of which men with variable and subjective pe are part of .", "the separate state of lifelong pe among the 3 other pe subtypes with regard to the ielt and its ielt distribution has consequences for the methodology and design of drug treatment studies . as has recently been argued by waldinger , the method and design for drug treatment studies of men with lifelong pe who ejaculate within seconds , are not required for drug treatment studies of men with variable and subjective pe with normal ielt values ." ], [ "the ielt distribution of men in the general population is most accurately fitted by the lognormal distribution , whereas the ielt distribution of men with lifelong pe is most accurately fitted by the gumbel max distribution .", "as there are significantly more men with lifelong pe who ejaculate within 30 and 60 seconds than can be extrapolated from the probability density curve of the ielt distribution of men in the general population it is concluded that men with lifelong pe have a distinct ielt distribution that can only be retrieved from the general male population ielt distribution when thousands of men would participate in a stopwatch study of the ielt .", "the cause of the difference in ielt distribution should be further investigated and may be due to genetic and epigenetic factors .", "application of the mathematical formula of the lognormal ielt distribution is useful for epidemiological research of the ielt in large populations of men ." ] ]

[ "opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration", "patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively .", "the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) .", "patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm .", "exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci .", "baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts .", "history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded .", "blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes .", "patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively .", "the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des .", "blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) .", "pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi", "iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained .", "type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus .", "type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present .", "type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions . mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis .", "cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) .", "mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent", "thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure .", "stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours .", "secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding .", "all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group .", "the academic research consortium definitions were used for stent thrombosis.14 results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables .", "the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables .", "all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows .", "opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration", "patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively .", "the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) .", "patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm .", "exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci .", "baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts .", "history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded .", "blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes .", "patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively .", "the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des .", "blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) .", "pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi", "iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained .", "type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus .", "type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present .", "type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions . mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis .", "cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) .", "mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent", "thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure .", "stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours .", "secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding .", "all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group .", "the academic research consortium definitions were used for stent thrombosis.14 results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables .", "the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables .", "all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows .", "opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration", "patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively .", "the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) .", "patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm .", "exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci .", "all the patients underwent complete history and physical examination . baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts .", "history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded .", "blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes .", "patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups . all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively .", "the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des .", "blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) .", "pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi", "iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained .", "type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus .", "type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present .", "type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions .", "mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis .", "cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) .", "mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent", "thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure .", "stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours .", "secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding .", "all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group .", "results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables .", "the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables .", "all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows .", "the total study population represents a group of 442 patients ( mean age 59 9.5 years ) with stable angina pectoris in whom pci was performed . among them , 225 participants implanted bms and the rest implanted des .", "a total of 224 patients treated with osvix ( 110 patients in bms and 114 patients in des ) and 218 subjects were treated with plavix ( 115 patients in bms and 103 patients in des ) .", "except male to female ratio that was higher in the osvix than the plavix group , the two drug groups were comparable with respect to baseline characteristics , clinical data especially left ventricular ejection fraction , and medical history .", "there were significantly more frequent hypertensive patients in the osvix than in the plavix group ( p < 0.01 ) , but no other significant differences were seen in patient characteristics between the two groups table 2 . besides , in both groups with des and bms , there was no significant difference in the type of coronary lesion , direct stenting , stent length and stent diameter ( table 3 ) .", "baseline characteristics and clinical data of all participants based on plavix or osvix use recorded characteristics and clinical data of all patients based on their stent type . coronary angiography and stent characteristics of patients and", "drug type used figure 1 shows the frequency and 95% confidence intervals of events such as death , instant thrombosis , mace rate , and mi following osvix or plavix placement in bare - metal or drug eluting stents patients . frequency and 95% confidence intervals of events following osvix or plavix placement in bare - metal or drug eluting stents patients", "there was no significant difference regarding mace rate in patients treated with osvix compared with plavix ( figure 1 ) .", "the study groups had the same frequency of mi , thrombosis , stroke and tvr ( figure 1 ) .", "regarding changes in laboratory variable , no significant changes were found concerning liver enzymes , platelet and neutrophil counts between the two drug regimens in both des and bms groups ( figures 2 and 3 ) .", "changes of serum platelet and neutrophil counts following osvix and plavix regimens in patients who underwent bare - metal or drug eluting stenting changes of serum aspartate aminotransferase and alanine aminotransferase levels following osvix and plavix regimens in patients who underwent bare - metal or drug eluting stenting", "opcess is the first randomized trial to compare the effect of a generic form of clopidogrel on mace after coronary stenting in iran .", "increased platelet activity has been shown to increase cardiovascular complications in acute coronary syndrome ( acs ) , and subsequent to pci.15 various studies have demonstrated that administration of antiplatelet drugs such as aspirin , ticlopidine and clopidogrel reduce short and long - term cardiovascular events in patients suffering acs or undergoing pci.16 we studied the short and long - term cardiovascular complications , including death , myocardial infarction , stroke , tlr and tvr in patients treated with osvix ( 300 mg bolus , and 75 mg / daily maintenance ) or plavix ( 300 mg bolus , and 75 mg / daily maintenance ) .", "assessment of secondary end - points such as angina , heart failure ( hf ) , major hemorrhagic episodes , and reduction of platelet or neutrophil counts and liver enzyme changes revealed no significant difference between the two treated groups . reduced systolic", "performance and ageing are among important factors underlying mace in these patients . however , the frequency distribution of age , sex and left ventricular systolic function were similar in osvix and plavix groups .", "tavazzi showed that concomitant disease and cvd risk factors can increase mace.17 whereas these risk factors were evenly distributed in the two groups in this randomized clinical trial ; a higher frequency of hypertension in the osvix group was seen ( p = 0.001 ) ( table 2 ) .", "kang and colleagues demonstrated that hypertension does not affect short and long - term cardiovascular complications of pci.18 patients in osvix group showed nonsignificant lower mace rate than the plavix group ( p < 0.05 ) .", "thus , it can be concluded that osvix has at least similar cvd complications as plavix .", "ali - doosti and colleagues showed that the type of vessel selected for pci did not affect the short and long term complications of the procedure.19 in our study , the frequency distribution according to the vessel elected for angioplasty was the same in both groups .", "various studies have demonstrated an increase in short and long term post - angioplasty complications in patients with complex lesions : the worse the anatomy and morphology of the lesion , the more difficult the procedure and the poorer the outcomes.2021 in our study , the frequency distribution of the type of coronary lesion elected for angioplasty was not significantly different in the osvix and plavix groups . in separate studies , singh and", "pompa showed that the length and diameter of stents are directly , and inversely proportional to tlr.2223 in our study , the stent length and diameter , as well as angioplasty technique had similar distribution in both groups , although the rate of tvr was low in our study as shown by results .", "this may have been due to the short follow - up period ( one month for non - pharmacological stents and six months for pharmacological stents ) .", "tvr was seen in two of our patients who underwent cabg a day after unsuccessful angioplasty .", "based on comparison of frequency distribution of patients according to mace in the preceding one and six months for bms and des stents , respectively , the incidence of mace including death was similar in the osvix and plavix groups .", "the incidence of mi was 0.4% in the osvix and 1.4% in the pla - vix groups with no significant difference .", "no significant difference was observed between the two groups in regard to episodes of angina , hf and bleeding which suggest no inferiority of osvix compared to plavix . although the occurrence of events was not significantly different in the two groups , the distribution of findings revealed an up - sloping trend in complications in the plavix group .", "thus , it may be concluded that osvix was not associated with more complications than plavix in patients who underwent pci .", "safety of antiplatelet drugs is of great importance and must be assessed against possible side - effects .", "gurbel demonstrated that including clopidogrel in the therapy of unstable angina patients , significantly reduced mortality from mace .", "although clopidogrel may significantly increase the risk of bleeding , its benefits have been shown to outweigh the risks.24 yusuf and colleagues showed that adding clopidogrel to aspirin reduced mace but increased the risk of bleeding . however , in our study , the rate of major bleeding in osvix and plavix groups who were treated with aspirin too , were not significant . during our follow - up period ,", "mean platelet counts did not change significantly in either groups ( plavix , p = 0.80 and osvix , p = 0.99 ) or between them ( p = 0.87 ) .", "however , neutrophil counts were reduced significantly in the two groups ( p < 0.001 ) , but the trend of change in counts was not significantly different between the two groups .", "the above findings showed that adverse hematological effects ( thrombopenia or neutropenia ) did not increase in the osvix group as compared to the plavix group .", "we noted some limitations to our study , the most important of which was the small study population .", "however , the power of the study was enough to achieve the main and secondary objectives of the study .", "the other important limitation was that the angiographies were performed by different cardiologists in different centers .", "thus , interobserver variability was inevitable . in conclusion , our data demonstrated the safety of osvix in improving clinical outcome compared to plavix after coronary stenting in bms or des using patients .", "the frequency of mace , non - cardiac events or adverse hematological changes did not increase with osvix compared to plavix patients which suggest it 's safety .", "however , due to more cost advantages of osvix regimen over plavix , administration of osvix among iranian patients is preferable .", "akh designed the study and drafted this manuscript , mp designed the study , mo , gkm , mn , akh , mh , fr , shs , and jk carried the study , mgh drafted the manuscript and tables , hz helped in designing the study and carried the study , aat helped in designing the study , bs data analysis and drafted the tables , mgh helped in designing the study , mrkh and fn helped in statistical analysis , ekh and mj helped in data gathering , and ns helped in designing the study , revising the manuscript procedures , and drafting the manuscript ." ]

[ "<S> background : to compare the early and late cardiovascular events as well as side effects of osvix , a generic form of clopidogrel versus plavix regimens in patients with chronic stable angina , undergoing bare metal stent ( bms ) or drug eluting stent ( des ) placement , this study was carried out.methods:a total of 442 patients with chronic stable angina who were scheduled for elective percutaneous coronary intervention ( pci ) were included in a randomized , double blind , multi - centric clinical trial being performed in 6 distinct university hospitals in 5 cities of iran from march 2007 to november 2009 . </S>", "<S> baseline , demographic and history of risk factors were recorded using the patients medical charts . </S>", "<S> stenting procedure was performed via transfemoral approach using low osmolar contrast agents . </S>", "<S> patients underwent bms or des placements based on the physician selection and were randomly assigned to osvix or plavix groups . </S>", "<S> patients were followed by telephone in 0 and 6 months intervals regarding the major adverse cardiovascular events ( mace ) including death , myocardial infarction , in - stent thrombosis , stroke , target lesion revascularization , and target vascular revascularization . </S>", "<S> angina episodes , bleeding , liver enzymes , neutrophils and platelets count were also assessed in these intervals.results:there was not any significant difference between these two groups regarding the baseline characteristics . in the des group , </S>", "<S> the 6-month mortality rate and the incidence of mace in osvix and plavix groups were 0.9% and 1.9% ( p = 0.61 ) and 1.8% and 4.9% ( p = 0.26 ) , respectively . during the follow up period after des or bms placement , there was nt any significant difference regarding neutrophil and platelet counts or liver enzymes between study groups.conclusions:using osvix and plavix are followed by similar major cardiovascular events and side - effect profile in patients undergoing pci . </S>" ]

[ "Methods", "None", "Study Population", "Study participants", "Study Protocol", "Angioplasty procedure", "Study end points", "Statistical analysis", "Results", "Discussion", "Authors Contributions" ]

[ [ "opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration", "patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively .", "the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) .", "patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm .", "exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci .", "baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts .", "history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded .", "blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes .", "patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively .", "the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des .", "blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) .", "pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi", "iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained .", "type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus .", "type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present .", "type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions . mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis .", "cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) .", "mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent", "thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure .", "stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours .", "secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding .", "all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group .", "the academic research consortium definitions were used for stent thrombosis.14 results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables .", "the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables .", "all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows ." ], [ "opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration", "patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively .", "the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) .", "patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm .", "exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci .", "baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts .", "history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded .", "blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes .", "patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively .", "the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des .", "blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) .", "pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi", "iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained .", "type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus .", "type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present .", "type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions . mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis .", "cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) .", "mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent", "thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure .", "stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours .", "secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding .", "all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group .", "the academic research consortium definitions were used for stent thrombosis.14 results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables .", "the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables .", "all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows ." ], [ "opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration", "patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups .", "all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively .", "the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) ." ], [ "patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm .", "exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci ." ], [ "all the patients underwent complete history and physical examination . baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts .", "history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded .", "blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes .", "patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups . all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively .", "the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des .", "blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) ." ], [ "pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi", "iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained .", "type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus .", "type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present .", "type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions ." ], [ "mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis .", "cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) .", "mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent", "thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure .", "stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours .", "secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding .", "all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group ." ], [ "results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables .", "the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables .", "all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows ." ], [ "the total study population represents a group of 442 patients ( mean age 59 9.5 years ) with stable angina pectoris in whom pci was performed . among them , 225 participants implanted bms and the rest implanted des .", "a total of 224 patients treated with osvix ( 110 patients in bms and 114 patients in des ) and 218 subjects were treated with plavix ( 115 patients in bms and 103 patients in des ) .", "except male to female ratio that was higher in the osvix than the plavix group , the two drug groups were comparable with respect to baseline characteristics , clinical data especially left ventricular ejection fraction , and medical history .", "there were significantly more frequent hypertensive patients in the osvix than in the plavix group ( p < 0.01 ) , but no other significant differences were seen in patient characteristics between the two groups table 2 . besides , in both groups with des and bms , there was no significant difference in the type of coronary lesion , direct stenting , stent length and stent diameter ( table 3 ) .", "baseline characteristics and clinical data of all participants based on plavix or osvix use recorded characteristics and clinical data of all patients based on their stent type . coronary angiography and stent characteristics of patients and", "drug type used figure 1 shows the frequency and 95% confidence intervals of events such as death , instant thrombosis , mace rate , and mi following osvix or plavix placement in bare - metal or drug eluting stents patients . frequency and 95% confidence intervals of events following osvix or plavix placement in bare - metal or drug eluting stents patients", "there was no significant difference regarding mace rate in patients treated with osvix compared with plavix ( figure 1 ) .", "the study groups had the same frequency of mi , thrombosis , stroke and tvr ( figure 1 ) .", "regarding changes in laboratory variable , no significant changes were found concerning liver enzymes , platelet and neutrophil counts between the two drug regimens in both des and bms groups ( figures 2 and 3 ) .", "changes of serum platelet and neutrophil counts following osvix and plavix regimens in patients who underwent bare - metal or drug eluting stenting changes of serum aspartate aminotransferase and alanine aminotransferase levels following osvix and plavix regimens in patients who underwent bare - metal or drug eluting stenting" ], [ "opcess is the first randomized trial to compare the effect of a generic form of clopidogrel on mace after coronary stenting in iran .", "increased platelet activity has been shown to increase cardiovascular complications in acute coronary syndrome ( acs ) , and subsequent to pci.15 various studies have demonstrated that administration of antiplatelet drugs such as aspirin , ticlopidine and clopidogrel reduce short and long - term cardiovascular events in patients suffering acs or undergoing pci.16 we studied the short and long - term cardiovascular complications , including death , myocardial infarction , stroke , tlr and tvr in patients treated with osvix ( 300 mg bolus , and 75 mg / daily maintenance ) or plavix ( 300 mg bolus , and 75 mg / daily maintenance ) .", "assessment of secondary end - points such as angina , heart failure ( hf ) , major hemorrhagic episodes , and reduction of platelet or neutrophil counts and liver enzyme changes revealed no significant difference between the two treated groups . reduced systolic", "performance and ageing are among important factors underlying mace in these patients . however , the frequency distribution of age , sex and left ventricular systolic function were similar in osvix and plavix groups .", "tavazzi showed that concomitant disease and cvd risk factors can increase mace.17 whereas these risk factors were evenly distributed in the two groups in this randomized clinical trial ; a higher frequency of hypertension in the osvix group was seen ( p = 0.001 ) ( table 2 ) .", "kang and colleagues demonstrated that hypertension does not affect short and long - term cardiovascular complications of pci.18 patients in osvix group showed nonsignificant lower mace rate than the plavix group ( p < 0.05 ) .", "thus , it can be concluded that osvix has at least similar cvd complications as plavix .", "ali - doosti and colleagues showed that the type of vessel selected for pci did not affect the short and long term complications of the procedure.19 in our study , the frequency distribution according to the vessel elected for angioplasty was the same in both groups .", "various studies have demonstrated an increase in short and long term post - angioplasty complications in patients with complex lesions : the worse the anatomy and morphology of the lesion , the more difficult the procedure and the poorer the outcomes.2021 in our study , the frequency distribution of the type of coronary lesion elected for angioplasty was not significantly different in the osvix and plavix groups . in separate studies , singh and", "pompa showed that the length and diameter of stents are directly , and inversely proportional to tlr.2223 in our study , the stent length and diameter , as well as angioplasty technique had similar distribution in both groups , although the rate of tvr was low in our study as shown by results .", "this may have been due to the short follow - up period ( one month for non - pharmacological stents and six months for pharmacological stents ) .", "tvr was seen in two of our patients who underwent cabg a day after unsuccessful angioplasty .", "based on comparison of frequency distribution of patients according to mace in the preceding one and six months for bms and des stents , respectively , the incidence of mace including death was similar in the osvix and plavix groups .", "the incidence of mi was 0.4% in the osvix and 1.4% in the pla - vix groups with no significant difference .", "no significant difference was observed between the two groups in regard to episodes of angina , hf and bleeding which suggest no inferiority of osvix compared to plavix . although the occurrence of events was not significantly different in the two groups , the distribution of findings revealed an up - sloping trend in complications in the plavix group .", "thus , it may be concluded that osvix was not associated with more complications than plavix in patients who underwent pci .", "safety of antiplatelet drugs is of great importance and must be assessed against possible side - effects .", "gurbel demonstrated that including clopidogrel in the therapy of unstable angina patients , significantly reduced mortality from mace .", "although clopidogrel may significantly increase the risk of bleeding , its benefits have been shown to outweigh the risks.24 yusuf and colleagues showed that adding clopidogrel to aspirin reduced mace but increased the risk of bleeding . however , in our study , the rate of major bleeding in osvix and plavix groups who were treated with aspirin too , were not significant . during our follow - up period ,", "mean platelet counts did not change significantly in either groups ( plavix , p = 0.80 and osvix , p = 0.99 ) or between them ( p = 0.87 ) .", "however , neutrophil counts were reduced significantly in the two groups ( p < 0.001 ) , but the trend of change in counts was not significantly different between the two groups .", "the above findings showed that adverse hematological effects ( thrombopenia or neutropenia ) did not increase in the osvix group as compared to the plavix group .", "we noted some limitations to our study , the most important of which was the small study population .", "however , the power of the study was enough to achieve the main and secondary objectives of the study .", "the other important limitation was that the angiographies were performed by different cardiologists in different centers .", "thus , interobserver variability was inevitable . in conclusion , our data demonstrated the safety of osvix in improving clinical outcome compared to plavix after coronary stenting in bms or des using patients .", "the frequency of mace , non - cardiac events or adverse hematological changes did not increase with osvix compared to plavix patients which suggest it 's safety .", "however , due to more cost advantages of osvix regimen over plavix , administration of osvix among iranian patients is preferable ." ], [ "akh designed the study and drafted this manuscript , mp designed the study , mo , gkm , mn , akh , mh , fr , shs , and jk carried the study , mgh drafted the manuscript and tables , hz helped in designing the study and carried the study , aat helped in designing the study , bs data analysis and drafted the tables , mgh helped in designing the study , mrkh and fn helped in statistical analysis , ekh and mj helped in data gathering , and ns helped in designing the study , revising the manuscript procedures , and drafting the manuscript ." ] ]

[ "animals and treatment : all procedures in this experiment were conducted following protocols approved by the iwate university laboratory animal \n care and use committee . four primiparous non - lactating , rumen - cannulated healthy holstein cattle , weighing 645 62 ( mean sd ) kg , were housed in a stanchion \n barn at the cattle research center of iwate university . during the experimental period , the cattle were fed a sara - inducing diet 2 weeks before and 1 week after \n first administration of bp .", "the sara - inducing diet was composed of a mix of orchard grass and timothy hay with an equivalent amount of flaked barley and corn . \n", "the ratio of roughage - to - concentrate was 3:7 , and the percentages of total digestible nutrients , crude \n protein , neutral detergent fiber , non - fiber carbohydrates and starch in the dry matter were adjusted to 75.1 , 12.2 , 37.7 , 42.4 and 37.0% , respectively .", "cattle \n were fed daily at 9:00 and 17:00 and allowed free access to fresh water .", "cattle were assigned randomly to a 4 4 latin square experimental design containing \n three treatments and a control .", ", tokyo , japan ) included l. plantarum strain 220 ( 9 10 \n colony - forming units ( cfu)/g ) , e. faecium strain 26 ( 9 10 cfu / g ) and c. butyricum strain miyari ( 9 \n 10 cfu / g ) was administered as a daily single dose of 20 , 50 or 100 g per head for 7 consecutive days .", "cows fed the sara - inducing diet without bp \n served as a control . based on the effects of the treatment , the experimental design consisted of 2 weeks of an adaptation period to the respective bp treatment \n during which cattle were fed only hay .", "the bp was stored at 4c , and each dose was mixed with their diet during the morning feeding .", "ruminal ph measurements : ruminal ph was measured with a radio transmission ph measurement system ( ycow - s ; dkk - toa yamagata , yamagata , japan ) . \n", "the ph sensor was calibrated with ph 4 and 7 buffer solutions at the start of each experiment and was placed in the ventral sac of the rumen via the rumen \n cannula , as the ventral sac of the rumen has more stable ph values than the other ruminal site .", "ruminal fluid sampling and vfa , lactic acid and nh3-n assays : ruminal fluids were collected from the same location as the ph sensor \n through the rumen cannula at 8:00 , 11:00 , 14:00 , 17:00 and 20:00 on 7 days before ( day 7 ) and 7 days after ( day 7 ) the first bp administration ( day 0 ) . these \n sampling times ( 8:0020:00 )", "samples for the vfa , lactic acid and \n nh3-n measurements were filtered immediately through two layers of cheesecloth .", "for the vfa analysis , 10 ml of ruminal fluid was \n added to 2 ml of 25% metaphosphoric acid in 3 n sulfuric acid .", "total vfas and three individual vfas ( acetic acid , propionic acid and butyric \n acid ) were separated and quantified with gas chromatography ( model 135 , hitachi , tokyo , japan ) using a packed glass column ( 3% thermon-3000 ) on a shimalite tpa \n 6080 support ( shinwa chemical industries ltd . ,", "the ruminal fluid was centrifuged immediately at 2,000 \n g for 15 min , and concentrations in the supernatant were determined using a commercially available kit ( f - kit ; d - lactate / l - lactate , j. k. international \n co. , tokyo , japan ) . to measure nh3-n concentrations , ruminal", "fluid was analyzed using the steam distillation method with an nh3-n analyzer \n ( kjeltec auto sampler system 1035 analyzer , tecator , sweden ) .", "blood sampling and plasma metabolite profiles : blood samples were collected from the jugular vein into 10-ml evacuated \n serum - separating tubes and tubes containing sodium fluoride ( bd vacutainer , franklin lakes , nj , u.s.a . ) .", "blood samples were collected at the same time as the \n ruminal fluid samples ( days 7 and 7 ) , centrifuged ( 1,500 g , 15 min , 4c ) immediately to separate the serum and plasma , and preserved at 80c \n until the analysis . glucose ( glu ) , non - esterified fatty acids ( nefa ) and -hydroxybutyrate acid ( bhba ) in plasma , as well as total cholesterol ( t - chol ) , \n triglycerides ( tg ) and blood urea nitrogen ( bun ) in sera were analyzed using an automated biochemistry analyzer ( accute , toshiba ltd . , tokyo , japan ) . statistical analysis : quantitative data are expressed as means standard errors ( ses ) .", "the main effects included the sara challenge and bp \n treatment , day of the experiment , and hours after administration and feeding .", "diurnal measurements of the ruminal ph were analyzed as the 24-hr mean ph from day \n 7 to day 7 .", "the ph data collected in 10-min intervals were summarized as a 1-hr mean from 9:00 to 8:00 of the following day to assess circadian changes .", "the \n minimum and maximum ph in one day was determined for days 7 , 0 and 7 .", "5.01 ( la jolla , ca , u.s.a . ) was used for the statistical calculations , \n and two - way repeated - measures analysis of variance ( anova ) followed by the bonferroni post hoc test was used to evaluate the differences between the treatment and \n control groups .", "ruminal ph : sara was successfully induced in cattle fed the sara - inducing diet . according to representative ph data on 3 days after beginning \n the diet ,", "the 1-hr mean ruminal ph decreased rapidly and slowly after the morning and evening feedings , which was indicative of sara ( fig .", "1.circadian changes in the 1-hr mean ruminal ph at control day ( n=4 ; ) and 3 days after beginning the sara - inducing diet ( n=4 ; ) .", "the 1-hr mean ph in cattle fed sara - inducing diet decreased after the morning and evening feedings , which was indicative of \n successfully induced sara .", "data are means se . * significant difference compared with a control on the same day ( p<0.05 ) . \n", "considerable disparities in the ruminal ph among the treatment and control groups were observed and continued throughout the treatment period ( fig .", "2.changes in the 24-hr mean ruminal ph in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial probiotic for 7 consecutive days . \n", "data are means se . * significant difference compared with a control on \n the same day ( p<0.05 ) .", "the 24-hr mean ruminal ph in the control was generally lower , whereas that in the bp groups receiving 20 or 50 g was significantly higher from days 2 to \n 7 compared with the control .", "the 24-hr mean ph in the 100 g group was also significantly higher from days 2 to 4 compared with the control ; however , it decreased \n on days 5 to 7 . among the treatment groups , the 20 g bp group maintained a constant ph ( 6.46.5 ) from days 3 to 7 .", "circadian changes in the 1-hr mean ruminal ph \n were almost identical among the treatment groups on days 0 and 7 .", "however , the 1-hr mean phs in the treatment groups were slightly higher than those in the \n control group on day 7 ( fig . 3fig .", "3.circadian changes in the 1-hr mean ruminal ph on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial \n probiotic for 7 consecutive days .", "additionally , the mean minimum ph in the control on day 7 was < 5.0 , which was significantly lower than in the treatment groups ( fig .", "4.box plots of the maximum and minimum ruminal ph on days 7 , 0 and 7 in the treatment groups administered 20 g ( n=4 ; light gray boxes ) , 50 g ( n=4 ; gray \n boxes ) and 100 g ( n=4 ; dark boxes ) of a bacterial probiotic for 7 consecutive days .", "cattle not administered the probiotic served as a control ( n=4 ; white \n boxes ) . the median and quartiles", "the upper and lower bars represent the maximum and minimum values , respectively . * \n significant difference compared with a control on the same day ( p<0.05 ) . significant difference compared with \n the same group on day 7 ( p<0.05 ) . ) .", "the mean maximum ph approached > 6.8 in the treatment groups , which did not differ from that in the control .", "circadian changes in the 1-hr mean ruminal ph at control day ( n=4 ; ) and 3 days after beginning the sara - inducing diet ( n=4 ; ) .", "the 1-hr mean ph in cattle fed sara - inducing diet decreased after the morning and evening feedings , which was indicative of \n successfully induced sara .", "data are means se . * significant difference compared with a control on the same day ( p<0.05 ) . \n", "changes in the 24-hr mean ruminal ph in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial probiotic for 7 consecutive days . \n", "* significant difference compared with a control on \n the same day ( p<0.05 ) .", "circadian changes in the 1-hr mean ruminal ph on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial \n probiotic for 7 consecutive days .", "box plots of the maximum and minimum ruminal ph on days 7 , 0 and 7 in the treatment groups administered 20 g ( n=4 ; light gray boxes ) , 50 g ( n=4 ; gray \n boxes ) and 100 g ( n=4 ; dark boxes ) of a bacterial probiotic for 7 consecutive days .", "cattle not administered the probiotic served as a control ( n=4 ; white \n boxes ) . the median and quartiles", "the upper and lower bars represent the maximum and minimum values , respectively . * \n significant difference compared with a control on the same day ( p<0.05 ) . significant difference compared with \n the same group on day 7 ( p<0.05 ) .", "ruminal vfa , lactic acid and nh3-n concentrations : total vfa increased from 8:00 to 20:00 in the treatment and control groups on days \n 0 and 7 ( fig . 5fig .", "5.circadian changes in the ruminal vfa , a : p ratio , lactic acid and nh3-n concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g \n ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial probiotic for 7 consecutive days .", "* significant difference compared with a control on the same day ( p<0.05 ) . )", ". however , no difference was observed in the total or individual vfa concentrations between the treatment and control groups . the acetic acid - to - propionic \n acid ratio ( a : p ) was almost identical among the treatment groups .", "lactic acid concentrations remained stable in the treatment groups , and the concentrations at \n 20:00 on day 7 were significantly lower than that in the control . no difference was observed in nh3-n concentrations among the treatment groups , \n although the nh3-n concentrations in the 50 g and 100 g groups at 8:00 on day 7 were significantly higher than that in the control .", "circadian changes in the ruminal vfa , a : p ratio , lactic acid and nh3-n concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g \n ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial probiotic for 7 consecutive days .", "data are \n means se . * significant difference compared with a control on the same day ( p<0.05 ) .", "blood metabolite profiles : glu concentrations decreased from 8:00 to 20:00 , but were almost the same among the treatment and control groups \n ( fig .", "6.circadian changes in the blood glu , nefa , bhba and bun concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) \n of a bacterial probiotic for 7 consecutive days .", "data are means se . * \n significant difference compared with a control on the same day ( p<0.05 ) . ) .", "nefa levels were affected by feeding time and were significantly lower at 20:00 compared with at 8:00 in the treatment groups on days 0 and 7 . \n", "furthermore , nefa level in the 50 g group at 20:00 on day 7 was significantly lower than that in the control .", "in contrast , bhba and bun concentrations were higher \n and lower , respectively , in all groups from 8:00 to 20:00 .", "bhba concentrations in the 20 g and 50 g at 17:00 and 20:00 , and bun concentrations in the treatment \n groups at 8:00 were significantly higher than that in the controls , respectively .", "circadian changes in the blood glu , nefa , bhba and bun concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) \n of a bacterial probiotic for 7 consecutive days .", "data are means se . * \n significant difference compared with a control on the same day ( p<0.05 ) .", "sara occurs when the ruminal ph decreases due to a combination of overproduction of vfa and lactic acid and a decrease in vfa absorption in the rumen [ 1 , 22 ] .", "sara is diagnosed when the ph in the ruminal fluid is < 5.6 for at least 3 hr \n per day [ 5 , 17 , 21 ] . in this study ,", "the \n ruminal ph measured continuously indicated that sara was successfully induced during the experimental period , especially during the first week of the experiment , \n by feeding a sara - inducing diet .", "studies have shown that the main ruminal bacteria affecting the ruminal fermentative capacity are classified as lactic acid - producers and consumers [ 4 , 15 ] , and both of these bacterial groups have been used as bps .", "( 2002 ) reported that a bp containing both lactate - consuming \n ( propionibacterium ) and lactate - producing ( enterococcus ) bacteria activated ruminal fermentation and reduced the risk of \n acidosis in cattle . in another study , a bp consisting of l. plantarum and e. faecium induced changes in the ruminal ph of cows \n fed a high - grain diet .", "however , the mechanism of the effects of bp on ruminal fermentation is unclear , and the effects \n of bp on sara in cattle are unknown . in this study , the 24-hr mean ruminal ph was higher in the bp - treated groups compared with in the control group during sara \n challenge .", "this was indicative of increased ruminal fermentation in the cows , as reported previously [ 12 , 27 , 28 ] .", "( 2012 ) examined the effects of administering bp in sara - challenged cows and found no effects on ruminal ph when bp was administered as a single strain ; \n however , ruminal ph increased compared over that in the control group when using a combination of e. faecium and yeast .", "we used the same bp in a \n previous study on weaned calves fed a high - concentrate diet , and the mean ruminal ph was significantly higher in the treatment group compared with in the control \n group . in this study , different doses of a multi - strain bp were administered to sara - challenged cows , and the 24-hr \n mean ruminal ph was notably higher on days 3 to 7 , whereas the minimum ph increased on day 7 in the treatment groups .", "bp appears to increase the ability of ruminal bacteria to metabolize lactic acid and regulate ruminal ph [ 20 , 21 ] .", "it has been hypothesized that the functionality and efficacy of bps can be determined based on their effects on the \n predominant rumen microbiota [ 7 , 12 ] . in this study , cattle receiving lower bp doses \n ( 20 g per head ) had a constant mean ph of 6.46.6 .", "in contrast , cattle receiving higher bp doses ( 100 g per head ) had higher mean ph on days 2 and 3 , which \n decreased on days 5 to 7 .", "previous reports have indicated that changes in rumen bacterial composition and diversity , increased activities of lactate - consuming \n bacteria and greater lactate absorption affect ruminal ph [ 13 , 15 ] .", "the higher bp \n dose might increase lab numbers in the rumen , which could be related to increased ruminal fermentation capacity , higher carbohydrate fermentation and increased \n ruminal ph .", "therefore , ruminal lab might have been overly increased due to the higher bp dose , causing the ruminal \n fermentation and ph to decrease on day 5 .", "these results are consistent with previous reports that lower bp doses improve gastrointestinal tract microbiota in \n calves .", "furthermore , weaned calves administered lower bp doses showed a higher mean ruminal ph compared with controls \n . based on our results , administration of an extremely high dose ( 100 g per head ) of lab could cause decreased \n ruminal fermentation and ruminal ph .", "decreases in ruminal ph are related to vfa production and lactic acid accumulation after feeding a high - grain diet [ 1 , \n 18 ] . in this study , although ruminal vfa concentrations were not affected by bp treatment , they were affected by feeding \n time .", "this is in agreement with previous reports in which ruminal vfas were not affected by probiotics including lab [ 6 , \n 20 ] .", "administration of lab probiotics is thought to help rumen microbiota adapt to the presence of lactic acid and prevent lactate accumulation in the rumen .", "( 1992 ) reported that lactate - consuming bacteria increased only when lactic acid accumulated and ruminal ph decreased . \n", "based on these results , lab probiotics might affect ruminal ph by increasing the activity of lactate - consuming bacteria . \n in our previous study , significantly lower lactic acid concentrations were observed in the ruminal fluid of weaned calves receiving bp compared with the control \n . in this study ,", "lactic acid concentrations were stable in the treatment groups ; however , lactic acid was higher and \n correlated with a lower mean ruminal ph in the control group . gradually increasing ruminal", "ph might be due to time after feeding and the absorption capacity of \n the rumen .", "bps may prevent a decline in ruminal ph by increasing the lactic acid consumption by some microbes [ 3 , 6 ] .", "a combination of certain probiotic bacteria that synthesize lactic acid may sustain \n a tonic level of lactic acid in the rumen , stimulating rumen microbial communities that consume lactic acid and reducing acidity , causing the ruminal ph to remain \n constant and stable .", "conversely , the nh3-n concentrations in the ruminal fluid of the bp groups remained stable in this study , although significant difference was observed \n at 8:00 on day 7 between the bp - treated and control groups .", "bp has no effect on the nh3-n concentrations in the rumen [ 7 , 12 ] .", "decreases in nh3-n levels after morning feeding in this study were in agreement with the \n previous study , which found that nh3-n concentrations decreased with decreasing ruminal ph [ 7 , 13 ] .", "it has been reported that nefa levels are \n lower in steers with sara compared with in the control ; therefore , the decrease in nefa levels indicates a more \n efficient use of dietary energy and greater dry matter intake in the bp - treated groups . in conclusion ,", "repeated administration of a bp comprised of l. plantarum , e. faecium and c. butyricum improved \n the 24-hr mean ruminal ph in cattle with experimentally induced sara at doses of 20 or 50 g per head .", "diurnal patterns of the 1-hr mean ruminal ph were identical \n among the treatment and control groups .", "ruminal vfa was not affected by bp treatment ; however , lactic acid was lower in the treatment groups than in the control \n group . based on these results", ", bp might affect ruminal ph by increasing lactate consumption and decreasing lactic acid concentrations , resulting in a consistently \n higher ruminal ph in sara cattle .", "these results suggest that repeated administration of a multi - strain bp might reduce the risk of sara in cattle and that \n consecutive treatment with 20 or 50 g of a bp containing lab during high - concentrate feeding might reduce the incidence of sara in dairy cattle ." ]

[ "<S> effects of a bacterial probiotic ( bp ) on ruminal fermentation and plasma metabolites were evaluated in four holstein cattle ( body weight , 645 62 kg ; mean \n sd ) with induced subacute ruminal acidosis ( sara ) . </S>", "<S> sara was induced by feeding a sara - inducing diet , and thereafter , 20 , 50 or 100 g per head of a commercial bp \n was administered for 7 consecutive days during the morning feeding . </S>", "<S> cattle without bp served as the control . </S>", "<S> the 24-hr mean ruminal ph in the control was lower , \n whereas those in the bp groups administered 20 or 50 g were significantly higher compared to the control from days 2 to 7 . </S>", "<S> circadian patterns of the 1-hr mean \n ruminal ph were identical ( 6.46.8 ) among all cattle receiving bp . although the mean minimum ph in the control on day 7 and day 0 was < 5.8 , the ph in the \n treatment groups on day 7 was > 5.8 and significantly higher than that of the control group ( > 5.2 ) . </S>", "<S> ruminal volatile fatty acid ( vfa ) concentrations were \n not affected by bp treatment ; however , the bp groups had lower lactic acid levels compared with the control group at 20:00 on day 7 . </S>", "<S> additionally , \n non - esterified fatty acid levels decreased from 8:00 to 20:00 in all bp groups on day 7 . </S>", "<S> these results suggest that administration of 20 to 50 g of a \n multi - strain bp for 7 days might improve the low ph and high lactic acid level of the ruminal fluid in sara cattle . </S>" ]

[ "MATERIALS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "animals and treatment : all procedures in this experiment were conducted following protocols approved by the iwate university laboratory animal \n care and use committee . four primiparous non - lactating , rumen - cannulated healthy holstein cattle , weighing 645 62 ( mean sd ) kg , were housed in a stanchion \n barn at the cattle research center of iwate university . during the experimental period , the cattle were fed a sara - inducing diet 2 weeks before and 1 week after \n first administration of bp .", "the sara - inducing diet was composed of a mix of orchard grass and timothy hay with an equivalent amount of flaked barley and corn . \n", "the ratio of roughage - to - concentrate was 3:7 , and the percentages of total digestible nutrients , crude \n protein , neutral detergent fiber , non - fiber carbohydrates and starch in the dry matter were adjusted to 75.1 , 12.2 , 37.7 , 42.4 and 37.0% , respectively .", "cattle \n were fed daily at 9:00 and 17:00 and allowed free access to fresh water .", "cattle were assigned randomly to a 4 4 latin square experimental design containing \n three treatments and a control .", ", tokyo , japan ) included l. plantarum strain 220 ( 9 10 \n colony - forming units ( cfu)/g ) , e. faecium strain 26 ( 9 10 cfu / g ) and c. butyricum strain miyari ( 9 \n 10 cfu / g ) was administered as a daily single dose of 20 , 50 or 100 g per head for 7 consecutive days .", "cows fed the sara - inducing diet without bp \n served as a control . based on the effects of the treatment , the experimental design consisted of 2 weeks of an adaptation period to the respective bp treatment \n during which cattle were fed only hay .", "the bp was stored at 4c , and each dose was mixed with their diet during the morning feeding .", "ruminal ph measurements : ruminal ph was measured with a radio transmission ph measurement system ( ycow - s ; dkk - toa yamagata , yamagata , japan ) . \n", "the ph sensor was calibrated with ph 4 and 7 buffer solutions at the start of each experiment and was placed in the ventral sac of the rumen via the rumen \n cannula , as the ventral sac of the rumen has more stable ph values than the other ruminal site .", "ruminal fluid sampling and vfa , lactic acid and nh3-n assays : ruminal fluids were collected from the same location as the ph sensor \n through the rumen cannula at 8:00 , 11:00 , 14:00 , 17:00 and 20:00 on 7 days before ( day 7 ) and 7 days after ( day 7 ) the first bp administration ( day 0 ) . these \n sampling times ( 8:0020:00 )", "samples for the vfa , lactic acid and \n nh3-n measurements were filtered immediately through two layers of cheesecloth .", "for the vfa analysis , 10 ml of ruminal fluid was \n added to 2 ml of 25% metaphosphoric acid in 3 n sulfuric acid .", "total vfas and three individual vfas ( acetic acid , propionic acid and butyric \n acid ) were separated and quantified with gas chromatography ( model 135 , hitachi , tokyo , japan ) using a packed glass column ( 3% thermon-3000 ) on a shimalite tpa \n 6080 support ( shinwa chemical industries ltd . ,", "the ruminal fluid was centrifuged immediately at 2,000 \n g for 15 min , and concentrations in the supernatant were determined using a commercially available kit ( f - kit ; d - lactate / l - lactate , j. k. international \n co. , tokyo , japan ) . to measure nh3-n concentrations , ruminal", "fluid was analyzed using the steam distillation method with an nh3-n analyzer \n ( kjeltec auto sampler system 1035 analyzer , tecator , sweden ) .", "blood sampling and plasma metabolite profiles : blood samples were collected from the jugular vein into 10-ml evacuated \n serum - separating tubes and tubes containing sodium fluoride ( bd vacutainer , franklin lakes , nj , u.s.a . ) .", "blood samples were collected at the same time as the \n ruminal fluid samples ( days 7 and 7 ) , centrifuged ( 1,500 g , 15 min , 4c ) immediately to separate the serum and plasma , and preserved at 80c \n until the analysis . glucose ( glu ) , non - esterified fatty acids ( nefa ) and -hydroxybutyrate acid ( bhba ) in plasma , as well as total cholesterol ( t - chol ) , \n triglycerides ( tg ) and blood urea nitrogen ( bun ) in sera were analyzed using an automated biochemistry analyzer ( accute , toshiba ltd . , tokyo , japan ) . statistical analysis : quantitative data are expressed as means standard errors ( ses ) .", "the main effects included the sara challenge and bp \n treatment , day of the experiment , and hours after administration and feeding .", "diurnal measurements of the ruminal ph were analyzed as the 24-hr mean ph from day \n 7 to day 7 .", "the ph data collected in 10-min intervals were summarized as a 1-hr mean from 9:00 to 8:00 of the following day to assess circadian changes .", "the \n minimum and maximum ph in one day was determined for days 7 , 0 and 7 .", "5.01 ( la jolla , ca , u.s.a . ) was used for the statistical calculations , \n and two - way repeated - measures analysis of variance ( anova ) followed by the bonferroni post hoc test was used to evaluate the differences between the treatment and \n control groups ." ], [ "ruminal ph : sara was successfully induced in cattle fed the sara - inducing diet . according to representative ph data on 3 days after beginning \n the diet ,", "the 1-hr mean ruminal ph decreased rapidly and slowly after the morning and evening feedings , which was indicative of sara ( fig .", "1.circadian changes in the 1-hr mean ruminal ph at control day ( n=4 ; ) and 3 days after beginning the sara - inducing diet ( n=4 ; ) .", "the 1-hr mean ph in cattle fed sara - inducing diet decreased after the morning and evening feedings , which was indicative of \n successfully induced sara .", "data are means se . * significant difference compared with a control on the same day ( p<0.05 ) . \n", "considerable disparities in the ruminal ph among the treatment and control groups were observed and continued throughout the treatment period ( fig .", "2.changes in the 24-hr mean ruminal ph in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial probiotic for 7 consecutive days . \n", "data are means se . * significant difference compared with a control on \n the same day ( p<0.05 ) .", "the 24-hr mean ruminal ph in the control was generally lower , whereas that in the bp groups receiving 20 or 50 g was significantly higher from days 2 to \n 7 compared with the control .", "the 24-hr mean ph in the 100 g group was also significantly higher from days 2 to 4 compared with the control ; however , it decreased \n on days 5 to 7 . among the treatment groups , the 20 g bp group maintained a constant ph ( 6.46.5 ) from days 3 to 7 .", "circadian changes in the 1-hr mean ruminal ph \n were almost identical among the treatment groups on days 0 and 7 .", "however , the 1-hr mean phs in the treatment groups were slightly higher than those in the \n control group on day 7 ( fig . 3fig .", "3.circadian changes in the 1-hr mean ruminal ph on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial \n probiotic for 7 consecutive days .", "additionally , the mean minimum ph in the control on day 7 was < 5.0 , which was significantly lower than in the treatment groups ( fig .", "4.box plots of the maximum and minimum ruminal ph on days 7 , 0 and 7 in the treatment groups administered 20 g ( n=4 ; light gray boxes ) , 50 g ( n=4 ; gray \n boxes ) and 100 g ( n=4 ; dark boxes ) of a bacterial probiotic for 7 consecutive days .", "cattle not administered the probiotic served as a control ( n=4 ; white \n boxes ) . the median and quartiles", "the upper and lower bars represent the maximum and minimum values , respectively . * \n significant difference compared with a control on the same day ( p<0.05 ) . significant difference compared with \n the same group on day 7 ( p<0.05 ) . ) .", "the mean maximum ph approached > 6.8 in the treatment groups , which did not differ from that in the control .", "circadian changes in the 1-hr mean ruminal ph at control day ( n=4 ; ) and 3 days after beginning the sara - inducing diet ( n=4 ; ) .", "the 1-hr mean ph in cattle fed sara - inducing diet decreased after the morning and evening feedings , which was indicative of \n successfully induced sara .", "data are means se . * significant difference compared with a control on the same day ( p<0.05 ) . \n", "changes in the 24-hr mean ruminal ph in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial probiotic for 7 consecutive days . \n", "* significant difference compared with a control on \n the same day ( p<0.05 ) .", "circadian changes in the 1-hr mean ruminal ph on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial \n probiotic for 7 consecutive days .", "box plots of the maximum and minimum ruminal ph on days 7 , 0 and 7 in the treatment groups administered 20 g ( n=4 ; light gray boxes ) , 50 g ( n=4 ; gray \n boxes ) and 100 g ( n=4 ; dark boxes ) of a bacterial probiotic for 7 consecutive days .", "cattle not administered the probiotic served as a control ( n=4 ; white \n boxes ) . the median and quartiles", "the upper and lower bars represent the maximum and minimum values , respectively . * \n significant difference compared with a control on the same day ( p<0.05 ) . significant difference compared with \n the same group on day 7 ( p<0.05 ) .", "ruminal vfa , lactic acid and nh3-n concentrations : total vfa increased from 8:00 to 20:00 in the treatment and control groups on days \n 0 and 7 ( fig . 5fig .", "5.circadian changes in the ruminal vfa , a : p ratio , lactic acid and nh3-n concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g \n ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial probiotic for 7 consecutive days .", "* significant difference compared with a control on the same day ( p<0.05 ) . )", ". however , no difference was observed in the total or individual vfa concentrations between the treatment and control groups . the acetic acid - to - propionic \n acid ratio ( a : p ) was almost identical among the treatment groups .", "lactic acid concentrations remained stable in the treatment groups , and the concentrations at \n 20:00 on day 7 were significantly lower than that in the control . no difference was observed in nh3-n concentrations among the treatment groups , \n although the nh3-n concentrations in the 50 g and 100 g groups at 8:00 on day 7 were significantly higher than that in the control .", "circadian changes in the ruminal vfa , a : p ratio , lactic acid and nh3-n concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g \n ( n=4 ; ) or 100 g ( n=4 ; ) of a bacterial probiotic for 7 consecutive days .", "data are \n means se . * significant difference compared with a control on the same day ( p<0.05 ) .", "blood metabolite profiles : glu concentrations decreased from 8:00 to 20:00 , but were almost the same among the treatment and control groups \n ( fig .", "6.circadian changes in the blood glu , nefa , bhba and bun concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) \n of a bacterial probiotic for 7 consecutive days .", "data are means se . * \n significant difference compared with a control on the same day ( p<0.05 ) . ) .", "nefa levels were affected by feeding time and were significantly lower at 20:00 compared with at 8:00 in the treatment groups on days 0 and 7 . \n", "furthermore , nefa level in the 50 g group at 20:00 on day 7 was significantly lower than that in the control .", "in contrast , bhba and bun concentrations were higher \n and lower , respectively , in all groups from 8:00 to 20:00 .", "bhba concentrations in the 20 g and 50 g at 17:00 and 20:00 , and bun concentrations in the treatment \n groups at 8:00 were significantly higher than that in the controls , respectively .", "circadian changes in the blood glu , nefa , bhba and bun concentrations on days 0 and 7 in cattle administered 20 g ( n=4 ; ) , 50 g ( n=4 ; ) or 100 g ( n=4 ; ) \n of a bacterial probiotic for 7 consecutive days .", "data are means se . * \n significant difference compared with a control on the same day ( p<0.05 ) ." ], [ "sara occurs when the ruminal ph decreases due to a combination of overproduction of vfa and lactic acid and a decrease in vfa absorption in the rumen [ 1 , 22 ] .", "sara is diagnosed when the ph in the ruminal fluid is < 5.6 for at least 3 hr \n per day [ 5 , 17 , 21 ] . in this study ,", "the \n ruminal ph measured continuously indicated that sara was successfully induced during the experimental period , especially during the first week of the experiment , \n by feeding a sara - inducing diet .", "studies have shown that the main ruminal bacteria affecting the ruminal fermentative capacity are classified as lactic acid - producers and consumers [ 4 , 15 ] , and both of these bacterial groups have been used as bps .", "( 2002 ) reported that a bp containing both lactate - consuming \n ( propionibacterium ) and lactate - producing ( enterococcus ) bacteria activated ruminal fermentation and reduced the risk of \n acidosis in cattle . in another study , a bp consisting of l. plantarum and e. faecium induced changes in the ruminal ph of cows \n fed a high - grain diet .", "however , the mechanism of the effects of bp on ruminal fermentation is unclear , and the effects \n of bp on sara in cattle are unknown . in this study , the 24-hr mean ruminal ph was higher in the bp - treated groups compared with in the control group during sara \n challenge .", "this was indicative of increased ruminal fermentation in the cows , as reported previously [ 12 , 27 , 28 ] .", "( 2012 ) examined the effects of administering bp in sara - challenged cows and found no effects on ruminal ph when bp was administered as a single strain ; \n however , ruminal ph increased compared over that in the control group when using a combination of e. faecium and yeast .", "we used the same bp in a \n previous study on weaned calves fed a high - concentrate diet , and the mean ruminal ph was significantly higher in the treatment group compared with in the control \n group . in this study , different doses of a multi - strain bp were administered to sara - challenged cows , and the 24-hr \n mean ruminal ph was notably higher on days 3 to 7 , whereas the minimum ph increased on day 7 in the treatment groups .", "bp appears to increase the ability of ruminal bacteria to metabolize lactic acid and regulate ruminal ph [ 20 , 21 ] .", "it has been hypothesized that the functionality and efficacy of bps can be determined based on their effects on the \n predominant rumen microbiota [ 7 , 12 ] . in this study , cattle receiving lower bp doses \n ( 20 g per head ) had a constant mean ph of 6.46.6 .", "in contrast , cattle receiving higher bp doses ( 100 g per head ) had higher mean ph on days 2 and 3 , which \n decreased on days 5 to 7 .", "previous reports have indicated that changes in rumen bacterial composition and diversity , increased activities of lactate - consuming \n bacteria and greater lactate absorption affect ruminal ph [ 13 , 15 ] .", "the higher bp \n dose might increase lab numbers in the rumen , which could be related to increased ruminal fermentation capacity , higher carbohydrate fermentation and increased \n ruminal ph .", "therefore , ruminal lab might have been overly increased due to the higher bp dose , causing the ruminal \n fermentation and ph to decrease on day 5 .", "these results are consistent with previous reports that lower bp doses improve gastrointestinal tract microbiota in \n calves .", "furthermore , weaned calves administered lower bp doses showed a higher mean ruminal ph compared with controls \n . based on our results , administration of an extremely high dose ( 100 g per head ) of lab could cause decreased \n ruminal fermentation and ruminal ph .", "decreases in ruminal ph are related to vfa production and lactic acid accumulation after feeding a high - grain diet [ 1 , \n 18 ] . in this study , although ruminal vfa concentrations were not affected by bp treatment , they were affected by feeding \n time .", "this is in agreement with previous reports in which ruminal vfas were not affected by probiotics including lab [ 6 , \n 20 ] .", "administration of lab probiotics is thought to help rumen microbiota adapt to the presence of lactic acid and prevent lactate accumulation in the rumen .", "( 1992 ) reported that lactate - consuming bacteria increased only when lactic acid accumulated and ruminal ph decreased . \n", "based on these results , lab probiotics might affect ruminal ph by increasing the activity of lactate - consuming bacteria . \n in our previous study , significantly lower lactic acid concentrations were observed in the ruminal fluid of weaned calves receiving bp compared with the control \n . in this study ,", "lactic acid concentrations were stable in the treatment groups ; however , lactic acid was higher and \n correlated with a lower mean ruminal ph in the control group . gradually increasing ruminal", "ph might be due to time after feeding and the absorption capacity of \n the rumen .", "bps may prevent a decline in ruminal ph by increasing the lactic acid consumption by some microbes [ 3 , 6 ] .", "a combination of certain probiotic bacteria that synthesize lactic acid may sustain \n a tonic level of lactic acid in the rumen , stimulating rumen microbial communities that consume lactic acid and reducing acidity , causing the ruminal ph to remain \n constant and stable .", "conversely , the nh3-n concentrations in the ruminal fluid of the bp groups remained stable in this study , although significant difference was observed \n at 8:00 on day 7 between the bp - treated and control groups .", "bp has no effect on the nh3-n concentrations in the rumen [ 7 , 12 ] .", "decreases in nh3-n levels after morning feeding in this study were in agreement with the \n previous study , which found that nh3-n concentrations decreased with decreasing ruminal ph [ 7 , 13 ] .", "it has been reported that nefa levels are \n lower in steers with sara compared with in the control ; therefore , the decrease in nefa levels indicates a more \n efficient use of dietary energy and greater dry matter intake in the bp - treated groups . in conclusion ,", "repeated administration of a bp comprised of l. plantarum , e. faecium and c. butyricum improved \n the 24-hr mean ruminal ph in cattle with experimentally induced sara at doses of 20 or 50 g per head .", "diurnal patterns of the 1-hr mean ruminal ph were identical \n among the treatment and control groups .", "ruminal vfa was not affected by bp treatment ; however , lactic acid was lower in the treatment groups than in the control \n group . based on these results", ", bp might affect ruminal ph by increasing lactate consumption and decreasing lactic acid concentrations , resulting in a consistently \n higher ruminal ph in sara cattle .", "these results suggest that repeated administration of a multi - strain bp might reduce the risk of sara in cattle and that \n consecutive treatment with 20 or 50 g of a bp containing lab during high - concentrate feeding might reduce the incidence of sara in dairy cattle ." ] ]

[ "the importance of early and intensive control of blood glucose levels in improving outcomes for people with type 2 diabetes mellitus ( t2 dm ) was established in the uk prospective diabetes study ( ukpds ) . despite differences in glucose levels between the intensive and standard treatment groups disappearing soon after completion of the active study intervention", ", the benefits of earlier intensive glycaemic control in reducing diabetic complications were found to persist in long - term ( 10-year ) follow - up . combined with evidence that even short periods ( i.e. weeks ) of hyperglycaemia increase the risk for developing diabetic complications", ", this highlights the importance of trying to establish good glycaemic control as early as possible .", "subsequent large - scale intervention studies have confirmed the benefit of intensive glycaemic control in reducing microvascular risk , but the findings regarding cardiovascular disease and overall mortality have been less conclusive . of note ,", "what constitutes intensive therapy has evolved over the two decades since the ukpds as new antihyperglycaemic agents have become available .", "the newer agents , particularly those associated with a low risk of hypoglycaemia compared with sulphonylureas and insulin , offer new options for combination therapy aimed at achieving better glycaemic control .", "the global partnership for effective diabetes management was an early advocate for adoption of tailored targets and treatment in t2 dm , and subsequent guidelines have evolved to reflect the evidence , with universal glycaemic targets and treatment algorithms replaced with recommendations to individualize targets and treatment . while it seems clear that individualized targets are needed to optimize care , it is critical that good glycaemic control , as defined by individualized hba1c targets , is pursued without delay and with commitment .", "however , it is widely recognized that glucose levels are not well controlled in many people with t2 dm . through a review of recently published data , we aimed to summarize current standards of glycaemic control globally .", "we also sought to identify the factors that may contribute to poor rates of glycaemic control .", "searches were performed , last updated in july 2015 , in the us national library of medicine / national institutes of health pubmed database .", "abstracts of the publications identified by the searches were reviewed manually and included in the review if they reported ( 1 ) original data related to proportions of people with t2 dm achieving glycaemic control or ( 2 ) obstacles or barriers contributing to suboptimal glycaemic control .", "publications were excluded from the review if they were ( 1 ) published before 2011 or ( 2 ) published in a language other than english .", "manuscripts written originally in a language other than english but where an english translation is available were included .", "the search of the pubmed database was complemented by a manual search of online abstracts from recent ( 20132015 ) diabetes conferences including those from the european association for the study of diabetes and the american diabetes association annual meetings .", "terms used in the pubmed search were also entered into the google search engine , and the results were reviewed manually to identify any additional relevant data from credible sources that might inform the analysis and review .", "a meta - analysis was not performed owing to the diverse nature of the data collected .", "a total of 45 relevant publications were identified by the search strategy and included in the literature review and analysis .", "the results of the literature review suggest that suboptimal glycaemic control is a common and widespread problem .", "the proportion of patients not achieving hba1c targets in 12 studies published between 2011 and 2015 inclusive is summarized in table 1 . even in the study reporting the best rate of control , one in every three patients was above the hba1c target .", "a multi - centre , cross - sectional study of ~5800 people with t2 dm from nine european countries found that hba1c exceeded the target of 7.0% ( 53 mmol / mol ) in 37% of cases , with rates for individual countries ranging from 26% in the netherlands up to 52% in turkey .", "tidm : type 1 diabetes mellitus ; t2 dm : type 2 diabetes mellitus . target hba1c 7.0% ( 53 mmol / mol ) , other than for the study of laiteerapong et al . which used individualized targets of hba1c < ", "6.5% , < 7.0% or < 8.0% ( < 48 mmol / mol , < 53 mmol / mol or < 64 mmol / mol ) based on patient characteristics .", "nine european countries : belgium , france , germany , greece , italy , the netherlands , spain , turkey and the united kingdom .", "other publications show that hba1c levels are markedly above target in a large proportion of people with t2 dm . in a registry - based study of > 10,000 people undergoing care for t2 dm in specialist clinics in canada ,", "62% were not at target [ hba1c 7.0% ( 53 mmol / mol ) ] and 15% had an hba1c level of 9.0% ( 75 mmol / mol ) or above . a puerto rican study of 600 adults with type 1 or type 2 diabetes reported that just 37% were at the recommended hba1c target . a survey of > 200,000 people with diabetes in taiwan found hba1c > 9.0% ( 75 mmol / mol ) in nearly one in every four people and 30% of outpatients with t2 dm attending a medical college in rural india had hba1c 9.5% ( 80 mmol / mol ) .", "there was some evidence to suggest that suboptimal control occurred more frequently in low- and middle - income countries compared with wealthier countries , with the highest rate of 87% for non - achievement of hba1c target reported in rural india . however , high proportions of people in high - income , resource - rich settings also had inadequate control of hyperglycaemia .", "suboptimal glycaemic control is often accompanied by poor control of other cardiometabolic risk factors . in a prospective cohort study of more than 3000 people with t2 dm in canada ,", "the proportion of people with hba1c , blood pressure and low - density lipoprotein ( ldl ) cholesterol at target on study entry was 47% , 46% and 36% , respectively .", "all three risk factors were controlled to target in just 19% of people , and only 7% maintained good control of glycaemia , blood pressure and ldl cholesterol throughout the 12-month study .", "a canadian study of more than 5000 people with t2 dm seen by 479 primary care physicians found that the proportions achieving targets were 50% for hba1c , 36% for blood pressure and 57% for ldl cholesterol , with only 13% reaching all three targets .", "similar results were found in more than 25,000 outpatients with t2 dm in china , with the proportions of patients achieving targets for hba1c , blood pressure and lipids of 48% , 28% and 36% , respectively .", "importantly , nearly all of the studies reviewed assessed the quality of glycaemic control against a single target of hba1c 7.0% ( 53 mmol / mol ) . within the context of individualized targets now recommended in guidelines , a somewhat higher target", "[ e.g. hba1c 7.5% ( 59 mmol / mol ) ] may have represented optimal management based on factors such as age , presence of cardiovascular comorbidities and duration of diabetes .", "this is unlikely to account for the large proportion of patients not controlled to hba1c 7.0% ( 53 mmol / mol ) and certainly should not justify the sizeable proportion of people with hba1c levels exceeding 9.0% ( 75 mmol / mol ) .", "however , it highlights that a single target is unlikely to represent optimal management for all people .", "the review provides strong evidence that in all regions of the world , many people with t2 dm are not achieving good glycaemic control . although there is considerable heterogeneity in the studies and the data generated , rates of uncontrolled glycaemia are typically in the range of 40%60% .", "consequently , many people are left at increased risk of diabetic complications and the associated burden of illness and mortality , as well as consequent economic costs .", "recent studies examining barriers to the achievement of good glycaemic control are summarized in table 2 .", "tidm : type 1 diabetes mellitus ; t2 dm : type 2 diabetes mellitus ; hcps : healthcare professionals .", "prescribers often identify poor patient motivation and self - management as leading barriers to achieving glycaemic targets .", "the second diabetes , attitudes , wishes and needs ( dawn2 ) study , included an online survey of 4785 healthcare professionals ( hcps ) ( ~40% primary care clinicians , 30% diabetes specialists , 20% nurses and 10% dieticians ) from 17 countries across four continents .", "the majority of the respondents ( 60% ) indicated a need for improvements in self - management by people with diabetes , particularly in the area of diet and exercise .", "resistance to lifestyle measures was also identified as a leading barrier by 252 primary care physicians , nurse practitioners and physician assistants in the united states , with 52% of hcps indicating that it was often or almost always a factor contributing to chronic lack of glycaemic control .", "other leading patient - related barriers identified by the hcps included psychosocial issues ( 34% ) , reluctance to use insulin ( 31% ) and non - compliance ( 30% ) . a study in the united kingdom examining the reasons for very poor glycaemic control [ hba1c 10% ( 86 mmol / mol ) ] in 128 people with t2 dm attending three primary care centres found poor adherence to lifestyle measures to be most frequently involved .", "other factors included infrequent clinic attendance ( 16% ) , side effects ( 16% ) , poor compliance with pharmacotherapy ( 14% ) , lack of knowledge of diabetes ( 14% ) , lack of titration of insulin ( 13% ) and insulin refusal ( 12% ) .", "poor patient motivation and adherence were also identified as barriers to good glycaemic control in qualitative studies with hcps .", "interviews with a small number ( n = 9 ) of hcps in the united arab emirates revealed a tendency to view patient attitudes and behaviours as the origin of poor glycaemic control and to believe that if patients were more knowledgeable about diabetes , cooperated with hcps and were adherent with treatment , then they could achieve their glycaemic targets .", "the studies reviewed also revealed that a number of healthcare system - level factors are often seen as important barriers to good glycaemic control .", "the dawn2 study , for example , found that only 30% of the hcps surveyed believed that healthcare was well organized for the management of chronic conditions .", "participants from western european countries and china were more likely to feel that healthcare was well organized than those from north or south america , eastern europe , india or japan ( figure 1 ) .", "approximately half of the participants considered the healthcare remuneration system to be a barrier and most ( ~60% ) indicated a need for better access to qualified nurse educators / specialist diabetes nurses and psychologists / psychiatrists .", "proportion of healthcare professionals reporting that healthcare in their country is well organized for the management of chronic conditions , including debates .", "a survey of 47 european countries found that while all had guidelines in place for managing diabetes , only 7 ( 15% ) had protocols for monitoring their implementation . the majority ( 83% ) of countries had or were developing national plans for diabetes , but these were often not comprehensive .", "there was substantial variability in the provision of multidisciplinary care . while 80% reported that nurses play an important role in providing education for self - management , diabetes specialist nursing was recognized as a speciality in only 19 countries ( 40% ) .", "despite the widespread belief that patients need to be more engaged and effective in self - management , only 25% of the countries recommended continuous education for all people with diabetes .", "lack of effective self - management education was also identified as a barrier in a qualitative study involving focus group discussions with 25 primary care clinicians in the united states , along with a persistent focus on acute care , poor integration of diabetes care , lack of clinical information and inadequate public health support .", "lack of coordination in care and limited access to secondary resources were also identified as important barriers by studies in the netherlands and ireland .", "limitations on the time available to manage patients are often identified as an obstacle to good glycaemic control . in the survey of 252 us primary care physicians , lack of time for treating complex patients", "was reported as often or always a factor in failure to achieve good glycaemic control by 41% of the physicians .", "treatment costs were much less frequently seen as a barrier , with 25% of the hcps indicating that it made no contribution and most ( 58% ) saying that it was a factor in only a minority ( 20%40% ) of cases of poor glycaemic control .", "other studies have also suggested that hcps in primary care often feel that they lack the time or other resources needed to manage diabetes effectively .", "this raises particular concerns , given that health policy in some countries involves shifting more of the responsibility for diabetes management onto primary care . compared with patient- and system - related barriers to effective glycaemic control , there is limited information available regarding physician - related barriers", ". however , there is evidence to suggest that a lack of diabetes - focused education may be contributing to failure to achieve therapeutic targets .", "for example , one in five of the hcps surveyed in the dawn2 study indicated that they had received no formal postgraduate diabetes training , and in a uk survey , only 35% of 2149 trainee doctors felt that their postgraduate training had prepared them adequately to optimize treatment of diabetes and only 41% reported that they would take the initiative to optimize glycaemic control .", "a survey of 209 primary care clinicians in australia identified gaps in knowledge relating to the medical management of t2 dm , with 46% self - reporting a need for education on pharmacological management .", "it appears that prescribers often lack confidence in the use of insulin , particularly with more complex regimens .", "people with diabetes often continue in poor glycaemic control for extended periods of time without appropriate action being taken . a retrospective cohort study of > 80,000 people with t2 dm in the united kingdom revealed substantial delays in intensifying treatment when glycaemic control was suboptimal . for people who were inadequately controlled on one oral glucose ", "lowering agent , the median time from hba1c being above a threshold of 7.0% ( 53 mmol / mol ) to treatment intensification with an additional oral agent was nearly 3 years , and even when hba1c was 8.0% ( 64 mmol / mol ) , intensification was delayed by a median of 1.6 years .", "there were even longer delays ( median of ~67 years ) in intensifying treatment with the addition of a third oral agent or the initiation of insulin .", "mean hba1c at intensification ranged from 8.7%9.7% ( 7283 mmol / mol ) . in a study of nearly 20,000 people with t2 dm initiated on basal insulin at hospitals across china ,", "a study of primary care records of > 17,000 patients with t2 dm being treated with oral glucose lowering agents in france found that > 3000 of the patients were not at glycaemic target and required treatment intensification . however , treatment was actually intensified in only 39% of the patients requiring it and the majority ( ~60% ) of changes were delayed by 6 months or more and a substantial proportion ( ~40% ) by more than a year .", "the diattitude study ( table 3 ) also looked at therapeutic inertia in the care of > 2000 people with t2 dm by 236 primary care physicians and found that among patients with uncontrolled glycaemia , representing 41% of all the patients studied , only 7% had their treatment intensified .", "the most common ( 60% ) reason given for not intensifying treatment was that the patient s hba1c was satisfactory .", "other leading reasons given included that lifestyle advice was more of a priority than changing medication ( 20% ) , the decision was postponed until the next clinic visit ( 11% ) and that hba1c had decreased since the previous clinic visit ( 7% ) , although in the majority ( 58% ) of these cases , an hba1c reduction was not confirmed by the available records .", "the problem appears to be common and widespread , with further studies in bahrain , croatia , spain , taiwan and the united states reporting high rates ( typically 30%60% ) of clinical inertia when managing elevated glucose levels in people with diabetes .", "summary of studies reporting rates of and factors contributing to therapeutic inertia in the management of type 2 diabetes .", "ldl : low - density lipoprotein ; t2 dm : type 2 diabetes mellitus ; hcps : healthcare professionals .", "a number of studies have attempted to explore the factors that may act as obstacles to action in addressing inadequate glycaemic control . in an online survey completed by 508 primary care physicians who provided data on 770 elderly patients ( > 65 years ) ,", "the leading reason for not initiating glucose - lowering medication , given as the first - ranked reason for 58% of the patients and a contributory factor in nearly all patients , was that they were treated with diet and exercise , and presumably , this was believed to be adequate management ( figure 2 ) .", "however , 33% of the patients were not at target hba1c , suggesting substantial clinical inertia in this population .", "other reasons given for not starting drug treatment included that hyperglycaemia was mild ( 84% ) , patient concerns ( 61% ) , physician concerns about glucose - lowering agents ( 49% ) and presence of comorbidities and polypharmacy ( 37% ) . when asked to identify a threshold for initiating treatment with glucose - lowering agents in the elderly population ,", "the mean hba1c threshold value stated was 7.1% ( 54 mmol / mol ) . in approximately 20% of the cases", ", the physicians indicated that they were planning to initiate glucose - lowering therapy within the next month , but more than half ( 54% ) of these patients already had an hba1c level above the physician - stated threshold .", "results from an online survey of 508 us primary care physicians providing clinical date for 770 patients .", "a qualitative study of 20 hcps in the united kingdom found a pattern of seeking to lessen their own sense of accountability for clinical inertia by emphasizing patient - level barriers such as comorbidities , motivation and self - management capabilities as well as health system level barriers , especially time constraints .", "there was also a tendency for the hcps to overestimate the achievement of targets in their primary care centres .", "factors contributing to therapeutic inertia in the initiation of insulin treatment for t2 dm were investigated in the translating research into action for diabetes ( triad ) project using structured interviews with primary care hcps in the united states .", "the majority of the 83 clinicians interviewed supported guideline recommendations on glycaemic targets , with 69% choosing an hba1c < 7% ( < 53 mmol / mol ) as the ideal target for good glycaemic control . at the same time ,", "the most frequently cited reasons for setting higher individualized hba1c targets included advanced age or short life expectancy ( 54% ) , poor self - management capacity due to poor cognitive abilities ( 35% ) , presence of comorbidities ( 34% ) , low educational level or poor health literacy ( 34% ) and patients unwillingness to self - manage their diabetes ( 33% ) .", "the most frequent reasons cited by the clinicians for not initiating insulin treatment were patient refusal or resistance ( 64% ) and lack of patient self - management skills ( 43% ) .", "factors determining initiation of insulin were also investigated using data for 1933 people with t2 dm from the french national health and wellness survey , an annual internet - based survey among french adults .", "early initiation of insulin therapy was almost 10 times more likely to be prescribed by an endocrinologist or diabetologist than by a primary care physician .", "early versus late insulin initiation was also more likely in patients who were younger , had diabetes - related complications or smoked .", "insulin - treated patients were more likely to be adherent , and there was no apparent deterioration in quality of life associated with insulin use . in focus group discussions with 114 primary care physicians in belgium ,", "many of the physicians found the concept of clinical inertia interesting , but some also found it insulting and felt it implied that they were passive in their work .", "they also noted that it was important to differentiate between cases where there was a conscious decision to not pursue lower targets based on a consideration of the individual patient factors and those where there was genuine inertia .", "it was acknowledged that genuine clinical inertia was a real risk in primary care , but there was a tendency to ascribe this risk to patient- and system - level barriers rather than physician - level behaviours and practices . in summary , recent studies suggest that clinical inertia is a common and widespread problem , typically affecting the care of 30%50% of people with t2 dm .", "the causes are diverse and complex , but surveys of hcps tend to emphasize patient - level barriers to treatment intensification , such as patient reluctance to start insulin therapy , or system - level barriers , such as disconnects between what is recommended in guidelines and what is reimbursed in practice .", "however , it also appears that hcps sometimes delay taking necessary action and may be willing to tolerate periods of ", "it also appears that many hcps , while recognizing the importance of good glycaemic control , have low expectations for their patients .", "the issue of clinical inertia needs to be better understood if it is to be addressed effectively , particularly regarding the patient- , hcp- and system - level factors that underlie it .", "this is challenging as clinical inertia as defined in studies typically captures a range of behaviours , some of which may not reflect suboptimal care .", "however , studies of clinical inertia often apply a single glycaemic threshold [ typically hba1c = 7% ( 53 mmol / mol ) ] across a broad range of patients .", "guidelines for management of t2 dm have traditionally advocated a stepwise approach to management , in which treatment is started with diet and exercise , followed by the addition of oral antihyperglycaemic monotherapy , then combination therapy and eventually insulin . however , when applied systematically without considering individual factors such as the patient s current hba1c level , it can lead to treatment futility with people receiving therapy that has little chance of lowering glucose levels sufficiently to achieve their glycaemic target .", "several recent publications have indicated potential benefits of early , intensive intervention with pharmacotherapy , including combinations of oral glucose lowering agents and/or insulin .", "the treatment options for type 2 diabetes in adolescents and youth ( today ) study compared metformin alone , metformin plus a thiazolidinedione and metformin plus intensive lifestyle intervention as initial therapy for new - onset t2 dm in people aged 1017 years and with obesity . the proportion of people who maintained glycaemic control over 5 years was significantly higher with the combination pharmacotherapy than either metformin alone or metformin plus intensive lifestyle measures .", "the combination pharmacotherapy was also associated with greater improvements in insulin sensitivity and -cell function compared with the other two approaches .", "the results lend support to early , intensive intervention with combination therapy , at least in this population of young people with new - onset t2 dm .", "early intensive therapy was also studied in older ( mean age = 45 years ) people with newly diagnosed t2 dm who started treatment for 3 months with metformin plus insulin and were then randomized to either continuing metformin plus insulin therapy or triple oral glucose lowering therapy with metformin , glyburide and pioglitazone .", "good glycaemic control was maintained for over 6 years with no significant difference between the groups ( end of study hba1c = 7.3% for metformin plus insulin and 6.4 for triple oral combination ; p = 0.4 ) .", "a review of the health records for nearly 3000 people with t2 dm in the united states found that early initiation of metformin ( within 6 months of diagnosis ) significantly reduced hba1c ( 0.36% ; p < 0.001 ) and bmi ( 0.46 kg / m ; p < ", "the likelihood of achieving hba1c 7% was doubled and the likelihood of requiring therapy intensification was reduced by 28% with early versus delayed initiation of metformin .", "another study in the united states looking at medical records for 5870 people with t2 dm found a similar pattern for early introduction of combination oral glucose ", "early intensification was defined as addition of a second oral drug to metformin therapy within 3 months of treatment failure ( hba1c 7.5% ) and late intensification as the introduction of combination therapy 1015 months after loss of glycaemic control .", "people who received early intensification were 38% more likely to achieve good glycaemic control at 1.52 years than those with late intensification . in the advance trial of 11,140 patients with t2 dm , therapeutic intensification with addition of an oral glucose ", "lowering agent doubled the chance of achieving good glycaemic control , and with intensification with insulin , the odds were increased 2.5-fold .", "a total of 45 relevant publications were identified by the search strategy and included in the literature review and analysis .", "the results of the literature review suggest that suboptimal glycaemic control is a common and widespread problem .", "the proportion of patients not achieving hba1c targets in 12 studies published between 2011 and 2015 inclusive is summarized in table 1 . even in the study reporting the best rate of control , one in every three patients was above the hba1c target . a multi - centre ,", "cross - sectional study of ~5800 people with t2 dm from nine european countries found that hba1c exceeded the target of 7.0% ( 53 mmol / mol ) in 37% of cases , with rates for individual countries ranging from 26% in the netherlands up to 52% in turkey .", "target hba1c 7.0% ( 53 mmol / mol ) , other than for the study of laiteerapong et al . which used individualized targets of hba1c < 6.5% , < 7.0% or < 8.0% ( < 48 mmol / mol , < 53 mmol / mol or < 64 mmol / mol ) based on patient characteristics .", "nine european countries : belgium , france , germany , greece , italy , the netherlands , spain , turkey and the united kingdom .", "other publications show that hba1c levels are markedly above target in a large proportion of people with t2 dm . in a registry - based study of > 10,000 people undergoing care for t2 dm in specialist clinics in canada ,", "62% were not at target [ hba1c 7.0% ( 53 mmol / mol ) ] and 15% had an hba1c level of 9.0% ( 75 mmol / mol ) or above . a puerto rican study of 600 adults with type 1 or type 2 diabetes reported that just 37% were at the recommended hba1c target . a survey of > 200,000 people with diabetes in taiwan found hba1c > 9.0% ( 75 mmol / mol ) in nearly one in every four people and 30% of outpatients with t2 dm attending a medical college in rural india had hba1c 9.5% ( 80 mmol / mol ) .", "there was some evidence to suggest that suboptimal control occurred more frequently in low- and middle - income countries compared with wealthier countries , with the highest rate of 87% for non - achievement of hba1c target reported in rural india . however , high proportions of people in high - income , resource - rich settings also had inadequate control of hyperglycaemia .", "suboptimal glycaemic control is often accompanied by poor control of other cardiometabolic risk factors . in a prospective cohort study of more than 3000 people with t2 dm in canada ,", "the proportion of people with hba1c , blood pressure and low - density lipoprotein ( ldl ) cholesterol at target on study entry was 47% , 46% and 36% , respectively .", "all three risk factors were controlled to target in just 19% of people , and only 7% maintained good control of glycaemia , blood pressure and ldl cholesterol throughout the 12-month study .", "a canadian study of more than 5000 people with t2 dm seen by 479 primary care physicians found that the proportions achieving targets were 50% for hba1c , 36% for blood pressure and 57% for ldl cholesterol , with only 13% reaching all three targets .", "similar results were found in more than 25,000 outpatients with t2 dm in china , with the proportions of patients achieving targets for hba1c , blood pressure and lipids of 48% , 28% and 36% , respectively .", "importantly , nearly all of the studies reviewed assessed the quality of glycaemic control against a single target of hba1c 7.0% ( 53 mmol / mol ) . within the context of individualized targets now recommended in guidelines , a somewhat higher target [", "e.g. hba1c 7.5% ( 59 mmol / mol ) ] may have represented optimal management based on factors such as age , presence of cardiovascular comorbidities and duration of diabetes .", "this is unlikely to account for the large proportion of patients not controlled to hba1c 7.0% ( 53 mmol / mol ) and certainly should not justify the sizeable proportion of people with hba1c levels exceeding 9.0% ( 75 mmol / mol ) .", "however , it highlights that a single target is unlikely to represent optimal management for all people .", "the review provides strong evidence that in all regions of the world , many people with t2 dm are not achieving good glycaemic control .", "although there is considerable heterogeneity in the studies and the data generated , rates of uncontrolled glycaemia are typically in the range of 40%60% .", "consequently , many people are left at increased risk of diabetic complications and the associated burden of illness and mortality , as well as consequent economic costs .", "recent studies examining barriers to the achievement of good glycaemic control are summarized in table 2 .", "tidm : type 1 diabetes mellitus ; t2 dm : type 2 diabetes mellitus ; hcps : healthcare professionals .", "prescribers often identify poor patient motivation and self - management as leading barriers to achieving glycaemic targets .", "the second diabetes , attitudes , wishes and needs ( dawn2 ) study , included an online survey of 4785 healthcare professionals ( hcps ) ( ~40% primary care clinicians , 30% diabetes specialists , 20% nurses and 10% dieticians ) from 17 countries across four continents .", "the majority of the respondents ( 60% ) indicated a need for improvements in self - management by people with diabetes , particularly in the area of diet and exercise .", "resistance to lifestyle measures was also identified as a leading barrier by 252 primary care physicians , nurse practitioners and physician assistants in the united states , with 52% of hcps indicating that it was often or almost always a factor contributing to chronic lack of glycaemic control .", "other leading patient - related barriers identified by the hcps included psychosocial issues ( 34% ) , reluctance to use insulin ( 31% ) and non - compliance ( 30% ) . a study in the united kingdom examining the reasons for very poor glycaemic control [ hba1c 10% ( 86 mmol / mol ) ] in 128 people with t2 dm attending three primary care centres found poor adherence to lifestyle measures to be most frequently involved .", "other factors included infrequent clinic attendance ( 16% ) , side effects ( 16% ) , poor compliance with pharmacotherapy ( 14% ) , lack of knowledge of diabetes ( 14% ) , lack of titration of insulin ( 13% ) and insulin refusal ( 12% ) .", "poor patient motivation and adherence were also identified as barriers to good glycaemic control in qualitative studies with hcps .", "interviews with a small number ( n = 9 ) of hcps in the united arab emirates revealed a tendency to view patient attitudes and behaviours as the origin of poor glycaemic control and to believe that if patients were more knowledgeable about diabetes , cooperated with hcps and were adherent with treatment , then they could achieve their glycaemic targets . the studies reviewed also revealed that a number of healthcare system - level factors are often seen as important barriers to good glycaemic control .", "the dawn2 study , for example , found that only 30% of the hcps surveyed believed that healthcare was well organized for the management of chronic conditions .", "participants from western european countries and china were more likely to feel that healthcare was well organized than those from north or south america , eastern europe , india or japan ( figure 1 ) .", "approximately half of the participants considered the healthcare remuneration system to be a barrier and most ( ~60% ) indicated a need for better access to qualified nurse educators / specialist diabetes nurses and psychologists / psychiatrists .", "proportion of healthcare professionals reporting that healthcare in their country is well organized for the management of chronic conditions , including debates .", "a survey of 47 european countries found that while all had guidelines in place for managing diabetes , only 7 ( 15% ) had protocols for monitoring their implementation . the majority ( 83% ) of countries had or were developing national plans for diabetes , but these were often not comprehensive", "there was substantial variability in the provision of multidisciplinary care . while 80% reported that nurses play an important role in providing education for self - management , diabetes specialist nursing was recognized as a speciality in only 19 countries ( 40% ) .", "despite the widespread belief that patients need to be more engaged and effective in self - management , only 25% of the countries recommended continuous education for all people with diabetes .", "lack of effective self - management education was also identified as a barrier in a qualitative study involving focus group discussions with 25 primary care clinicians in the united states , along with a persistent focus on acute care , poor integration of diabetes care , lack of clinical information and inadequate public health support .", "lack of coordination in care and limited access to secondary resources were also identified as important barriers by studies in the netherlands and ireland .", "limitations on the time available to manage patients are often identified as an obstacle to good glycaemic control . in the survey of 252 us primary care physicians , lack of time for treating complex patients", "was reported as often or always a factor in failure to achieve good glycaemic control by 41% of the physicians .", "treatment costs were much less frequently seen as a barrier , with 25% of the hcps indicating that it made no contribution and most ( 58% ) saying that it was a factor in only a minority ( 20%40% ) of cases of poor glycaemic control . other studies have also suggested that hcps in primary care often feel that they lack the time or other resources needed to manage diabetes effectively .", "this raises particular concerns , given that health policy in some countries involves shifting more of the responsibility for diabetes management onto primary care .", "compared with patient- and system - related barriers to effective glycaemic control , there is limited information available regarding physician - related barriers .", "however , there is evidence to suggest that a lack of diabetes - focused education may be contributing to failure to achieve therapeutic targets .", "for example , one in five of the hcps surveyed in the dawn2 study indicated that they had received no formal postgraduate diabetes training , and in a uk survey , only 35% of 2149 trainee doctors felt that their postgraduate training had prepared them adequately to optimize treatment of diabetes and only 41% reported that they would take the initiative to optimize glycaemic control .", "a survey of 209 primary care clinicians in australia identified gaps in knowledge relating to the medical management of t2 dm , with 46% self - reporting a need for education on pharmacological management .", "it appears that prescribers often lack confidence in the use of insulin , particularly with more complex regimens .", "prescribers often identify poor patient motivation and self - management as leading barriers to achieving glycaemic targets .", "the second diabetes , attitudes , wishes and needs ( dawn2 ) study , included an online survey of 4785 healthcare professionals ( hcps ) ( ~40% primary care clinicians , 30% diabetes specialists , 20% nurses and 10% dieticians ) from 17 countries across four continents .", "the majority of the respondents ( 60% ) indicated a need for improvements in self - management by people with diabetes , particularly in the area of diet and exercise .", "resistance to lifestyle measures was also identified as a leading barrier by 252 primary care physicians , nurse practitioners and physician assistants in the united states , with 52% of hcps indicating that it was often or almost always a factor contributing to chronic lack of glycaemic control .", "other leading patient - related barriers identified by the hcps included psychosocial issues ( 34% ) , reluctance to use insulin ( 31% ) and non - compliance ( 30% ) . a study in the united kingdom examining the reasons for very poor glycaemic control [ hba1c 10% ( 86 mmol / mol ) ] in 128 people with t2 dm attending three primary care centres found poor adherence to lifestyle measures to be most frequently involved .", "other factors included infrequent clinic attendance ( 16% ) , side effects ( 16% ) , poor compliance with pharmacotherapy ( 14% ) , lack of knowledge of diabetes ( 14% ) , lack of titration of insulin ( 13% ) and insulin refusal ( 12% ) .", "poor patient motivation and adherence were also identified as barriers to good glycaemic control in qualitative studies with hcps .", "interviews with a small number ( n = 9 ) of hcps in the united arab emirates revealed a tendency to view patient attitudes and behaviours as the origin of poor glycaemic control and to believe that if patients were more knowledgeable about diabetes , cooperated with hcps and were adherent with treatment , then they could achieve their glycaemic targets .", "the studies reviewed also revealed that a number of healthcare system - level factors are often seen as important barriers to good glycaemic control . the dawn2 study ,", "for example , found that only 30% of the hcps surveyed believed that healthcare was well organized for the management of chronic conditions .", "participants from western european countries and china were more likely to feel that healthcare was well organized than those from north or south america , eastern europe , india or japan ( figure 1 ) .", "approximately half of the participants considered the healthcare remuneration system to be a barrier and most ( ~60% ) indicated a need for better access to qualified nurse educators / specialist diabetes nurses and psychologists / psychiatrists .", "proportion of healthcare professionals reporting that healthcare in their country is well organized for the management of chronic conditions , including debates .", "a survey of 47 european countries found that while all had guidelines in place for managing diabetes , only 7 ( 15% ) had protocols for monitoring their implementation . the majority ( 83% ) of countries had or were developing national plans for diabetes , but these were often not comprehensive .", "there was substantial variability in the provision of multidisciplinary care . while 80% reported that nurses play an important role in providing education for self - management , diabetes specialist nursing was recognized as a speciality in only 19 countries ( 40% ) .", "despite the widespread belief that patients need to be more engaged and effective in self - management , only 25% of the countries recommended continuous education for all people with diabetes .", "lack of effective self - management education was also identified as a barrier in a qualitative study involving focus group discussions with 25 primary care clinicians in the united states , along with a persistent focus on acute care , poor integration of diabetes care , lack of clinical information and inadequate public health support .", "lack of coordination in care and limited access to secondary resources were also identified as important barriers by studies in the netherlands and ireland .", "limitations on the time available to manage patients are often identified as an obstacle to good glycaemic control . in the survey of 252 us primary care physicians , lack of time for treating complex patients", "was reported as often or always a factor in failure to achieve good glycaemic control by 41% of the physicians .", "treatment costs were much less frequently seen as a barrier , with 25% of the hcps indicating that it made no contribution and most ( 58% ) saying that it was a factor in only a minority ( 20%40% ) of cases of poor glycaemic control .", "other studies have also suggested that hcps in primary care often feel that they lack the time or other resources needed to manage diabetes effectively .", "this raises particular concerns , given that health policy in some countries involves shifting more of the responsibility for diabetes management onto primary care .", "compared with patient- and system - related barriers to effective glycaemic control , there is limited information available regarding physician - related barriers", ". however , there is evidence to suggest that a lack of diabetes - focused education may be contributing to failure to achieve therapeutic targets .", "for example , one in five of the hcps surveyed in the dawn2 study indicated that they had received no formal postgraduate diabetes training , and in a uk survey , only 35% of 2149 trainee doctors felt that their postgraduate training had prepared them adequately to optimize treatment of diabetes and only 41% reported that they would take the initiative to optimize glycaemic control .", "a survey of 209 primary care clinicians in australia identified gaps in knowledge relating to the medical management of t2 dm , with 46% self - reporting a need for education on pharmacological management .", "it appears that prescribers often lack confidence in the use of insulin , particularly with more complex regimens .", "people with diabetes often continue in poor glycaemic control for extended periods of time without appropriate action being taken . a retrospective cohort study of > 80,000 people with t2 dm in the united kingdom revealed substantial delays in intensifying treatment when glycaemic control was suboptimal . for people who were inadequately controlled on one oral glucose ", "lowering agent , the median time from hba1c being above a threshold of 7.0% ( 53 mmol / mol ) to treatment intensification with an additional oral agent was nearly 3 years , and even when hba1c was 8.0% ( 64 mmol / mol ) , intensification was delayed by a median of 1.6 years .", "there were even longer delays ( median of ~67 years ) in intensifying treatment with the addition of a third oral agent or the initiation of insulin .", "mean hba1c at intensification ranged from 8.7%9.7% ( 7283 mmol / mol ) . in a study of nearly 20,000 people with t2", "dm initiated on basal insulin at hospitals across china , mean hba1c at the time of insulin initiation was 9.6% .", "a study of primary care records of > 17,000 patients with t2 dm being treated with oral glucose lowering agents in france found that > 3000 of the patients were not at glycaemic target and required treatment intensification .", "however , treatment was actually intensified in only 39% of the patients requiring it and the majority ( ~60% ) of changes were delayed by 6 months or more and a substantial proportion ( ~40% ) by more than a year .", "the diattitude study ( table 3 ) also looked at therapeutic inertia in the care of > 2000 people with t2 dm by 236 primary care physicians and found that among patients with uncontrolled glycaemia , representing 41% of all the patients studied , only 7% had their treatment intensified .", "the most common ( 60% ) reason given for not intensifying treatment was that the patient s hba1c was satisfactory .", "other leading reasons given included that lifestyle advice was more of a priority than changing medication ( 20% ) , the decision was postponed until the next clinic visit ( 11% ) and that hba1c had decreased since the previous clinic visit ( 7% ) , although in the majority ( 58% ) of these cases , an hba1c reduction was not confirmed by the available records .", "the problem appears to be common and widespread , with further studies in bahrain , croatia , spain , taiwan and the united states reporting high rates ( typically 30%60% ) of clinical inertia when managing elevated glucose levels in people with diabetes .", "summary of studies reporting rates of and factors contributing to therapeutic inertia in the management of type 2 diabetes .", "ldl : low - density lipoprotein ; t2 dm : type 2 diabetes mellitus ; hcps : healthcare professionals .", "a number of studies have attempted to explore the factors that may act as obstacles to action in addressing inadequate glycaemic control . in an online survey completed by 508 primary care physicians who provided data on 770 elderly patients ( > 65 years ) ,", "the leading reason for not initiating glucose - lowering medication , given as the first - ranked reason for 58% of the patients and a contributory factor in nearly all patients , was that they were treated with diet and exercise , and presumably , this was believed to be adequate management ( figure 2 ) .", "however , 33% of the patients were not at target hba1c , suggesting substantial clinical inertia in this population .", "other reasons given for not starting drug treatment included that hyperglycaemia was mild ( 84% ) , patient concerns ( 61% ) , physician concerns about glucose - lowering agents ( 49% ) and presence of comorbidities and polypharmacy ( 37% ) .", "when asked to identify a threshold for initiating treatment with glucose - lowering agents in the elderly population , the mean hba1c threshold value stated was 7.1% ( 54 mmol / mol ) . in approximately 20% of the cases", ", the physicians indicated that they were planning to initiate glucose - lowering therapy within the next month , but more than half ( 54% ) of these patients already had an hba1c level above the physician - stated threshold .", "results from an online survey of 508 us primary care physicians providing clinical date for 770 patients .", "a qualitative study of 20 hcps in the united kingdom found a pattern of seeking to lessen their own sense of accountability for clinical inertia by emphasizing patient - level barriers such as comorbidities , motivation and self - management capabilities as well as health system level barriers , especially time constraints .", "there was also a tendency for the hcps to overestimate the achievement of targets in their primary care centres .", "factors contributing to therapeutic inertia in the initiation of insulin treatment for t2 dm were investigated in the translating research into action for diabetes ( triad ) project using structured interviews with primary care hcps in the united states .", "the majority of the 83 clinicians interviewed supported guideline recommendations on glycaemic targets , with 69% choosing an hba1c < 7% ( < 53 mmol / mol ) as the ideal target for good glycaemic control . at the same time ,", "the most frequently cited reasons for setting higher individualized hba1c targets included advanced age or short life expectancy ( 54% ) , poor self - management capacity due to poor cognitive abilities ( 35% ) , presence of comorbidities ( 34% ) , low educational level or poor health literacy ( 34% ) and patients unwillingness to self - manage their diabetes ( 33% ) .", "the most frequent reasons cited by the clinicians for not initiating insulin treatment were patient refusal or resistance ( 64% ) and lack of patient self - management skills ( 43% ) .", "factors determining initiation of insulin were also investigated using data for 1933 people with t2 dm from the french national health and wellness survey , an annual internet - based survey among french adults .", "early initiation of insulin therapy was almost 10 times more likely to be prescribed by an endocrinologist or diabetologist than by a primary care physician .", "early versus late insulin initiation was also more likely in patients who were younger , had diabetes - related complications or smoked .", "insulin - treated patients were more likely to be adherent , and there was no apparent deterioration in quality of life associated with insulin use . in focus group discussions with 114 primary care physicians in belgium ,", "many of the physicians found the concept of clinical inertia interesting , but some also found it insulting and felt it implied that they were passive in their work .", "they also noted that it was important to differentiate between cases where there was a conscious decision to not pursue lower targets based on a consideration of the individual patient factors and those where there was genuine inertia .", "it was acknowledged that genuine clinical inertia was a real risk in primary care , but there was a tendency to ascribe this risk to patient- and system - level barriers rather than physician - level behaviours and practices . in summary , recent studies suggest that clinical inertia is a common and widespread problem , typically affecting the care of 30%50% of people with t2 dm .", "the causes are diverse and complex , but surveys of hcps tend to emphasize patient - level barriers to treatment intensification , such as patient reluctance to start insulin therapy , or system - level barriers , such as disconnects between what is recommended in guidelines and what is reimbursed in practice .", "however , it also appears that hcps sometimes delay taking necessary action and may be willing to tolerate periods of ", "it also appears that many hcps , while recognizing the importance of good glycaemic control , have low expectations for their patients .", "the issue of clinical inertia needs to be better understood if it is to be addressed effectively , particularly regarding the patient- , hcp- and system - level factors that underlie it .", "this is challenging as clinical inertia as defined in studies typically captures a range of behaviours , some of which may not reflect suboptimal care .", "however , studies of clinical inertia often apply a single glycaemic threshold [ typically hba1c = 7% ( 53 mmol / mol ) ] across a broad range of patients .", "guidelines for management of t2 dm have traditionally advocated a stepwise approach to management , in which treatment is started with diet and exercise , followed by the addition of oral antihyperglycaemic monotherapy , then combination therapy and eventually insulin . however , when applied systematically without considering individual factors such as the patient s current hba1c level , it can lead to treatment futility with people receiving therapy that has little chance of lowering glucose levels sufficiently to achieve their glycaemic target .", "several recent publications have indicated potential benefits of early , intensive intervention with pharmacotherapy , including combinations of oral glucose lowering agents and/or insulin .", "the treatment options for type 2 diabetes in adolescents and youth ( today ) study compared metformin alone , metformin plus a thiazolidinedione and metformin plus intensive lifestyle intervention as initial therapy for new - onset t2 dm in people aged 1017 years and with obesity . the proportion of people who maintained glycaemic control over 5 years was significantly higher with the combination pharmacotherapy than either metformin alone or metformin plus intensive lifestyle measures .", "the combination pharmacotherapy was also associated with greater improvements in insulin sensitivity and -cell function compared with the other two approaches .", "the results lend support to early , intensive intervention with combination therapy , at least in this population of young people with new - onset t2 dm .", "early intensive therapy was also studied in older ( mean age = 45 years ) people with newly diagnosed t2 dm who started treatment for 3 months with metformin plus insulin and were then randomized to either continuing metformin plus insulin therapy or triple oral glucose ", "good glycaemic control was maintained for over 6 years with no significant difference between the groups ( end of study hba1c = 7.3% for metformin plus insulin and 6.4 for triple oral combination ; p = 0.4 ) .", "a review of the health records for nearly 3000 people with t2 dm in the united states found that early initiation of metformin ( within 6 months of diagnosis ) significantly reduced hba1c ( 0.36% ; p < 0.001 ) and bmi ( 0.46 kg / m ; p < 0.001 ) compared with when introduction of pharmacotherapy with metformin was delayed .", "the likelihood of achieving hba1c 7% was doubled and the likelihood of requiring therapy intensification was reduced by 28% with early versus delayed initiation of metformin .", "another study in the united states looking at medical records for 5870 people with t2 dm found a similar pattern for early introduction of combination oral glucose ", "early intensification was defined as addition of a second oral drug to metformin therapy within 3 months of treatment failure ( hba1c 7.5% ) and late intensification as the introduction of combination therapy 1015 months after loss of glycaemic control .", "people who received early intensification were 38% more likely to achieve good glycaemic control at 1.52 years than those with late intensification . in the advance trial of 11,140 patients with t2 dm , therapeutic intensification with addition of an oral glucose ", "lowering agent doubled the chance of achieving good glycaemic control , and with intensification with insulin , the odds were increased 2.5-fold .", "recently published evidence highlights that many people with t2 dm do not achieve good control of blood glucose leaving them at increased and avoidable risk of serious complications .", "a tendency to focus on patient - related obstacles may fail to consider issues arising from the attitudes , perceptions and behaviours of healthcare providers .", "in particular , there are often delays in the implementation of appropriate interventions to achieve glycaemic targets due to a complex range of negative factors that exacerbate the situation .", "an improved understanding of these factors would better inform strategies to assist hcps in more timely treatment of inadequately controlled glycaemia . despite a wide range of therapeutic options available", ", insufficient numbers of people with type 2 diabetes are reaching their glycaemic targets and large numbers are exposed to prolonged periods of hyperglycaemia .", "a number of obstacles exist globally that must be addressed and overcome if we are to improve the outcomes for patients .", "despite a wide range of therapeutic options available , insufficient numbers of people with type 2 diabetes are reaching their glycaemic targets and large numbers are exposed to prolonged periods of hyperglycaemia .", "a number of obstacles exist globally that must be addressed and overcome if we are to improve the outcomes for patients ." ]

[ "<S> background : glycaemic control is suboptimal in a large proportion of people with type 2 diabetes who are consequently at an increased and avoidable risk of potentially severe complications . we sought to explore attitudes and practices among healthcare professionals that may contribute to suboptimal glycaemic control through a review of recent relevant publications in the scientific literature.methods:an electronic search of the pubmed database was performed to identify relevant publications from january 2011 to july 2015 . </S>", "<S> the electronic search was complemented by a manual search of abstracts from key diabetes conferences in 2014/2015 available online.results:recently published data indicate that glycaemic control is suboptimal in a substantial proportion ( typically 40%60% ) of people with diabetes . </S>", "<S> this is the case across geographic regions and in both low- and higher - income countries . </S>", "<S> therapeutic inertia appears to be an important contributor to poor glycaemic control in up to half of people with type 2 diabetes . in particular , </S>", "<S> prescribers are often willing to tolerate extended periods of </S>", "<S> mild </S>", "<S> hyperglycaemia as well as having low expectations for their patients . </S>", "<S> there are often delays of 3 years or longer in initiating or intensifying glucose - lowering therapy when needed.conclusion:many people with type 2 diabetes are failed by current management , with approximately half not achieving or maintaining appropriate target blood glucose levels , leaving these patients at increased and avoidable risk of serious complications.review criteria : the methodology of this review article is detailed in the methods section . </S>" ]

[ "Review criteria:", "Introduction", "Methods", "Results and discussion", "Information sources", "Standards of glycaemic control", "Barriers to effective glycaemic control", "Patient-level barriers", "Healthcare systemrelated barriers", "Physician-level barriers", "Barriers to effective intensification of treatment", "Potential benefits of early, intensive intervention", "Conclusion", "Take home message for the clinician" ]

[ [ "" ], [ "the importance of early and intensive control of blood glucose levels in improving outcomes for people with type 2 diabetes mellitus ( t2 dm ) was established in the uk prospective diabetes study ( ukpds ) . despite differences in glucose levels between the intensive and standard treatment groups disappearing soon after completion of the active study intervention", ", the benefits of earlier intensive glycaemic control in reducing diabetic complications were found to persist in long - term ( 10-year ) follow - up . combined with evidence that even short periods ( i.e. weeks ) of hyperglycaemia increase the risk for developing diabetic complications", ", this highlights the importance of trying to establish good glycaemic control as early as possible .", "subsequent large - scale intervention studies have confirmed the benefit of intensive glycaemic control in reducing microvascular risk , but the findings regarding cardiovascular disease and overall mortality have been less conclusive . of note ,", "what constitutes intensive therapy has evolved over the two decades since the ukpds as new antihyperglycaemic agents have become available .", "the newer agents , particularly those associated with a low risk of hypoglycaemia compared with sulphonylureas and insulin , offer new options for combination therapy aimed at achieving better glycaemic control .", "the global partnership for effective diabetes management was an early advocate for adoption of tailored targets and treatment in t2 dm , and subsequent guidelines have evolved to reflect the evidence , with universal glycaemic targets and treatment algorithms replaced with recommendations to individualize targets and treatment . while it seems clear that individualized targets are needed to optimize care , it is critical that good glycaemic control , as defined by individualized hba1c targets , is pursued without delay and with commitment .", "however , it is widely recognized that glucose levels are not well controlled in many people with t2 dm . through a review of recently published data , we aimed to summarize current standards of glycaemic control globally .", "we also sought to identify the factors that may contribute to poor rates of glycaemic control ." ], [ "searches were performed , last updated in july 2015 , in the us national library of medicine / national institutes of health pubmed database .", "abstracts of the publications identified by the searches were reviewed manually and included in the review if they reported ( 1 ) original data related to proportions of people with t2 dm achieving glycaemic control or ( 2 ) obstacles or barriers contributing to suboptimal glycaemic control .", "publications were excluded from the review if they were ( 1 ) published before 2011 or ( 2 ) published in a language other than english .", "manuscripts written originally in a language other than english but where an english translation is available were included .", "the search of the pubmed database was complemented by a manual search of online abstracts from recent ( 20132015 ) diabetes conferences including those from the european association for the study of diabetes and the american diabetes association annual meetings .", "terms used in the pubmed search were also entered into the google search engine , and the results were reviewed manually to identify any additional relevant data from credible sources that might inform the analysis and review .", "a meta - analysis was not performed owing to the diverse nature of the data collected ." ], [ "a total of 45 relevant publications were identified by the search strategy and included in the literature review and analysis .", "the results of the literature review suggest that suboptimal glycaemic control is a common and widespread problem .", "the proportion of patients not achieving hba1c targets in 12 studies published between 2011 and 2015 inclusive is summarized in table 1 . even in the study reporting the best rate of control , one in every three patients was above the hba1c target .", "a multi - centre , cross - sectional study of ~5800 people with t2 dm from nine european countries found that hba1c exceeded the target of 7.0% ( 53 mmol / mol ) in 37% of cases , with rates for individual countries ranging from 26% in the netherlands up to 52% in turkey .", "tidm : type 1 diabetes mellitus ; t2 dm : type 2 diabetes mellitus . target hba1c 7.0% ( 53 mmol / mol ) , other than for the study of laiteerapong et al . which used individualized targets of hba1c < ", "6.5% , < 7.0% or < 8.0% ( < 48 mmol / mol , < 53 mmol / mol or < 64 mmol / mol ) based on patient characteristics .", "nine european countries : belgium , france , germany , greece , italy , the netherlands , spain , turkey and the united kingdom .", "other publications show that hba1c levels are markedly above target in a large proportion of people with t2 dm . in a registry - based study of > 10,000 people undergoing care for t2 dm in specialist clinics in canada ,", "62% were not at target [ hba1c 7.0% ( 53 mmol / mol ) ] and 15% had an hba1c level of 9.0% ( 75 mmol / mol ) or above . a puerto rican study of 600 adults with type 1 or type 2 diabetes reported that just 37% were at the recommended hba1c target . a survey of > 200,000 people with diabetes in taiwan found hba1c > 9.0% ( 75 mmol / mol ) in nearly one in every four people and 30% of outpatients with t2 dm attending a medical college in rural india had hba1c 9.5% ( 80 mmol / mol ) .", "there was some evidence to suggest that suboptimal control occurred more frequently in low- and middle - income countries compared with wealthier countries , with the highest rate of 87% for non - achievement of hba1c target reported in rural india . however , high proportions of people in high - income , resource - rich settings also had inadequate control of hyperglycaemia .", "suboptimal glycaemic control is often accompanied by poor control of other cardiometabolic risk factors . in a prospective cohort study of more than 3000 people with t2 dm in canada ,", "the proportion of people with hba1c , blood pressure and low - density lipoprotein ( ldl ) cholesterol at target on study entry was 47% , 46% and 36% , respectively .", "all three risk factors were controlled to target in just 19% of people , and only 7% maintained good control of glycaemia , blood pressure and ldl cholesterol throughout the 12-month study .", "a canadian study of more than 5000 people with t2 dm seen by 479 primary care physicians found that the proportions achieving targets were 50% for hba1c , 36% for blood pressure and 57% for ldl cholesterol , with only 13% reaching all three targets .", "similar results were found in more than 25,000 outpatients with t2 dm in china , with the proportions of patients achieving targets for hba1c , blood pressure and lipids of 48% , 28% and 36% , respectively .", "importantly , nearly all of the studies reviewed assessed the quality of glycaemic control against a single target of hba1c 7.0% ( 53 mmol / mol ) . within the context of individualized targets now recommended in guidelines , a somewhat higher target", "[ e.g. hba1c 7.5% ( 59 mmol / mol ) ] may have represented optimal management based on factors such as age , presence of cardiovascular comorbidities and duration of diabetes .", "this is unlikely to account for the large proportion of patients not controlled to hba1c 7.0% ( 53 mmol / mol ) and certainly should not justify the sizeable proportion of people with hba1c levels exceeding 9.0% ( 75 mmol / mol ) .", "however , it highlights that a single target is unlikely to represent optimal management for all people .", "the review provides strong evidence that in all regions of the world , many people with t2 dm are not achieving good glycaemic control . although there is considerable heterogeneity in the studies and the data generated , rates of uncontrolled glycaemia are typically in the range of 40%60% .", "consequently , many people are left at increased risk of diabetic complications and the associated burden of illness and mortality , as well as consequent economic costs .", "recent studies examining barriers to the achievement of good glycaemic control are summarized in table 2 .", "tidm : type 1 diabetes mellitus ; t2 dm : type 2 diabetes mellitus ; hcps : healthcare professionals .", "prescribers often identify poor patient motivation and self - management as leading barriers to achieving glycaemic targets .", "the second diabetes , attitudes , wishes and needs ( dawn2 ) study , included an online survey of 4785 healthcare professionals ( hcps ) ( ~40% primary care clinicians , 30% diabetes specialists , 20% nurses and 10% dieticians ) from 17 countries across four continents .", "the majority of the respondents ( 60% ) indicated a need for improvements in self - management by people with diabetes , particularly in the area of diet and exercise .", "resistance to lifestyle measures was also identified as a leading barrier by 252 primary care physicians , nurse practitioners and physician assistants in the united states , with 52% of hcps indicating that it was often or almost always a factor contributing to chronic lack of glycaemic control .", "other leading patient - related barriers identified by the hcps included psychosocial issues ( 34% ) , reluctance to use insulin ( 31% ) and non - compliance ( 30% ) . a study in the united kingdom examining the reasons for very poor glycaemic control [ hba1c 10% ( 86 mmol / mol ) ] in 128 people with t2 dm attending three primary care centres found poor adherence to lifestyle measures to be most frequently involved .", "other factors included infrequent clinic attendance ( 16% ) , side effects ( 16% ) , poor compliance with pharmacotherapy ( 14% ) , lack of knowledge of diabetes ( 14% ) , lack of titration of insulin ( 13% ) and insulin refusal ( 12% ) .", "poor patient motivation and adherence were also identified as barriers to good glycaemic control in qualitative studies with hcps .", "interviews with a small number ( n = 9 ) of hcps in the united arab emirates revealed a tendency to view patient attitudes and behaviours as the origin of poor glycaemic control and to believe that if patients were more knowledgeable about diabetes , cooperated with hcps and were adherent with treatment , then they could achieve their glycaemic targets .", "the studies reviewed also revealed that a number of healthcare system - level factors are often seen as important barriers to good glycaemic control .", "the dawn2 study , for example , found that only 30% of the hcps surveyed believed that healthcare was well organized for the management of chronic conditions .", "participants from western european countries and china were more likely to feel that healthcare was well organized than those from north or south america , eastern europe , india or japan ( figure 1 ) .", "approximately half of the participants considered the healthcare remuneration system to be a barrier and most ( ~60% ) indicated a need for better access to qualified nurse educators / specialist diabetes nurses and psychologists / psychiatrists .", "proportion of healthcare professionals reporting that healthcare in their country is well organized for the management of chronic conditions , including debates .", "a survey of 47 european countries found that while all had guidelines in place for managing diabetes , only 7 ( 15% ) had protocols for monitoring their implementation . the majority ( 83% ) of countries had or were developing national plans for diabetes , but these were often not comprehensive .", "there was substantial variability in the provision of multidisciplinary care . while 80% reported that nurses play an important role in providing education for self - management , diabetes specialist nursing was recognized as a speciality in only 19 countries ( 40% ) .", "despite the widespread belief that patients need to be more engaged and effective in self - management , only 25% of the countries recommended continuous education for all people with diabetes .", "lack of effective self - management education was also identified as a barrier in a qualitative study involving focus group discussions with 25 primary care clinicians in the united states , along with a persistent focus on acute care , poor integration of diabetes care , lack of clinical information and inadequate public health support .", "lack of coordination in care and limited access to secondary resources were also identified as important barriers by studies in the netherlands and ireland .", "limitations on the time available to manage patients are often identified as an obstacle to good glycaemic control . in the survey of 252 us primary care physicians , lack of time for treating complex patients", "was reported as often or always a factor in failure to achieve good glycaemic control by 41% of the physicians .", "treatment costs were much less frequently seen as a barrier , with 25% of the hcps indicating that it made no contribution and most ( 58% ) saying that it was a factor in only a minority ( 20%40% ) of cases of poor glycaemic control .", "other studies have also suggested that hcps in primary care often feel that they lack the time or other resources needed to manage diabetes effectively .", "this raises particular concerns , given that health policy in some countries involves shifting more of the responsibility for diabetes management onto primary care . compared with patient- and system - related barriers to effective glycaemic control , there is limited information available regarding physician - related barriers", ". however , there is evidence to suggest that a lack of diabetes - focused education may be contributing to failure to achieve therapeutic targets .", "for example , one in five of the hcps surveyed in the dawn2 study indicated that they had received no formal postgraduate diabetes training , and in a uk survey , only 35% of 2149 trainee doctors felt that their postgraduate training had prepared them adequately to optimize treatment of diabetes and only 41% reported that they would take the initiative to optimize glycaemic control .", "a survey of 209 primary care clinicians in australia identified gaps in knowledge relating to the medical management of t2 dm , with 46% self - reporting a need for education on pharmacological management .", "it appears that prescribers often lack confidence in the use of insulin , particularly with more complex regimens .", "people with diabetes often continue in poor glycaemic control for extended periods of time without appropriate action being taken . a retrospective cohort study of > 80,000 people with t2 dm in the united kingdom revealed substantial delays in intensifying treatment when glycaemic control was suboptimal . for people who were inadequately controlled on one oral glucose ", "lowering agent , the median time from hba1c being above a threshold of 7.0% ( 53 mmol / mol ) to treatment intensification with an additional oral agent was nearly 3 years , and even when hba1c was 8.0% ( 64 mmol / mol ) , intensification was delayed by a median of 1.6 years .", "there were even longer delays ( median of ~67 years ) in intensifying treatment with the addition of a third oral agent or the initiation of insulin .", "mean hba1c at intensification ranged from 8.7%9.7% ( 7283 mmol / mol ) . in a study of nearly 20,000 people with t2 dm initiated on basal insulin at hospitals across china ,", "a study of primary care records of > 17,000 patients with t2 dm being treated with oral glucose lowering agents in france found that > 3000 of the patients were not at glycaemic target and required treatment intensification . however , treatment was actually intensified in only 39% of the patients requiring it and the majority ( ~60% ) of changes were delayed by 6 months or more and a substantial proportion ( ~40% ) by more than a year .", "the diattitude study ( table 3 ) also looked at therapeutic inertia in the care of > 2000 people with t2 dm by 236 primary care physicians and found that among patients with uncontrolled glycaemia , representing 41% of all the patients studied , only 7% had their treatment intensified .", "the most common ( 60% ) reason given for not intensifying treatment was that the patient s hba1c was satisfactory .", "other leading reasons given included that lifestyle advice was more of a priority than changing medication ( 20% ) , the decision was postponed until the next clinic visit ( 11% ) and that hba1c had decreased since the previous clinic visit ( 7% ) , although in the majority ( 58% ) of these cases , an hba1c reduction was not confirmed by the available records .", "the problem appears to be common and widespread , with further studies in bahrain , croatia , spain , taiwan and the united states reporting high rates ( typically 30%60% ) of clinical inertia when managing elevated glucose levels in people with diabetes .", "summary of studies reporting rates of and factors contributing to therapeutic inertia in the management of type 2 diabetes .", "ldl : low - density lipoprotein ; t2 dm : type 2 diabetes mellitus ; hcps : healthcare professionals .", "a number of studies have attempted to explore the factors that may act as obstacles to action in addressing inadequate glycaemic control . in an online survey completed by 508 primary care physicians who provided data on 770 elderly patients ( > 65 years ) ,", "the leading reason for not initiating glucose - lowering medication , given as the first - ranked reason for 58% of the patients and a contributory factor in nearly all patients , was that they were treated with diet and exercise , and presumably , this was believed to be adequate management ( figure 2 ) .", "however , 33% of the patients were not at target hba1c , suggesting substantial clinical inertia in this population .", "other reasons given for not starting drug treatment included that hyperglycaemia was mild ( 84% ) , patient concerns ( 61% ) , physician concerns about glucose - lowering agents ( 49% ) and presence of comorbidities and polypharmacy ( 37% ) . when asked to identify a threshold for initiating treatment with glucose - lowering agents in the elderly population ,", "the mean hba1c threshold value stated was 7.1% ( 54 mmol / mol ) . in approximately 20% of the cases", ", the physicians indicated that they were planning to initiate glucose - lowering therapy within the next month , but more than half ( 54% ) of these patients already had an hba1c level above the physician - stated threshold .", "results from an online survey of 508 us primary care physicians providing clinical date for 770 patients .", "a qualitative study of 20 hcps in the united kingdom found a pattern of seeking to lessen their own sense of accountability for clinical inertia by emphasizing patient - level barriers such as comorbidities , motivation and self - management capabilities as well as health system level barriers , especially time constraints .", "there was also a tendency for the hcps to overestimate the achievement of targets in their primary care centres .", "factors contributing to therapeutic inertia in the initiation of insulin treatment for t2 dm were investigated in the translating research into action for diabetes ( triad ) project using structured interviews with primary care hcps in the united states .", "the majority of the 83 clinicians interviewed supported guideline recommendations on glycaemic targets , with 69% choosing an hba1c < 7% ( < 53 mmol / mol ) as the ideal target for good glycaemic control . at the same time ,", "the most frequently cited reasons for setting higher individualized hba1c targets included advanced age or short life expectancy ( 54% ) , poor self - management capacity due to poor cognitive abilities ( 35% ) , presence of comorbidities ( 34% ) , low educational level or poor health literacy ( 34% ) and patients unwillingness to self - manage their diabetes ( 33% ) .", "the most frequent reasons cited by the clinicians for not initiating insulin treatment were patient refusal or resistance ( 64% ) and lack of patient self - management skills ( 43% ) .", "factors determining initiation of insulin were also investigated using data for 1933 people with t2 dm from the french national health and wellness survey , an annual internet - based survey among french adults .", "early initiation of insulin therapy was almost 10 times more likely to be prescribed by an endocrinologist or diabetologist than by a primary care physician .", "early versus late insulin initiation was also more likely in patients who were younger , had diabetes - related complications or smoked .", "insulin - treated patients were more likely to be adherent , and there was no apparent deterioration in quality of life associated with insulin use . in focus group discussions with 114 primary care physicians in belgium ,", "many of the physicians found the concept of clinical inertia interesting , but some also found it insulting and felt it implied that they were passive in their work .", "they also noted that it was important to differentiate between cases where there was a conscious decision to not pursue lower targets based on a consideration of the individual patient factors and those where there was genuine inertia .", "it was acknowledged that genuine clinical inertia was a real risk in primary care , but there was a tendency to ascribe this risk to patient- and system - level barriers rather than physician - level behaviours and practices . in summary , recent studies suggest that clinical inertia is a common and widespread problem , typically affecting the care of 30%50% of people with t2 dm .", "the causes are diverse and complex , but surveys of hcps tend to emphasize patient - level barriers to treatment intensification , such as patient reluctance to start insulin therapy , or system - level barriers , such as disconnects between what is recommended in guidelines and what is reimbursed in practice .", "however , it also appears that hcps sometimes delay taking necessary action and may be willing to tolerate periods of ", "it also appears that many hcps , while recognizing the importance of good glycaemic control , have low expectations for their patients .", "the issue of clinical inertia needs to be better understood if it is to be addressed effectively , particularly regarding the patient- , hcp- and system - level factors that underlie it .", "this is challenging as clinical inertia as defined in studies typically captures a range of behaviours , some of which may not reflect suboptimal care .", "however , studies of clinical inertia often apply a single glycaemic threshold [ typically hba1c = 7% ( 53 mmol / mol ) ] across a broad range of patients .", "guidelines for management of t2 dm have traditionally advocated a stepwise approach to management , in which treatment is started with diet and exercise , followed by the addition of oral antihyperglycaemic monotherapy , then combination therapy and eventually insulin . however , when applied systematically without considering individual factors such as the patient s current hba1c level , it can lead to treatment futility with people receiving therapy that has little chance of lowering glucose levels sufficiently to achieve their glycaemic target .", "several recent publications have indicated potential benefits of early , intensive intervention with pharmacotherapy , including combinations of oral glucose lowering agents and/or insulin .", "the treatment options for type 2 diabetes in adolescents and youth ( today ) study compared metformin alone , metformin plus a thiazolidinedione and metformin plus intensive lifestyle intervention as initial therapy for new - onset t2 dm in people aged 1017 years and with obesity . the proportion of people who maintained glycaemic control over 5 years was significantly higher with the combination pharmacotherapy than either metformin alone or metformin plus intensive lifestyle measures .", "the combination pharmacotherapy was also associated with greater improvements in insulin sensitivity and -cell function compared with the other two approaches .", "the results lend support to early , intensive intervention with combination therapy , at least in this population of young people with new - onset t2 dm .", "early intensive therapy was also studied in older ( mean age = 45 years ) people with newly diagnosed t2 dm who started treatment for 3 months with metformin plus insulin and were then randomized to either continuing metformin plus insulin therapy or triple oral glucose lowering therapy with metformin , glyburide and pioglitazone .", "good glycaemic control was maintained for over 6 years with no significant difference between the groups ( end of study hba1c = 7.3% for metformin plus insulin and 6.4 for triple oral combination ; p = 0.4 ) .", "a review of the health records for nearly 3000 people with t2 dm in the united states found that early initiation of metformin ( within 6 months of diagnosis ) significantly reduced hba1c ( 0.36% ; p < 0.001 ) and bmi ( 0.46 kg / m ; p < ", "the likelihood of achieving hba1c 7% was doubled and the likelihood of requiring therapy intensification was reduced by 28% with early versus delayed initiation of metformin .", "another study in the united states looking at medical records for 5870 people with t2 dm found a similar pattern for early introduction of combination oral glucose ", "early intensification was defined as addition of a second oral drug to metformin therapy within 3 months of treatment failure ( hba1c 7.5% ) and late intensification as the introduction of combination therapy 1015 months after loss of glycaemic control .", "people who received early intensification were 38% more likely to achieve good glycaemic control at 1.52 years than those with late intensification . in the advance trial of 11,140 patients with t2 dm , therapeutic intensification with addition of an oral glucose ", "lowering agent doubled the chance of achieving good glycaemic control , and with intensification with insulin , the odds were increased 2.5-fold ." ], [ "a total of 45 relevant publications were identified by the search strategy and included in the literature review and analysis ." ], [ "the results of the literature review suggest that suboptimal glycaemic control is a common and widespread problem .", "the proportion of patients not achieving hba1c targets in 12 studies published between 2011 and 2015 inclusive is summarized in table 1 . even in the study reporting the best rate of control , one in every three patients was above the hba1c target . a multi - centre ,", "cross - sectional study of ~5800 people with t2 dm from nine european countries found that hba1c exceeded the target of 7.0% ( 53 mmol / mol ) in 37% of cases , with rates for individual countries ranging from 26% in the netherlands up to 52% in turkey .", "target hba1c 7.0% ( 53 mmol / mol ) , other than for the study of laiteerapong et al . which used individualized targets of hba1c < 6.5% , < 7.0% or < 8.0% ( < 48 mmol / mol , < 53 mmol / mol or < 64 mmol / mol ) based on patient characteristics .", "nine european countries : belgium , france , germany , greece , italy , the netherlands , spain , turkey and the united kingdom .", "other publications show that hba1c levels are markedly above target in a large proportion of people with t2 dm . in a registry - based study of > 10,000 people undergoing care for t2 dm in specialist clinics in canada ,", "62% were not at target [ hba1c 7.0% ( 53 mmol / mol ) ] and 15% had an hba1c level of 9.0% ( 75 mmol / mol ) or above . a puerto rican study of 600 adults with type 1 or type 2 diabetes reported that just 37% were at the recommended hba1c target . a survey of > 200,000 people with diabetes in taiwan found hba1c > 9.0% ( 75 mmol / mol ) in nearly one in every four people and 30% of outpatients with t2 dm attending a medical college in rural india had hba1c 9.5% ( 80 mmol / mol ) .", "there was some evidence to suggest that suboptimal control occurred more frequently in low- and middle - income countries compared with wealthier countries , with the highest rate of 87% for non - achievement of hba1c target reported in rural india . however , high proportions of people in high - income , resource - rich settings also had inadequate control of hyperglycaemia .", "suboptimal glycaemic control is often accompanied by poor control of other cardiometabolic risk factors . in a prospective cohort study of more than 3000 people with t2 dm in canada ,", "the proportion of people with hba1c , blood pressure and low - density lipoprotein ( ldl ) cholesterol at target on study entry was 47% , 46% and 36% , respectively .", "all three risk factors were controlled to target in just 19% of people , and only 7% maintained good control of glycaemia , blood pressure and ldl cholesterol throughout the 12-month study .", "a canadian study of more than 5000 people with t2 dm seen by 479 primary care physicians found that the proportions achieving targets were 50% for hba1c , 36% for blood pressure and 57% for ldl cholesterol , with only 13% reaching all three targets .", "similar results were found in more than 25,000 outpatients with t2 dm in china , with the proportions of patients achieving targets for hba1c , blood pressure and lipids of 48% , 28% and 36% , respectively .", "importantly , nearly all of the studies reviewed assessed the quality of glycaemic control against a single target of hba1c 7.0% ( 53 mmol / mol ) . within the context of individualized targets now recommended in guidelines , a somewhat higher target [", "e.g. hba1c 7.5% ( 59 mmol / mol ) ] may have represented optimal management based on factors such as age , presence of cardiovascular comorbidities and duration of diabetes .", "this is unlikely to account for the large proportion of patients not controlled to hba1c 7.0% ( 53 mmol / mol ) and certainly should not justify the sizeable proportion of people with hba1c levels exceeding 9.0% ( 75 mmol / mol ) .", "however , it highlights that a single target is unlikely to represent optimal management for all people .", "the review provides strong evidence that in all regions of the world , many people with t2 dm are not achieving good glycaemic control .", "although there is considerable heterogeneity in the studies and the data generated , rates of uncontrolled glycaemia are typically in the range of 40%60% .", "consequently , many people are left at increased risk of diabetic complications and the associated burden of illness and mortality , as well as consequent economic costs ." ], [ "recent studies examining barriers to the achievement of good glycaemic control are summarized in table 2 .", "tidm : type 1 diabetes mellitus ; t2 dm : type 2 diabetes mellitus ; hcps : healthcare professionals .", "prescribers often identify poor patient motivation and self - management as leading barriers to achieving glycaemic targets .", "the second diabetes , attitudes , wishes and needs ( dawn2 ) study , included an online survey of 4785 healthcare professionals ( hcps ) ( ~40% primary care clinicians , 30% diabetes specialists , 20% nurses and 10% dieticians ) from 17 countries across four continents .", "the majority of the respondents ( 60% ) indicated a need for improvements in self - management by people with diabetes , particularly in the area of diet and exercise .", "resistance to lifestyle measures was also identified as a leading barrier by 252 primary care physicians , nurse practitioners and physician assistants in the united states , with 52% of hcps indicating that it was often or almost always a factor contributing to chronic lack of glycaemic control .", "other leading patient - related barriers identified by the hcps included psychosocial issues ( 34% ) , reluctance to use insulin ( 31% ) and non - compliance ( 30% ) . a study in the united kingdom examining the reasons for very poor glycaemic control [ hba1c 10% ( 86 mmol / mol ) ] in 128 people with t2 dm attending three primary care centres found poor adherence to lifestyle measures to be most frequently involved .", "other factors included infrequent clinic attendance ( 16% ) , side effects ( 16% ) , poor compliance with pharmacotherapy ( 14% ) , lack of knowledge of diabetes ( 14% ) , lack of titration of insulin ( 13% ) and insulin refusal ( 12% ) .", "poor patient motivation and adherence were also identified as barriers to good glycaemic control in qualitative studies with hcps .", "interviews with a small number ( n = 9 ) of hcps in the united arab emirates revealed a tendency to view patient attitudes and behaviours as the origin of poor glycaemic control and to believe that if patients were more knowledgeable about diabetes , cooperated with hcps and were adherent with treatment , then they could achieve their glycaemic targets . the studies reviewed also revealed that a number of healthcare system - level factors are often seen as important barriers to good glycaemic control .", "the dawn2 study , for example , found that only 30% of the hcps surveyed believed that healthcare was well organized for the management of chronic conditions .", "participants from western european countries and china were more likely to feel that healthcare was well organized than those from north or south america , eastern europe , india or japan ( figure 1 ) .", "approximately half of the participants considered the healthcare remuneration system to be a barrier and most ( ~60% ) indicated a need for better access to qualified nurse educators / specialist diabetes nurses and psychologists / psychiatrists .", "proportion of healthcare professionals reporting that healthcare in their country is well organized for the management of chronic conditions , including debates .", "a survey of 47 european countries found that while all had guidelines in place for managing diabetes , only 7 ( 15% ) had protocols for monitoring their implementation . the majority ( 83% ) of countries had or were developing national plans for diabetes , but these were often not comprehensive", "there was substantial variability in the provision of multidisciplinary care . while 80% reported that nurses play an important role in providing education for self - management , diabetes specialist nursing was recognized as a speciality in only 19 countries ( 40% ) .", "despite the widespread belief that patients need to be more engaged and effective in self - management , only 25% of the countries recommended continuous education for all people with diabetes .", "lack of effective self - management education was also identified as a barrier in a qualitative study involving focus group discussions with 25 primary care clinicians in the united states , along with a persistent focus on acute care , poor integration of diabetes care , lack of clinical information and inadequate public health support .", "lack of coordination in care and limited access to secondary resources were also identified as important barriers by studies in the netherlands and ireland .", "limitations on the time available to manage patients are often identified as an obstacle to good glycaemic control . in the survey of 252 us primary care physicians , lack of time for treating complex patients", "was reported as often or always a factor in failure to achieve good glycaemic control by 41% of the physicians .", "treatment costs were much less frequently seen as a barrier , with 25% of the hcps indicating that it made no contribution and most ( 58% ) saying that it was a factor in only a minority ( 20%40% ) of cases of poor glycaemic control . other studies have also suggested that hcps in primary care often feel that they lack the time or other resources needed to manage diabetes effectively .", "this raises particular concerns , given that health policy in some countries involves shifting more of the responsibility for diabetes management onto primary care .", "compared with patient- and system - related barriers to effective glycaemic control , there is limited information available regarding physician - related barriers .", "however , there is evidence to suggest that a lack of diabetes - focused education may be contributing to failure to achieve therapeutic targets .", "for example , one in five of the hcps surveyed in the dawn2 study indicated that they had received no formal postgraduate diabetes training , and in a uk survey , only 35% of 2149 trainee doctors felt that their postgraduate training had prepared them adequately to optimize treatment of diabetes and only 41% reported that they would take the initiative to optimize glycaemic control .", "a survey of 209 primary care clinicians in australia identified gaps in knowledge relating to the medical management of t2 dm , with 46% self - reporting a need for education on pharmacological management .", "it appears that prescribers often lack confidence in the use of insulin , particularly with more complex regimens ." ], [ "prescribers often identify poor patient motivation and self - management as leading barriers to achieving glycaemic targets .", "the second diabetes , attitudes , wishes and needs ( dawn2 ) study , included an online survey of 4785 healthcare professionals ( hcps ) ( ~40% primary care clinicians , 30% diabetes specialists , 20% nurses and 10% dieticians ) from 17 countries across four continents .", "the majority of the respondents ( 60% ) indicated a need for improvements in self - management by people with diabetes , particularly in the area of diet and exercise .", "resistance to lifestyle measures was also identified as a leading barrier by 252 primary care physicians , nurse practitioners and physician assistants in the united states , with 52% of hcps indicating that it was often or almost always a factor contributing to chronic lack of glycaemic control .", "other leading patient - related barriers identified by the hcps included psychosocial issues ( 34% ) , reluctance to use insulin ( 31% ) and non - compliance ( 30% ) . a study in the united kingdom examining the reasons for very poor glycaemic control [ hba1c 10% ( 86 mmol / mol ) ] in 128 people with t2 dm attending three primary care centres found poor adherence to lifestyle measures to be most frequently involved .", "other factors included infrequent clinic attendance ( 16% ) , side effects ( 16% ) , poor compliance with pharmacotherapy ( 14% ) , lack of knowledge of diabetes ( 14% ) , lack of titration of insulin ( 13% ) and insulin refusal ( 12% ) .", "poor patient motivation and adherence were also identified as barriers to good glycaemic control in qualitative studies with hcps .", "interviews with a small number ( n = 9 ) of hcps in the united arab emirates revealed a tendency to view patient attitudes and behaviours as the origin of poor glycaemic control and to believe that if patients were more knowledgeable about diabetes , cooperated with hcps and were adherent with treatment , then they could achieve their glycaemic targets ." ], [ "the studies reviewed also revealed that a number of healthcare system - level factors are often seen as important barriers to good glycaemic control . the dawn2 study ,", "for example , found that only 30% of the hcps surveyed believed that healthcare was well organized for the management of chronic conditions .", "participants from western european countries and china were more likely to feel that healthcare was well organized than those from north or south america , eastern europe , india or japan ( figure 1 ) .", "approximately half of the participants considered the healthcare remuneration system to be a barrier and most ( ~60% ) indicated a need for better access to qualified nurse educators / specialist diabetes nurses and psychologists / psychiatrists .", "proportion of healthcare professionals reporting that healthcare in their country is well organized for the management of chronic conditions , including debates .", "a survey of 47 european countries found that while all had guidelines in place for managing diabetes , only 7 ( 15% ) had protocols for monitoring their implementation . the majority ( 83% ) of countries had or were developing national plans for diabetes , but these were often not comprehensive .", "there was substantial variability in the provision of multidisciplinary care . while 80% reported that nurses play an important role in providing education for self - management , diabetes specialist nursing was recognized as a speciality in only 19 countries ( 40% ) .", "despite the widespread belief that patients need to be more engaged and effective in self - management , only 25% of the countries recommended continuous education for all people with diabetes .", "lack of effective self - management education was also identified as a barrier in a qualitative study involving focus group discussions with 25 primary care clinicians in the united states , along with a persistent focus on acute care , poor integration of diabetes care , lack of clinical information and inadequate public health support .", "lack of coordination in care and limited access to secondary resources were also identified as important barriers by studies in the netherlands and ireland .", "limitations on the time available to manage patients are often identified as an obstacle to good glycaemic control . in the survey of 252 us primary care physicians , lack of time for treating complex patients", "was reported as often or always a factor in failure to achieve good glycaemic control by 41% of the physicians .", "treatment costs were much less frequently seen as a barrier , with 25% of the hcps indicating that it made no contribution and most ( 58% ) saying that it was a factor in only a minority ( 20%40% ) of cases of poor glycaemic control .", "other studies have also suggested that hcps in primary care often feel that they lack the time or other resources needed to manage diabetes effectively .", "this raises particular concerns , given that health policy in some countries involves shifting more of the responsibility for diabetes management onto primary care ." ], [ "compared with patient- and system - related barriers to effective glycaemic control , there is limited information available regarding physician - related barriers", ". however , there is evidence to suggest that a lack of diabetes - focused education may be contributing to failure to achieve therapeutic targets .", "for example , one in five of the hcps surveyed in the dawn2 study indicated that they had received no formal postgraduate diabetes training , and in a uk survey , only 35% of 2149 trainee doctors felt that their postgraduate training had prepared them adequately to optimize treatment of diabetes and only 41% reported that they would take the initiative to optimize glycaemic control .", "a survey of 209 primary care clinicians in australia identified gaps in knowledge relating to the medical management of t2 dm , with 46% self - reporting a need for education on pharmacological management .", "it appears that prescribers often lack confidence in the use of insulin , particularly with more complex regimens ." ], [ "people with diabetes often continue in poor glycaemic control for extended periods of time without appropriate action being taken . a retrospective cohort study of > 80,000 people with t2 dm in the united kingdom revealed substantial delays in intensifying treatment when glycaemic control was suboptimal . for people who were inadequately controlled on one oral glucose ", "lowering agent , the median time from hba1c being above a threshold of 7.0% ( 53 mmol / mol ) to treatment intensification with an additional oral agent was nearly 3 years , and even when hba1c was 8.0% ( 64 mmol / mol ) , intensification was delayed by a median of 1.6 years .", "there were even longer delays ( median of ~67 years ) in intensifying treatment with the addition of a third oral agent or the initiation of insulin .", "mean hba1c at intensification ranged from 8.7%9.7% ( 7283 mmol / mol ) . in a study of nearly 20,000 people with t2", "dm initiated on basal insulin at hospitals across china , mean hba1c at the time of insulin initiation was 9.6% .", "a study of primary care records of > 17,000 patients with t2 dm being treated with oral glucose lowering agents in france found that > 3000 of the patients were not at glycaemic target and required treatment intensification .", "however , treatment was actually intensified in only 39% of the patients requiring it and the majority ( ~60% ) of changes were delayed by 6 months or more and a substantial proportion ( ~40% ) by more than a year .", "the diattitude study ( table 3 ) also looked at therapeutic inertia in the care of > 2000 people with t2 dm by 236 primary care physicians and found that among patients with uncontrolled glycaemia , representing 41% of all the patients studied , only 7% had their treatment intensified .", "the most common ( 60% ) reason given for not intensifying treatment was that the patient s hba1c was satisfactory .", "other leading reasons given included that lifestyle advice was more of a priority than changing medication ( 20% ) , the decision was postponed until the next clinic visit ( 11% ) and that hba1c had decreased since the previous clinic visit ( 7% ) , although in the majority ( 58% ) of these cases , an hba1c reduction was not confirmed by the available records .", "the problem appears to be common and widespread , with further studies in bahrain , croatia , spain , taiwan and the united states reporting high rates ( typically 30%60% ) of clinical inertia when managing elevated glucose levels in people with diabetes .", "summary of studies reporting rates of and factors contributing to therapeutic inertia in the management of type 2 diabetes .", "ldl : low - density lipoprotein ; t2 dm : type 2 diabetes mellitus ; hcps : healthcare professionals .", "a number of studies have attempted to explore the factors that may act as obstacles to action in addressing inadequate glycaemic control . in an online survey completed by 508 primary care physicians who provided data on 770 elderly patients ( > 65 years ) ,", "the leading reason for not initiating glucose - lowering medication , given as the first - ranked reason for 58% of the patients and a contributory factor in nearly all patients , was that they were treated with diet and exercise , and presumably , this was believed to be adequate management ( figure 2 ) .", "however , 33% of the patients were not at target hba1c , suggesting substantial clinical inertia in this population .", "other reasons given for not starting drug treatment included that hyperglycaemia was mild ( 84% ) , patient concerns ( 61% ) , physician concerns about glucose - lowering agents ( 49% ) and presence of comorbidities and polypharmacy ( 37% ) .", "when asked to identify a threshold for initiating treatment with glucose - lowering agents in the elderly population , the mean hba1c threshold value stated was 7.1% ( 54 mmol / mol ) . in approximately 20% of the cases", ", the physicians indicated that they were planning to initiate glucose - lowering therapy within the next month , but more than half ( 54% ) of these patients already had an hba1c level above the physician - stated threshold .", "results from an online survey of 508 us primary care physicians providing clinical date for 770 patients .", "a qualitative study of 20 hcps in the united kingdom found a pattern of seeking to lessen their own sense of accountability for clinical inertia by emphasizing patient - level barriers such as comorbidities , motivation and self - management capabilities as well as health system level barriers , especially time constraints .", "there was also a tendency for the hcps to overestimate the achievement of targets in their primary care centres .", "factors contributing to therapeutic inertia in the initiation of insulin treatment for t2 dm were investigated in the translating research into action for diabetes ( triad ) project using structured interviews with primary care hcps in the united states .", "the majority of the 83 clinicians interviewed supported guideline recommendations on glycaemic targets , with 69% choosing an hba1c < 7% ( < 53 mmol / mol ) as the ideal target for good glycaemic control . at the same time ,", "the most frequently cited reasons for setting higher individualized hba1c targets included advanced age or short life expectancy ( 54% ) , poor self - management capacity due to poor cognitive abilities ( 35% ) , presence of comorbidities ( 34% ) , low educational level or poor health literacy ( 34% ) and patients unwillingness to self - manage their diabetes ( 33% ) .", "the most frequent reasons cited by the clinicians for not initiating insulin treatment were patient refusal or resistance ( 64% ) and lack of patient self - management skills ( 43% ) .", "factors determining initiation of insulin were also investigated using data for 1933 people with t2 dm from the french national health and wellness survey , an annual internet - based survey among french adults .", "early initiation of insulin therapy was almost 10 times more likely to be prescribed by an endocrinologist or diabetologist than by a primary care physician .", "early versus late insulin initiation was also more likely in patients who were younger , had diabetes - related complications or smoked .", "insulin - treated patients were more likely to be adherent , and there was no apparent deterioration in quality of life associated with insulin use . in focus group discussions with 114 primary care physicians in belgium ,", "many of the physicians found the concept of clinical inertia interesting , but some also found it insulting and felt it implied that they were passive in their work .", "they also noted that it was important to differentiate between cases where there was a conscious decision to not pursue lower targets based on a consideration of the individual patient factors and those where there was genuine inertia .", "it was acknowledged that genuine clinical inertia was a real risk in primary care , but there was a tendency to ascribe this risk to patient- and system - level barriers rather than physician - level behaviours and practices . in summary , recent studies suggest that clinical inertia is a common and widespread problem , typically affecting the care of 30%50% of people with t2 dm .", "the causes are diverse and complex , but surveys of hcps tend to emphasize patient - level barriers to treatment intensification , such as patient reluctance to start insulin therapy , or system - level barriers , such as disconnects between what is recommended in guidelines and what is reimbursed in practice .", "however , it also appears that hcps sometimes delay taking necessary action and may be willing to tolerate periods of ", "it also appears that many hcps , while recognizing the importance of good glycaemic control , have low expectations for their patients .", "the issue of clinical inertia needs to be better understood if it is to be addressed effectively , particularly regarding the patient- , hcp- and system - level factors that underlie it .", "this is challenging as clinical inertia as defined in studies typically captures a range of behaviours , some of which may not reflect suboptimal care .", "however , studies of clinical inertia often apply a single glycaemic threshold [ typically hba1c = 7% ( 53 mmol / mol ) ] across a broad range of patients ." ], [ "guidelines for management of t2 dm have traditionally advocated a stepwise approach to management , in which treatment is started with diet and exercise , followed by the addition of oral antihyperglycaemic monotherapy , then combination therapy and eventually insulin . however , when applied systematically without considering individual factors such as the patient s current hba1c level , it can lead to treatment futility with people receiving therapy that has little chance of lowering glucose levels sufficiently to achieve their glycaemic target .", "several recent publications have indicated potential benefits of early , intensive intervention with pharmacotherapy , including combinations of oral glucose lowering agents and/or insulin .", "the treatment options for type 2 diabetes in adolescents and youth ( today ) study compared metformin alone , metformin plus a thiazolidinedione and metformin plus intensive lifestyle intervention as initial therapy for new - onset t2 dm in people aged 1017 years and with obesity . the proportion of people who maintained glycaemic control over 5 years was significantly higher with the combination pharmacotherapy than either metformin alone or metformin plus intensive lifestyle measures .", "the combination pharmacotherapy was also associated with greater improvements in insulin sensitivity and -cell function compared with the other two approaches .", "the results lend support to early , intensive intervention with combination therapy , at least in this population of young people with new - onset t2 dm .", "early intensive therapy was also studied in older ( mean age = 45 years ) people with newly diagnosed t2 dm who started treatment for 3 months with metformin plus insulin and were then randomized to either continuing metformin plus insulin therapy or triple oral glucose ", "good glycaemic control was maintained for over 6 years with no significant difference between the groups ( end of study hba1c = 7.3% for metformin plus insulin and 6.4 for triple oral combination ; p = 0.4 ) .", "a review of the health records for nearly 3000 people with t2 dm in the united states found that early initiation of metformin ( within 6 months of diagnosis ) significantly reduced hba1c ( 0.36% ; p < 0.001 ) and bmi ( 0.46 kg / m ; p < 0.001 ) compared with when introduction of pharmacotherapy with metformin was delayed .", "the likelihood of achieving hba1c 7% was doubled and the likelihood of requiring therapy intensification was reduced by 28% with early versus delayed initiation of metformin .", "another study in the united states looking at medical records for 5870 people with t2 dm found a similar pattern for early introduction of combination oral glucose ", "early intensification was defined as addition of a second oral drug to metformin therapy within 3 months of treatment failure ( hba1c 7.5% ) and late intensification as the introduction of combination therapy 1015 months after loss of glycaemic control .", "people who received early intensification were 38% more likely to achieve good glycaemic control at 1.52 years than those with late intensification . in the advance trial of 11,140 patients with t2 dm , therapeutic intensification with addition of an oral glucose ", "lowering agent doubled the chance of achieving good glycaemic control , and with intensification with insulin , the odds were increased 2.5-fold ." ], [ "recently published evidence highlights that many people with t2 dm do not achieve good control of blood glucose leaving them at increased and avoidable risk of serious complications .", "a tendency to focus on patient - related obstacles may fail to consider issues arising from the attitudes , perceptions and behaviours of healthcare providers .", "in particular , there are often delays in the implementation of appropriate interventions to achieve glycaemic targets due to a complex range of negative factors that exacerbate the situation .", "an improved understanding of these factors would better inform strategies to assist hcps in more timely treatment of inadequately controlled glycaemia . despite a wide range of therapeutic options available", ", insufficient numbers of people with type 2 diabetes are reaching their glycaemic targets and large numbers are exposed to prolonged periods of hyperglycaemia .", "a number of obstacles exist globally that must be addressed and overcome if we are to improve the outcomes for patients ." ], [ "despite a wide range of therapeutic options available , insufficient numbers of people with type 2 diabetes are reaching their glycaemic targets and large numbers are exposed to prolonged periods of hyperglycaemia .", "a number of obstacles exist globally that must be addressed and overcome if we are to improve the outcomes for patients ." ] ]

[ "calcium channels have not been identified previously in c. albicans , but high - affinity and low - affinity calcium - uptake systems ( hacs and lacs ) have been described and partially characterized in s. cerevisiae \n [ 8 , 9 ] .", "we identified and deleted in c albicans , homologs of components of these systems the voltage - gated cch1p channel and the stretch - activated channel mid1p .", "together , these two channels are thought to form a complex that defines the hacs .", "cacch1 encodes a putative 2254 amino acid protein with 38.4% identity to its s. cerevisiae homolog . the 24 predicted transmembrane ( tm ) regions in cacch1p are arranged in four repeated units ( i to iv ) of six tm domains , as they are in mammalian calcium channels where they tetramerize to form the core 1-subunit of l - type ca channels .", "the tm regions include segments responsible for voltage - dependency , channel - specificity , and association with organic calcium - channel blockers .", "the c. albicans cch1p and the human voltage - gated calcium channel cav1.2 are 62.9% similar and 37.7% identical over a 20 amino acid region in the four ca selective , pore - forming p domains . in the voltage - sensitive s4 domains , 13 of the 23 basic residues in cav1.2", "the camid1 gene sequence had 36.9% and 34.4% identity to scmid1 and schizosaccharomyces pombe yam8(+ ) , respectively .", "the 559 amino acid protein it encodes contains four putative tm regions ( h1 - 4 ) , potential n - glycosylation sites , a helix - loop - helix domain and 10 conserved cysteines that , in s. cerevisiae , form the c1/c2 domains that are essential for activity and localization [ 11 , 16 ] . unlike in s. cerevisiae , the c1/c2 regions in c. albicans are located between h3 and the c - terminal h4 .", "cafig1p shares 48.5% identity with scfig1p , a putative homolog of mammalian pmp-22/emp / mp20/claudins , which are involved in the trafficking and assembly of membrane - associated proteins .", "consistent with emp homology , cafig1p has four predicted n - glycosylation sites in the first of its four tm domains .", "the role of scfig1p in s. cerevisiae is not well - defined , but it localizes predominantly to the plasma membrane and is required for low - affinity calcium transport and for the calcium - dependent fusion of mating projections .", "control strains were created by the generation of conditional mutants expressing a single remaining wild - type gene from the mrp1 maltose - regulatable promoter ( cch1 or mid1 ) or by reintegration of the gene at a high - expression locus ( fig1 or mid1 ) ( see the experimental procedures in the supplemental data available with this article online ) . in s. cerevisiae , hacs is activated by low ca conditions and is regulated by calcineurin , which controls calcium homeostasis and specific stress responses [ 811 , 18 ] .", "scfig1p ( lacs ) activity was only revealed under conditions when hacs was inhibited by rich media .", "in contrast to hacs , lacs is insensitive to calcineurin and its affinity for ca is 16-fold lower .", "however , both systems are activated on exposure of cells to -pheromone , which stimulates the formation of the polarized mating projection [ 8 , 19 ] .", "localization of scmid1p and scfig1p to the mating projection is dependent on scspa2 and scbni1 \n [ 19 , 8 ] . in c. albicans ,", "caspa2p and cabni1p are components of the hyphal polarisome and spitzenkrper , respectively , and are required for polarized hyphal extension . mid1p and fig1p may therefore be involved in polarized cell growth in both organisms . in c. albicans , mrna encoding hacs and lacs component proteins", "was detected in both yeast and hyphal growth conditions and in c. albicans - infected rabbit kidney ( data not shown ) . also , both hacs and lacs mutants were affected in the thigmotropic response ( see below ) . therefore ,", "both hacs and lacs are expressed in c. albicans during the normal in vitro and in vivo growth of this fungus . after prolonged incubation on ca - depleted solid minimal medium ( > 14 days ) , wild - type c. albicans colonies produced aberrant lobed margins that could be alleviated by the addition of 10 mm ca to the medium .", "emerging colonies of the cacch1 and camid1 mutants produced lobed colonies at 2 days ( see supplemental data ) .", "the aberrant morphologies of the colonies of cafig1 , cacna1 , and cacnb1 mutants were partially alleviated on supplementation with exogenous ca , supporting the view that these genes are involved in calcium signaling in c. albicans .", "the growth rates of the cacch1 , camid1 , and cacch1-mid1 mutants were reduced by 19% , 23% , and 25% , respectively , when they were grown in the yeast form , compared to the control strain ( p = < 0.037 ) , but the extension rates of hyphae were not affected by the mutations ( data not shown ) .", "consistent with the putative roles of camid1p and cch1p as ca channels , ca accumulation in the camid1 and cacch1 mutants was significantly reduced after 2 hr culture in ca - depleted medium supplemented with ca ( p = < 0.001 ; supplemental data ) .", "the double cacch1-mid1 mutant had similar ca accumulation and yeast growth rates to the single mutants , consistent with the model that these proteins operate within the same pathway .", "this is consistent with reports that , in s. cerevisiae , cch1-mid1p are involved in ca homeostasis under low - ca conditions , where fig1p activity is not detectable .", "tropic growth responses to applied external electric fields ( galvanotropism ) have been observed in migratory and tip - growing cells .", "growing c. albicans hyphae orient toward the cathode in such fields . to characterize hyphal orientation , we measured the angle at which germ tubes emerged from the mother cell ( emergence angle ) and the angle of the hyphal tip after 6 hr growth ( final angle ) relative to the cathode . to investigate the role of calcium ions and channels in galvanotropism , we measured the emergence and final angles of hyphae exposed to electrical fields in media of varying extracellular [ ca ] or in the presence of pharmacological agents that block the activity of l - type voltage - gated cation channels . in c. albicans ,", "site selection of germ tubes is not strictly controlled by the bud proteins that regulate bud - site selection during axial and bipolar budding of s. cerevisiae \n .", "we observed that , in electric fields , germ tubes were formed almost exclusively on the cathode - facing pole of cells , suggesting that imposed electrical fields can override cortical bud evagination markers .", "the percentage cathodal germ - tube emergence was positively correlated with extracellular [ ca ] .", "cathodal orientation of wild - type cells was attenuated in ca - depleted medium ( figures 1a and 1b ) and was further significantly reduced in the presence of calcium - channel blockers ( p = < 0.05 ) ( figure 2a ) .", "therefore , ca influx is important for cathodal evagination of the germ tube in c. albicans . localized ca uptake correlates with sites of germination of other cell types , such as the zygotes of the brown alga silvetia compressa \n .", "even in low ca medium , extending hyphae grew toward the cathode and reoriented their direction of growth when the field polarity was reversed ( figure 1c ) .", "deletion of cacch1 resulted in a significant reduction in the cathodal orientation of germ - tube emergence ( p = < 0.001 ) ( figure 2b ) .", "cathodal orientation was restored in the conditional cacch1 strain when cacch1 expression was induced by growth on maltose but not when camid1 was induced in the cch1/mid1/mrp1-mid1 strain .", "we hypothesize that the voltage - gated cacch1p ca channel is activated by membrane depolarization at the cathodal face of yeast cells and that this results in localized ca uptake and subsequent induction of localized polarized growth .", "the effects of extracellular [ ca ] deprivation and cacch1 deletion primarily affected the site of germ - tube emergence . in low [ ca ] ,", "in the cacch1 mutant and in the presence of l - type ca - channel blockers , cathodal germ - tube emergence was approximately half that of the control strain , but after 6 hr exposure to an electric field , this effect was almost lost ( figure 2a ) .", "even hyphae that had emerged from the anodal face of the cacch1 mother cell eventually responded to the electric field .", "this suggested that differences exist between the mechanism that establishes cathodal growth and that which maintains it . only the former appears to depend on calcium influx and cch1p .", "the ability of fungal hyphae to exhibit tropic growth responses in relation to changes in substratum topography is well - known in plant pathogens and has been demonstrated previously for c. albicans and certain dermatophytes [ 24 , 25 ] .", "some plant pathogenic fungi also use topographical features to trigger formation of the appressorium infection structure ( thigmodifferentiation ) .", "we tested whether treatments and mutations that attenuated galvanotropism also influenced thigmotropic orientation in c. albicans hyphae . in wild - type cells , 60% of hyphae that contacted a 0.79 m ridge responded by reorienting their growth axis ( figures 3a and 3b ) .", "this is consistent with previous observations that thigmotropism was attenuated by inhibitors of stretch - activated ca channels and supports a model whereby changes in the underlying topography induce stresses in the membrane that are sensed by mid1p ( figure 4b ) .", "reorientation of the cacch1 strain was also significantly reduced ( p = < 0.001 ) compared to the control strain ( figure 3 ) .", "thigmotropism was attenuated in the regulatable cacch1/mrp1-cch1 under repressing conditions and restored to normal under mrp1p - inducing conditions .", "expression of camid1 in the cacch1/camid1/mrp1-mid1 conditional mutant did not restore the reorientation response , confirming that cacch1p is required for thigmotropism .", "we propose that stretch activation of camid1p , and subsequent opening of the cacch1p channel , results in localized ca influx that exerts an influence on the molecular machinery involved in polarized growth of the hyphal tip .", "this influence overrides or repositions , or both , the molecular markers that defined the original axis of growth .", "however , because hyphal reorientation was observed in almost 30% of ridge interactions in the camid1 mutant , either cacch1p is activated independently of camid1p or other sensing elements also contribute to the regulation of thigmotropism .", "the function of fig1p in c. albicans is not known , but in s. cerevisiae its deletion resulted in defective cell - cell fusion during mating , suggesting it could be involved in the delivery of components to the fusion site . because its deletion in c. albicans reduces hyphal reorientation during contact - sensing , cafig1p may again be involved in targeted delivery of secretory vesicles to the cell surface .", "no instances of tip bifurcation were observed when a hyphal tip contacted a ridge , even when the angle of approach was 90 ( figure 3c ) , suggesting that orientation - determining factors are a nondivisible entity or discrete protein complex .", "calcium - dependent gene transcription in eukaryotes involves activation of a transcription factor by the phosphatase , calcineurin .", "calcineurin regulates fungal morphogenesis , calcium flux , and homeostasis . in c. albicans , calcineurin acts on the transcription factor ,", "the deletion of cacrz1 affected both cathodal germ - tube emergence ( figure 2b ) and thigmotropism ( figure 3 ) , whereas the inhibition of calcineurin with fk506 or deletion of genes encoding either the catalytic cacna1 or regulatory cacnb1 calcineurin subunits only affected cathodal emergence ( p = 0.006 ) .", "our results suggest that calcineurin is required for the establishment of cathodal cell polarity in an electric field but not for the reorientation of already - polarized hyphal tips during contact - sensing .", "although cacrz1p influences cacch1 expression , it does not appear to be essential for basal expression or activation of camid1p or cacch1p because the morphology of cacrz1 mutant colonies was the same as the wild - type strain , whereas the camid1 and cacch1 mutants formed aberrant colonies ( see supplemental data ) .", "other targets of cacrz1p have been identified in c. albicans and are required for resistance to membrane damage and alkaline stress .", "it is not known whether cacrz1p - mediated gene expression regulates events that are upstream or downstream of calcium - influx induced tropisms .", "the ability of external cues to influence the orientation of hyphal growth of the human pathogenic fungus , candida albicans , may be relevant to their capacity to infiltrate between human cells during tissue invasion . we have found that reorientation of c. albicans hyphae in relation to electrical fields and topographical signals is ca dependent and is mediated by ca channels and a ca - dependent transcription factor , cacrz1p .", "calcineurin , the primary regulator of cacrz1p , was required for galvanotropism but not thigmotropism .", "we observed that the hyphae of mutants lacking the stretch - activated camid1p or voltage - activated cacch1p proteins grew normally but were attenuated in orientation responses resulting from physical contact or imposed electric fields , respectively .", "we propose a model whereby localized ca - channel activation , caused by localized changes in membrane potential or membrane stretch , results in calcium influx that directs polarized growth ( figure 4 ) .", "stretch - activated ion channel activity has been described in patch - clamp analysis of c. albicans membranes , but the ion selectivity of these channels is not known . in plant cells ,", "mechanosensory ca channels produce high cytosolic ca and initiation of localized cell - wall expansion at sites of shear stress .", "similarly , in mammalian synapses , localized channel activation produces intracellular ca microdomains where the spatial boundary of the domain correlates with the capacity for efficient vesicle exocytosis .", "localized ca influx may result in an asymmetry in the tip - high ca gradient in hyphae of c. albicans and other fungi .", "this alters the axis of growth by increasing the rate of vesicle fusion within a local ca - high microdomain or by affecting the activity of calcium - binding proteins that are involved in polarized cell extension .", "ca influx may therefore override existing polarity determinants at the cortex of evaginating mother cells and growing hyphae .", "thus , both thigmotropic and galvanotropic responses of c. albicans hyphae are dependent on a single ca - regulated orientation mechanism .", "for strains used in this study , mutant - strain construction , growth conditions , and determination of ca accumulation , see the supplemental experimental procedures .", "yeast cells were adhered to poly - l - lysine - coated microscope slides and placed on the flat bed of a biorad midi - sub cell electrophoresis tank and cultured in modified soll 's medium at 37c 1c for 6 hr at 10 v / cm and a current of 33 2 ma .", "this field strength could be applied without affecting germ - tube formation or hyphal extension rate , yet it is sufficient for inducing cytoplasmic [ ca ] increases in other cell systems [ 3 , 4 ] .", "hyphal orientation at the site of germ - tube emergence and at the germ - tube tip relative to the cathode were measured with improvision openlab 2.0 software . the percentage cathodal orientation ( p )", "was calculated with p = ( sin /n ) 100 , for n measurements .", "a minimum of 100 cells was measured in each of three independent experiments for each treatment .", "yeast cells were adhered to poly - l - lysine - coated quartz slides featuring ridges of 0.79 m 40 nm and a pitch of 25 m ( kelvin nanotechnology , glasgow , uk ) .", "this ridge height caused maximal hypha reorientation in a preliminary trial of five heights ( data not shown ) .", "slides were placed in 20 ml prewarmed 20% ( v / v ) newborn - calf serum and 2% ( w / v ) glucose at 37c for 6 hr for inducing hyphae .", "the number of hyphae reorienting on contact with a ridge was expressed as a percentage of the total observed interactions .", "a minimum of 100 interactions was observed per strain in each experiment , and results were reported as the mean value from three independent experiments sd ." ]

[ "<S> summaryeukaryotic cells from fungal hyphae to neurites that grow by polarized extension must coordinate cell growth and cell orientation to enable them to exhibit growth tropisms and to respond to relevant environmental cues . </S>", "<S> such cells generally maintain a tip - high ca2 + cytoplasmic gradient , which is correlated with their ability to exhibit polarized tip growth and to respond to growth - directing extracellular signals [ 15 ] . in yeast and other fungi , </S>", "<S> the polarisome , exocyst , arp2/3 , and spitzenkrper protein complexes collectively orchestrate tip growth and cell polarity , but it is not clear whether these molecular complexes also regulate cell orientation or whether they are influenced by cytoplasmic ca2 + gradients . </S>", "<S> hyphae of the human pathogenic fungus candida albicans reorient their growth axis in response to underlying surface topography ( thigmotropism ) [ 6 ] and imposed electric fields ( galvanotropism ) [ 7 ] . </S>", "<S> the establishment and maintenance of directional growth in relation to these environmental cues was ca2 + dependent . </S>", "<S> tropisms were attenuated in media containing low ca2 + , or calcium - channel blockers , and in mutants where calcium channels or elements of the calcium signaling pathway were deleted </S>", "<S> . therefore galvanotropism and thigmotropism may both be mediated by localized ca2 + influx at sites of polarized growth via ca2 + channels that are activated by appropriate environmental signals . </S>" ]

[ "Results and Discussion", "Conclusion", "Experimental Procedures" ]

[ [ "calcium channels have not been identified previously in c. albicans , but high - affinity and low - affinity calcium - uptake systems ( hacs and lacs ) have been described and partially characterized in s. cerevisiae \n [ 8 , 9 ] .", "we identified and deleted in c albicans , homologs of components of these systems the voltage - gated cch1p channel and the stretch - activated channel mid1p .", "together , these two channels are thought to form a complex that defines the hacs .", "cacch1 encodes a putative 2254 amino acid protein with 38.4% identity to its s. cerevisiae homolog . the 24 predicted transmembrane ( tm ) regions in cacch1p are arranged in four repeated units ( i to iv ) of six tm domains , as they are in mammalian calcium channels where they tetramerize to form the core 1-subunit of l - type ca channels .", "the tm regions include segments responsible for voltage - dependency , channel - specificity , and association with organic calcium - channel blockers .", "the c. albicans cch1p and the human voltage - gated calcium channel cav1.2 are 62.9% similar and 37.7% identical over a 20 amino acid region in the four ca selective , pore - forming p domains . in the voltage - sensitive s4 domains , 13 of the 23 basic residues in cav1.2", "the camid1 gene sequence had 36.9% and 34.4% identity to scmid1 and schizosaccharomyces pombe yam8(+ ) , respectively .", "the 559 amino acid protein it encodes contains four putative tm regions ( h1 - 4 ) , potential n - glycosylation sites , a helix - loop - helix domain and 10 conserved cysteines that , in s. cerevisiae , form the c1/c2 domains that are essential for activity and localization [ 11 , 16 ] . unlike in s. cerevisiae , the c1/c2 regions in c. albicans are located between h3 and the c - terminal h4 .", "cafig1p shares 48.5% identity with scfig1p , a putative homolog of mammalian pmp-22/emp / mp20/claudins , which are involved in the trafficking and assembly of membrane - associated proteins .", "consistent with emp homology , cafig1p has four predicted n - glycosylation sites in the first of its four tm domains .", "the role of scfig1p in s. cerevisiae is not well - defined , but it localizes predominantly to the plasma membrane and is required for low - affinity calcium transport and for the calcium - dependent fusion of mating projections .", "control strains were created by the generation of conditional mutants expressing a single remaining wild - type gene from the mrp1 maltose - regulatable promoter ( cch1 or mid1 ) or by reintegration of the gene at a high - expression locus ( fig1 or mid1 ) ( see the experimental procedures in the supplemental data available with this article online ) . in s. cerevisiae , hacs is activated by low ca conditions and is regulated by calcineurin , which controls calcium homeostasis and specific stress responses [ 811 , 18 ] .", "scfig1p ( lacs ) activity was only revealed under conditions when hacs was inhibited by rich media .", "in contrast to hacs , lacs is insensitive to calcineurin and its affinity for ca is 16-fold lower .", "however , both systems are activated on exposure of cells to -pheromone , which stimulates the formation of the polarized mating projection [ 8 , 19 ] .", "localization of scmid1p and scfig1p to the mating projection is dependent on scspa2 and scbni1 \n [ 19 , 8 ] . in c. albicans ,", "caspa2p and cabni1p are components of the hyphal polarisome and spitzenkrper , respectively , and are required for polarized hyphal extension . mid1p and fig1p may therefore be involved in polarized cell growth in both organisms . in c. albicans , mrna encoding hacs and lacs component proteins", "was detected in both yeast and hyphal growth conditions and in c. albicans - infected rabbit kidney ( data not shown ) . also , both hacs and lacs mutants were affected in the thigmotropic response ( see below ) . therefore ,", "both hacs and lacs are expressed in c. albicans during the normal in vitro and in vivo growth of this fungus . after prolonged incubation on ca - depleted solid minimal medium ( > 14 days ) , wild - type c. albicans colonies produced aberrant lobed margins that could be alleviated by the addition of 10 mm ca to the medium .", "emerging colonies of the cacch1 and camid1 mutants produced lobed colonies at 2 days ( see supplemental data ) .", "the aberrant morphologies of the colonies of cafig1 , cacna1 , and cacnb1 mutants were partially alleviated on supplementation with exogenous ca , supporting the view that these genes are involved in calcium signaling in c. albicans .", "the growth rates of the cacch1 , camid1 , and cacch1-mid1 mutants were reduced by 19% , 23% , and 25% , respectively , when they were grown in the yeast form , compared to the control strain ( p = < 0.037 ) , but the extension rates of hyphae were not affected by the mutations ( data not shown ) .", "consistent with the putative roles of camid1p and cch1p as ca channels , ca accumulation in the camid1 and cacch1 mutants was significantly reduced after 2 hr culture in ca - depleted medium supplemented with ca ( p = < 0.001 ; supplemental data ) .", "the double cacch1-mid1 mutant had similar ca accumulation and yeast growth rates to the single mutants , consistent with the model that these proteins operate within the same pathway .", "this is consistent with reports that , in s. cerevisiae , cch1-mid1p are involved in ca homeostasis under low - ca conditions , where fig1p activity is not detectable .", "tropic growth responses to applied external electric fields ( galvanotropism ) have been observed in migratory and tip - growing cells .", "growing c. albicans hyphae orient toward the cathode in such fields . to characterize hyphal orientation , we measured the angle at which germ tubes emerged from the mother cell ( emergence angle ) and the angle of the hyphal tip after 6 hr growth ( final angle ) relative to the cathode . to investigate the role of calcium ions and channels in galvanotropism , we measured the emergence and final angles of hyphae exposed to electrical fields in media of varying extracellular [ ca ] or in the presence of pharmacological agents that block the activity of l - type voltage - gated cation channels . in c. albicans ,", "site selection of germ tubes is not strictly controlled by the bud proteins that regulate bud - site selection during axial and bipolar budding of s. cerevisiae \n .", "we observed that , in electric fields , germ tubes were formed almost exclusively on the cathode - facing pole of cells , suggesting that imposed electrical fields can override cortical bud evagination markers .", "the percentage cathodal germ - tube emergence was positively correlated with extracellular [ ca ] .", "cathodal orientation of wild - type cells was attenuated in ca - depleted medium ( figures 1a and 1b ) and was further significantly reduced in the presence of calcium - channel blockers ( p = < 0.05 ) ( figure 2a ) .", "therefore , ca influx is important for cathodal evagination of the germ tube in c. albicans . localized ca uptake correlates with sites of germination of other cell types , such as the zygotes of the brown alga silvetia compressa \n .", "even in low ca medium , extending hyphae grew toward the cathode and reoriented their direction of growth when the field polarity was reversed ( figure 1c ) .", "deletion of cacch1 resulted in a significant reduction in the cathodal orientation of germ - tube emergence ( p = < 0.001 ) ( figure 2b ) .", "cathodal orientation was restored in the conditional cacch1 strain when cacch1 expression was induced by growth on maltose but not when camid1 was induced in the cch1/mid1/mrp1-mid1 strain .", "we hypothesize that the voltage - gated cacch1p ca channel is activated by membrane depolarization at the cathodal face of yeast cells and that this results in localized ca uptake and subsequent induction of localized polarized growth .", "the effects of extracellular [ ca ] deprivation and cacch1 deletion primarily affected the site of germ - tube emergence . in low [ ca ] ,", "in the cacch1 mutant and in the presence of l - type ca - channel blockers , cathodal germ - tube emergence was approximately half that of the control strain , but after 6 hr exposure to an electric field , this effect was almost lost ( figure 2a ) .", "even hyphae that had emerged from the anodal face of the cacch1 mother cell eventually responded to the electric field .", "this suggested that differences exist between the mechanism that establishes cathodal growth and that which maintains it . only the former appears to depend on calcium influx and cch1p .", "the ability of fungal hyphae to exhibit tropic growth responses in relation to changes in substratum topography is well - known in plant pathogens and has been demonstrated previously for c. albicans and certain dermatophytes [ 24 , 25 ] .", "some plant pathogenic fungi also use topographical features to trigger formation of the appressorium infection structure ( thigmodifferentiation ) .", "we tested whether treatments and mutations that attenuated galvanotropism also influenced thigmotropic orientation in c. albicans hyphae . in wild - type cells , 60% of hyphae that contacted a 0.79 m ridge responded by reorienting their growth axis ( figures 3a and 3b ) .", "this is consistent with previous observations that thigmotropism was attenuated by inhibitors of stretch - activated ca channels and supports a model whereby changes in the underlying topography induce stresses in the membrane that are sensed by mid1p ( figure 4b ) .", "reorientation of the cacch1 strain was also significantly reduced ( p = < 0.001 ) compared to the control strain ( figure 3 ) .", "thigmotropism was attenuated in the regulatable cacch1/mrp1-cch1 under repressing conditions and restored to normal under mrp1p - inducing conditions .", "expression of camid1 in the cacch1/camid1/mrp1-mid1 conditional mutant did not restore the reorientation response , confirming that cacch1p is required for thigmotropism .", "we propose that stretch activation of camid1p , and subsequent opening of the cacch1p channel , results in localized ca influx that exerts an influence on the molecular machinery involved in polarized growth of the hyphal tip .", "this influence overrides or repositions , or both , the molecular markers that defined the original axis of growth .", "however , because hyphal reorientation was observed in almost 30% of ridge interactions in the camid1 mutant , either cacch1p is activated independently of camid1p or other sensing elements also contribute to the regulation of thigmotropism .", "the function of fig1p in c. albicans is not known , but in s. cerevisiae its deletion resulted in defective cell - cell fusion during mating , suggesting it could be involved in the delivery of components to the fusion site . because its deletion in c. albicans reduces hyphal reorientation during contact - sensing , cafig1p may again be involved in targeted delivery of secretory vesicles to the cell surface .", "no instances of tip bifurcation were observed when a hyphal tip contacted a ridge , even when the angle of approach was 90 ( figure 3c ) , suggesting that orientation - determining factors are a nondivisible entity or discrete protein complex .", "calcium - dependent gene transcription in eukaryotes involves activation of a transcription factor by the phosphatase , calcineurin .", "calcineurin regulates fungal morphogenesis , calcium flux , and homeostasis . in c. albicans , calcineurin acts on the transcription factor ,", "the deletion of cacrz1 affected both cathodal germ - tube emergence ( figure 2b ) and thigmotropism ( figure 3 ) , whereas the inhibition of calcineurin with fk506 or deletion of genes encoding either the catalytic cacna1 or regulatory cacnb1 calcineurin subunits only affected cathodal emergence ( p = 0.006 ) .", "our results suggest that calcineurin is required for the establishment of cathodal cell polarity in an electric field but not for the reorientation of already - polarized hyphal tips during contact - sensing .", "although cacrz1p influences cacch1 expression , it does not appear to be essential for basal expression or activation of camid1p or cacch1p because the morphology of cacrz1 mutant colonies was the same as the wild - type strain , whereas the camid1 and cacch1 mutants formed aberrant colonies ( see supplemental data ) .", "other targets of cacrz1p have been identified in c. albicans and are required for resistance to membrane damage and alkaline stress .", "it is not known whether cacrz1p - mediated gene expression regulates events that are upstream or downstream of calcium - influx induced tropisms ." ], [ "the ability of external cues to influence the orientation of hyphal growth of the human pathogenic fungus , candida albicans , may be relevant to their capacity to infiltrate between human cells during tissue invasion . we have found that reorientation of c. albicans hyphae in relation to electrical fields and topographical signals is ca dependent and is mediated by ca channels and a ca - dependent transcription factor , cacrz1p .", "calcineurin , the primary regulator of cacrz1p , was required for galvanotropism but not thigmotropism .", "we observed that the hyphae of mutants lacking the stretch - activated camid1p or voltage - activated cacch1p proteins grew normally but were attenuated in orientation responses resulting from physical contact or imposed electric fields , respectively .", "we propose a model whereby localized ca - channel activation , caused by localized changes in membrane potential or membrane stretch , results in calcium influx that directs polarized growth ( figure 4 ) .", "stretch - activated ion channel activity has been described in patch - clamp analysis of c. albicans membranes , but the ion selectivity of these channels is not known . in plant cells ,", "mechanosensory ca channels produce high cytosolic ca and initiation of localized cell - wall expansion at sites of shear stress .", "similarly , in mammalian synapses , localized channel activation produces intracellular ca microdomains where the spatial boundary of the domain correlates with the capacity for efficient vesicle exocytosis .", "localized ca influx may result in an asymmetry in the tip - high ca gradient in hyphae of c. albicans and other fungi .", "this alters the axis of growth by increasing the rate of vesicle fusion within a local ca - high microdomain or by affecting the activity of calcium - binding proteins that are involved in polarized cell extension .", "ca influx may therefore override existing polarity determinants at the cortex of evaginating mother cells and growing hyphae .", "thus , both thigmotropic and galvanotropic responses of c. albicans hyphae are dependent on a single ca - regulated orientation mechanism ." ], [ "for strains used in this study , mutant - strain construction , growth conditions , and determination of ca accumulation , see the supplemental experimental procedures .", "yeast cells were adhered to poly - l - lysine - coated microscope slides and placed on the flat bed of a biorad midi - sub cell electrophoresis tank and cultured in modified soll 's medium at 37c 1c for 6 hr at 10 v / cm and a current of 33 2 ma .", "this field strength could be applied without affecting germ - tube formation or hyphal extension rate , yet it is sufficient for inducing cytoplasmic [ ca ] increases in other cell systems [ 3 , 4 ] .", "hyphal orientation at the site of germ - tube emergence and at the germ - tube tip relative to the cathode were measured with improvision openlab 2.0 software . the percentage cathodal orientation ( p )", "was calculated with p = ( sin /n ) 100 , for n measurements .", "a minimum of 100 cells was measured in each of three independent experiments for each treatment .", "yeast cells were adhered to poly - l - lysine - coated quartz slides featuring ridges of 0.79 m 40 nm and a pitch of 25 m ( kelvin nanotechnology , glasgow , uk ) .", "this ridge height caused maximal hypha reorientation in a preliminary trial of five heights ( data not shown ) .", "slides were placed in 20 ml prewarmed 20% ( v / v ) newborn - calf serum and 2% ( w / v ) glucose at 37c for 6 hr for inducing hyphae .", "the number of hyphae reorienting on contact with a ridge was expressed as a percentage of the total observed interactions .", "a minimum of 100 interactions was observed per strain in each experiment , and results were reported as the mean value from three independent experiments sd ." ] ]

[ "periodontitis causes loss of tooth supporting bone . the bone loss may be vertical , horizontal , or combined .", "regenerative periodontal therapy aims to predictably restore the structure and function of these lost tissues . for this purpose ,", "these grafting materials are available in different forms . among the graft materials , autogenous bone grafts", "have been considered the gold standard and can be harvested in either particulate or in the form of bone blocks . with the advancement in techniques and materials used for regeneration ,", "sources of bone include bone from healing extraction wounds , bone from edentulous ridges , bone trephined from within the jaw , exostoses , and bone removed during osteoplasty and ostectomy .", "bone removed from the donor site during osteoplasty has the advantage of eliminating the need for additional donor site while accomplishing the objective of obtaining desirable bone contour in the surgical site .", "the obvious advantage of this technique is the ease of obtaining bone from already exposed surgical sites , but the disadvantages are its relatively low predictability and inability to procure adequate material for large defects . in this case report ,", "bone from ledges present on the mandibular posterior area was removed with the simple technique of scrapping with a sharp sickle scaler and the resulting graft was used to fill a two wall defect distal to first molar .", "a 37-year - old male patient reported to the department of periodontology government dental college , calicut with a chief complaint of bleeding from gums and bad breath of 3 months duration . on clinical examination , generalized moderate to deep periodontal pockets and bleeding on probing was noticed .", "based on the history , clinical and radiographic findings , the patient was diagnosed with chronic generalized periodontitis .", "six weeks after performing thorough scaling and root planning and following a strict plaque control regime , the case was reevaluated .", "since the pocket depth was 7 mm distal to 36 , it was decided to proceed with flap surgery [ figure 1 ] . after giving block anesthesia in the left mandibular posterior area ,", "meticulous defect debridement and root planing was done to remove subgingival plaque , calculus , and inflammatory granulation tissue .", "a deep two wall defect was observed distal to first molar , heavy ledges were noticed extending bucally from second premolar to the second molar area [ figure 2 ] .", "first it was decided to remove the ledges as a part of osteoplasty procedure and then utilize the removed bone to fill the two wall defect . for this purpose , a sharp sickle scaler ( pdt cruise scaler u15 - 33 r113 , pdt inc . , us ) [ figure 3 ] was used in a scrapping motion with sufficient force such that the bone was essentially shaved off [ figure 4 ] .", "the harvested bone was carefully collected in a dappen dish and then transferred to the defect with a periosteal elevator [ figures 5 and 6 ] .", "the flap was repositioned , single interrupted sutures were placed and periodontal dressing was given [ figures 7 and 8 ] . the patient was reviewed after 1-week , the healing was found to be uneventful .", "the patient was put on the maintenance phase with oral prophylaxis performed every 3 months .", "clinically , there was a reduction in probing pocket depth from 7 mm to 3 mm [ figure 9 ] .", "radiographs revealed radio opacity in the site distal to first molar , suggestive of bone fill in the defect [ figure 10 ] .", "defect exposed , ledges seen ( a and b ) pdt cruise scaler u15 - 33 r113 used to remove ledge autograft obtained from ledge removal with sickle scaler autogenous graft placed in the defect distal to first molar postoperative view after 1-year ( a ) preoperative radiograph , ( b ) postoperative radiograph after 1-year", "there is ample evidence that autogenous bone grafts shore up the formation of new attachment .", "autogenous bone , long considered the gold standard of grafting materials , is currently the only osteogenic graft available to clinical practitioners .", "grafted autogenous bone heals into growing bone through the processes of osteogenesis , osteoinduction , and osteoconduction .", "moreover , auto - grafts are bioabsorbable , nonallergenic , easy to handle , and not costly .", "rapid revascularization occurs around autogenous bone graft particles , and the graft can release growth and differentiation factors .", "although iliac crest is one of the most preferred site , it is not always recommended due to its associated problems , such as postoperative infection ; exfoliation and sequestration ; varying rates of healing ; root resorption and rapid recurrence of the defect , in addition to increased patient expense and patient morbidity .", "intramembranous ( craniofacial origin ) bones have been found to have more survival potential than endo - chondral ( long bones ) origin .", "insulin - like growth factor - ii and transforming growth factor- have been found to be in a greater concentration in calvarial bone than in bone from the iliac crest or vertebral body .", "this increase in concentration of growth factors leads to a greater capacity for bone repair and graft retention .", "the use of intraoral graft material is , however , limited by the restricted donor sites in the oral cavity for extensive grafting .", "the use of bone removed from ledges as a part of osteoplasty procedure offers the added advantage of eliminating the nonphysiologic bony architecture .", "the goal of osseous respective therapy is to reshape the marginal bone to resemble that of the alveolar process undamaged by periodontal disease .", "the reshaping process is an attempt to gradualize the bone sufficiently to allow soft tissue structures to follow the contour of the bone . in this case ,", "radicular blending was done using the sharp blade of scaler to gradualize the bone over the entire radicular surface providing a smooth , blended surface for good flap adaptation .", "many techniques and devices are available to harvest intraoral autogenous bone grafts , such as : bone scraper , rotary instruments , bone chisels , rongeur pliers and piezoelectric devices .", "particulate graft material is easy to procure and is best suited for two , three , or more walled defect , as they usually provide adequate space for placing and retaining the bone graft material for a sufficient period .", "the commonly used technique to procure particulate grafts is the use of carbide bur at speeds between 5000 and 30,000 rpm .", "the inability to procure adequate material for large defects is a major disadvantage of using rotary instruments .", "conventional osteotomy or milling procedures has some limitations like overheating of the bone when water cooling is insufficient , which leads to possible structural bone changes on living cells .", "a simple technique for removal of bone using a sharp sickle was done , in this case , which turned out to be effective means for harvesting sufficient amounts of bone graft .", "although specialized bone scrapers have been used for obtaining grafts from intraoral sites before . to the best of our knowledge , there are no previous reports of bone harvesting done with scaler alone .", "reported higher percentages of cultured osteoblast cells for both rotary drill and the piezoelectric device , 88.9% and 87.9% , respectively .", "they concluded that the harvesting methods are not different concerning the detrimental effect on the viability .", "another study by bacci et al . showed contradictory results , their histological findings revealed that smaller particle size bone chips , obtained with a piezoelectric device , had less vital bone ( 64.83% ) compared with the small particle size bone chips harvested with a bone scraper ( 75.34% ) . in this case , sufficient amounts of autogenous particulate bone were obtained during the ledge removal process to fill the two wall defect distal to first molar .", "instead the sharp blade of the sickle scaler was utilized , which was also less time consuming .", "autogenous grafts have long remained and will continue to remain as the first preference for bone grafting procedures because of its superior biologic properties .", "ledges are frequently found in posterior regions and if properly managed can act as a source of autogenous graft .", "routinely used scaling instruments may be utilized in certain situations instead of using specialized bone scrapping instrument ." ]

[ "<S> autogenous bone grafts have been considered the gold standard for bone grafting procedures . </S>", "<S> this case report describes the management of a two wall defect by utilizing the autogenous bone graft obtained during removal of ledges as a part of osteoplasty procedure . </S>", "<S> the bone was removed with a sickle scaler , and sufficient amounts of bone graft material were obtained to fill a two wall defect distal to left mandibular first molar . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "periodontitis causes loss of tooth supporting bone . the bone loss may be vertical , horizontal , or combined .", "regenerative periodontal therapy aims to predictably restore the structure and function of these lost tissues . for this purpose ,", "these grafting materials are available in different forms . among the graft materials , autogenous bone grafts", "have been considered the gold standard and can be harvested in either particulate or in the form of bone blocks . with the advancement in techniques and materials used for regeneration ,", "sources of bone include bone from healing extraction wounds , bone from edentulous ridges , bone trephined from within the jaw , exostoses , and bone removed during osteoplasty and ostectomy .", "bone removed from the donor site during osteoplasty has the advantage of eliminating the need for additional donor site while accomplishing the objective of obtaining desirable bone contour in the surgical site .", "the obvious advantage of this technique is the ease of obtaining bone from already exposed surgical sites , but the disadvantages are its relatively low predictability and inability to procure adequate material for large defects . in this case report ,", "bone from ledges present on the mandibular posterior area was removed with the simple technique of scrapping with a sharp sickle scaler and the resulting graft was used to fill a two wall defect distal to first molar ." ], [ "a 37-year - old male patient reported to the department of periodontology government dental college , calicut with a chief complaint of bleeding from gums and bad breath of 3 months duration . on clinical examination , generalized moderate to deep periodontal pockets and bleeding on probing was noticed .", "based on the history , clinical and radiographic findings , the patient was diagnosed with chronic generalized periodontitis .", "six weeks after performing thorough scaling and root planning and following a strict plaque control regime , the case was reevaluated .", "since the pocket depth was 7 mm distal to 36 , it was decided to proceed with flap surgery [ figure 1 ] . after giving block anesthesia in the left mandibular posterior area ,", "meticulous defect debridement and root planing was done to remove subgingival plaque , calculus , and inflammatory granulation tissue .", "a deep two wall defect was observed distal to first molar , heavy ledges were noticed extending bucally from second premolar to the second molar area [ figure 2 ] .", "first it was decided to remove the ledges as a part of osteoplasty procedure and then utilize the removed bone to fill the two wall defect . for this purpose , a sharp sickle scaler ( pdt cruise scaler u15 - 33 r113 , pdt inc . , us ) [ figure 3 ] was used in a scrapping motion with sufficient force such that the bone was essentially shaved off [ figure 4 ] .", "the harvested bone was carefully collected in a dappen dish and then transferred to the defect with a periosteal elevator [ figures 5 and 6 ] .", "the flap was repositioned , single interrupted sutures were placed and periodontal dressing was given [ figures 7 and 8 ] . the patient was reviewed after 1-week , the healing was found to be uneventful .", "the patient was put on the maintenance phase with oral prophylaxis performed every 3 months .", "clinically , there was a reduction in probing pocket depth from 7 mm to 3 mm [ figure 9 ] .", "radiographs revealed radio opacity in the site distal to first molar , suggestive of bone fill in the defect [ figure 10 ] .", "defect exposed , ledges seen ( a and b ) pdt cruise scaler u15 - 33 r113 used to remove ledge autograft obtained from ledge removal with sickle scaler autogenous graft placed in the defect distal to first molar postoperative view after 1-year ( a ) preoperative radiograph , ( b ) postoperative radiograph after 1-year" ], [ "there is ample evidence that autogenous bone grafts shore up the formation of new attachment .", "autogenous bone , long considered the gold standard of grafting materials , is currently the only osteogenic graft available to clinical practitioners .", "grafted autogenous bone heals into growing bone through the processes of osteogenesis , osteoinduction , and osteoconduction .", "moreover , auto - grafts are bioabsorbable , nonallergenic , easy to handle , and not costly .", "rapid revascularization occurs around autogenous bone graft particles , and the graft can release growth and differentiation factors .", "although iliac crest is one of the most preferred site , it is not always recommended due to its associated problems , such as postoperative infection ; exfoliation and sequestration ; varying rates of healing ; root resorption and rapid recurrence of the defect , in addition to increased patient expense and patient morbidity .", "intramembranous ( craniofacial origin ) bones have been found to have more survival potential than endo - chondral ( long bones ) origin .", "insulin - like growth factor - ii and transforming growth factor- have been found to be in a greater concentration in calvarial bone than in bone from the iliac crest or vertebral body .", "this increase in concentration of growth factors leads to a greater capacity for bone repair and graft retention .", "the use of intraoral graft material is , however , limited by the restricted donor sites in the oral cavity for extensive grafting .", "the use of bone removed from ledges as a part of osteoplasty procedure offers the added advantage of eliminating the nonphysiologic bony architecture .", "the goal of osseous respective therapy is to reshape the marginal bone to resemble that of the alveolar process undamaged by periodontal disease .", "the reshaping process is an attempt to gradualize the bone sufficiently to allow soft tissue structures to follow the contour of the bone . in this case ,", "radicular blending was done using the sharp blade of scaler to gradualize the bone over the entire radicular surface providing a smooth , blended surface for good flap adaptation .", "many techniques and devices are available to harvest intraoral autogenous bone grafts , such as : bone scraper , rotary instruments , bone chisels , rongeur pliers and piezoelectric devices .", "particulate graft material is easy to procure and is best suited for two , three , or more walled defect , as they usually provide adequate space for placing and retaining the bone graft material for a sufficient period .", "the commonly used technique to procure particulate grafts is the use of carbide bur at speeds between 5000 and 30,000 rpm .", "the inability to procure adequate material for large defects is a major disadvantage of using rotary instruments .", "conventional osteotomy or milling procedures has some limitations like overheating of the bone when water cooling is insufficient , which leads to possible structural bone changes on living cells .", "a simple technique for removal of bone using a sharp sickle was done , in this case , which turned out to be effective means for harvesting sufficient amounts of bone graft .", "although specialized bone scrapers have been used for obtaining grafts from intraoral sites before . to the best of our knowledge , there are no previous reports of bone harvesting done with scaler alone .", "reported higher percentages of cultured osteoblast cells for both rotary drill and the piezoelectric device , 88.9% and 87.9% , respectively .", "they concluded that the harvesting methods are not different concerning the detrimental effect on the viability .", "another study by bacci et al . showed contradictory results , their histological findings revealed that smaller particle size bone chips , obtained with a piezoelectric device , had less vital bone ( 64.83% ) compared with the small particle size bone chips harvested with a bone scraper ( 75.34% ) . in this case , sufficient amounts of autogenous particulate bone were obtained during the ledge removal process to fill the two wall defect distal to first molar .", "instead the sharp blade of the sickle scaler was utilized , which was also less time consuming .", "autogenous grafts have long remained and will continue to remain as the first preference for bone grafting procedures because of its superior biologic properties .", "ledges are frequently found in posterior regions and if properly managed can act as a source of autogenous graft .", "routinely used scaling instruments may be utilized in certain situations instead of using specialized bone scrapping instrument ." ] ]

[ "tuberculosis remains a frequent and important infectious disease worldwide , it occurs with , in approximately 30% of all cases , extrapulmonary involvement in the form of tuberculous pleurisy ( tp ) .", "and tp increases the morbidity and mortality associated with tuberculosis , and it is a major economic and health burden all over the world . the differential diagnosis of tp reliably is challenging although many conventional diagnostic methods are used , including microscopic analysis of pleural fluid or sputum smears , culturing for mycobacteria and even performing a pleural biopsy .", "each of these methods has significant limitations : microscopic analysis of pleural fluid is rarely positive ( < 5% ) , culture of pleural fluid shows poor sensitivity ( 24 - 58% ) , culturing for mycobacterium tuberculosis requires several weeks , and pleural biopsy , which is considered the best method for confirming tp diagnosis , is invasive and technically difficult .", "even more invasive procedures such as thoracoscopy or thoracotomy have been used for differential diagnosis of tp , but these complex procedures can cause complications and even increase morbidity . these led investigators to explore several biomarkers as possible diagnostic indicators .", "its release by mesothelial cells in pleura contributes to the occurrence and development of tp through three mechanisms : it acts as a proinflammatory cytokine to attract neutrophils to kill mycobacteria directly as part of an innate immune response ; it acts synergistically with interferon ( ifn- ) to activate macrophages as part of an adaptive immune response ; and it recruits nave t cells to the granuloma .", "tumor necrosis factor- ( tnf- ) levels in the pleural fluid are significantly higher in patients with tuberculosis than in patients with pulmonary malignancy .", "some studies have reported that levels of tnf- in pleural fluid provide high diagnostic sensitivity ( 96.0% ) and specificity ( 93.0% ) .", "other studies , however , have reported much lower corresponding values of 70% and 66% . however , the comprehensive picture of the diagnostic usefulness of tnf- levels in pleural fluid is not obvious .", "therefore in this article we will product a meta - analysis to explore the diagnostic accuracy of pleural fluid tnf- in tp .", "we conducted this meta - analysis according to the guidelines of the prisma and the quality assessment of diagnostic accuracy studies ( quadas ) checklist .", "the following search terms were used : tuberculosis or tb disease or active tuberculosis or ", "tnf - alpha or cytokines and sensitivity and specificity and diagnosis .", "reference lists of articles identified in these searches were also searched manually . to be included in our study , studies had to \n evaluate the sensitivity and/or specificity of pleural fluid tnf- for diagnosis of tp using.a case - control design involving.case and control groups classified using clear diagnostic criteria and.the same method to assay tnf-.apply an adequate experimental method . \n ", "evaluate the sensitivity and/or specificity of pleural fluid tnf- for diagnosis of tp using . a case - control design involving . case and", "control groups classified using clear diagnostic criteria and . the same method to assay tnf-. apply an adequate experimental method .", "unpublished data , abstracts , review articles , and letters to the editor were excluded .", "two reviewers ( z.l . and w.q . ) independently assembled a final set of eligible studies , and a third author ( x.c . )", "the same procedure was followed to extract data from the included studies using a standardized form .", "extracted data included first author , publication year , country of the study , number and characteristics of participants , tnf- assay method , cut - off value for tnf- detection , sensitivity and specificity , and numbers of true positives , false positives , true negatives and false negatives .", "we assessed the methodological quality of the studies using the quadas-2 checklist , with a maximum score of 11 .", "analyses were performed using stata and meta - disc ( xi cochrane colloquium , barcelona , spain ) .", "the following measures of test accuracy were computed for each study : sensitivity , specificity , positive likelihood ratio ( plr ) , negative likelihood ratio ( nlr ) and diagnostic odds ratio ( dor ) .", "these curves were plotted for each study using the sensitivity and specificity based on the single - test threshold identified within the same study .", "then we performed a meta - regression analysis to assess the effect of the baseline in each study on the relative dor of pleural fluid tnf- in tp diagnosis .", "we used a random - effect model to meta - analyze sensitivity , specificity , and other diagnostic measures across multiple studies . to assess statistically significant variability ( heterogeneity ) across studies , we used chi - squared and fisher 's exact tests .", "the following search terms were used : tuberculosis or tb disease or active tuberculosis or ", "tnf - alpha or cytokines and sensitivity and specificity and diagnosis . only english - language articles were considered .", "reference lists of articles identified in these searches were also searched manually . to be included in our study ,", "studies had to \n evaluate the sensitivity and/or specificity of pleural fluid tnf- for diagnosis of tp using.a case - control design involving.case and control groups classified using clear diagnostic criteria and.the same method to assay tnf-.apply an adequate experimental method . \n ", "evaluate the sensitivity and/or specificity of pleural fluid tnf- for diagnosis of tp using . a case - control design involving . case and", "control groups classified using clear diagnostic criteria and . the same method to assay tnf-. apply an adequate experimental method .", "unpublished data , abstracts , review articles , and letters to the editor were excluded .", "two reviewers ( z.l . and w.q . ) independently assembled a final set of eligible studies , and a third author ( x.c . )", "the same procedure was followed to extract data from the included studies using a standardized form . extracted data", "included first author , publication year , country of the study , number and characteristics of participants , tnf- assay method , cut - off value for tnf- detection , sensitivity and specificity , and numbers of true positives , false positives , true negatives and false negatives .", "we assessed the methodological quality of the studies using the quadas-2 checklist , with a maximum score of 11 .", "analyses were performed using stata and meta - disc ( xi cochrane colloquium , barcelona , spain ) .", "the following measures of test accuracy were computed for each study : sensitivity , specificity , positive likelihood ratio ( plr ) , negative likelihood ratio ( nlr ) and diagnostic odds ratio ( dor ) .", "these curves were plotted for each study using the sensitivity and specificity based on the single - test threshold identified within the same study .", "then we performed a meta - regression analysis to assess the effect of the baseline in each study on the relative dor of pleural fluid tnf- in tp diagnosis .", "we used a random - effect model to meta - analyze sensitivity , specificity , and other diagnostic measures across multiple studies . to assess statistically significant variability ( heterogeneity ) across studies , we used chi - squared and fisher 's exact tests .", "literature searches turned up 96 potentially eligible studies , and 86 were excluded based on review of titles and abstracts .", "the remaining 10 articles were read in full , and 4 were excluded because they did not apply an adequate experimental method . in the end , six publications assessing the diagnostic performance of pleural fluid tnf- assay in tp were included in our analysis .", "one study involved two control groups , and sufficient data were reported for both that we were able to treat the groups as two independent studies in the meta - analysis [ figure 1 ] .", "flow diagram of included and excluded studies characteristics of studies included in the meta - analysis the seven studies involved 159 patients with tp and 338 without it .", "average sample size in the seven studies was 71 ( range : 50 - 97 ) .", "tnf- levels in all studies were assayed by enzyme - linked immunosorbent assay ( elisa ) .", "sensitivity for pleural fluid tnf- in tp diagnosis ranged from 0.71 to 0.96 in the seven studies , and meta - analysis of sensitivity and specificity indicated a pooled sensitivity of 0.89 ( 95% confidence interval [ ci ] : 0.83 - 0.93 ) [ figure 2 ] .", "specificity ranged from 0.71 to 0.93 , and meta - analysis showed a pooled specificity of 0.82 ( 95% ci : 0.78 - 0.86 ) [ figure 2 ] .", "plr was 4.78 ( 95% ci : 3.32 - 6.89 ) and nlr was 0.16 ( 95% ci : 0.1 - 0.27 ) [ figure 3 ] .", "dor was 32.43 ( 95% ci : 14.48 - 72.6 ) [ figure 4 ] .", "significant heterogeneity ( i > 50% ) was present among the seven studies with respect to plr data but not data for other diagnostic measures : i was 0% for sensitivity , 47.5% for specificity , 53.8% for plr , 25% for nlr , and 46.9% for dor .", "forest plot of estimates of sensitivity / specificity for pleural fluid tnf- in the diagnosis of tuberculous pleurisy .", "point estimates of sensitivity and specificity from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "numbers indicate the reference numbers of the studies forest plot of estimates of positive and negative likelihood ratios ( plr and nlr ) for pleural fluid tnf- in the diagnosis of tuberculous pleurisy .", "point estimates of plr and nlr from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "numbers indicate the reference numbers of studies ( a ) forest plot of estimates of diagnostic odds ratios for pleural fluid tumor necrosis factor- ( tnf- ) in the diagnosis of tuberculous pleurisy .", "point estimates of diagnostic odds ratios from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "( b ) summary receiver operating characteristic curves for pleural fluid tnf-. each study is depicted as a solid circle , the size of which reflects the total number of cases and controls summary receiver operating characteristic curves were generated by plotting sensitivity against ( 1-specificity ) for individual studies [ figure 4 ] .", "the curves did not lie near the desired upper left corner , and the maximum joint sensitivity and specificity was 0.89 , with an area under the curve ( auc ) of 0.8556 ( standard error of mean 0.0559 ) . on the other hand , dor was 32.43 , suggesting relatively high overall accuracy .", "across the seven studies , tnf- cut - off values in the elisa and different diseases in the control group were different significantly [ table 1 ] .", "weighted meta - regression of the effects of diseases in control groups and cut - off values on diagnostic accuracy of pleural fluid tnf- funnel plots showed some asymmetry [ figure 5 ] , presumably reflecting the small number of studies included in our meta - analysis .", "nevertheless , deeks test gave a p = 0.11 , suggesting that our analysis did not carry significant risk of publication bias .", "funnel plot for evaluating publication bias among the six articles ( seven studies ) included in the meta - analysis .", "the log of the diagnostic odds ratio ( dor ) is plotted against the standard error of log dor ; the latter serves as an indicator of sample size .", "average sample size in the seven studies was 71 ( range : 50 - 97 ) .", "tnf- levels in all studies were assayed by enzyme - linked immunosorbent assay ( elisa ) .", "sensitivity for pleural fluid tnf- in tp diagnosis ranged from 0.71 to 0.96 in the seven studies , and meta - analysis of sensitivity and specificity indicated a pooled sensitivity of 0.89 ( 95% confidence interval [ ci ] : 0.83 - 0.93 ) [ figure 2 ] .", "specificity ranged from 0.71 to 0.93 , and meta - analysis showed a pooled specificity of 0.82 ( 95% ci : 0.78 - 0.86 ) [ figure 2 ] .", "plr was 4.78 ( 95% ci : 3.32 - 6.89 ) and nlr was 0.16 ( 95% ci : 0.1 - 0.27 ) [ figure 3 ] .", "dor was 32.43 ( 95% ci : 14.48 - 72.6 ) [ figure 4 ] .", "significant heterogeneity ( i > 50% ) was present among the seven studies with respect to plr data but not data for other diagnostic measures : i was 0% for sensitivity , 47.5% for specificity , 53.8% for plr , 25% for nlr , and 46.9% for dor .", "forest plot of estimates of sensitivity / specificity for pleural fluid tnf- in the diagnosis of tuberculous pleurisy . point", "estimates of sensitivity and specificity from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "numbers indicate the reference numbers of the studies forest plot of estimates of positive and negative likelihood ratios ( plr and nlr ) for pleural fluid tnf- in the diagnosis of tuberculous pleurisy .", "point estimates of plr and nlr from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "numbers indicate the reference numbers of studies ( a ) forest plot of estimates of diagnostic odds ratios for pleural fluid tumor necrosis factor- ( tnf- ) in the diagnosis of tuberculous pleurisy .", "point estimates of diagnostic odds ratios from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "( b ) summary receiver operating characteristic curves for pleural fluid tnf-. each study is depicted as a solid circle , the size of which reflects the total number of cases and controls summary receiver operating characteristic curves were generated by plotting sensitivity against ( 1-specificity ) for individual studies [ figure 4 ] .", "the curves did not lie near the desired upper left corner , and the maximum joint sensitivity and specificity was 0.89 , with an area under the curve ( auc ) of 0.8556 ( standard error of mean 0.0559 ) . on the other hand", "across the seven studies , tnf- cut - off values in the elisa and different diseases in the control group were different significantly [ table 1 ] .", "weighted meta - regression of the effects of diseases in control groups and cut - off values on diagnostic accuracy of pleural fluid tnf-", "funnel plots showed some asymmetry [ figure 5 ] , presumably reflecting the small number of studies included in our meta - analysis .", "nevertheless , deeks test gave a p = 0.11 , suggesting that our analysis did not carry significant risk of publication bias .", "funnel plot for evaluating publication bias among the six articles ( seven studies ) included in the meta - analysis .", "the log of the diagnostic odds ratio ( dor ) is plotted against the standard error of log dor ; the latter serves as an indicator of sample size .", "given the numerous limitations associated with current methods for diagnosing tp , researchers have explored whether pleural fluid biomarkers such as tnf- can serve as diagnostic markers .", "these studies have given conflicting results about the diagnostic performance of pleural fluid tnf- , so here we meta - analyzed the available evidence .", "our analysis suggests that pleural fluid tnf- measurements by themselves are not sufficiently sensitivity ( 0.89 ) and specificity ( 0.82 ) to diagnose tp , but they can provide complementary diagnostic information when used in combination with assays of other pleural fluid biomarkers and conventional tests such as bacteriological examination or pleural biopsy .", "a meta - analysis of the seven included studies indicated a pooled dor of 32.4 for the pleural fluid tnf- assay , indicating a relatively high accuracy .", "dor , which combines sensitivity and specificity data that serves as an aggregate indicator of test accuracy , is the ratio of the odds of positive test results in individuals with disease relative to the odds of positive test results in individuals without disease .", "the sroc curve and its auc present an overall summary of test performance and display the trade - off between sensitivity and specificity . in the present meta - analysis", ", we found the sensitivity of the pleural fluid tnf- assay to be 0.89 ; specificity , 0.82 ; maximum joint sensitivity and specificity , 0.89 ; and auc , 0.8556 .", "diagnostic odds ratio and sroc curve analysis are not easy to interpret and use in clinical practice , and likelihood ratios are considered more clinically meaningful .", "therefore , we meta - analyzed the data to determine pooled plr and nlr as measures of diagnostic accuracy .", "the plr value of 4.78 suggested that the probability of being positive for pleural fluid tnf- was nearly 5-fold higher for patients with tp than for patients without it .", "although this is insufficient to serve as the sole basis for diagnosing tp , it is likely to be sufficient to allow a clinician to decide whether to initiate or continue anti - tuberculosis treatment of tp in individuals who do not present evidence of malignancy or inflammation . at the same time , nlr was 0.16 in our meta - analysis , indicating that patients negative for pleural fluid tnf- still have a 16% chance of having tp .", "this provides further evidence that such an assay is inadequate , on its own , for ruling out tp .", "thus , a negative pleural fluid tnf- assay is not sufficient cause to deny or discontinue anti - tuberculosis therapy .", "the reliability of meta - analyses depends on heterogeneity among the included studies , and we found significant heterogeneity in the data for plr . since the causes of heterogeneity can reveal systematic factors affecting the accuracy and reliability of meta - analyses , we examined the seven studies more carefully . in all studies ,", "tp was diagnosed based on bacteriology , histology or both ; tnf- was determined using an elisa kit according to the manufacturer 's guidelines ( oxfordimmunotec ltd . , abingdon , uk ) ; and the quadus-2 score in each study was relatively high .", "in addition , inter - study variation in tnf- cut - off values and baseline tnf- levels in the control groups did not substantially affect diagnostic accuracy .", "therefore , the basis for the heterogeneity in plr data in our meta - analysis is unclear , and in any case , further large studies are needed to verify our findings , especially since we excluded possibly relevant studies that were not published in english or that were published only as conference abstracts or letters to the editor .", "analyses of other pleural fluid biomarkers suggest that they , like tnf- , can not be used alone as diagnostic indicators .", "a meta - analysis of the diagnostic accuracy of ifn - g in tp reported a sensitivity of 0.75 , specificity of 0.82 , plr of 3.49 , nlr of 0.24 , and dor of 19.04 .", "diagnostic performance was better in meta - analysis of adenosine deaminase , which reported a sensitivity of 0.86 , specificity of 0.88 , plr of 6.32 , nlr of 0.15 , and dor of 45.25 .", "these results suggest none of the biomarkers is sufficient on its own . assaying for multiple biomarkers may improve the accuracy of tp diagnosis , and", "such combined approaches should include tnf- because the peptide plays such an important role in tuberculosis pathogenesis , especially in patients with auto - immune diseases who are taking tnf- antagonists .", "the available evidence suggests that the pleural fluid tnf- assay should not be used on its own to diagnose tp or guide treatment decisions , but it can be used to complement other tests including microscopic smear examination , culture for m. tuberculosis , pleural tissue histology and response to anti - tuberculosis therapy .", "the diagnostic performance of tnf- may improve by changing the detection platform from the current elisa to more advanced flow cytometry , elispot and luminex methods .", "lz searched the literature and assessed articles , extracted data , performed meta - analyses , and wrote the draft .", "cx contributed in the conception of the work , revised the draft , approval of the final version of the manuscript , and agreed for all aspects of the work ." ]

[ "<S> background : pleurisy is a common extra pulmonary complication of tuberculosis , but current methods for diagnosing it are fairly crude . here we product a meta - analysis for the available evidence on the ability of tumor necrosis factor- ( tnf- ) in pleural fluid to serve as a diagnostic marker of tuberculous pleurisy ( tp).materials and methods : we searched the pubmed , embase , and google scholar databases systematically for studies measuring sensitivity , specificity and other measures of diagnostic accuracy of pleural fluid tnf- in the diagnosis of tp were meta - analyzed by stata , version 12 and meta-disc.results:a total of six publications reporting seven case - control studies were identified . </S>", "<S> pooled results indicated that pleural fluid tnf- showed a diagnostic sensitivity of 0.89 ( 95% confidence interval [ 95% ci ] 0.83 - 0.93 ; range , 0.42 - 1.0 ) and a diagnostic specificity of 0.82 ( 95% ci : 0.78 - 0.86 ; range , 0.58 - 0.98 ) . </S>", "<S> the pooled positive likelihood ratio was 4.78 ( 95% ci : 3.32 - 6.89 ) ; the negative likelihood ratio , 0.16 ( 95% ci : 0.1 - 0.27 ) ; the diagnostic odds ratio , 32.43 ( 95% ci : 14.48 - 72.6 ) ; and the area under the curve was 0.8556 ( standard error of mean 0.0559).conclusion : pleural fluid tnf- levels shows relatively high sensitivity but insufficient specificity for diagnosing tp . </S>", "<S> pleural fluid tnf- measurement may be useful in combination with clinical manifestations and conventional tests such as microbiological examination or pleural biopsy . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Search strategy and selection criteria", "Data extraction and quality assessment", "Statistical analysis", "RESULTS", "Study characteristics", "Diagnostic accuracy", "Multiple regression analysis", "Publication bias", "DISCUSSION", "CONCLUSION", "Financial support and sponsorship", "Conflicts of interest", "AUTHOR'S CONTRIBUTIONS" ]

[ [ "tuberculosis remains a frequent and important infectious disease worldwide , it occurs with , in approximately 30% of all cases , extrapulmonary involvement in the form of tuberculous pleurisy ( tp ) .", "and tp increases the morbidity and mortality associated with tuberculosis , and it is a major economic and health burden all over the world . the differential diagnosis of tp reliably is challenging although many conventional diagnostic methods are used , including microscopic analysis of pleural fluid or sputum smears , culturing for mycobacteria and even performing a pleural biopsy .", "each of these methods has significant limitations : microscopic analysis of pleural fluid is rarely positive ( < 5% ) , culture of pleural fluid shows poor sensitivity ( 24 - 58% ) , culturing for mycobacterium tuberculosis requires several weeks , and pleural biopsy , which is considered the best method for confirming tp diagnosis , is invasive and technically difficult .", "even more invasive procedures such as thoracoscopy or thoracotomy have been used for differential diagnosis of tp , but these complex procedures can cause complications and even increase morbidity . these led investigators to explore several biomarkers as possible diagnostic indicators .", "its release by mesothelial cells in pleura contributes to the occurrence and development of tp through three mechanisms : it acts as a proinflammatory cytokine to attract neutrophils to kill mycobacteria directly as part of an innate immune response ; it acts synergistically with interferon ( ifn- ) to activate macrophages as part of an adaptive immune response ; and it recruits nave t cells to the granuloma .", "tumor necrosis factor- ( tnf- ) levels in the pleural fluid are significantly higher in patients with tuberculosis than in patients with pulmonary malignancy .", "some studies have reported that levels of tnf- in pleural fluid provide high diagnostic sensitivity ( 96.0% ) and specificity ( 93.0% ) .", "other studies , however , have reported much lower corresponding values of 70% and 66% . however , the comprehensive picture of the diagnostic usefulness of tnf- levels in pleural fluid is not obvious .", "therefore in this article we will product a meta - analysis to explore the diagnostic accuracy of pleural fluid tnf- in tp ." ], [ "we conducted this meta - analysis according to the guidelines of the prisma and the quality assessment of diagnostic accuracy studies ( quadas ) checklist .", "the following search terms were used : tuberculosis or tb disease or active tuberculosis or ", "tnf - alpha or cytokines and sensitivity and specificity and diagnosis .", "reference lists of articles identified in these searches were also searched manually . to be included in our study , studies had to \n evaluate the sensitivity and/or specificity of pleural fluid tnf- for diagnosis of tp using.a case - control design involving.case and control groups classified using clear diagnostic criteria and.the same method to assay tnf-.apply an adequate experimental method . \n ", "evaluate the sensitivity and/or specificity of pleural fluid tnf- for diagnosis of tp using . a case - control design involving . case and", "control groups classified using clear diagnostic criteria and . the same method to assay tnf-. apply an adequate experimental method .", "unpublished data , abstracts , review articles , and letters to the editor were excluded .", "two reviewers ( z.l . and w.q . ) independently assembled a final set of eligible studies , and a third author ( x.c . )", "the same procedure was followed to extract data from the included studies using a standardized form .", "extracted data included first author , publication year , country of the study , number and characteristics of participants , tnf- assay method , cut - off value for tnf- detection , sensitivity and specificity , and numbers of true positives , false positives , true negatives and false negatives .", "we assessed the methodological quality of the studies using the quadas-2 checklist , with a maximum score of 11 .", "analyses were performed using stata and meta - disc ( xi cochrane colloquium , barcelona , spain ) .", "the following measures of test accuracy were computed for each study : sensitivity , specificity , positive likelihood ratio ( plr ) , negative likelihood ratio ( nlr ) and diagnostic odds ratio ( dor ) .", "these curves were plotted for each study using the sensitivity and specificity based on the single - test threshold identified within the same study .", "then we performed a meta - regression analysis to assess the effect of the baseline in each study on the relative dor of pleural fluid tnf- in tp diagnosis .", "we used a random - effect model to meta - analyze sensitivity , specificity , and other diagnostic measures across multiple studies . to assess statistically significant variability ( heterogeneity ) across studies , we used chi - squared and fisher 's exact tests ." ], [ "the following search terms were used : tuberculosis or tb disease or active tuberculosis or ", "tnf - alpha or cytokines and sensitivity and specificity and diagnosis . only english - language articles were considered .", "reference lists of articles identified in these searches were also searched manually . to be included in our study ,", "studies had to \n evaluate the sensitivity and/or specificity of pleural fluid tnf- for diagnosis of tp using.a case - control design involving.case and control groups classified using clear diagnostic criteria and.the same method to assay tnf-.apply an adequate experimental method . \n ", "evaluate the sensitivity and/or specificity of pleural fluid tnf- for diagnosis of tp using . a case - control design involving . case and", "control groups classified using clear diagnostic criteria and . the same method to assay tnf-. apply an adequate experimental method .", "unpublished data , abstracts , review articles , and letters to the editor were excluded ." ], [ "two reviewers ( z.l . and w.q . ) independently assembled a final set of eligible studies , and a third author ( x.c . )", "the same procedure was followed to extract data from the included studies using a standardized form . extracted data", "included first author , publication year , country of the study , number and characteristics of participants , tnf- assay method , cut - off value for tnf- detection , sensitivity and specificity , and numbers of true positives , false positives , true negatives and false negatives .", "we assessed the methodological quality of the studies using the quadas-2 checklist , with a maximum score of 11 ." ], [ "analyses were performed using stata and meta - disc ( xi cochrane colloquium , barcelona , spain ) .", "the following measures of test accuracy were computed for each study : sensitivity , specificity , positive likelihood ratio ( plr ) , negative likelihood ratio ( nlr ) and diagnostic odds ratio ( dor ) .", "these curves were plotted for each study using the sensitivity and specificity based on the single - test threshold identified within the same study .", "then we performed a meta - regression analysis to assess the effect of the baseline in each study on the relative dor of pleural fluid tnf- in tp diagnosis .", "we used a random - effect model to meta - analyze sensitivity , specificity , and other diagnostic measures across multiple studies . to assess statistically significant variability ( heterogeneity ) across studies , we used chi - squared and fisher 's exact tests ." ], [ "literature searches turned up 96 potentially eligible studies , and 86 were excluded based on review of titles and abstracts .", "the remaining 10 articles were read in full , and 4 were excluded because they did not apply an adequate experimental method . in the end , six publications assessing the diagnostic performance of pleural fluid tnf- assay in tp were included in our analysis .", "one study involved two control groups , and sufficient data were reported for both that we were able to treat the groups as two independent studies in the meta - analysis [ figure 1 ] .", "flow diagram of included and excluded studies characteristics of studies included in the meta - analysis the seven studies involved 159 patients with tp and 338 without it .", "average sample size in the seven studies was 71 ( range : 50 - 97 ) .", "tnf- levels in all studies were assayed by enzyme - linked immunosorbent assay ( elisa ) .", "sensitivity for pleural fluid tnf- in tp diagnosis ranged from 0.71 to 0.96 in the seven studies , and meta - analysis of sensitivity and specificity indicated a pooled sensitivity of 0.89 ( 95% confidence interval [ ci ] : 0.83 - 0.93 ) [ figure 2 ] .", "specificity ranged from 0.71 to 0.93 , and meta - analysis showed a pooled specificity of 0.82 ( 95% ci : 0.78 - 0.86 ) [ figure 2 ] .", "plr was 4.78 ( 95% ci : 3.32 - 6.89 ) and nlr was 0.16 ( 95% ci : 0.1 - 0.27 ) [ figure 3 ] .", "dor was 32.43 ( 95% ci : 14.48 - 72.6 ) [ figure 4 ] .", "significant heterogeneity ( i > 50% ) was present among the seven studies with respect to plr data but not data for other diagnostic measures : i was 0% for sensitivity , 47.5% for specificity , 53.8% for plr , 25% for nlr , and 46.9% for dor .", "forest plot of estimates of sensitivity / specificity for pleural fluid tnf- in the diagnosis of tuberculous pleurisy .", "point estimates of sensitivity and specificity from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "numbers indicate the reference numbers of the studies forest plot of estimates of positive and negative likelihood ratios ( plr and nlr ) for pleural fluid tnf- in the diagnosis of tuberculous pleurisy .", "point estimates of plr and nlr from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "numbers indicate the reference numbers of studies ( a ) forest plot of estimates of diagnostic odds ratios for pleural fluid tumor necrosis factor- ( tnf- ) in the diagnosis of tuberculous pleurisy .", "point estimates of diagnostic odds ratios from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "( b ) summary receiver operating characteristic curves for pleural fluid tnf-. each study is depicted as a solid circle , the size of which reflects the total number of cases and controls summary receiver operating characteristic curves were generated by plotting sensitivity against ( 1-specificity ) for individual studies [ figure 4 ] .", "the curves did not lie near the desired upper left corner , and the maximum joint sensitivity and specificity was 0.89 , with an area under the curve ( auc ) of 0.8556 ( standard error of mean 0.0559 ) . on the other hand , dor was 32.43 , suggesting relatively high overall accuracy .", "across the seven studies , tnf- cut - off values in the elisa and different diseases in the control group were different significantly [ table 1 ] .", "weighted meta - regression of the effects of diseases in control groups and cut - off values on diagnostic accuracy of pleural fluid tnf- funnel plots showed some asymmetry [ figure 5 ] , presumably reflecting the small number of studies included in our meta - analysis .", "nevertheless , deeks test gave a p = 0.11 , suggesting that our analysis did not carry significant risk of publication bias .", "funnel plot for evaluating publication bias among the six articles ( seven studies ) included in the meta - analysis .", "the log of the diagnostic odds ratio ( dor ) is plotted against the standard error of log dor ; the latter serves as an indicator of sample size ." ], [ "average sample size in the seven studies was 71 ( range : 50 - 97 ) .", "tnf- levels in all studies were assayed by enzyme - linked immunosorbent assay ( elisa ) ." ], [ "sensitivity for pleural fluid tnf- in tp diagnosis ranged from 0.71 to 0.96 in the seven studies , and meta - analysis of sensitivity and specificity indicated a pooled sensitivity of 0.89 ( 95% confidence interval [ ci ] : 0.83 - 0.93 ) [ figure 2 ] .", "specificity ranged from 0.71 to 0.93 , and meta - analysis showed a pooled specificity of 0.82 ( 95% ci : 0.78 - 0.86 ) [ figure 2 ] .", "plr was 4.78 ( 95% ci : 3.32 - 6.89 ) and nlr was 0.16 ( 95% ci : 0.1 - 0.27 ) [ figure 3 ] .", "dor was 32.43 ( 95% ci : 14.48 - 72.6 ) [ figure 4 ] .", "significant heterogeneity ( i > 50% ) was present among the seven studies with respect to plr data but not data for other diagnostic measures : i was 0% for sensitivity , 47.5% for specificity , 53.8% for plr , 25% for nlr , and 46.9% for dor .", "forest plot of estimates of sensitivity / specificity for pleural fluid tnf- in the diagnosis of tuberculous pleurisy . point", "estimates of sensitivity and specificity from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "numbers indicate the reference numbers of the studies forest plot of estimates of positive and negative likelihood ratios ( plr and nlr ) for pleural fluid tnf- in the diagnosis of tuberculous pleurisy .", "point estimates of plr and nlr from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "numbers indicate the reference numbers of studies ( a ) forest plot of estimates of diagnostic odds ratios for pleural fluid tumor necrosis factor- ( tnf- ) in the diagnosis of tuberculous pleurisy .", "point estimates of diagnostic odds ratios from each study are shown as solid circles , the size of which reflects the total number of cases and controls .", "( b ) summary receiver operating characteristic curves for pleural fluid tnf-. each study is depicted as a solid circle , the size of which reflects the total number of cases and controls summary receiver operating characteristic curves were generated by plotting sensitivity against ( 1-specificity ) for individual studies [ figure 4 ] .", "the curves did not lie near the desired upper left corner , and the maximum joint sensitivity and specificity was 0.89 , with an area under the curve ( auc ) of 0.8556 ( standard error of mean 0.0559 ) . on the other hand" ], [ "across the seven studies , tnf- cut - off values in the elisa and different diseases in the control group were different significantly [ table 1 ] .", "weighted meta - regression of the effects of diseases in control groups and cut - off values on diagnostic accuracy of pleural fluid tnf-" ], [ "funnel plots showed some asymmetry [ figure 5 ] , presumably reflecting the small number of studies included in our meta - analysis .", "nevertheless , deeks test gave a p = 0.11 , suggesting that our analysis did not carry significant risk of publication bias .", "funnel plot for evaluating publication bias among the six articles ( seven studies ) included in the meta - analysis .", "the log of the diagnostic odds ratio ( dor ) is plotted against the standard error of log dor ; the latter serves as an indicator of sample size ." ], [ "given the numerous limitations associated with current methods for diagnosing tp , researchers have explored whether pleural fluid biomarkers such as tnf- can serve as diagnostic markers .", "these studies have given conflicting results about the diagnostic performance of pleural fluid tnf- , so here we meta - analyzed the available evidence .", "our analysis suggests that pleural fluid tnf- measurements by themselves are not sufficiently sensitivity ( 0.89 ) and specificity ( 0.82 ) to diagnose tp , but they can provide complementary diagnostic information when used in combination with assays of other pleural fluid biomarkers and conventional tests such as bacteriological examination or pleural biopsy .", "a meta - analysis of the seven included studies indicated a pooled dor of 32.4 for the pleural fluid tnf- assay , indicating a relatively high accuracy .", "dor , which combines sensitivity and specificity data that serves as an aggregate indicator of test accuracy , is the ratio of the odds of positive test results in individuals with disease relative to the odds of positive test results in individuals without disease .", "the sroc curve and its auc present an overall summary of test performance and display the trade - off between sensitivity and specificity . in the present meta - analysis", ", we found the sensitivity of the pleural fluid tnf- assay to be 0.89 ; specificity , 0.82 ; maximum joint sensitivity and specificity , 0.89 ; and auc , 0.8556 .", "diagnostic odds ratio and sroc curve analysis are not easy to interpret and use in clinical practice , and likelihood ratios are considered more clinically meaningful .", "therefore , we meta - analyzed the data to determine pooled plr and nlr as measures of diagnostic accuracy .", "the plr value of 4.78 suggested that the probability of being positive for pleural fluid tnf- was nearly 5-fold higher for patients with tp than for patients without it .", "although this is insufficient to serve as the sole basis for diagnosing tp , it is likely to be sufficient to allow a clinician to decide whether to initiate or continue anti - tuberculosis treatment of tp in individuals who do not present evidence of malignancy or inflammation . at the same time , nlr was 0.16 in our meta - analysis , indicating that patients negative for pleural fluid tnf- still have a 16% chance of having tp .", "this provides further evidence that such an assay is inadequate , on its own , for ruling out tp .", "thus , a negative pleural fluid tnf- assay is not sufficient cause to deny or discontinue anti - tuberculosis therapy .", "the reliability of meta - analyses depends on heterogeneity among the included studies , and we found significant heterogeneity in the data for plr . since the causes of heterogeneity can reveal systematic factors affecting the accuracy and reliability of meta - analyses , we examined the seven studies more carefully . in all studies ,", "tp was diagnosed based on bacteriology , histology or both ; tnf- was determined using an elisa kit according to the manufacturer 's guidelines ( oxfordimmunotec ltd . , abingdon , uk ) ; and the quadus-2 score in each study was relatively high .", "in addition , inter - study variation in tnf- cut - off values and baseline tnf- levels in the control groups did not substantially affect diagnostic accuracy .", "therefore , the basis for the heterogeneity in plr data in our meta - analysis is unclear , and in any case , further large studies are needed to verify our findings , especially since we excluded possibly relevant studies that were not published in english or that were published only as conference abstracts or letters to the editor .", "analyses of other pleural fluid biomarkers suggest that they , like tnf- , can not be used alone as diagnostic indicators .", "a meta - analysis of the diagnostic accuracy of ifn - g in tp reported a sensitivity of 0.75 , specificity of 0.82 , plr of 3.49 , nlr of 0.24 , and dor of 19.04 .", "diagnostic performance was better in meta - analysis of adenosine deaminase , which reported a sensitivity of 0.86 , specificity of 0.88 , plr of 6.32 , nlr of 0.15 , and dor of 45.25 .", "these results suggest none of the biomarkers is sufficient on its own . assaying for multiple biomarkers may improve the accuracy of tp diagnosis , and", "such combined approaches should include tnf- because the peptide plays such an important role in tuberculosis pathogenesis , especially in patients with auto - immune diseases who are taking tnf- antagonists ." ], [ "the available evidence suggests that the pleural fluid tnf- assay should not be used on its own to diagnose tp or guide treatment decisions , but it can be used to complement other tests including microscopic smear examination , culture for m. tuberculosis , pleural tissue histology and response to anti - tuberculosis therapy .", "the diagnostic performance of tnf- may improve by changing the detection platform from the current elisa to more advanced flow cytometry , elispot and luminex methods ." ], [ "" ], [ "" ], [ "lz searched the literature and assessed articles , extracted data , performed meta - analyses , and wrote the draft .", "cx contributed in the conception of the work , revised the draft , approval of the final version of the manuscript , and agreed for all aspects of the work ." ] ]

[ "specific learning disabilities ( slds ) are a generic term that refers to a heterogeneous group of neurobehavioral disorders manifested by significant unexpected , specific , and persistent difficulties in the acquisition and use of efficient reading ( dyslexia ) , writing ( dysgraphia ) , or mathematical ( dyscalculia ) abilities despite conventional instruction , intact senses , normal intelligence , proper motivation , and adequate sociocultural opportunity .", "international classification of diseases-10 describes them as specific developmental disorders of scholastic skills and diagnostic and statistical manual 4 edition - text revision ( dsm - iv ) as learning disorders and dsm-5 as specific learning disorders .", "dyslexia ( reading disorder ) is the most common among the learning disorders , affecting 80% of all those identified as learning - disabled .", "the incidence of dyslexia in primary school children in india has been reported to be 218% , dysgraphia 14% , and dyscalculia 5.5% .", "they are found most commonly associated with other cortical - based disorders such as other learning disorders , language disorders , motor disorders , and organization and executive function disability .", "the most common comorbidities include attention deficit hyperactivity disorder ( adhd ) , conduct disorder , oppositional defiant disorder , anxiety disorder , and depression . keeping the paucity of literature regarding slds in india", ", this study aims to document the sociodemographic profile and psychiatric comorbidities in school children . \n to study the number of children having slds and comorbidities in school settingto study the comorbidities in children with slds who are identified and on remedial educationto study the comorbidities in children who do not have sldsto study the difference in sociodemographic variables as well as a comorbidity in the three groups . \n to study the number of children having slds and comorbidities in school setting to study the comorbidities in children with slds who are identified and on remedial education to study the comorbidities in children who do not have slds to study the difference in sociodemographic variables as well as a comorbidity in the three groups", "keeping the paucity of literature regarding slds in india , this study aims to document the sociodemographic profile and psychiatric comorbidities in school children .", "\n to study the number of children having slds and comorbidities in school settingto study the comorbidities in children with slds who are identified and on remedial educationto study the comorbidities in children who do not have sldsto study the difference in sociodemographic variables as well as a comorbidity in the three groups . \n to study the number of children having slds and comorbidities in school setting to study the comorbidities in children with slds who are identified and on remedial education to study the comorbidities in children who do not have slds to study the difference in sociodemographic variables as well as a comorbidity in the three groups", "the data are collected from a group of sld children in remedial schools ( dyslexia association of andhra pradesh , hyderabad ) .", "the other in two urban and two semi - urban schools all of them are nongovernment , private organized english medium schools .", "they were divided into three groups : group 1 consists of thirty children who are already diagnosed as having an sld by a psychiatrist and are on remedial education . after taking written informed consent from the parents , the details as per the intake pro forma", "are taken and parent is interviewed according to the developmental psychopathology checklist ( dpcl ) .", "subsequently , the child is administered colored progressive matrices ( cpm)/standard progressive matrices ( spm ) , malin 's intelligence scale for indian children ( misic ) and nimhans sld index .", "group 2 consists of 32 children diagnosed for the first time at schools during the study . after taking permission from the school management ,", "following the screening , children were administered spm / cpm and those who scored more than the 50 percentile were shortlisted . after taking written informed consent from the parents and using the inclusion and exclusion criteria and information obtained from parents interview and intake pro forma and dpcl , the children were then administered misic and nimhans sld index .", "group 3 constituted of 34 children who were not included in the list after administering the checklist ; they were administered spm / cpm .", "the parents of those children who scored more than the 50 percentile were interviewed and information was collected in intake pro forma and dpcl after taking consent .", "means and standard deviation , chi - square test , and anova were used for analysis . \n", "semi - structured intake pro formaintelligence tests : \n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n misic \n the misic is an indian adaptation of wechsler intelligence scale for children \n\n the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld .", "it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years)checklist to identify learning disabilitiesdpcl for children . \n ", "semi - structured intake pro forma \n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n misic \n the misic is an indian adaptation of wechsler intelligence scale for children \n\n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years ) colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n the misic is an indian adaptation of wechsler intelligence scale for children \n the misic is an indian adaptation of wechsler intelligence scale for children the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld .", "it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years ) checklist to identify learning disabilities dpcl for children .", "means and standard deviation , chi - square test , and anova were used for analysis .", "\n semi - structured intake pro formaintelligence tests : \n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n misic \n the misic is an indian adaptation of wechsler intelligence scale for children \n\n the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld .", "it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years)checklist to identify learning disabilitiesdpcl for children . \n ", "semi - structured intake pro forma \n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n misic \n the misic is an indian adaptation of wechsler intelligence scale for children \n\n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years ) colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n the misic is an indian adaptation of wechsler intelligence scale for children \n the misic is an indian adaptation of wechsler intelligence scale for children the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld .", "it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years ) checklist to identify learning disabilities dpcl for children .", "they comprised 40 males ( m ) and 22 females ( f ) with the gender distribution ratio ( m / f ) of 1.8:1 [ table 1 ] .", "sociodemographic and personal data prematurity ( sld special education group 6 ( 20% ) and sld schools group 1 ( 3.1% ) ( p = 0.006 ) and cesarean section ( sld special education group 63.4% and sld schools group ( 31.3% ) ( p = 0.02 ) are found to be related to sld in children .", "sld children have delayed milestones particularly problems with speech , which is predominant in sld special education group ( 36.7% ) ( p = 0.02 ) .", "sld children have significant school problems in the form of school refusal , poor school performance , reading , writing , spelling , and arithmetic when compared to normal children . in sld children ,", "the problems with reading were found to be about 56 ( 90.3% ) , with comprehension 55 ( 88.7% ) , with arithmetic 17 ( 27.4% ) , with spelling 47 ( 75.8% ) , and with writing 28 ( 45.2% ) [ table 2 ] .", "break up of learning disabilities ( nimhans specific learning disabilities index ) table 3 shows various presentations of learning disabilities , pure reading disability is found in 22 ( 35.48% ) , writing disability in 1 ( 1.61% ) , dyscalculia in 3 ( 4.83% ) whereas combined learning disability in 36 ( 58.06% ) , which is found to be the most common type of learning disability .", "mathematic disability is found to occur less frequently in sld special education group ( 16.7% ) than in sld schools group ( 34.4% ) .", "presentation of learning disabilities overall , the prevalence of sld cases in schools is found to be 6.6% . among the subtypes of learning disability ,", "the prevalence of dyslexia is 4.58% , dysgraphia 0.2% , dyscalculia 0.63% , and combined learning disability in 7.5% respectively in school children .", "children with sld committed significant errors on bender gestalt test ( bgt ) ( p = 0.001 ) as well as number cancellation test ( p = 0.001 ) .", "no statistical difference was found ( p = 0.37 ) on scores on recall of bgt designs ( memory ) among three groups indicating that there is no statistical difference in memory in the three groups .", "pre- , peri- , and post - natal problems in mother , epilepsy , head injury or infections in infancy , poor vision , speech and language delay during childhood were found to be significant in children with sld in both groups .", "developmental psychopathology checklist - developmental history table 5 shows various developmental problems . only speech and articulation problems were found to be of highly statistically significant ( p = 0.001 ) in children with sld .", "developmental psychopathology checklist - developmental problems adhd [ table 6 ] is found to be the most common comorbidity associated with sld amounting to 26 ( 41.9% ) with trends toward inattentive subtype .", "attention deficit hyperactivity disorder conduct disorder is found to be about 1 ( 3% ) in sld schools group with trends toward symptoms of disobedience .", "emotional disorders are found to be about 2 ( 6.3% ) in sld schools group and 4 ( 13.3% ) in sld special education group .", "none of the children in both sld groups met the criteria for somatic problems , psychosis , or obsessive - compulsive disorder .", "the family history of alcoholism was found to be significantly higher ( p = 0.001 ) in normal children ( 50% ) [ table 7 ] .", "the family history of learning problems has been found to be occurring more frequently in both sld groups , which is found to be statistically significant ( p = 0.02 ) .", "developmental psychopathology checklist - family history stressors such as over expectation ( 26.7% ) ( p = 0.04 ) and over - involvement ( 36.7% ) p = 0.001 ) by parents are found in children with sld special education group .", "the prevalence of sld in schools is found to be 6.6% with combined learning disability being most common 7.5% . the male : female ratio in children with sld if 1.8:1 , indicating male preponderance .", "the similar findings were found by karande et al . in his study with a gender distribution ratio of 2.1:1 . a higher representation of middle socioeconomic strata in sld schools group ( 84.4% ) and high socioeconomic strata in sld special education group ( 66.7% ) is observed .", "the higher representation of middle class is because the study sample is taken from urban and semi - urban areas , with english as the medium of instruction .", "it was being done due to the lack of standardized assessment tools for sld in vernacular languages .", "study which states that sld is higher ( 82% ) in middle socioeconomic strata . the higher representation of high socioeconomic strata in sld special education could be because the remedial or special education which was received by the students was from special educators / special education centers which charge a particular amount per month . hence , most of the children who are receiving the special education are the ones whose parents could afford to pay them , apart from regular school fees .", "the findings are not in continuation of the previous study by murnane who stated that sld are more common in a minority group and those belonging to low socioeconomic status .", "the parents of children with sld special education group belong to high education group ( mothers 73.3% and fathers 46.7% ) and most of them are professionals ( mothers 23.3% and fathers 46.7% ) while those parents of children with sld schools group are educated up to secondary education ( mothers 46.9% and fathers 25% ) . the previous study conducted by kohli et al . has noticed that parents of sld children had 1215 years of education .", "mean age of children with sld in schools group is 9.88 years while the mean age of children with sld in special education group is 9.43 years , respectively .", "the mean age of children with sld as observed by karande et al . was 11.4 years .", "children with sld in special education group are joined in schools at an early age ( i.e. , 3.02 years ) when compared to that of children with sld schools group ( 3.81 years ) .", "the difference is statistically significant ( p = 0.001 ) , indicating that children in sld with special education group are being joined in the school at an earlier age than others and due to the high socioeconomic status of these parents of these children who keep them in special education schools for remedial methods .", "it is to be noted that none of the previous studies compared the age of the child at the time of joining school .", "prematurity ( sld special education group 20% and sld schools group 3.1% ) and cesarean section ( sld special education group 63.4% and sld schools group 31.3% ) are found to be related to sld in children .", "the findings go with the result of previous study by wood et al . , where prematurity is found to be a risk factor with sld .", "furthermore , cesarean section also appears to be a risk factor . however , there are no such reports from the previous studies .", "sld children have delayed milestones particularly problems with speech , which is predominant in sld special education group ( 36.7% ) indicates that sld special education group has developmental immaturity .", "significant pre- , peri- , post - natal problems ( 40% ) were found in mothers of children with sld in special education group along with epilepsy , head injury or infections in infancy , poor vision , speech and language delay during childhood are found to be significant in both groups of sld , clearly indicating that sld is a developmental problem .", "sld children have significant school problems in the form of school refusal , poor school performance , reading , writing , spelling , and arithmetic when compared to normal children . in sld children ,", "the problems with reading were found to be about 90.3% , with comprehension 88.7% , with arithmetic 27.4% , with spelling 75.8% , and with writing 45.2% .", "pure reading disability is found in 35.48% , writing disability in 1.61% , dyscalculia in 4.83% while combined learning disability in 58.06% , which is found to be the most common type of learning disability .", "mathematic disability is found to occur less frequently in sld special education group ( 16.7% ) than in sld schools group ( 34.4% ) .", "these findings are similar to the findings of karande et al . where combined subtype was represented more .", "however , compared to the study by ramaa where mathematic disability is found to be 5.98% , mathematic disabilities have found to occur less frequently in special education group ; this could be probably due to the reason that most of the children brought to the special educators are having problems with reading and writing . in this study ,", "most common type of learning disability was found to be combined ( reading + writing ) 35.48% which was similar to a finding by karande et al . , 2007 .", "but as compared to the previous indian studies , mathematic disabilities have found to occur less frequently in special education group , this could be probably due to the reason that most of the children brought to the special educators are having problems with reading , spelling , and writing .", "children with sld committed statistically significant errors on bgt ( p = 0.001 ) denoting perceptual and visuomotor integration problems in children with sld .", "this amounts to the high amount of inattentiveness seen in sld and also the influence of other comorbid conditions like adhd .", "adhd is found to be the most common comorbidity associated with sld amounting to 41.9% with trends toward inattentive subtype .", "analysis of the characteristics of the 26 ( 41.9% ) children with adhd in sld group , it is found that there are 3 ( 11.5% ) females and 23 ( 88.5% ) males . on subtyping", ", the number of sld children with adhd inattentive type is found to be 11 ( 42.3% ) , adhd hyperactive type 7 ( 26.9% ) , and combined type of adhd in 8 ( 30.8% ) .", "the comorbidity of adhd was about 35% . in the present study , trends are toward inattentive type , whereas in other studies on indian and pakistani data found that combined subtype was more common . conduct disorder is found to be about 3% in sld schools group with trends toward symptoms of disobedience .", "emotional disorders are found to be about 6.3% in sld schools group and 13.3% in sld special education group .", "none of the children in both sld groups met the criteria for somatic problems , psychosis or obsessive compulsive disorder . on the whole , the comorbidities as assessed by dpcl showed significance on adhd , conduct and emotional disorders which was not found in the study by karande et al . except for adhd .", "significant family history of learning disabilities is found in children with both groups of sld .", "learning disorders in family history is found to be significant as also observed by snowling et al .", "stressors like over expectation and over involvement by parents are found in children with sld special education group .", "the study was not conducted in government schools as the assessment of sld in vernacular languages was not available .", "the study was not conducted in government schools as the assessment of sld in vernacular languages was not available .", "there was a significant association with prematurity , cesarean section , delayed speech and family history of sld . among comorbidities of sld ," ]

[ "<S> background : specific learning disabilities ( slds ) are an important cause of academic underachievement in children and are also associated with comorbidities like attention deficit hyperactivity disorder ( adhd ) which further have an impact on the child 's education.aims:to estimate the prevalence and psychosocial profile and psychiatric comorbidities in children with sld in two settings , i.e. , on special ( remedial ) education and schools and to compare the findings with normal children.materials and methods : this study was carried out in schools situated in urban and semi - urban areas and special education schools . </S>", "<S> a total of 96 children were chosen for the study . after taking informed consent from the parents , the details about socioeconomic status , family , developmental history , and school history of the children </S>", "<S> were collected on a semi - structured pro forma and then the children were screened for sld . </S>", "<S> they were administered colored / standard progressive matrices and malin 's intelligence scale for assessing their intelligence quotient and nimhans sld index and developmental psychopathology checklist to study psychopathology . </S>", "<S> chi - square test and anova were done.results:the prevalence of sld in schools is found out to be 6.6% . </S>", "<S> there was a significant association with prematurity , cesarean section , delayed speech , and family history of sld . among comorbidities of sld , </S>", "<S> association with adhd alone has been found to be significant.conclusion:the most common type of sld is combined type comorbid with adhd . </S>", "<S> there is a need for early identification of learning disabilities in schools so that with early recognition and remedial intervention children can be helped with to cope with studies . </S>" ]

[ "INTRODUCTION", "Need for the study", "Aims and objectives of the study", "MATERIALS AND METHODS", "Inclusion criteria", "Exclusion criteria", "Statistics", "Tools used", "RESULTS", "DISCUSSION", "Limitations", "CONCLUSION", "Financial support and sponsorship", "Conflicts of interest" ]

[ [ "specific learning disabilities ( slds ) are a generic term that refers to a heterogeneous group of neurobehavioral disorders manifested by significant unexpected , specific , and persistent difficulties in the acquisition and use of efficient reading ( dyslexia ) , writing ( dysgraphia ) , or mathematical ( dyscalculia ) abilities despite conventional instruction , intact senses , normal intelligence , proper motivation , and adequate sociocultural opportunity .", "international classification of diseases-10 describes them as specific developmental disorders of scholastic skills and diagnostic and statistical manual 4 edition - text revision ( dsm - iv ) as learning disorders and dsm-5 as specific learning disorders .", "dyslexia ( reading disorder ) is the most common among the learning disorders , affecting 80% of all those identified as learning - disabled .", "the incidence of dyslexia in primary school children in india has been reported to be 218% , dysgraphia 14% , and dyscalculia 5.5% .", "they are found most commonly associated with other cortical - based disorders such as other learning disorders , language disorders , motor disorders , and organization and executive function disability .", "the most common comorbidities include attention deficit hyperactivity disorder ( adhd ) , conduct disorder , oppositional defiant disorder , anxiety disorder , and depression . keeping the paucity of literature regarding slds in india", ", this study aims to document the sociodemographic profile and psychiatric comorbidities in school children . \n to study the number of children having slds and comorbidities in school settingto study the comorbidities in children with slds who are identified and on remedial educationto study the comorbidities in children who do not have sldsto study the difference in sociodemographic variables as well as a comorbidity in the three groups . \n to study the number of children having slds and comorbidities in school setting to study the comorbidities in children with slds who are identified and on remedial education to study the comorbidities in children who do not have slds to study the difference in sociodemographic variables as well as a comorbidity in the three groups" ], [ "keeping the paucity of literature regarding slds in india , this study aims to document the sociodemographic profile and psychiatric comorbidities in school children ." ], [ "\n to study the number of children having slds and comorbidities in school settingto study the comorbidities in children with slds who are identified and on remedial educationto study the comorbidities in children who do not have sldsto study the difference in sociodemographic variables as well as a comorbidity in the three groups . \n to study the number of children having slds and comorbidities in school setting to study the comorbidities in children with slds who are identified and on remedial education to study the comorbidities in children who do not have slds to study the difference in sociodemographic variables as well as a comorbidity in the three groups" ], [ "the data are collected from a group of sld children in remedial schools ( dyslexia association of andhra pradesh , hyderabad ) .", "the other in two urban and two semi - urban schools all of them are nongovernment , private organized english medium schools .", "they were divided into three groups : group 1 consists of thirty children who are already diagnosed as having an sld by a psychiatrist and are on remedial education . after taking written informed consent from the parents , the details as per the intake pro forma", "are taken and parent is interviewed according to the developmental psychopathology checklist ( dpcl ) .", "subsequently , the child is administered colored progressive matrices ( cpm)/standard progressive matrices ( spm ) , malin 's intelligence scale for indian children ( misic ) and nimhans sld index .", "group 2 consists of 32 children diagnosed for the first time at schools during the study . after taking permission from the school management ,", "following the screening , children were administered spm / cpm and those who scored more than the 50 percentile were shortlisted . after taking written informed consent from the parents and using the inclusion and exclusion criteria and information obtained from parents interview and intake pro forma and dpcl , the children were then administered misic and nimhans sld index .", "group 3 constituted of 34 children who were not included in the list after administering the checklist ; they were administered spm / cpm .", "the parents of those children who scored more than the 50 percentile were interviewed and information was collected in intake pro forma and dpcl after taking consent .", "means and standard deviation , chi - square test , and anova were used for analysis . \n", "semi - structured intake pro formaintelligence tests : \n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n misic \n the misic is an indian adaptation of wechsler intelligence scale for children \n\n the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld .", "it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years)checklist to identify learning disabilitiesdpcl for children . \n ", "semi - structured intake pro forma \n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n misic \n the misic is an indian adaptation of wechsler intelligence scale for children \n\n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years ) colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n the misic is an indian adaptation of wechsler intelligence scale for children \n the misic is an indian adaptation of wechsler intelligence scale for children the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld .", "it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years ) checklist to identify learning disabilities dpcl for children ." ], [ "" ], [ "" ], [ "means and standard deviation , chi - square test , and anova were used for analysis ." ], [ "\n semi - structured intake pro formaintelligence tests : \n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n misic \n the misic is an indian adaptation of wechsler intelligence scale for children \n\n the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld .", "it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years)checklist to identify learning disabilitiesdpcl for children . \n ", "semi - structured intake pro forma \n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n misic \n the misic is an indian adaptation of wechsler intelligence scale for children \n\n raven 's progressive matrices \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years)colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n standard progressive matrices : the booklet comprises five sets ( a to e ) of 12 items each.(children : 1112 years ) colored progressive matrices : the booklet comprises three sets ( a , ab , b ) of 12 items each ( children up to 11 years ) .", "progressive matrices are used as a screening instrument to rule out children with low iq \n the misic is an indian adaptation of wechsler intelligence scale for children \n the misic is an indian adaptation of wechsler intelligence scale for children the nimhans index for slds is a battery of tests used for confirming the diagnosis of sld .", "it consists of two levels : level 1 : ( 57 years of age ) ; level 2 : ( 812 years ) checklist to identify learning disabilities dpcl for children ." ], [ "they comprised 40 males ( m ) and 22 females ( f ) with the gender distribution ratio ( m / f ) of 1.8:1 [ table 1 ] .", "sociodemographic and personal data prematurity ( sld special education group 6 ( 20% ) and sld schools group 1 ( 3.1% ) ( p = 0.006 ) and cesarean section ( sld special education group 63.4% and sld schools group ( 31.3% ) ( p = 0.02 ) are found to be related to sld in children .", "sld children have delayed milestones particularly problems with speech , which is predominant in sld special education group ( 36.7% ) ( p = 0.02 ) .", "sld children have significant school problems in the form of school refusal , poor school performance , reading , writing , spelling , and arithmetic when compared to normal children . in sld children ,", "the problems with reading were found to be about 56 ( 90.3% ) , with comprehension 55 ( 88.7% ) , with arithmetic 17 ( 27.4% ) , with spelling 47 ( 75.8% ) , and with writing 28 ( 45.2% ) [ table 2 ] .", "break up of learning disabilities ( nimhans specific learning disabilities index ) table 3 shows various presentations of learning disabilities , pure reading disability is found in 22 ( 35.48% ) , writing disability in 1 ( 1.61% ) , dyscalculia in 3 ( 4.83% ) whereas combined learning disability in 36 ( 58.06% ) , which is found to be the most common type of learning disability .", "mathematic disability is found to occur less frequently in sld special education group ( 16.7% ) than in sld schools group ( 34.4% ) .", "presentation of learning disabilities overall , the prevalence of sld cases in schools is found to be 6.6% . among the subtypes of learning disability ,", "the prevalence of dyslexia is 4.58% , dysgraphia 0.2% , dyscalculia 0.63% , and combined learning disability in 7.5% respectively in school children .", "children with sld committed significant errors on bender gestalt test ( bgt ) ( p = 0.001 ) as well as number cancellation test ( p = 0.001 ) .", "no statistical difference was found ( p = 0.37 ) on scores on recall of bgt designs ( memory ) among three groups indicating that there is no statistical difference in memory in the three groups .", "pre- , peri- , and post - natal problems in mother , epilepsy , head injury or infections in infancy , poor vision , speech and language delay during childhood were found to be significant in children with sld in both groups .", "developmental psychopathology checklist - developmental history table 5 shows various developmental problems . only speech and articulation problems were found to be of highly statistically significant ( p = 0.001 ) in children with sld .", "developmental psychopathology checklist - developmental problems adhd [ table 6 ] is found to be the most common comorbidity associated with sld amounting to 26 ( 41.9% ) with trends toward inattentive subtype .", "attention deficit hyperactivity disorder conduct disorder is found to be about 1 ( 3% ) in sld schools group with trends toward symptoms of disobedience .", "emotional disorders are found to be about 2 ( 6.3% ) in sld schools group and 4 ( 13.3% ) in sld special education group .", "none of the children in both sld groups met the criteria for somatic problems , psychosis , or obsessive - compulsive disorder .", "the family history of alcoholism was found to be significantly higher ( p = 0.001 ) in normal children ( 50% ) [ table 7 ] .", "the family history of learning problems has been found to be occurring more frequently in both sld groups , which is found to be statistically significant ( p = 0.02 ) .", "developmental psychopathology checklist - family history stressors such as over expectation ( 26.7% ) ( p = 0.04 ) and over - involvement ( 36.7% ) p = 0.001 ) by parents are found in children with sld special education group ." ], [ "the prevalence of sld in schools is found to be 6.6% with combined learning disability being most common 7.5% . the male : female ratio in children with sld if 1.8:1 , indicating male preponderance .", "the similar findings were found by karande et al . in his study with a gender distribution ratio of 2.1:1 . a higher representation of middle socioeconomic strata in sld schools group ( 84.4% ) and high socioeconomic strata in sld special education group ( 66.7% ) is observed .", "the higher representation of middle class is because the study sample is taken from urban and semi - urban areas , with english as the medium of instruction .", "it was being done due to the lack of standardized assessment tools for sld in vernacular languages .", "study which states that sld is higher ( 82% ) in middle socioeconomic strata . the higher representation of high socioeconomic strata in sld special education could be because the remedial or special education which was received by the students was from special educators / special education centers which charge a particular amount per month . hence , most of the children who are receiving the special education are the ones whose parents could afford to pay them , apart from regular school fees .", "the findings are not in continuation of the previous study by murnane who stated that sld are more common in a minority group and those belonging to low socioeconomic status .", "the parents of children with sld special education group belong to high education group ( mothers 73.3% and fathers 46.7% ) and most of them are professionals ( mothers 23.3% and fathers 46.7% ) while those parents of children with sld schools group are educated up to secondary education ( mothers 46.9% and fathers 25% ) . the previous study conducted by kohli et al . has noticed that parents of sld children had 1215 years of education .", "mean age of children with sld in schools group is 9.88 years while the mean age of children with sld in special education group is 9.43 years , respectively .", "the mean age of children with sld as observed by karande et al . was 11.4 years .", "children with sld in special education group are joined in schools at an early age ( i.e. , 3.02 years ) when compared to that of children with sld schools group ( 3.81 years ) .", "the difference is statistically significant ( p = 0.001 ) , indicating that children in sld with special education group are being joined in the school at an earlier age than others and due to the high socioeconomic status of these parents of these children who keep them in special education schools for remedial methods .", "it is to be noted that none of the previous studies compared the age of the child at the time of joining school .", "prematurity ( sld special education group 20% and sld schools group 3.1% ) and cesarean section ( sld special education group 63.4% and sld schools group 31.3% ) are found to be related to sld in children .", "the findings go with the result of previous study by wood et al . , where prematurity is found to be a risk factor with sld .", "furthermore , cesarean section also appears to be a risk factor . however , there are no such reports from the previous studies .", "sld children have delayed milestones particularly problems with speech , which is predominant in sld special education group ( 36.7% ) indicates that sld special education group has developmental immaturity .", "significant pre- , peri- , post - natal problems ( 40% ) were found in mothers of children with sld in special education group along with epilepsy , head injury or infections in infancy , poor vision , speech and language delay during childhood are found to be significant in both groups of sld , clearly indicating that sld is a developmental problem .", "sld children have significant school problems in the form of school refusal , poor school performance , reading , writing , spelling , and arithmetic when compared to normal children . in sld children ,", "the problems with reading were found to be about 90.3% , with comprehension 88.7% , with arithmetic 27.4% , with spelling 75.8% , and with writing 45.2% .", "pure reading disability is found in 35.48% , writing disability in 1.61% , dyscalculia in 4.83% while combined learning disability in 58.06% , which is found to be the most common type of learning disability .", "mathematic disability is found to occur less frequently in sld special education group ( 16.7% ) than in sld schools group ( 34.4% ) .", "these findings are similar to the findings of karande et al . where combined subtype was represented more .", "however , compared to the study by ramaa where mathematic disability is found to be 5.98% , mathematic disabilities have found to occur less frequently in special education group ; this could be probably due to the reason that most of the children brought to the special educators are having problems with reading and writing . in this study ,", "most common type of learning disability was found to be combined ( reading + writing ) 35.48% which was similar to a finding by karande et al . , 2007 .", "but as compared to the previous indian studies , mathematic disabilities have found to occur less frequently in special education group , this could be probably due to the reason that most of the children brought to the special educators are having problems with reading , spelling , and writing .", "children with sld committed statistically significant errors on bgt ( p = 0.001 ) denoting perceptual and visuomotor integration problems in children with sld .", "this amounts to the high amount of inattentiveness seen in sld and also the influence of other comorbid conditions like adhd .", "adhd is found to be the most common comorbidity associated with sld amounting to 41.9% with trends toward inattentive subtype .", "analysis of the characteristics of the 26 ( 41.9% ) children with adhd in sld group , it is found that there are 3 ( 11.5% ) females and 23 ( 88.5% ) males . on subtyping", ", the number of sld children with adhd inattentive type is found to be 11 ( 42.3% ) , adhd hyperactive type 7 ( 26.9% ) , and combined type of adhd in 8 ( 30.8% ) .", "the comorbidity of adhd was about 35% . in the present study , trends are toward inattentive type , whereas in other studies on indian and pakistani data found that combined subtype was more common . conduct disorder is found to be about 3% in sld schools group with trends toward symptoms of disobedience .", "emotional disorders are found to be about 6.3% in sld schools group and 13.3% in sld special education group .", "none of the children in both sld groups met the criteria for somatic problems , psychosis or obsessive compulsive disorder . on the whole , the comorbidities as assessed by dpcl showed significance on adhd , conduct and emotional disorders which was not found in the study by karande et al . except for adhd .", "significant family history of learning disabilities is found in children with both groups of sld .", "learning disorders in family history is found to be significant as also observed by snowling et al .", "stressors like over expectation and over involvement by parents are found in children with sld special education group .", "the study was not conducted in government schools as the assessment of sld in vernacular languages was not available ." ], [ "the study was not conducted in government schools as the assessment of sld in vernacular languages was not available ." ], [ "there was a significant association with prematurity , cesarean section , delayed speech and family history of sld . among comorbidities of sld ," ], [ "" ], [ "" ] ]

[ "hypoxaemia in critical illness may be caused by hypo - ventilation , ventilation / perfusion mismatch , right - to - left shunting or limitation of diffusion across the alveolar capillary membrane .", "hypoxaemia my also occur as a result of breathing a low fractional inspired oxygen tension ; for example , at high altitude .", "tissue hypoxia ( reduced cellular or mitochondrial oxygen availability ) may arise as a consequence of hypoxaemia or as a result of reduced oxygen delivery due to decreased cardiac output or decreased red - cell concentration ( anaemia ) .", "this may be due to decreased tissue oxygen delivery associated with microcirculatory dysfunction , or may occur via alterations in cellular energy pathways and mitochondrial function , resulting in a decreased ability to utilise the available oxygen a phenomenon termed cellular dysoxia .", "hypoxic epithelial cell activation releases tumour necrosis factor alpha and il-8 , resulting in pathological increases in vascular permeability and in the release of il-6 , the main cytokine of the acute - phase response .", "hypoxia - mediated cell death will generate an inflammatory response , further perpetuating the cascade of critical illness .", "furthermore , myocardial tissue hypoxia may impair contractile function , thus reducing the total blood flow and further exacerbating global tissue hypoxia .", "for example , in monge 's disease ( chronic mountain sickness ) occurring in natives or long - life residents living above 2,500 metres , excessive erythrocytosis coupled with hypoxic pulmonary vaso - constriction may result in high pulmonary artery pressures and cor pulmonale , leading to congestive heart failure .", "time may , however , also allow beneficial adaptive processes that permit an individual to survive severe tissue hypoxia at levels that , encountered more acutely , might prove fatal .", "critically ill patients form a heterogeneous group ; preadmission patient characteristics ( for example , age , fitness , comorbidities ) and precipitating illnesses ( for example , trauma , infection , ischaemic event ) vary considerably .", "in addition , many pathological and physiological processes occur concurrently , and separating the cause and effect of just one feature of the disease ( tissue hypoxia ) can prove extremely difficult .", "hypoxic adaptive processes are likely to be common to tissue hypoxia whatever the cause , however , and studying healthy individuals progressively exposed to hypoxia through ascent to high altitude may inform of the nature of the hypoxic adaptive processes occurring in critically ill patients .", "it might be possible , in other words , to take knowledge ' from mountainside to bedside ' .", "this approach offers the advantages of a relatively homogeneous study population and environmental challenge , in contrast to those observed on critical care units , as well as the availability of ' premorbid ' information and levels of function .", "finally , the approach also offers an ethical alternative to hypoxia experimentation in patients ; all individuals involved are willing participants in climbing or trekking ventures , as a consequence of which they expose themselves to a hypoxic environment .", "the troposphere is the lowest portion of the atmosphere and envelopes the earth 's entire surface . within the troposphere", "the concentration of oxygen in air remains constant so , as the barometric pressure decreases , the partial pressure of oxygen decreases proportionately .", "the partial pressure of oxygen at the altitude of everest base camp ( 5,300 metres altitude ) is about one - half of the sealevel value .", "the summit of mount everest is the highest point above sea level on the earth 's surface , at 8,850 metres altitude , and has a partial pressure of oxygen about one - third of the sea - level value .", "high - altitude physiology may be divided into the study of short - term changes that occur with exposure to hypobaric hypoxia ( the acute response to hypoxia ) and studies of longer - term acclimatisation and adaptation .", "acute exposure to the ambient atmosphere at extreme altitude ( for example , above 8,000 metres ) is rapidly fatal .", "acclimatisation is the set of beneficial processes whereby lowland humans respond to a reduced inspired partial pressure of oxygen .", "these changes tend to reduce the gradient of oxygen partial pressure from ambient air to tissues ( classical oxygen cascade ) and are distinct from the pathological changes that lead to altitude illness .", "adaptation to high altitude describes changes that have occurred over a number of generations as a result of natural selection in a hypobaric hypoxic environment , and this can be observed in some groups of high - altitude residents .", "high - altitude illness may be divided into the acute syndromes that affect lowland or highland residents ascending to altitudes greater than those to which they are accustomed and the chronic conditions that affect individuals resident at high altitude for long periods .", "the acute adult syndromes of high altitude are acute mountain sickness , high - altitude pulmonary oedema ( hape ) and high - altitude cerebral oedema .", "hypoxia fulfils criteria as a causative agent for the acute high - altitude illnesses .", "the incidence and severity of acute mountain sickness , hape and high - altitude cerebral oedema are related to the speed of ascent and the maximum height gained , suggesting a dose response type of relationship in susceptible individuals .", "a number of studies also suggest , however , that inflammation may be contributory in the pathogenesis of altitude illness [ 8 - 16 ] . several studies have failed to convincingly demonstrate any association between an acute inflammatory response and the development of acute mountain sickness . on the other hand ,", "a number of studies have shown that individuals with pre - existing inflammatory conditions ( for example , diarrhoeal illness , upper respiratory tract infection ) have an increased predisposition to acute high - altitude illnesses ( that is , acute high - altitude illnesses occur at lower altitudes or with slower ascents ) .", "this pattern is consistent with moore and moore 's ' two hit model ' of critical illness initiation , whereby a single insult primes the body for a much more severe response to a secondary insult ( for example , major trauma followed by hypoxia and hypovolaemia ) .", "the initial pathogenesis in hape is thought to be nonuniform hypoxic pulmonary vasoconstriction leading to pulmonary capillary stress failure and a high - permeability type of oedema in the face of a normal left atrial pressure .", "although alveolar fluid in early hape does not demonstrate inflammatory activation , bronchoalveolar lavage fluid from individuals with established hape has high levels of inflammatory cells and mediators .", "when broncho - alveolar lavage was performed in the field , in patients suffering from hape for 24 hours or less , there was a marked increase in total cells , with macrophages being predominant , along with elevated levels of cytokines including il-6 , il-8 and tumour necrosis factor alpha .", "in hospitalised patients later in the course of hape , the proportion of neutrophils increases and the observation is an inflammatory process similar in magnitude and pattern to acute respiratory distress syndrome in the critical care unit .", "the development of an inflammatory component may modify the natural history of hape ; anecdotally , the recovery time for hape ( and high - altitude cerebral oedema ) seems to be related to the duration of illness .", "this might be explained by postulating that in early hape , where mechanical capillary stress failure leads to accumulation of intra - alveolar fluid , relief of hypoxia will reduce pulmonary hypertension and the oedema will rapidly resolve . in more established hape , where significant inflammation has developed , resolution requires reversal of the inflammatory state , and takes more time .", "a similar pattern of injury may occur with critical illness : patients who remain poorly resuscitated on the wards for prolonged periods prior to their admission to critical care commonly have a much more prolonged recovery duration than those who are rapidly resuscitated following an initial insult .", "the study of the interaction between hypoxia and inflammation as mechanisms of injury occurring in healthy individuals in a hypoxic environment has the potential to increase our understanding of these interactions in the critically ill .", "parallels exist between the pattern of responses seen following acute , in comparison with subacute , hypobaric hypoxia and those responses that occur during different phases of critical illness .", "the physiological response to acute hypobaric hypoxia serves to increase oxygen delivery to the tissues : ventilation , cardiac output and haemoglobin concentrations increase ( haemoglobin concentration increases initially by the haemo - concentration and later as a result of increased erythro - poiesis ) .", "similarly , the textbook paradigm of acclimatisation to hypobaric hypoxia emphasises the development of mechanisms to increase oxygen flux ( increase in ventilation , cardiac output , oxygen carriage , and capillarity ) .", "these observations , however , do not adequately explain the observed differences between individuals in their tolerance of hypobaric hypoxic environments . neither the baseline cardiorespiratory performance ( maximal oxygen consumption ) nor changes in the response to chronic hypoxia account for differences between individuals in acclimatisation to prolonged hypoxia or performance at altitude .", "maximal oxygen consumption , maximal heart rate and stroke volume are all reduced after acclimatisation despite normalisation of the blood oxygen content to sea - level values ( by an increase in haemoglobin concentration ) .", "furthermore , pure oxygen breathing by acclimatised individuals ( which results in an oxygen content greater than that at sea level ) does not return maximal oxygen consumption to sealevel values .", "these surprising findings suggest that oxygen carriage is not a limiting factor for maximal oxygen consumption at altitude", ". this could be consistent with central nervous system limitation of the maximal exercise capacity , with limitation of oxygen flux within the tissues , or with a downregulation of cellular metabolism .", "an alternative model supported by empirical evidence suggests that mechanisms not related to oxygen delivery may play an even greater role : this alternative model proposes that acclimatisation is achieved not solely by increasing the oxygen flux , but also by decreasing utilisation .", "acclimatisation may therefore be mediated in part by alterations in oxygen delivery , but also by reductions in cellular oxygen demand , perhaps through hibernation / stunning or preconditioning pathways , or through improvements in efficiency of use of metabolic substrates .", "indeed , hypoxia - tolerant systems rarely activate the anaerobic metabolism but tend to favour a reduced energy turnover state and reduce costly cellular activities such as ion - pumping and protein turnover . in this regard , it is interesting to note that other hypoxia - tolerant species tend to adapt to hypoxia by reducing demand ( hibernation , reduced metabolic rate ) rather than increasing supply .", "the available empirical data suggest that this might be so . early in critical illness ( for example , severe sepsis , the immediate perioperative setting or major trauma ) , when the mitochondrial and metabolic activity is high , increasing oxygen delivery ( or maintaining normal oxygen delivery in the face of evidence that the level is reduced ) decreases subsequent mortality [ 29 - 35 ] .", "conversely , in established critical illness , increasing oxygen delivery is at best of no benefit and may even increase mortality [ 34 - 37 ] . in established critical illness ,", "mitochondrial activity and oxidative phosphorylation are reduced ; just as such effects may be beneficial in acclimatisation to hypobaric hypoxia , singer and colleagues have proposed that such ' reduced metabolic demand ' may offer protection against the cellular hypoxia of critical illness . in both critical illness and during exposure to hypoxia at altitude the acute response seems to be to overcome tissue hypoxia by compensating with increased oxygen delivery ( consistent with a fight or flight response ) whereas the longer term response seems focused on reducing utilisation , perhaps through hibernation / stunning , through ' preconditioning ' phenomena or through enhanced efficiency of oxygen utilisation . exploring these differences by studying humans exposed to the hypoxia of high altitude has the potential to identify mechanisms important in established critical illness and perhaps to alter our therapeutic focus towards increasing the efficiency of oxygen utilisation rather than improving delivery .", "consistent with this model there is a suggestion that improved performance at altitude associated with genotype ii of the angiotensin - converting enzyme ( ace ) polymorphism ( see below ) may be related to alterations in the efficiency of oxygen utilisation .", "just as in the outcome from critical illness , there is wide variation between individuals regarding performance at high altitude and susceptibility to high - altitude illness .", "this phenotypic variation occurs as a result of genetic variation , and the selection of ' advantageous genetic variants ' may underpin fundamental differences in the physiology of long - term highland dwellers when compared with lowland populations .", "native tibetan populations have been resident at high altitude for hundreds of generations , whereas the han chinese residents of tibet have migrated from lowland regions during the past 60 years .", "when compared with the han chinese , native tibetans have greater maximal oxygen uptakes and vital capacities , a reduced alveolar arterial oxygen gradient , a higher arterial oxygen saturation at birth and during the first 4 months of life , and an increased uterine artery blood flow leading to a reduction in the incidence of intrauterine growth rate and of low - birth - weight babies .", "some of these changes have also been noted in south american andean natives when compared with lowland residents . among lowlanders ascending to high altitude , genetic differences", "individuals homozygous for the insertion variant of the human ace gene , which is associated with reduced ace levels , seem to perform better at altitude .", "the association of ace polymorphisms with acute and chronic high - altitude illnesses is more complex . in a japanese ( lowland resident )", "population there was no difference in the insertion / deletion allele distribution between hape - resistant and hape - susceptible groups , although pulmonary vascular resistance was higher in those individuals with the d allele when they developed hape .", "this contrasts with studies in kyrghyz ( highland resident ) populations suggesting that high - altitude pulmonary hypertension is associated with the ace gene insertion ( i ) allele .", "there are also data suggesting that variants of the endothelial nitric oxide synthase gene could be involved in adaptation to altitude .", "nitric oxide is synthesised in the lungs and is involved in the regulation of pulmonary blood flow .", "exhaled nitric oxide levels are higher in native populations resident at high altitude . in japanese subjects ,", "polymorphisms of the endothelial nitric oxide synthase gene resulting in decreased nitric oxide synthesis were associated with an increased susceptibility to hape . in caucasians , however , no difference in nitric oxide synthase genotype frequencies was found when comparing hape - susceptible and hape - resistant individuals , and there was also no association between pulmonary artery systolic pressure in acute hypoxia and the nitric oxide synthase genotype .", "the nitric oxide synthase gene polymorphisms associated with lower nitric oxide activity were found to have an increased frequency in nepalese sherpas when compared with nonsherpa lowland residents .", "interactions with other gene systems will probably be responsible for the contrasting effect of the ace gene insertion allele and nitric oxide synthase gene alleles observed in different groups .", "if the paradigm proposed at the outset of the present review has validity , then it would be expected that genes conferring benefit for high - altitude performance might be related to improved outcomes in critical illness . in keeping with this hypothesis ,", "the ace gene insertion allele is associated not only with improved performance at high altitude , but with a lower mortality from acute respiratory distress syndrome , improved outcomes in childhood meningococcal septicaemia and improved cardiorespiratory response to premature birth .", "studying human responses to hypobaric hypoxia may offer important insights into the pathophysiology of critical illness .", "ace = angiotensin - converting enzyme ; hape = high - altitude pulmonary oedema ; il = interleukin ." ]

[ "<S> cellular hypoxia is a fundamental mechanism of injury in the critically ill . </S>", "<S> the study of human responses to hypoxia occurring as a consequence of hypobaria defines the fields of high - altitude medicine and physiology . </S>", "<S> a new paradigm suggests that the physiological and pathophysiological responses to extreme environmental challenges ( for example , hypobaric hypoxia , hyper - baria , microgravity , cold , heat ) may be similar to responses seen in critical illness . </S>", "<S> the present review explores the idea that human responses to the hypoxia of high altitude may be used as a means of exploring elements of the pathophysiology of critical illness . </S>" ]

[ "Introduction", "High-altitude physiology and pathophysiology", "Hypoxia and inflammation as mechanisms of injury", "Physiological and metabolic responses to hypoxia: increased delivery or decreased utilisation", "Genes, hypoxia and adaptation", "Conclusion", "Abbreviations", "Competing interests" ]

[ [ "hypoxaemia in critical illness may be caused by hypo - ventilation , ventilation / perfusion mismatch , right - to - left shunting or limitation of diffusion across the alveolar capillary membrane .", "hypoxaemia my also occur as a result of breathing a low fractional inspired oxygen tension ; for example , at high altitude .", "tissue hypoxia ( reduced cellular or mitochondrial oxygen availability ) may arise as a consequence of hypoxaemia or as a result of reduced oxygen delivery due to decreased cardiac output or decreased red - cell concentration ( anaemia ) .", "this may be due to decreased tissue oxygen delivery associated with microcirculatory dysfunction , or may occur via alterations in cellular energy pathways and mitochondrial function , resulting in a decreased ability to utilise the available oxygen a phenomenon termed cellular dysoxia .", "hypoxic epithelial cell activation releases tumour necrosis factor alpha and il-8 , resulting in pathological increases in vascular permeability and in the release of il-6 , the main cytokine of the acute - phase response .", "hypoxia - mediated cell death will generate an inflammatory response , further perpetuating the cascade of critical illness .", "furthermore , myocardial tissue hypoxia may impair contractile function , thus reducing the total blood flow and further exacerbating global tissue hypoxia .", "for example , in monge 's disease ( chronic mountain sickness ) occurring in natives or long - life residents living above 2,500 metres , excessive erythrocytosis coupled with hypoxic pulmonary vaso - constriction may result in high pulmonary artery pressures and cor pulmonale , leading to congestive heart failure .", "time may , however , also allow beneficial adaptive processes that permit an individual to survive severe tissue hypoxia at levels that , encountered more acutely , might prove fatal .", "critically ill patients form a heterogeneous group ; preadmission patient characteristics ( for example , age , fitness , comorbidities ) and precipitating illnesses ( for example , trauma , infection , ischaemic event ) vary considerably .", "in addition , many pathological and physiological processes occur concurrently , and separating the cause and effect of just one feature of the disease ( tissue hypoxia ) can prove extremely difficult .", "hypoxic adaptive processes are likely to be common to tissue hypoxia whatever the cause , however , and studying healthy individuals progressively exposed to hypoxia through ascent to high altitude may inform of the nature of the hypoxic adaptive processes occurring in critically ill patients .", "it might be possible , in other words , to take knowledge ' from mountainside to bedside ' .", "this approach offers the advantages of a relatively homogeneous study population and environmental challenge , in contrast to those observed on critical care units , as well as the availability of ' premorbid ' information and levels of function .", "finally , the approach also offers an ethical alternative to hypoxia experimentation in patients ; all individuals involved are willing participants in climbing or trekking ventures , as a consequence of which they expose themselves to a hypoxic environment ." ], [ "the troposphere is the lowest portion of the atmosphere and envelopes the earth 's entire surface . within the troposphere", "the concentration of oxygen in air remains constant so , as the barometric pressure decreases , the partial pressure of oxygen decreases proportionately .", "the partial pressure of oxygen at the altitude of everest base camp ( 5,300 metres altitude ) is about one - half of the sealevel value .", "the summit of mount everest is the highest point above sea level on the earth 's surface , at 8,850 metres altitude , and has a partial pressure of oxygen about one - third of the sea - level value .", "high - altitude physiology may be divided into the study of short - term changes that occur with exposure to hypobaric hypoxia ( the acute response to hypoxia ) and studies of longer - term acclimatisation and adaptation .", "acute exposure to the ambient atmosphere at extreme altitude ( for example , above 8,000 metres ) is rapidly fatal .", "acclimatisation is the set of beneficial processes whereby lowland humans respond to a reduced inspired partial pressure of oxygen .", "these changes tend to reduce the gradient of oxygen partial pressure from ambient air to tissues ( classical oxygen cascade ) and are distinct from the pathological changes that lead to altitude illness .", "adaptation to high altitude describes changes that have occurred over a number of generations as a result of natural selection in a hypobaric hypoxic environment , and this can be observed in some groups of high - altitude residents .", "high - altitude illness may be divided into the acute syndromes that affect lowland or highland residents ascending to altitudes greater than those to which they are accustomed and the chronic conditions that affect individuals resident at high altitude for long periods .", "the acute adult syndromes of high altitude are acute mountain sickness , high - altitude pulmonary oedema ( hape ) and high - altitude cerebral oedema ." ], [ "hypoxia fulfils criteria as a causative agent for the acute high - altitude illnesses .", "the incidence and severity of acute mountain sickness , hape and high - altitude cerebral oedema are related to the speed of ascent and the maximum height gained , suggesting a dose response type of relationship in susceptible individuals .", "a number of studies also suggest , however , that inflammation may be contributory in the pathogenesis of altitude illness [ 8 - 16 ] . several studies have failed to convincingly demonstrate any association between an acute inflammatory response and the development of acute mountain sickness . on the other hand ,", "a number of studies have shown that individuals with pre - existing inflammatory conditions ( for example , diarrhoeal illness , upper respiratory tract infection ) have an increased predisposition to acute high - altitude illnesses ( that is , acute high - altitude illnesses occur at lower altitudes or with slower ascents ) .", "this pattern is consistent with moore and moore 's ' two hit model ' of critical illness initiation , whereby a single insult primes the body for a much more severe response to a secondary insult ( for example , major trauma followed by hypoxia and hypovolaemia ) .", "the initial pathogenesis in hape is thought to be nonuniform hypoxic pulmonary vasoconstriction leading to pulmonary capillary stress failure and a high - permeability type of oedema in the face of a normal left atrial pressure .", "although alveolar fluid in early hape does not demonstrate inflammatory activation , bronchoalveolar lavage fluid from individuals with established hape has high levels of inflammatory cells and mediators .", "when broncho - alveolar lavage was performed in the field , in patients suffering from hape for 24 hours or less , there was a marked increase in total cells , with macrophages being predominant , along with elevated levels of cytokines including il-6 , il-8 and tumour necrosis factor alpha .", "in hospitalised patients later in the course of hape , the proportion of neutrophils increases and the observation is an inflammatory process similar in magnitude and pattern to acute respiratory distress syndrome in the critical care unit .", "the development of an inflammatory component may modify the natural history of hape ; anecdotally , the recovery time for hape ( and high - altitude cerebral oedema ) seems to be related to the duration of illness .", "this might be explained by postulating that in early hape , where mechanical capillary stress failure leads to accumulation of intra - alveolar fluid , relief of hypoxia will reduce pulmonary hypertension and the oedema will rapidly resolve . in more established hape , where significant inflammation has developed , resolution requires reversal of the inflammatory state , and takes more time .", "a similar pattern of injury may occur with critical illness : patients who remain poorly resuscitated on the wards for prolonged periods prior to their admission to critical care commonly have a much more prolonged recovery duration than those who are rapidly resuscitated following an initial insult .", "the study of the interaction between hypoxia and inflammation as mechanisms of injury occurring in healthy individuals in a hypoxic environment has the potential to increase our understanding of these interactions in the critically ill ." ], [ "parallels exist between the pattern of responses seen following acute , in comparison with subacute , hypobaric hypoxia and those responses that occur during different phases of critical illness .", "the physiological response to acute hypobaric hypoxia serves to increase oxygen delivery to the tissues : ventilation , cardiac output and haemoglobin concentrations increase ( haemoglobin concentration increases initially by the haemo - concentration and later as a result of increased erythro - poiesis ) .", "similarly , the textbook paradigm of acclimatisation to hypobaric hypoxia emphasises the development of mechanisms to increase oxygen flux ( increase in ventilation , cardiac output , oxygen carriage , and capillarity ) .", "these observations , however , do not adequately explain the observed differences between individuals in their tolerance of hypobaric hypoxic environments . neither the baseline cardiorespiratory performance ( maximal oxygen consumption ) nor changes in the response to chronic hypoxia account for differences between individuals in acclimatisation to prolonged hypoxia or performance at altitude .", "maximal oxygen consumption , maximal heart rate and stroke volume are all reduced after acclimatisation despite normalisation of the blood oxygen content to sea - level values ( by an increase in haemoglobin concentration ) .", "furthermore , pure oxygen breathing by acclimatised individuals ( which results in an oxygen content greater than that at sea level ) does not return maximal oxygen consumption to sealevel values .", "these surprising findings suggest that oxygen carriage is not a limiting factor for maximal oxygen consumption at altitude", ". this could be consistent with central nervous system limitation of the maximal exercise capacity , with limitation of oxygen flux within the tissues , or with a downregulation of cellular metabolism .", "an alternative model supported by empirical evidence suggests that mechanisms not related to oxygen delivery may play an even greater role : this alternative model proposes that acclimatisation is achieved not solely by increasing the oxygen flux , but also by decreasing utilisation .", "acclimatisation may therefore be mediated in part by alterations in oxygen delivery , but also by reductions in cellular oxygen demand , perhaps through hibernation / stunning or preconditioning pathways , or through improvements in efficiency of use of metabolic substrates .", "indeed , hypoxia - tolerant systems rarely activate the anaerobic metabolism but tend to favour a reduced energy turnover state and reduce costly cellular activities such as ion - pumping and protein turnover . in this regard , it is interesting to note that other hypoxia - tolerant species tend to adapt to hypoxia by reducing demand ( hibernation , reduced metabolic rate ) rather than increasing supply .", "the available empirical data suggest that this might be so . early in critical illness ( for example , severe sepsis , the immediate perioperative setting or major trauma ) , when the mitochondrial and metabolic activity is high , increasing oxygen delivery ( or maintaining normal oxygen delivery in the face of evidence that the level is reduced ) decreases subsequent mortality [ 29 - 35 ] .", "conversely , in established critical illness , increasing oxygen delivery is at best of no benefit and may even increase mortality [ 34 - 37 ] . in established critical illness ,", "mitochondrial activity and oxidative phosphorylation are reduced ; just as such effects may be beneficial in acclimatisation to hypobaric hypoxia , singer and colleagues have proposed that such ' reduced metabolic demand ' may offer protection against the cellular hypoxia of critical illness . in both critical illness and during exposure to hypoxia at altitude the acute response seems to be to overcome tissue hypoxia by compensating with increased oxygen delivery ( consistent with a fight or flight response ) whereas the longer term response seems focused on reducing utilisation , perhaps through hibernation / stunning , through ' preconditioning ' phenomena or through enhanced efficiency of oxygen utilisation . exploring these differences by studying humans exposed to the hypoxia of high altitude has the potential to identify mechanisms important in established critical illness and perhaps to alter our therapeutic focus towards increasing the efficiency of oxygen utilisation rather than improving delivery .", "consistent with this model there is a suggestion that improved performance at altitude associated with genotype ii of the angiotensin - converting enzyme ( ace ) polymorphism ( see below ) may be related to alterations in the efficiency of oxygen utilisation ." ], [ "just as in the outcome from critical illness , there is wide variation between individuals regarding performance at high altitude and susceptibility to high - altitude illness .", "this phenotypic variation occurs as a result of genetic variation , and the selection of ' advantageous genetic variants ' may underpin fundamental differences in the physiology of long - term highland dwellers when compared with lowland populations .", "native tibetan populations have been resident at high altitude for hundreds of generations , whereas the han chinese residents of tibet have migrated from lowland regions during the past 60 years .", "when compared with the han chinese , native tibetans have greater maximal oxygen uptakes and vital capacities , a reduced alveolar arterial oxygen gradient , a higher arterial oxygen saturation at birth and during the first 4 months of life , and an increased uterine artery blood flow leading to a reduction in the incidence of intrauterine growth rate and of low - birth - weight babies .", "some of these changes have also been noted in south american andean natives when compared with lowland residents . among lowlanders ascending to high altitude , genetic differences", "individuals homozygous for the insertion variant of the human ace gene , which is associated with reduced ace levels , seem to perform better at altitude .", "the association of ace polymorphisms with acute and chronic high - altitude illnesses is more complex . in a japanese ( lowland resident )", "population there was no difference in the insertion / deletion allele distribution between hape - resistant and hape - susceptible groups , although pulmonary vascular resistance was higher in those individuals with the d allele when they developed hape .", "this contrasts with studies in kyrghyz ( highland resident ) populations suggesting that high - altitude pulmonary hypertension is associated with the ace gene insertion ( i ) allele .", "there are also data suggesting that variants of the endothelial nitric oxide synthase gene could be involved in adaptation to altitude .", "nitric oxide is synthesised in the lungs and is involved in the regulation of pulmonary blood flow .", "exhaled nitric oxide levels are higher in native populations resident at high altitude . in japanese subjects ,", "polymorphisms of the endothelial nitric oxide synthase gene resulting in decreased nitric oxide synthesis were associated with an increased susceptibility to hape . in caucasians , however , no difference in nitric oxide synthase genotype frequencies was found when comparing hape - susceptible and hape - resistant individuals , and there was also no association between pulmonary artery systolic pressure in acute hypoxia and the nitric oxide synthase genotype .", "the nitric oxide synthase gene polymorphisms associated with lower nitric oxide activity were found to have an increased frequency in nepalese sherpas when compared with nonsherpa lowland residents .", "interactions with other gene systems will probably be responsible for the contrasting effect of the ace gene insertion allele and nitric oxide synthase gene alleles observed in different groups .", "if the paradigm proposed at the outset of the present review has validity , then it would be expected that genes conferring benefit for high - altitude performance might be related to improved outcomes in critical illness . in keeping with this hypothesis ,", "the ace gene insertion allele is associated not only with improved performance at high altitude , but with a lower mortality from acute respiratory distress syndrome , improved outcomes in childhood meningococcal septicaemia and improved cardiorespiratory response to premature birth ." ], [ "studying human responses to hypobaric hypoxia may offer important insights into the pathophysiology of critical illness ." ], [ "ace = angiotensin - converting enzyme ; hape = high - altitude pulmonary oedema ; il = interleukin ." ], [ "" ] ]

[ "increasing prevalence of morbid obesity has augmented the demand for bariatric surgery , which is so far the most effective and durable therapy for the treatment of morbidly obese patients .", "deep vein thrombosis ( dvt ) and pulmonary embolism ( pe ) are the major risk factors of mortality after bariatric surgery [ 24 ] ; venous thrombosis of mesenteric vessels may also contribute to significant morbidity .", "although the risk of postoperative dvt following bariatric surgery does not exceed 2% in most studies , there are currently no strategies to identify individuals who have the highest risk for dvt .", "diagnosis of dvt in morbidly obese patients is a challenging clinical task because a considerable proportion of cases remains asymptomatic . to date", ", there are different prophylactic strategies available for prevention of dvt ; however , optimal personalized prophylactic regimes in individual patients are yet not defined .", "anticoagulation with low molecular weight heparin , anti - factor xa inhibitors , and unfractionated heparin are widely used in bariatric surgery for prevention of dvt and pe , but appropriate dosing of anticoagulation is still controversial .", "furthermore , the risk of dvt among morbidly obese patients undergoing bariatric surgery appears to persist despite the use of aggressive prophylactic anticoagulation therapy .", "identification of individuals with increased risk for dvt is also crucial for the balance between dvt prevention and the risk of bleeding , which is another major complication of bariatric surgery .", "recently , many different and complex changes in plasma coagulation factors have been described in patients with obesity .", "obesity is linked to elevated expression of the prothrombotic molecules plasminogen activator inhibitor-1 and tissue factor ( tf ) and increased platelet activation . a significant increase in median levels of circulating microparticles", "was observed in obese patients compared to controls , including annexin v - mp , endothelial - derived , leukocyte - derived , and tissue factor bearing mp compared to controls .", "the same study also showed that obese patients had a significantly higher median peak thrombin and increased median endogenous thrombin potential compared to controls .", "the same group of researchers has shown that after sleeve gastrectomy , the decrease of bmi in morbidly obese subjects was matched with a decrease of circulating microparticles of endothelial , platelet , and leukocyte origin .", "thromboelastography ( teg ) is used to monitor viscoelastic properties of blood clots , from formation to lysis . over the last decade", ", teg has gained significant importance in the management of high - risk bleeding populations , liver transplantation , and certain other clinical conditions .", "although teg is mostly used to monitor hypocoagulability states , recent evidence shows the importance of teg in evaluation of hypercoagulability in the perioperative setting .", "thromboelastography has recently gained wide scientific attention in obesity and hypercoagulation trends observed in these individuals . a clear relationship between hypercoagulability detected by whole - blood thromboelastometry and aggregometry and", "several studies addressed the role of hypercoagulability monitoring in the perioperative period of bariatric surgery .", "a recent systematic review about hypercoagulable state and teg indicated that further studies are needed to determine the ultimate role of this method for prediction of hypercoagulable states and postoperative thrombosis .", "the aim of this study was to determine hypercoagulability states in obese patients undergoing bariatric surgery and to assess the alterations of coagulation parameters in the perioperative setting using teg .", "we selected the cutoff values of teg parameters for defining hypercoagulability based on the studies discussed above .", "we also evaluated the potential of clinical and standard laboratory tests to predict hypercoagulable states in patients referred to bariatric surgery .", "sixty consecutive patients undergoing laparoscopic bariatric surgery ( gastric bypass or gastric plication ) were recruited at the department of surgery , lithuanian university of health sciences hospital from october 2014 to june 2015 .", "all the patients underwent general anesthesia in normothermal conditions and were given standardized thromboembolic prophylaxis with 500 ml 6% dextran 70 ( fresenius , poland ) after induction of anesthesia and fraxiparine 0.3 ml sc ( glaxosmithkline , poland ) starting in the morning of pod1 until discharge from the hospital .", "we excluded patients on anticoagulation therapy , as well as those with chronic kidney / liver failure , coagulation disorders ( liver cirrhosis , known hematological , or hereditary coagulation disorder ) , previous history of dvt , or using oral contraception .", "the patients were followed for clinically evident thrombotic complications for 1 year after the study by arranging clinical appointments 1 month and 1 year after the surgery .", "the study was approved by the regional kaunas ethics committee ( be-2 - 10 ) .", "thromboelastography was performed using the teg 5000 thromboelastograph hemostasis analyzer system ( braintree , usa ) .", "thromboelastographic alterations were assessed at 4 time points : at baseline prior to induction of anesthesia , immediately after the end of the surgery , and on the morning of postoperative days 1 ( pod1 ) and 2 ( pod2 ) .", "the following parameters of thromboelastography were assessed : r - time ( reaction time , min . ; normal range [ nr ] : 510 ) ; k - time ( clotting time , min ; nr : 13 ) ; -angle ( degrees ; nr : 5372 ) ; ma parameter ( maximum amplitude , millimeters ; nr : 5070 ) ; and g ( clot strength , dynes per square centimeter ; nr : 4.610.9 ) .", "briefly , mixing 1 ml of citrated blood with kaolin by an inversion technique was performed by the physician conducting teg .", "then , 20 l of 0.2 m calcium chloride was added to the plain teg cup , followed by 340 l of the blood / kaolin mix .", "data ( age , bmi , coagulation parameters , and teg values ) are presented as mean values and standard deviation ( sd ) .", "qualitative variables were compared using the chi - square or fisher s exact test , as appropriate .", "spearman correlation analysis was performed to determine the correlation between clinical or standard laboratory test values ( crp , esr , d - dimers , and fibrinogen ) and teg parameters .", "receiver operating characteristic ( roc ) curve analysis was performed to determine optimal fibrinogen levels for prediction of hypercoagulable states g 11 dynes / cm and ma 68 mm .", "statistical analysis was performed using the spss 20.0 software package ( chicago , il ) .", "sixty consecutive patients undergoing laparoscopic bariatric surgery ( gastric bypass or gastric plication ) were recruited at the department of surgery , lithuanian university of health sciences hospital from october 2014 to june 2015 .", "all the patients underwent general anesthesia in normothermal conditions and were given standardized thromboembolic prophylaxis with 500 ml 6% dextran 70 ( fresenius , poland ) after induction of anesthesia and fraxiparine 0.3 ml sc ( glaxosmithkline , poland ) starting in the morning of pod1 until discharge from the hospital .", "we excluded patients on anticoagulation therapy , as well as those with chronic kidney / liver failure , coagulation disorders ( liver cirrhosis , known hematological , or hereditary coagulation disorder ) , previous history of dvt , or using oral contraception .", "the patients were followed for clinically evident thrombotic complications for 1 year after the study by arranging clinical appointments 1 month and 1 year after the surgery .", "the study was approved by the regional kaunas ethics committee ( be-2 - 10 ) .", "thromboelastography was performed using the teg 5000 thromboelastograph hemostasis analyzer system ( braintree , usa ) .", "thromboelastographic alterations were assessed at 4 time points : at baseline prior to induction of anesthesia , immediately after the end of the surgery , and on the morning of postoperative days 1 ( pod1 ) and 2 ( pod2 ) .", "the following parameters of thromboelastography were assessed : r - time ( reaction time , min . ; normal range [ nr ] : 510 ) ; k - time ( clotting time , min ; nr : 13 ) ; -angle ( degrees ; nr : 5372 ) ; ma parameter ( maximum amplitude , millimeters ; nr : 5070 ) ; and g ( clot strength , dynes per square centimeter ; nr : 4.610.9 ) .", "briefly , mixing 1 ml of citrated blood with kaolin by an inversion technique was performed by the physician conducting teg .", "then , 20 l of 0.2 m calcium chloride was added to the plain teg cup , followed by 340 l of the blood / kaolin mix .", "data normality was assessed using kolmogorov - smirnov test . data ( age , bmi , coagulation parameters , and teg values )", "qualitative variables were compared using the chi - square or fisher s exact test , as appropriate .", "spearman correlation analysis was performed to determine the correlation between clinical or standard laboratory test values ( crp , esr , d - dimers , and fibrinogen ) and teg parameters .", "receiver operating characteristic ( roc ) curve analysis was performed to determine optimal fibrinogen levels for prediction of hypercoagulable states g 11 dynes / cm and ma 68 mm .", "statistical analysis was performed using the spss 20.0 software package ( chicago , il ) .", "the study included 60 consecutive patients ( 18 males and 42 females ) referred to bariatric surgery , with a mean age of 39.111.9 years ( age range 1862 ) and a mean bmi of 47.58.5 years ( bmi range 34.384.9 ) .", "gastric bypass surgery was performed in 38 ( 63.3% ) patients and gastric plication in 22 ( 36.7% ) patients .", "twenty - one patients ( 35% ) were active smokers at the time of the surgery , and 39 patients ( 65% ) had previous diagnosis of arterial hypertension .", "we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period ( data not shown ) .", "forty - six patients ( 76.7% ) had g parameter < 11 and 14 patients ( 23.3% ) exhibited hypercoagulability prior to surgery , as defined by g value 11 ( table 1 ) .", "comparison of clinical parameters and standard laboratory tests between the 2 groups ( g < 11 vs. g 11 ) did not reveal significant differences in bmi and crp levels ( table 1 ) .", "the age of patients in the g < 11 group was lower ( 37.8 ) than in the g 11 group ( 43.6 ) , but the difference was not significant ( p=0.117 ) .", "the only parameter that was significantly higher in the g 11 group compared to the g < 11 group was fibrinogen levels , which were 4.23 and 3.8", "g / l , respectively ( p=0.021 ; table 1 ) . among all study participants , 17 patients ( 28.3% )", "had ma 68 mm and 43 patients ( 71.7% ) had ma < 68 mm .", "patients with ma 68 were significantly older ( 44.2 years ) than in the group of patients with ma < 68 mm ( 37.1 years ; p=0.046 ; table 1 ) .", "fibrinogen levels were also higher in the hypercoagulable group ( ma 68 mm ) than in the remaining subjects with ma < 68 mm ( table 1 , p=0.025 ) mean values of teg parameters in the perioperative period of study participants are summarized in table 2 .", "there was an increase in r - time from preoperative levels ( 6.74 s ) to pod1 ( 6.96 s ) , but it reached significance only at pod2 ( 7.43 s , p=0.022 ; table 2 ) .", "k - time and a - angle remained at levels similar to the initial teg results recorded on pod1 and pod2 ( table 2 ) .", "there was a trend towards hypercoagulability and higher values of ma and g compared with initial teg values and results obtained on pod2 ; however , the differences were not significant ( table 2 ) .", "dynamics of fibrinogen and d - dimer during the perioperative period are also presented in table 2 .", "there was a significant increase in d - dimer levels in the postoperative period after bariatric surgery .", "average fibrinogen levels also increased significantly from basal values prior to surgery , from 3.90 to 4.16", "we also aimed to assess the changes in hypercoagulability status from baseline to pod2 . at baseline , 46 patients ( 76.7% ) had g parameter < 11 ( table 3 ) , but on pod2 13.0% of this group had g 11 .", "out 14 patients ( 23.3% ) showing hypercoagulability prior to surgery , as defined by g value 11 , 12 patients remained hypercoagulable on pod2 ( table 3 ) .", "prior to surgery , 17 patients ( 28.3% ) had ma 68 mm and 14 of them remained hypercoagulable on pod2 ( table 3 ) .", "forty - three patients ( 71.7% ) had ma < 68 mm at baseline , but 7 of them became hypercoagulable by pod2 . to assess the relationship between clinical variables and standard laboratory parameters with baseline teg values , we performed correlation analysis ( table 4 ) .", "similarly , markers of inflammation , crp , and erythrocyte sedimentation rate ( esr ) also did not correlate with coagulation parameters recorded by teg ( table 4 ) .", "preoperative fibrinogen levels were associated with k - time ( r=0.313 , p=0.018 ) , -angle ( r=0.333 , p=0.011 ) , ma ( r=0.431 , p=0.001 ) , and g ( r=0.387 , p=0.003 ; table 4 ) .", "interestingly , we also observed a weak but significant correlation between age and r - time ( r=0.280 , p=0.03 ) , k - time ( r=0.259 , p=0.046 ) , and -angle ( r=0.260 , p=0.045 , table 4 ) . to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg", ", we performed roc curve analysis . figure 1 shows that plasma fibrinogen level could predict clot strength ( g ) 11 dynes / cm with area under the curve ( auc)=0.680 ( 95% confidence interval ( ci ) : 0.530.80 , p=0.044 ) .", "roc curve analysis of plasma fibrinogen levels to predict ma 68 mm produced an auc of 0.679 ( 95% ci : 0.530.83 , p=0.041 , figure 2 ) .", "g / l , with sensitivity of 80.0% and specificity of 64.3% ( figure 2 ) .", "the study included 60 consecutive patients ( 18 males and 42 females ) referred to bariatric surgery , with a mean age of 39.111.9 years ( age range 1862 ) and a mean bmi of 47.58.5 years ( bmi range 34.384.9 ) .", "gastric bypass surgery was performed in 38 ( 63.3% ) patients and gastric plication in 22 ( 36.7% ) patients .", "twenty - one patients ( 35% ) were active smokers at the time of the surgery , and 39 patients ( 65% ) had previous diagnosis of arterial hypertension .", "we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period ( data not shown ) .", "forty - six patients ( 76.7% ) had g parameter < 11 and 14 patients ( 23.3% ) exhibited hypercoagulability prior to surgery , as defined by g value 11 ( table 1 ) .", "comparison of clinical parameters and standard laboratory tests between the 2 groups ( g < 11 vs. g 11 ) did not reveal significant differences in bmi and crp levels ( table 1 ) .", "the age of patients in the g < 11 group was lower ( 37.8 ) than in the g 11 group ( 43.6 ) , but the difference was not significant ( p=0.117 ) .", "the only parameter that was significantly higher in the g 11 group compared to the g < 11 group was fibrinogen levels , which were 4.23 and 3.8 g / l , respectively ( p=0.021 ; table 1 ) . among all study participants , 17 patients ( 28.3% )", "had ma 68 mm and 43 patients ( 71.7% ) had ma < 68 mm .", "patients with ma 68 were significantly older ( 44.2 years ) than in the group of patients with ma < 68 mm ( 37.1 years ; p=0.046 ; table 1 ) .", "fibrinogen levels were also higher in the hypercoagulable group ( ma 68 mm ) than in the remaining subjects with ma < 68 mm ( table 1 , p=0.025 )", "mean values of teg parameters in the perioperative period of study participants are summarized in table 2 .", "there was an increase in r - time from preoperative levels ( 6.74 s ) to pod1 ( 6.96 s ) , but it reached significance only at pod2 ( 7.43 s , p=0.022 ; table 2 ) .", "k - time and a - angle remained at levels similar to the initial teg results recorded on pod1 and pod2 ( table 2 ) .", "there was a trend towards hypercoagulability and higher values of ma and g compared with initial teg values and results obtained on pod2 ; however , the differences were not significant ( table 2 ) .", "dynamics of fibrinogen and d - dimer during the perioperative period are also presented in table 2 .", "there was a significant increase in d - dimer levels in the postoperative period after bariatric surgery .", "average fibrinogen levels also increased significantly from basal values prior to surgery , from 3.90 to 4.16", "we also aimed to assess the changes in hypercoagulability status from baseline to pod2 . at baseline , 46 patients ( 76.7% ) had g parameter < 11 ( table 3 ) , but on pod2 13.0% of this group had g 11 .", "out 14 patients ( 23.3% ) showing hypercoagulability prior to surgery , as defined by g value 11 , 12 patients remained hypercoagulable on pod2 ( table 3 ) .", "prior to surgery , 17 patients ( 28.3% ) had ma 68 mm and 14 of them remained hypercoagulable on pod2 ( table 3 ) .", "forty - three patients ( 71.7% ) had ma < 68 mm at baseline , but 7 of them became hypercoagulable by pod2 .", "to assess the relationship between clinical variables and standard laboratory parameters with baseline teg values , we performed correlation analysis ( table 4 ) .", "similarly , markers of inflammation , crp , and erythrocyte sedimentation rate ( esr ) also did not correlate with coagulation parameters recorded by teg ( table 4 ) .", "preoperative fibrinogen levels were associated with k - time ( r=0.313 , p=0.018 ) , -angle ( r=0.333 , p=0.011 ) , ma ( r=0.431 , p=0.001 ) , and g ( r=0.387 , p=0.003 ; table 4 ) .", "interestingly , we also observed a weak but significant correlation between age and r - time ( r=0.280 , p=0.03 ) , k - time ( r=0.259 , p=0.046 ) , and -angle ( r=0.260 , p=0.045 , table 4 ) .", "to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg , we performed roc curve analysis . figure 1 shows that plasma fibrinogen level could predict clot strength ( g ) 11 dynes / cm with area under the curve ( auc)=0.680 ( 95% confidence interval ( ci ) : 0.530.80 , p=0.044 ) . the best cutoff point for fibrinogen to predict g 11 was 3.97", "roc curve analysis of plasma fibrinogen levels to predict ma 68 mm produced an auc of 0.679 ( 95% ci : 0.530.83 , p=0.041 , figure 2 ) .", "g / l , with sensitivity of 80.0% and specificity of 64.3% ( figure 2 ) .", "much research has been targeted at establishment of optimal prophylactic anticoagulation regimens in bariatric surgery ; however , a consensus over this strategy has not been achieved .", "although the incidence of thromboembolic complications in patients undergoing bariatric surgery is not very high , the mortality rates associated with pe in this subgroup of patients is striking .", "routine coagulation tests , including aptt , inr , and platelet count , are not able to predict hypercoagulative status in the perioperative period . therefore , alternative testing and monitoring of coagulation status for identification of individuals at increased risk of vte is needed . in the present study we aimed to evaluate coagulation status in patients undergoing bariatric surgery and to assess dynamics of coagulation parameters in the perioperative setting using teg .", "the major finding of our study is that many patients referred to bariatric surgery show a trend towards hypercoagulability on teg .", "we used teg cutoff values of g 11 dynes / cm and ma 68 mm to define hypercoagulability based on previous studies that have shown these thresholds to be linked with thrombotic events .", "our results also show that fibrinogen level is the only indicator of hypercoagulative state among the routinely used laboratory parameters ; however , used alone , it is not able accurately predict hypercoagulative status .", "we believe that the results of our study add new insights into the monitoring of coagulation status in bariatric surgery .", "an increasing number of studies show the importance of teg for monitoring of coagulation status in the perioperative period .", "the major finding of the present study is that many patients referred to bariatric surgery show hypercoagulability as determined by teg in the perioperative setting .", "based on results of previous studies , we used g 11 dynes / cm to define a hypercoagulable state", "found that strongest predictor of vte in surgical patients was elevated g value ; for every 1 dyne / cm increase in g , the odds of a vte increased by 25% . a significant proportion ( 23% ) of our patients were in hypercoagulable state prior to surgery , which supports previous data obtained by rotem analysis .", "it is important to point out that 18 patients ( 30% ) also had g 11 at pod2 .", "we routinely stop low molecular weight heparins ( lmwh ) on discharge and this group of patients might benefit from extended antithrombotic prophylaxis , but this remains to be assessed in further studies .", "interestingly , we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period .", "the other teg parameter that may be used to identify hypercoagulable states and to predict dvt in the postoperative period is ma .", "recent studies have also shown that ma parameter 68 mm is linked with increased risk of thrombotic complications and postoperative myocardial infarction .", "it is worth pointing out that in our study 17 out 60 patients had ma 68 mm prior to surgery , but this proportion did not increase dramatically at pod2 ( 28.3% and 35.0% , respectively ) ; however , forfori et al", ". showed a substantial ( from 20% to 50% ) increase in the percentage of patients with ma 68 mm by pod3 .", "a potential reason why we did not observe a significant increase in the proportion of patients with ma 68 on pod2 could be the shorter overall surgery time ( 91.420.0 min ) as compared to the forfori et al .", "another reason might be related to the different regimens used for thrombosis prophylaxis . in our study , patients received dextran solution during induction of anesthesia .", "dextran is known for its anti - platelet activity realized through reduction of platelet adhesion to von willebrand factor and impairment of platelet activation by thrombin .", "we also observed a significant increase in r - time from preoperative levels to pod2 .", "these results indicate that a prophylactic regimen of 0.3 ml fraxiparine qd started on pod1 seems to reduce activation of the coagulation system in the postoperative period , similar to the prophylaxis regimen based solely on fraxiparine .", "were the first to show that morbidly obese patients have accelerated fibrin formation and higher ma values than lean patients referred to elective surgery , as determined by teg . in our study , the patients with clot strength g 11 dynes / cm had higher average plasma fibrinogen concentration than patients in the g < 11 group , and these findings support previously published results .", "we observed significant correlations between fibrinogen concentration and k - time , -angle , ma , and g values as determined by teg . to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg", "plasma fibrinogen level predicted clot strength ( g ) 11 dynes / cm with an auc=0.680 and the optimal cutoff point for fibrinogen to predict g 11 was 3.97", "similarly , roc curve analysis of plasma fibrinogen levels to predict ma 68 mm resulted in auc of 0.679 with an optimal cutoff point for fibrinogen at 3.85", "similar cutoff values of fibrinogen for prediction of hypercoagulability were observed in a spanish study .", "our results show that fibrinogen level is the only indicator of hypercoagulative state among routinely used laboratory parameters ; however , moderate auc values obtained in the study should be reassessed in larger studies .", "it is worth pointing out that some studies have shown that hyperfibrinogenemia is a potential factor in heparin resistance , and these patients might need enhanced vte prophylaxis regimens .", "obese patients have a higher prevalence of inherited and acquired thrombophilias compared to the general population .", "diabetes , which is closely linked to obesity , is also related to increased incidence of recurrent dvt .", "a recent study has reported that the inflammation biomarker crp was associated with hypercoagulable states evaluated by rotem in a group of patients referred to bariatric surgery . in the present study", "we did not observe a significant correlation between teg parameters and crp or esr levels .", "several previous studies have shown that bmi increases the risk of vte . in the present study ,", "bmi did not correlate with teg values , which was also observed previously by a research group .", "a recent study showed that central obesity , but now overall weight , is linked with increased thrombin generation and is a better predictor of complications .", "firstly , it was not aimed at determining the optimal anticoagulation regimes for bariatric surgery , and that remains a challenging task in further studies .", "we have selected the teg cutoff values based on some previous studies that have shown these thresholds to be linked with the risk of thrombotic events and used them in earlier studies : g 11 dynes / cm and ma parameter ma 68 mm ; however , we agree that the prediction of prothrombotic states based on teg results only is difficult and further research in the field is required .", "we also did not use doppler ultrasound to monitor subclinical vein thrombosis ; therefore , we might have missed some cases of obscure vein thrombosis in the follow - up . to date , it is not clear whether patients showing hypercoagulation parameters on teg should be managed differently , and the optimal strategies for dvt prevention in this group have yet to be established in well - designed randomized clinical trials .", "interestingly , some studies have suggested the need for extended anticoagulation regimens after bariatric surgery because some dvt occurs later after surgery .", "we speculate that patients showing hypercoagulability on pod2 by teg might benefit from receiving a prolonged prophylactic anticoagulation , but this needs to be assessed in further studies .", "potentially , a combination of teg results and other coagulation markers could provide more accurate prediction models , and this needs to be established in the future .", "bariatric surgery is clearly associated with significant weight loss and will retain a major role in treatment of morbid obesity .", "furthermore , the balance between the risk of bleeding and risk of thrombotic complications remains very important , posing further challenges in the field .", "a considerable proportion of patients referred to bariatric surgery show a trend towards hypercoagulability as determined by teg .", "this study shows the potential of hypercoagulation monitoring by teg in the perioperative setting of bariatric surgery ." ]

[ "<S> backgroundobesity is known as a major risk factor for postoperative vein thrombosis . </S>", "<S> thromboelastography ( teg ) is used to monitor viscoelastic features of blood clots . </S>", "<S> the aim of this study was to determine hypercoagulable states in patients undergoing bariatric surgery and to assess dynamics of coagulation parameters in the perioperative setting using teg.material/methodswe included 60 consecutive patients undergoing bariatric surgery . </S>", "<S> teg alterations were assessed at 4 time points : at baseline , after the surgery , and on postoperative day 1 ( pod1 ) and 2 ( pod2 ) . </S>", "<S> hypercoagulable state was defined when patients showed clot strength ( g ) of 11 dynes / cm2 or maximum amplitude ( ma ) 68 mm.resultsfourteen patients ( 23.3% ) out of 60 showed hypercoagulability prior to surgery on teg . </S>", "<S> fibrinogen levels were significantly higher in the g 11 group compared to the g < 11 group , at 4.2 and 3.8 </S>", "<S> g / l , respectively ( p=0.02 ) . </S>", "<S> seventeen patients ( 28.3% ) had ma 68 mm at baseline . </S>", "<S> fibrinogen levels increased significantly from 3.90 at baseline to 4.16 </S>", "<S> g / l in pod2 ( p<0.001 ) . </S>", "<S> there was an increase in mean reaction time from baseline ( 6.74 s ) to pod2 ( 7.43 s , p=0.022 ) . </S>", "<S> we found a correlation between baseline fibrinogen levels and ma ( r=0.431 , p=0.001 ) or g ( r=0.387 , p=0.003 ) . </S>", "<S> roc curve analysis showed that fibrinogen levels can predict clot strength ( g ) 11 dynes / cm2 with auc=0.680 ( p=0.044).conclusionsa considerable proportion of patients referred to bariatric surgery show a trend towards hypercoagulability on teg . </S>", "<S> this study shows the potential of hypercoagulation monitoring by teg in the perioperative setting of bariatric surgery . </S>" ]

[ "Background", "Material and Methods", "Study population", "Thromboelastography measurements", "Statistical analysis", "Results", "Characteristics of the study group", "Preoperative hypercoagulability rates determined by TEG", "Dynamics of TEG parameters, fibrinogen, and d-dimers in the perioperative period of bariatric surgery", "Dynamics of coagulation status in the perioperative period of bariatric surgery", "Analysis of correlation between TEG parameters and clinical or laboratory parameters", "Plasma fibrinogen levels show moderate predictive value for identifying individuals with G 11 dynes/cm", "Discussion", "Conclusions" ]

[ [ "increasing prevalence of morbid obesity has augmented the demand for bariatric surgery , which is so far the most effective and durable therapy for the treatment of morbidly obese patients .", "deep vein thrombosis ( dvt ) and pulmonary embolism ( pe ) are the major risk factors of mortality after bariatric surgery [ 24 ] ; venous thrombosis of mesenteric vessels may also contribute to significant morbidity .", "although the risk of postoperative dvt following bariatric surgery does not exceed 2% in most studies , there are currently no strategies to identify individuals who have the highest risk for dvt .", "diagnosis of dvt in morbidly obese patients is a challenging clinical task because a considerable proportion of cases remains asymptomatic . to date", ", there are different prophylactic strategies available for prevention of dvt ; however , optimal personalized prophylactic regimes in individual patients are yet not defined .", "anticoagulation with low molecular weight heparin , anti - factor xa inhibitors , and unfractionated heparin are widely used in bariatric surgery for prevention of dvt and pe , but appropriate dosing of anticoagulation is still controversial .", "furthermore , the risk of dvt among morbidly obese patients undergoing bariatric surgery appears to persist despite the use of aggressive prophylactic anticoagulation therapy .", "identification of individuals with increased risk for dvt is also crucial for the balance between dvt prevention and the risk of bleeding , which is another major complication of bariatric surgery .", "recently , many different and complex changes in plasma coagulation factors have been described in patients with obesity .", "obesity is linked to elevated expression of the prothrombotic molecules plasminogen activator inhibitor-1 and tissue factor ( tf ) and increased platelet activation . a significant increase in median levels of circulating microparticles", "was observed in obese patients compared to controls , including annexin v - mp , endothelial - derived , leukocyte - derived , and tissue factor bearing mp compared to controls .", "the same study also showed that obese patients had a significantly higher median peak thrombin and increased median endogenous thrombin potential compared to controls .", "the same group of researchers has shown that after sleeve gastrectomy , the decrease of bmi in morbidly obese subjects was matched with a decrease of circulating microparticles of endothelial , platelet , and leukocyte origin .", "thromboelastography ( teg ) is used to monitor viscoelastic properties of blood clots , from formation to lysis . over the last decade", ", teg has gained significant importance in the management of high - risk bleeding populations , liver transplantation , and certain other clinical conditions .", "although teg is mostly used to monitor hypocoagulability states , recent evidence shows the importance of teg in evaluation of hypercoagulability in the perioperative setting .", "thromboelastography has recently gained wide scientific attention in obesity and hypercoagulation trends observed in these individuals . a clear relationship between hypercoagulability detected by whole - blood thromboelastometry and aggregometry and", "several studies addressed the role of hypercoagulability monitoring in the perioperative period of bariatric surgery .", "a recent systematic review about hypercoagulable state and teg indicated that further studies are needed to determine the ultimate role of this method for prediction of hypercoagulable states and postoperative thrombosis .", "the aim of this study was to determine hypercoagulability states in obese patients undergoing bariatric surgery and to assess the alterations of coagulation parameters in the perioperative setting using teg .", "we selected the cutoff values of teg parameters for defining hypercoagulability based on the studies discussed above .", "we also evaluated the potential of clinical and standard laboratory tests to predict hypercoagulable states in patients referred to bariatric surgery ." ], [ "sixty consecutive patients undergoing laparoscopic bariatric surgery ( gastric bypass or gastric plication ) were recruited at the department of surgery , lithuanian university of health sciences hospital from october 2014 to june 2015 .", "all the patients underwent general anesthesia in normothermal conditions and were given standardized thromboembolic prophylaxis with 500 ml 6% dextran 70 ( fresenius , poland ) after induction of anesthesia and fraxiparine 0.3 ml sc ( glaxosmithkline , poland ) starting in the morning of pod1 until discharge from the hospital .", "we excluded patients on anticoagulation therapy , as well as those with chronic kidney / liver failure , coagulation disorders ( liver cirrhosis , known hematological , or hereditary coagulation disorder ) , previous history of dvt , or using oral contraception .", "the patients were followed for clinically evident thrombotic complications for 1 year after the study by arranging clinical appointments 1 month and 1 year after the surgery .", "the study was approved by the regional kaunas ethics committee ( be-2 - 10 ) .", "thromboelastography was performed using the teg 5000 thromboelastograph hemostasis analyzer system ( braintree , usa ) .", "thromboelastographic alterations were assessed at 4 time points : at baseline prior to induction of anesthesia , immediately after the end of the surgery , and on the morning of postoperative days 1 ( pod1 ) and 2 ( pod2 ) .", "the following parameters of thromboelastography were assessed : r - time ( reaction time , min . ; normal range [ nr ] : 510 ) ; k - time ( clotting time , min ; nr : 13 ) ; -angle ( degrees ; nr : 5372 ) ; ma parameter ( maximum amplitude , millimeters ; nr : 5070 ) ; and g ( clot strength , dynes per square centimeter ; nr : 4.610.9 ) .", "briefly , mixing 1 ml of citrated blood with kaolin by an inversion technique was performed by the physician conducting teg .", "then , 20 l of 0.2 m calcium chloride was added to the plain teg cup , followed by 340 l of the blood / kaolin mix .", "data ( age , bmi , coagulation parameters , and teg values ) are presented as mean values and standard deviation ( sd ) .", "qualitative variables were compared using the chi - square or fisher s exact test , as appropriate .", "spearman correlation analysis was performed to determine the correlation between clinical or standard laboratory test values ( crp , esr , d - dimers , and fibrinogen ) and teg parameters .", "receiver operating characteristic ( roc ) curve analysis was performed to determine optimal fibrinogen levels for prediction of hypercoagulable states g 11 dynes / cm and ma 68 mm .", "statistical analysis was performed using the spss 20.0 software package ( chicago , il ) ." ], [ "sixty consecutive patients undergoing laparoscopic bariatric surgery ( gastric bypass or gastric plication ) were recruited at the department of surgery , lithuanian university of health sciences hospital from october 2014 to june 2015 .", "all the patients underwent general anesthesia in normothermal conditions and were given standardized thromboembolic prophylaxis with 500 ml 6% dextran 70 ( fresenius , poland ) after induction of anesthesia and fraxiparine 0.3 ml sc ( glaxosmithkline , poland ) starting in the morning of pod1 until discharge from the hospital .", "we excluded patients on anticoagulation therapy , as well as those with chronic kidney / liver failure , coagulation disorders ( liver cirrhosis , known hematological , or hereditary coagulation disorder ) , previous history of dvt , or using oral contraception .", "the patients were followed for clinically evident thrombotic complications for 1 year after the study by arranging clinical appointments 1 month and 1 year after the surgery .", "the study was approved by the regional kaunas ethics committee ( be-2 - 10 ) ." ], [ "thromboelastography was performed using the teg 5000 thromboelastograph hemostasis analyzer system ( braintree , usa ) .", "thromboelastographic alterations were assessed at 4 time points : at baseline prior to induction of anesthesia , immediately after the end of the surgery , and on the morning of postoperative days 1 ( pod1 ) and 2 ( pod2 ) .", "the following parameters of thromboelastography were assessed : r - time ( reaction time , min . ; normal range [ nr ] : 510 ) ; k - time ( clotting time , min ; nr : 13 ) ; -angle ( degrees ; nr : 5372 ) ; ma parameter ( maximum amplitude , millimeters ; nr : 5070 ) ; and g ( clot strength , dynes per square centimeter ; nr : 4.610.9 ) .", "briefly , mixing 1 ml of citrated blood with kaolin by an inversion technique was performed by the physician conducting teg .", "then , 20 l of 0.2 m calcium chloride was added to the plain teg cup , followed by 340 l of the blood / kaolin mix ." ], [ "data normality was assessed using kolmogorov - smirnov test . data ( age , bmi , coagulation parameters , and teg values )", "qualitative variables were compared using the chi - square or fisher s exact test , as appropriate .", "spearman correlation analysis was performed to determine the correlation between clinical or standard laboratory test values ( crp , esr , d - dimers , and fibrinogen ) and teg parameters .", "receiver operating characteristic ( roc ) curve analysis was performed to determine optimal fibrinogen levels for prediction of hypercoagulable states g 11 dynes / cm and ma 68 mm .", "statistical analysis was performed using the spss 20.0 software package ( chicago , il ) ." ], [ "the study included 60 consecutive patients ( 18 males and 42 females ) referred to bariatric surgery , with a mean age of 39.111.9 years ( age range 1862 ) and a mean bmi of 47.58.5 years ( bmi range 34.384.9 ) .", "gastric bypass surgery was performed in 38 ( 63.3% ) patients and gastric plication in 22 ( 36.7% ) patients .", "twenty - one patients ( 35% ) were active smokers at the time of the surgery , and 39 patients ( 65% ) had previous diagnosis of arterial hypertension .", "we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period ( data not shown ) .", "forty - six patients ( 76.7% ) had g parameter < 11 and 14 patients ( 23.3% ) exhibited hypercoagulability prior to surgery , as defined by g value 11 ( table 1 ) .", "comparison of clinical parameters and standard laboratory tests between the 2 groups ( g < 11 vs. g 11 ) did not reveal significant differences in bmi and crp levels ( table 1 ) .", "the age of patients in the g < 11 group was lower ( 37.8 ) than in the g 11 group ( 43.6 ) , but the difference was not significant ( p=0.117 ) .", "the only parameter that was significantly higher in the g 11 group compared to the g < 11 group was fibrinogen levels , which were 4.23 and 3.8", "g / l , respectively ( p=0.021 ; table 1 ) . among all study participants , 17 patients ( 28.3% )", "had ma 68 mm and 43 patients ( 71.7% ) had ma < 68 mm .", "patients with ma 68 were significantly older ( 44.2 years ) than in the group of patients with ma < 68 mm ( 37.1 years ; p=0.046 ; table 1 ) .", "fibrinogen levels were also higher in the hypercoagulable group ( ma 68 mm ) than in the remaining subjects with ma < 68 mm ( table 1 , p=0.025 ) mean values of teg parameters in the perioperative period of study participants are summarized in table 2 .", "there was an increase in r - time from preoperative levels ( 6.74 s ) to pod1 ( 6.96 s ) , but it reached significance only at pod2 ( 7.43 s , p=0.022 ; table 2 ) .", "k - time and a - angle remained at levels similar to the initial teg results recorded on pod1 and pod2 ( table 2 ) .", "there was a trend towards hypercoagulability and higher values of ma and g compared with initial teg values and results obtained on pod2 ; however , the differences were not significant ( table 2 ) .", "dynamics of fibrinogen and d - dimer during the perioperative period are also presented in table 2 .", "there was a significant increase in d - dimer levels in the postoperative period after bariatric surgery .", "average fibrinogen levels also increased significantly from basal values prior to surgery , from 3.90 to 4.16", "we also aimed to assess the changes in hypercoagulability status from baseline to pod2 . at baseline , 46 patients ( 76.7% ) had g parameter < 11 ( table 3 ) , but on pod2 13.0% of this group had g 11 .", "out 14 patients ( 23.3% ) showing hypercoagulability prior to surgery , as defined by g value 11 , 12 patients remained hypercoagulable on pod2 ( table 3 ) .", "prior to surgery , 17 patients ( 28.3% ) had ma 68 mm and 14 of them remained hypercoagulable on pod2 ( table 3 ) .", "forty - three patients ( 71.7% ) had ma < 68 mm at baseline , but 7 of them became hypercoagulable by pod2 . to assess the relationship between clinical variables and standard laboratory parameters with baseline teg values , we performed correlation analysis ( table 4 ) .", "similarly , markers of inflammation , crp , and erythrocyte sedimentation rate ( esr ) also did not correlate with coagulation parameters recorded by teg ( table 4 ) .", "preoperative fibrinogen levels were associated with k - time ( r=0.313 , p=0.018 ) , -angle ( r=0.333 , p=0.011 ) , ma ( r=0.431 , p=0.001 ) , and g ( r=0.387 , p=0.003 ; table 4 ) .", "interestingly , we also observed a weak but significant correlation between age and r - time ( r=0.280 , p=0.03 ) , k - time ( r=0.259 , p=0.046 ) , and -angle ( r=0.260 , p=0.045 , table 4 ) . to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg", ", we performed roc curve analysis . figure 1 shows that plasma fibrinogen level could predict clot strength ( g ) 11 dynes / cm with area under the curve ( auc)=0.680 ( 95% confidence interval ( ci ) : 0.530.80 , p=0.044 ) .", "roc curve analysis of plasma fibrinogen levels to predict ma 68 mm produced an auc of 0.679 ( 95% ci : 0.530.83 , p=0.041 , figure 2 ) .", "g / l , with sensitivity of 80.0% and specificity of 64.3% ( figure 2 ) ." ], [ "the study included 60 consecutive patients ( 18 males and 42 females ) referred to bariatric surgery , with a mean age of 39.111.9 years ( age range 1862 ) and a mean bmi of 47.58.5 years ( bmi range 34.384.9 ) .", "gastric bypass surgery was performed in 38 ( 63.3% ) patients and gastric plication in 22 ( 36.7% ) patients .", "twenty - one patients ( 35% ) were active smokers at the time of the surgery , and 39 patients ( 65% ) had previous diagnosis of arterial hypertension .", "we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period ( data not shown ) ." ], [ "forty - six patients ( 76.7% ) had g parameter < 11 and 14 patients ( 23.3% ) exhibited hypercoagulability prior to surgery , as defined by g value 11 ( table 1 ) .", "comparison of clinical parameters and standard laboratory tests between the 2 groups ( g < 11 vs. g 11 ) did not reveal significant differences in bmi and crp levels ( table 1 ) .", "the age of patients in the g < 11 group was lower ( 37.8 ) than in the g 11 group ( 43.6 ) , but the difference was not significant ( p=0.117 ) .", "the only parameter that was significantly higher in the g 11 group compared to the g < 11 group was fibrinogen levels , which were 4.23 and 3.8 g / l , respectively ( p=0.021 ; table 1 ) . among all study participants , 17 patients ( 28.3% )", "had ma 68 mm and 43 patients ( 71.7% ) had ma < 68 mm .", "patients with ma 68 were significantly older ( 44.2 years ) than in the group of patients with ma < 68 mm ( 37.1 years ; p=0.046 ; table 1 ) .", "fibrinogen levels were also higher in the hypercoagulable group ( ma 68 mm ) than in the remaining subjects with ma < 68 mm ( table 1 , p=0.025 )" ], [ "mean values of teg parameters in the perioperative period of study participants are summarized in table 2 .", "there was an increase in r - time from preoperative levels ( 6.74 s ) to pod1 ( 6.96 s ) , but it reached significance only at pod2 ( 7.43 s , p=0.022 ; table 2 ) .", "k - time and a - angle remained at levels similar to the initial teg results recorded on pod1 and pod2 ( table 2 ) .", "there was a trend towards hypercoagulability and higher values of ma and g compared with initial teg values and results obtained on pod2 ; however , the differences were not significant ( table 2 ) .", "dynamics of fibrinogen and d - dimer during the perioperative period are also presented in table 2 .", "there was a significant increase in d - dimer levels in the postoperative period after bariatric surgery .", "average fibrinogen levels also increased significantly from basal values prior to surgery , from 3.90 to 4.16" ], [ "we also aimed to assess the changes in hypercoagulability status from baseline to pod2 . at baseline , 46 patients ( 76.7% ) had g parameter < 11 ( table 3 ) , but on pod2 13.0% of this group had g 11 .", "out 14 patients ( 23.3% ) showing hypercoagulability prior to surgery , as defined by g value 11 , 12 patients remained hypercoagulable on pod2 ( table 3 ) .", "prior to surgery , 17 patients ( 28.3% ) had ma 68 mm and 14 of them remained hypercoagulable on pod2 ( table 3 ) .", "forty - three patients ( 71.7% ) had ma < 68 mm at baseline , but 7 of them became hypercoagulable by pod2 ." ], [ "to assess the relationship between clinical variables and standard laboratory parameters with baseline teg values , we performed correlation analysis ( table 4 ) .", "similarly , markers of inflammation , crp , and erythrocyte sedimentation rate ( esr ) also did not correlate with coagulation parameters recorded by teg ( table 4 ) .", "preoperative fibrinogen levels were associated with k - time ( r=0.313 , p=0.018 ) , -angle ( r=0.333 , p=0.011 ) , ma ( r=0.431 , p=0.001 ) , and g ( r=0.387 , p=0.003 ; table 4 ) .", "interestingly , we also observed a weak but significant correlation between age and r - time ( r=0.280 , p=0.03 ) , k - time ( r=0.259 , p=0.046 ) , and -angle ( r=0.260 , p=0.045 , table 4 ) ." ], [ "to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg , we performed roc curve analysis . figure 1 shows that plasma fibrinogen level could predict clot strength ( g ) 11 dynes / cm with area under the curve ( auc)=0.680 ( 95% confidence interval ( ci ) : 0.530.80 , p=0.044 ) . the best cutoff point for fibrinogen to predict g 11 was 3.97", "roc curve analysis of plasma fibrinogen levels to predict ma 68 mm produced an auc of 0.679 ( 95% ci : 0.530.83 , p=0.041 , figure 2 ) .", "g / l , with sensitivity of 80.0% and specificity of 64.3% ( figure 2 ) ." ], [ "much research has been targeted at establishment of optimal prophylactic anticoagulation regimens in bariatric surgery ; however , a consensus over this strategy has not been achieved .", "although the incidence of thromboembolic complications in patients undergoing bariatric surgery is not very high , the mortality rates associated with pe in this subgroup of patients is striking .", "routine coagulation tests , including aptt , inr , and platelet count , are not able to predict hypercoagulative status in the perioperative period . therefore , alternative testing and monitoring of coagulation status for identification of individuals at increased risk of vte is needed . in the present study we aimed to evaluate coagulation status in patients undergoing bariatric surgery and to assess dynamics of coagulation parameters in the perioperative setting using teg .", "the major finding of our study is that many patients referred to bariatric surgery show a trend towards hypercoagulability on teg .", "we used teg cutoff values of g 11 dynes / cm and ma 68 mm to define hypercoagulability based on previous studies that have shown these thresholds to be linked with thrombotic events .", "our results also show that fibrinogen level is the only indicator of hypercoagulative state among the routinely used laboratory parameters ; however , used alone , it is not able accurately predict hypercoagulative status .", "we believe that the results of our study add new insights into the monitoring of coagulation status in bariatric surgery .", "an increasing number of studies show the importance of teg for monitoring of coagulation status in the perioperative period .", "the major finding of the present study is that many patients referred to bariatric surgery show hypercoagulability as determined by teg in the perioperative setting .", "based on results of previous studies , we used g 11 dynes / cm to define a hypercoagulable state", "found that strongest predictor of vte in surgical patients was elevated g value ; for every 1 dyne / cm increase in g , the odds of a vte increased by 25% . a significant proportion ( 23% ) of our patients were in hypercoagulable state prior to surgery , which supports previous data obtained by rotem analysis .", "it is important to point out that 18 patients ( 30% ) also had g 11 at pod2 .", "we routinely stop low molecular weight heparins ( lmwh ) on discharge and this group of patients might benefit from extended antithrombotic prophylaxis , but this remains to be assessed in further studies .", "interestingly , we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period .", "the other teg parameter that may be used to identify hypercoagulable states and to predict dvt in the postoperative period is ma .", "recent studies have also shown that ma parameter 68 mm is linked with increased risk of thrombotic complications and postoperative myocardial infarction .", "it is worth pointing out that in our study 17 out 60 patients had ma 68 mm prior to surgery , but this proportion did not increase dramatically at pod2 ( 28.3% and 35.0% , respectively ) ; however , forfori et al", ". showed a substantial ( from 20% to 50% ) increase in the percentage of patients with ma 68 mm by pod3 .", "a potential reason why we did not observe a significant increase in the proportion of patients with ma 68 on pod2 could be the shorter overall surgery time ( 91.420.0 min ) as compared to the forfori et al .", "another reason might be related to the different regimens used for thrombosis prophylaxis . in our study , patients received dextran solution during induction of anesthesia .", "dextran is known for its anti - platelet activity realized through reduction of platelet adhesion to von willebrand factor and impairment of platelet activation by thrombin .", "we also observed a significant increase in r - time from preoperative levels to pod2 .", "these results indicate that a prophylactic regimen of 0.3 ml fraxiparine qd started on pod1 seems to reduce activation of the coagulation system in the postoperative period , similar to the prophylaxis regimen based solely on fraxiparine .", "were the first to show that morbidly obese patients have accelerated fibrin formation and higher ma values than lean patients referred to elective surgery , as determined by teg . in our study , the patients with clot strength g 11 dynes / cm had higher average plasma fibrinogen concentration than patients in the g < 11 group , and these findings support previously published results .", "we observed significant correlations between fibrinogen concentration and k - time , -angle , ma , and g values as determined by teg . to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg", "plasma fibrinogen level predicted clot strength ( g ) 11 dynes / cm with an auc=0.680 and the optimal cutoff point for fibrinogen to predict g 11 was 3.97", "similarly , roc curve analysis of plasma fibrinogen levels to predict ma 68 mm resulted in auc of 0.679 with an optimal cutoff point for fibrinogen at 3.85", "similar cutoff values of fibrinogen for prediction of hypercoagulability were observed in a spanish study .", "our results show that fibrinogen level is the only indicator of hypercoagulative state among routinely used laboratory parameters ; however , moderate auc values obtained in the study should be reassessed in larger studies .", "it is worth pointing out that some studies have shown that hyperfibrinogenemia is a potential factor in heparin resistance , and these patients might need enhanced vte prophylaxis regimens .", "obese patients have a higher prevalence of inherited and acquired thrombophilias compared to the general population .", "diabetes , which is closely linked to obesity , is also related to increased incidence of recurrent dvt .", "a recent study has reported that the inflammation biomarker crp was associated with hypercoagulable states evaluated by rotem in a group of patients referred to bariatric surgery . in the present study", "we did not observe a significant correlation between teg parameters and crp or esr levels .", "several previous studies have shown that bmi increases the risk of vte . in the present study ,", "bmi did not correlate with teg values , which was also observed previously by a research group .", "a recent study showed that central obesity , but now overall weight , is linked with increased thrombin generation and is a better predictor of complications .", "firstly , it was not aimed at determining the optimal anticoagulation regimes for bariatric surgery , and that remains a challenging task in further studies .", "we have selected the teg cutoff values based on some previous studies that have shown these thresholds to be linked with the risk of thrombotic events and used them in earlier studies : g 11 dynes / cm and ma parameter ma 68 mm ; however , we agree that the prediction of prothrombotic states based on teg results only is difficult and further research in the field is required .", "we also did not use doppler ultrasound to monitor subclinical vein thrombosis ; therefore , we might have missed some cases of obscure vein thrombosis in the follow - up . to date , it is not clear whether patients showing hypercoagulation parameters on teg should be managed differently , and the optimal strategies for dvt prevention in this group have yet to be established in well - designed randomized clinical trials .", "interestingly , some studies have suggested the need for extended anticoagulation regimens after bariatric surgery because some dvt occurs later after surgery .", "we speculate that patients showing hypercoagulability on pod2 by teg might benefit from receiving a prolonged prophylactic anticoagulation , but this needs to be assessed in further studies .", "potentially , a combination of teg results and other coagulation markers could provide more accurate prediction models , and this needs to be established in the future .", "bariatric surgery is clearly associated with significant weight loss and will retain a major role in treatment of morbid obesity .", "furthermore , the balance between the risk of bleeding and risk of thrombotic complications remains very important , posing further challenges in the field ." ], [ "a considerable proportion of patients referred to bariatric surgery show a trend towards hypercoagulability as determined by teg .", "this study shows the potential of hypercoagulation monitoring by teg in the perioperative setting of bariatric surgery ." ] ]

[ "urinary tract infections ( utis ) \n affect millions of women each year , with an annual societal cost of billions of \n dollars .", "importantly , more than one quarter of women with a uti will have a \n recurrent infection within six months . \n", "there are few established options for prevention of uti other than the \n use of prophylactic antibiotics . \n", "however , resistance to commonly used antibiotics is increasing among \n bacterial cystitis isolates . \n", "a growing body of evidence suggests that vaginal h202-producing \n lactobacilli may have a protective effect against urogenital infections , \n including uti [ 511 ] .", "it is hypothesized \n that lactobacilli prevent uropathogen colonization of the vagina , a necessary \n step in ascending infection of the bladder . \n", "several clinical trials have demonstrated that certain lactobacillus species , mainly l. rhamnosus and l. fermentum , can be given orally or vaginally with resulting \n colonization of the vagina , reduction in vaginal coliform counts , and even \n reduction in uti recurrence \n [ 1218 ] .", "specific \n strains were chosen for their abilities to produce h202 , adhere to uroepithelial cells , interfere with uropathogen attachment and growth , and persist in the vagina [ 18 , 19 ] . \n", "the most frequently existing vaginal lactobacillus species , l. \n crispatus [ 20 , 21 ] , also produces h202 and possesses the adherence and inhibitory qualities essential for a good probiotic \n candidate [ 22 , 23 ] .", "l. crispatus strain ctv-05 has been \n developed as a vaginal suppository ( lactin - v ) for the prevention of recurrent \n uti in women .", "l. crispatus ctv-05 is \n highly adherent to vaginal epithelial cells , and when given as a suppository \n can colonize the vagina . \n furthermore , l. crispatus ctv-05 has been reported to have a specific dna fingerprint that distinguishes \n it from endogenous vaginal lactobacilli . \n to further evaluate the safety of this new formulation of l. crispatus ctv-05 and its effect on \n the vaginal microbial flora", ", we conducted a phase i , randomized , double - blind , \n placebo - controlled trial of vaginal l. \n crispatus ctv-05 in premenopausal women with a history of recurrent uti .", "we recruited premenopausal women aged 1835 \n years with a history of three or more uncomplicated utis diagnosed in the past \n year , or two uncomplicated \n utis diagnosed in the past six months , from ( student health center , university \n of washington , wash , usa . ) additional eligibility requirements \n included regular menstrual cycles or amenorrhea for at least six months \n secondary to use of a hormonal contraceptive , a normal pap smear documented in \n the last year or at the baseline clinic visit , abstinence \n from sexual activity or participation in \n a mutually monogamous sexual relationship , use of birth control , agreement not \n to use other intravaginal products , agreement not to have sexual intercourse or \n use tampons between the baseline and first follow - up visit , and capability to \n understand english and provide informed consent .", "exclusion criteria included history of \n urologic abnormality , recent urologic surgery or urinary catheterization , \n history of complicated pyelonephritis or renal calculi , hysterectomy , recent sexually \n transmitted infection ( sti ) or bacterial vaginosis , risk factors for sti and \n hiv , history of recurrent genital herpes , menses anticipated within ten days , pregnancy , \n lactation , recent antibiotic or antifungal use , diabetes or other immunocompromised state , drug \n or alcohol abuse , use of the ( nuvaring ) , prior use of the study drug or allergy \n to any of its components , and abnormal initial pelvic examination .", "the study was approved by ( institutional \n review board ( irb ) , university of washington , wash , \n usa . ) subjects were randomized in a double - blinded \n fashion to l. crispatus ctv-05 at a \n dose of 5 10 colony forming units \n ( cfu ) or placebo vaginal suppository to be inserted daily for five days .", "l. crispatus ctv-05 and placebo \n suppositories were similar in appearance and consisted of a preservation matrix \n and maltodextran .", ", palo alto , \n calif , usa ) according to a randomization \n schedule and supplied to the study site sequentially labeled with a subject \n number .", "subjects \n were seen at three clinic visits over a period of one month . at the first ( baseline )", "visit , eligible subjects \n provided informed consent before undergoing a structured medical and \n gynecologic history and physical examination . \n", "subjects inserted the first dose of study \n drug in the clinic and were instructed to record any symptoms occurring during \n the first week of study on a prepared diary card .", "subjects were seen in follow - up 68 days ( 1-week ) \n visit and 2634 days ( 4-week ) visit after enrollment . during these visits , new symptoms , new \n urogenital infections , and any changes in clinical history were recorded .", "finally , subjects were \n contacted by telephone six months after enrollment to assess symptoms , \n pregnancy , new diagnoses , or major medical events .", "laboratory \n testing included urine dipstick testing , urinalysis , urine culture , vaginal \n fluid wet preparation slides , gram stains , and culture for facultative isolates \n and lactobacillus species at all \n visits .", "repetitive element \n sequence - based polymerase chain reaction ( rep - pcr ) for l. crispatus ctv-05 was performed on three lactobacillus colonies \n from culture at each visit .", "testing for neisseria gonorrhoeae and chlamydia \n trachomatis by dna amplification was done at the baseline visit .", "a \n sample size of 30 was chosen to evaluate the primary outcome of safety as \n assessed through self - reported symptoms , physical exam findings , and laboratory \n studies .", "secondary outcomes included \n shifts in the vaginal flora assessed by vaginal culture and vaginal \n colonization with l. crispatus ctv-05 \n assessed by rep - pcr .", "two women randomized to l. \n crispatus ctv-05 completed treatment over six days , and one woman \n randomized to placebo used six suppositories over six days . all women remained on study through the \n 4-week visit ( figure 1 ) .", "baseline \n characteristics of subjects were similar in the two treatment groups ( table 1 ) .", "the majority of women were young , white , \n healthy , university students in their 20s . \n", "mild to moderate adverse events were \n relatively common , however . of those \n felt by the investigators to be related to study drug use ,", "abnormal vaginal \n discharge was the most frequently occurring , followed by external genital \n irritation and vaginal candidiasis ( table 2 ) .", "these adverse events occurred with \n similar or greater frequency in women randomized to placebo as compared to women \n randomized to l. crispatus ctv-05 .", "three women reporting vaginal candidiasis also \n reported multiple prior episodes in the preceding 12 months .", "two women randomized to l. crispatus ctv-05 reported episodes of cystitis during the \n study .", "all subjects completed a diary card of symptoms that occurred during the period of \n study drug insertion .", "recorded symptoms generally \n confirmed the reported adverse events and were similar to symptoms reported at the \n 1-week clinic visit ( below ) .", "abnormal \n vaginal discharge was the symptom most frequently reported by subjects at \n clinic visits following study drug use ( figure 2 ) .", "while no women reported abnormal \n vaginal discharge at baseline , six randomized to l. crispatus ctv-05 and five randomized to placebo reported abnormal \n vaginal discharge at the 1-week visit .", "external genital or vaginal irritation was \n also reported by six women in each treatment arm at follow - up visits .", "vaginal odor was reported at the 1-week visit \n by one woman randomized to l. crispatus ctv-05 , \n and at the 4-week visit by two women randomized to placebo .", "dysuria was reported by one woman randomized \n to l. crispatus ctv-05 at the 1-week visit \n and by one woman randomized to placebo at the 1- and 4-week visits .", "headache , abdominal or pelvic cramps / abdominal \n pain , and low back pain were frequently reported at baseline as well as at \n follow - up visits .", "there were no \n significant differences in reported symptoms at any visit between women in the \n two treatment arms .", "external genital erythema was found at the 1- \n and 4-week visits in more women randomized to l. crispatus ctv-05 than in women randomized to placebo ( 4 versus 1 , p=.33 ) .", "moderate - to - profuse vaginal \n discharge was frequently found in women from both treatment arms during \n follow - up examinations \n ( 12 versus 8 , p = 1.00 ) , though this finding was also often apparent at the \n baseline visit ( 6 women in each arm ) . \n", "seven \n ( 47% ) women randomized to l. crispatus ctv-05 had nine episodes of pyuria with a urine white blood cell count 8 per mm ( range 8203 ) during a follow - up clinic visit ( table 3 ) .", "in contrast , none \n of the women randomized to placebo had pyuria at 1 or 4 weeks ( p = .01 ) .", "pyuria was positively correlated with urine \n leukocyte - esterase test results ( spearman r = 0.57 , p<.01 ) .", "there \n were no significant associations with vaginal or urinary symptoms or exam \n findings .", "microbes isolated from the \n urine of women in the l. crispatus ctv-05 arm with pyuria included \n mixed gram - positive organisms ( 6 ) , lactobacillus ( 3 ) , e. coli ( 1 ) , enterococcus ( 1 ) , group b streptococcus ( 1 ) , and yeast \n ( 1 ) .", "one subject had a negative urine \n culture , and none had symptomatic infection . \n", "lactobacillus and mixed gram - positive rods were isolated at 10 - 10 cfu / ml from more women with pyuria ( 50% and 75% ) than without ( 19% and 46% , \n resp . ) at the 1-week visit ( p=.22 for lactobacillus , p=.60 for \n mixed gram - positive rods ) but not at the 4-week visit .", "none of the other isolated organisms was found \n at a significantly increased frequency in women randomized to l. crispatus ctv-05 versus placebo or \n in women with pyuria compared to those without pyuria at any visit .", "two women randomized to l. crispatus ctv-05 and one randomized to placebo had hematuria at \n the 4-week visit ( 30 red blood cells per mm ) .", "nine women in the l. crispatus ctv-05 arm and ten in the \n placebo arm completed the six - month follow - up telephone call .", "four women in the l. crispatus ctv-05 arm and one in the \n placebo arm reported one or more episodes of cystitis .", "28 of 30 women had vaginal \n colonization with h202-producing \n lactobacilli at baseline and all follow - up visits ( table 4 ) .", "two women \n randomized to placebo had no vaginal h202-producing \n lactobacilli detected at any clinic visit", ". \n one of these women had large quantities of gardnerella and bacteroides species morphotypes on gram stain .", "cultures \n from women with h202-producing \n lactobacilli yielded heavy ( 4 + ) growth .", "vaginal \n cultures from five women in each treatment arm yielded e. coli from one or more clinic visits .", "e. coli was isolated from vaginal cultures taken at the 1- and 4-week visits from 4 \n women randomized to l. crispatus ctv-05 and from one women randomized to placebo ( p=.33 ) .", "no inverse association between vaginal \n colonization with h202-producing \n lactobacilli and vaginal colonization with e. \n coli was detected .", "vaginal cultures \n frequently yielded candida at baseline \n as well as follow - up visits . using rep - pcr ,", "l. crispatus ctv-05 was detected in the vaginas of four women \n randomized to l. crispatus ctv-05 \n ( three women at each clinic visit ) .", "three \n women had a positive assay for l. \n crispatus ctv-05 detected at the baseline visit before administration of the \n study drug .", "no women randomized to \n placebo had a positive assay at any visit ( p = .22 each visit ) .", "we \n compared our rep - pcr results ( figure 3 ) \n to previously published rep - pcr patterns in order to identify the \n bacterial species . at baseline", ", 80% of \n subjects had l. crispatus , 37% had l. jensenii , 17% had l. gasseri , and 27% had other lactobacillus species detected in \n vaginal culture .", "the following \n combinations of lactobacillus species \n were found : l. crispatus ( 27% ) , l. crispatus and l. jensenii ( 20% ) , l. \n crispatus and l. gasseri ( 7% ) , l. crispatus and other lactobacillus species ( 13% ) , l. crispatus , l. jensenii , and l. gasseri ( 3% ) , l. crispatus , l. jensenii , and other lactobacillus species ( 7% ) , l. crispatus , l. gasseri , and other lactobacillus species ( 3% ) , l. jensenii ( 7% ) , l. gasseri ( 3% ) , other lactobacillus species ( 3% ) , and no lactobacillus ( 7% ) .", "the prevalences of individual species were \n relatively stable over time ( figure 4 ) .", "there were no significant differences in lactobacillus species prevalence between \n subjects in the two treatment arms with the exception of l. gasseri at the 1-week visit ( p = .02 ) .", "in phase i , placebo - controlled trial , use of a l. crispatus ctv-05 vaginal suppository , \n for the prevention of recurrent uti was well tolerated with minimal side effects .", "there were no serious adverse events , and \n despite the occurrence of abnormal vaginal discharge and external genital or \n vaginal irritation in several women , compliance was high .", "these mild to moderate symptoms appear to be \n secondary to the act of suppository use or a reaction to the preservation \n matrix rather than a consequence of l. \n crispatus ctv-05 as they occurred with similar frequency among women in \n each treatment arm .", "however , \n given the frequency of recurrent vaginal candidiasis experienced by subjects \n prior to enrollment and the prevalence of candida in baseline vaginal cultures , the relationship of this condition to study drug \n use is not compelling . ", "l. crispatus ctv-05 use was associated \n with pyuria ( detected by microscopy and urine leukocyte - esterase ) in seven women \n at either the 1- or 4-week visits ( p = .04 at 4 weeks ) .", "pyuria was not associated with urogenital \n symptoms , exam findings , or symptomatic uti . \n while lactobacillus was not isolated from the urine during every episode \n of pyuria , it is possible that some lactobacilli were misclassified as mixed gram - positive \n rods , as both were more frequently found in women with pyuria than in women \n without .", "our data suggest that vaginal \n instillation of l. crispatus ctv-05 induces \n a mild inflammatory response in the bladder or vaginal mucosa of some subjects \n without causing prolonged urogenital infection . \n", "the importance of this phenomenon with respect to safety or potential efficacy \n is unclear .", "it is possible that induction of an asymptomatic inflammatory \n response by lactobacilli protects against uropathogen colonization of the \n vagina or infection in the bladder .", "limited \n existing data from prior studies suggest that l. crispatus therapy is safe . \n", "women surveyed after participation in a clinical trial of a vaginal \n capsule formulation of l. crispatus ctv-05 \n for treatment of bacterial vaginosis rarely reported adverse effects , and those \n reported were largely related to a perceived difference in vaginal \n discharge . satisfaction with the vaginal \n capsule was high .", "no adverse effects were reported in a pilot study of l. crispatus ctv-05 given \n intravaginally to a small group of healthy women .", "nine women with a history of recurrent uti \n reported no adverse effects when given l. \n crispatus strain ga198332 as a vaginal suppository every other day for one \n year .", "studies of other probiotic \n strains of lactobacillus have \n indicated that they can be given to women safely with minimal or no side \n effects [ 1218 ] . to our knowledge , pyuria \n resulting from vaginal instillation of lactobacillus has not been previously reported , and should be evaluated further in future \n studies .", "we \n did not see an effect of l. crispatus ctv-05 use on vaginal lactobacillus growth . however , most study participants had heavy \n vaginal growth of h2o2-producing \n lactobacilli at baseline , making detection of subsequent changes difficult .", "data from a pilot study indicated that l. crispatus ctv-05 did not displace other \n endogenous vaginal lactobacilli .", "vaginal \n colonization was most successful in women lacking vaginal h2o2-producing \n lactobacilli at the outset , suggesting that a lactobacillus probiotic may be \n most effective at establishing vaginal colonization in women with abnormal \n flora , such as those with bacterial vaginosis or recurrent uti .", "to our knowledge , our study represents the first attempt to use rep - pcr to identify the genetic fingerprint of the probiotic strain l. crispatus ctv-05 in a larger - scale clinical study .", "although none of the placebo recipients were \n colonized with lactobacillus strains having the characteristic rep - pcr pattern of l. crispatus ctv-05 at any time , three \n women randomized to l. crispatus ctv-05 had at least one isolate in their vaginal culture at baseline that bore \n the characteristic fingerprint .", ". as there are no \n large - scale surveys of the prevalence and distribution of lactobacilli bearing this fingerprint , we do not know whether the rep - pcr technique failed to \n distinguish l. crispatus ctv-05 from \n other genetically closely related strains of l. crispatus naturally colonizing the vagina at baseline , or \n whether the identical strain was prevalent in our study population . of note ,", "subjects in our study had a much \n higher prevalence of vaginal h2o2-producing \n lactobacilli in baseline vaginal cultures than found in previous studies . by comparing our rep - pcr results", "to previous \n published rep - pcr patterns , we characterized lactobacillus isolates at the \n species level and found l. crispatus , l. jensenii , and l. gasseri to be the most prevalent species , with relatively stable \n prevalences over time .", "our prevalence \n data are consistent with what has been previously documented [ 20 , 21 ] .", "additional tests are under way to compare \n quantitative pcr results for l. crispatus in women before and after the introduction of active suppositories or placebo .", "our study was not \n designed nor statistically powered to evaluate the effect of l. crispatus ctv-05 on the rate of uti \n recurrence .", "therefore , it is difficult \n to interpret the report of cystitis in two women randomized to l. crispatus ctv-05 .", "other small studies have suggested that use of \n vaginally administered l. crispatus may \n be associated with a lower rate of uti recurrence [ 16 , 29 ] .", "the major strengths \n of our study include the placebo - controlled , randomized study design , excellent \n compliance , and extensive and complete followup .", "use of a study diary provided finer details \n of our subjects experiences that confirmed adverse events and symptoms \n reported from memory at clinic visits .", "weaknesses \n that should be mentioned include the inability to accurately document vaginal \n colonization with the probiotic strain , and the inherent lack of statistical \n power due to the small sample size typical of phase i studies .", "we are currently conducting a phase ii trial \n that will have more power to clarify some of the issues raised in this \n study . in \n conclusion ,", "l. crispatus ctv-05 is \n well tolerated when given as a vaginal suppository to healthy women with a \n history of recurrent uti .", "mild to \n moderate side effects related to suppository use occur but do not affect \n compliance .", "timing of administration and efficacy in \n preventing recurrent cystitis should be evaluated further in larger studies ." ]

[ "<S> objectives : we performed a phase i trial to assess the safety and tolerance of a lactobacillus vaginal suppository for prevention of recurrent uti . \n methods : premenopausal women with a history of recurrent uti were randomized to use l. crispatus ctv-05 or placebo vaginal suppositories daily for five days . \n results : 30 women were randomized ( 15 to l. crispatus ctv-05 ) . </S>", "<S> no severe adverse events occurred . </S>", "<S> mild to moderate vaginal discharge and genital irritation were reported by women in both study arms . </S>", "<S> seven women randomized to l. crispatus ctv-05 developed pyuria without associated symptoms . </S>", "<S> most women had high concentrations of vaginal \n h202-producing lactobacilli before randomization </S>", "<S> . l. crispatus , \n l. jensenii , and l. gasseri were the most common \n lactobacillus species identified , with stable prevalence over time . \n </S>", "<S> conclusions : l. crispatus ctv-05 can be given as a vaginal suppository with minimal sideeffects to healthy women with a history of recurrent uti . </S>", "<S> mild inflammation of the urinary tract was noted in some women . </S>" ]

[ "1. INTRODUCTION", "2. SUBJECTS AND METHODS", "3. RESULTS", "4. DISCUSSION" ]

[ [ "urinary tract infections ( utis ) \n affect millions of women each year , with an annual societal cost of billions of \n dollars .", "importantly , more than one quarter of women with a uti will have a \n recurrent infection within six months . \n", "there are few established options for prevention of uti other than the \n use of prophylactic antibiotics . \n", "however , resistance to commonly used antibiotics is increasing among \n bacterial cystitis isolates . \n", "a growing body of evidence suggests that vaginal h202-producing \n lactobacilli may have a protective effect against urogenital infections , \n including uti [ 511 ] .", "it is hypothesized \n that lactobacilli prevent uropathogen colonization of the vagina , a necessary \n step in ascending infection of the bladder . \n", "several clinical trials have demonstrated that certain lactobacillus species , mainly l. rhamnosus and l. fermentum , can be given orally or vaginally with resulting \n colonization of the vagina , reduction in vaginal coliform counts , and even \n reduction in uti recurrence \n [ 1218 ] .", "specific \n strains were chosen for their abilities to produce h202 , adhere to uroepithelial cells , interfere with uropathogen attachment and growth , and persist in the vagina [ 18 , 19 ] . \n", "the most frequently existing vaginal lactobacillus species , l. \n crispatus [ 20 , 21 ] , also produces h202 and possesses the adherence and inhibitory qualities essential for a good probiotic \n candidate [ 22 , 23 ] .", "l. crispatus strain ctv-05 has been \n developed as a vaginal suppository ( lactin - v ) for the prevention of recurrent \n uti in women .", "l. crispatus ctv-05 is \n highly adherent to vaginal epithelial cells , and when given as a suppository \n can colonize the vagina . \n furthermore , l. crispatus ctv-05 has been reported to have a specific dna fingerprint that distinguishes \n it from endogenous vaginal lactobacilli . \n to further evaluate the safety of this new formulation of l. crispatus ctv-05 and its effect on \n the vaginal microbial flora", ", we conducted a phase i , randomized , double - blind , \n placebo - controlled trial of vaginal l. \n crispatus ctv-05 in premenopausal women with a history of recurrent uti ." ], [ "we recruited premenopausal women aged 1835 \n years with a history of three or more uncomplicated utis diagnosed in the past \n year , or two uncomplicated \n utis diagnosed in the past six months , from ( student health center , university \n of washington , wash , usa . ) additional eligibility requirements \n included regular menstrual cycles or amenorrhea for at least six months \n secondary to use of a hormonal contraceptive , a normal pap smear documented in \n the last year or at the baseline clinic visit , abstinence \n from sexual activity or participation in \n a mutually monogamous sexual relationship , use of birth control , agreement not \n to use other intravaginal products , agreement not to have sexual intercourse or \n use tampons between the baseline and first follow - up visit , and capability to \n understand english and provide informed consent .", "exclusion criteria included history of \n urologic abnormality , recent urologic surgery or urinary catheterization , \n history of complicated pyelonephritis or renal calculi , hysterectomy , recent sexually \n transmitted infection ( sti ) or bacterial vaginosis , risk factors for sti and \n hiv , history of recurrent genital herpes , menses anticipated within ten days , pregnancy , \n lactation , recent antibiotic or antifungal use , diabetes or other immunocompromised state , drug \n or alcohol abuse , use of the ( nuvaring ) , prior use of the study drug or allergy \n to any of its components , and abnormal initial pelvic examination .", "the study was approved by ( institutional \n review board ( irb ) , university of washington , wash , \n usa . ) subjects were randomized in a double - blinded \n fashion to l. crispatus ctv-05 at a \n dose of 5 10 colony forming units \n ( cfu ) or placebo vaginal suppository to be inserted daily for five days .", "l. crispatus ctv-05 and placebo \n suppositories were similar in appearance and consisted of a preservation matrix \n and maltodextran .", ", palo alto , \n calif , usa ) according to a randomization \n schedule and supplied to the study site sequentially labeled with a subject \n number .", "subjects \n were seen at three clinic visits over a period of one month . at the first ( baseline )", "visit , eligible subjects \n provided informed consent before undergoing a structured medical and \n gynecologic history and physical examination . \n", "subjects inserted the first dose of study \n drug in the clinic and were instructed to record any symptoms occurring during \n the first week of study on a prepared diary card .", "subjects were seen in follow - up 68 days ( 1-week ) \n visit and 2634 days ( 4-week ) visit after enrollment . during these visits , new symptoms , new \n urogenital infections , and any changes in clinical history were recorded .", "finally , subjects were \n contacted by telephone six months after enrollment to assess symptoms , \n pregnancy , new diagnoses , or major medical events .", "laboratory \n testing included urine dipstick testing , urinalysis , urine culture , vaginal \n fluid wet preparation slides , gram stains , and culture for facultative isolates \n and lactobacillus species at all \n visits .", "repetitive element \n sequence - based polymerase chain reaction ( rep - pcr ) for l. crispatus ctv-05 was performed on three lactobacillus colonies \n from culture at each visit .", "testing for neisseria gonorrhoeae and chlamydia \n trachomatis by dna amplification was done at the baseline visit .", "a \n sample size of 30 was chosen to evaluate the primary outcome of safety as \n assessed through self - reported symptoms , physical exam findings , and laboratory \n studies .", "secondary outcomes included \n shifts in the vaginal flora assessed by vaginal culture and vaginal \n colonization with l. crispatus ctv-05 \n assessed by rep - pcr ." ], [ "two women randomized to l. \n crispatus ctv-05 completed treatment over six days , and one woman \n randomized to placebo used six suppositories over six days . all women remained on study through the \n 4-week visit ( figure 1 ) .", "baseline \n characteristics of subjects were similar in the two treatment groups ( table 1 ) .", "the majority of women were young , white , \n healthy , university students in their 20s . \n", "mild to moderate adverse events were \n relatively common , however . of those \n felt by the investigators to be related to study drug use ,", "abnormal vaginal \n discharge was the most frequently occurring , followed by external genital \n irritation and vaginal candidiasis ( table 2 ) .", "these adverse events occurred with \n similar or greater frequency in women randomized to placebo as compared to women \n randomized to l. crispatus ctv-05 .", "three women reporting vaginal candidiasis also \n reported multiple prior episodes in the preceding 12 months .", "two women randomized to l. crispatus ctv-05 reported episodes of cystitis during the \n study .", "all subjects completed a diary card of symptoms that occurred during the period of \n study drug insertion .", "recorded symptoms generally \n confirmed the reported adverse events and were similar to symptoms reported at the \n 1-week clinic visit ( below ) .", "abnormal \n vaginal discharge was the symptom most frequently reported by subjects at \n clinic visits following study drug use ( figure 2 ) .", "while no women reported abnormal \n vaginal discharge at baseline , six randomized to l. crispatus ctv-05 and five randomized to placebo reported abnormal \n vaginal discharge at the 1-week visit .", "external genital or vaginal irritation was \n also reported by six women in each treatment arm at follow - up visits .", "vaginal odor was reported at the 1-week visit \n by one woman randomized to l. crispatus ctv-05 , \n and at the 4-week visit by two women randomized to placebo .", "dysuria was reported by one woman randomized \n to l. crispatus ctv-05 at the 1-week visit \n and by one woman randomized to placebo at the 1- and 4-week visits .", "headache , abdominal or pelvic cramps / abdominal \n pain , and low back pain were frequently reported at baseline as well as at \n follow - up visits .", "there were no \n significant differences in reported symptoms at any visit between women in the \n two treatment arms .", "external genital erythema was found at the 1- \n and 4-week visits in more women randomized to l. crispatus ctv-05 than in women randomized to placebo ( 4 versus 1 , p=.33 ) .", "moderate - to - profuse vaginal \n discharge was frequently found in women from both treatment arms during \n follow - up examinations \n ( 12 versus 8 , p = 1.00 ) , though this finding was also often apparent at the \n baseline visit ( 6 women in each arm ) . \n", "seven \n ( 47% ) women randomized to l. crispatus ctv-05 had nine episodes of pyuria with a urine white blood cell count 8 per mm ( range 8203 ) during a follow - up clinic visit ( table 3 ) .", "in contrast , none \n of the women randomized to placebo had pyuria at 1 or 4 weeks ( p = .01 ) .", "pyuria was positively correlated with urine \n leukocyte - esterase test results ( spearman r = 0.57 , p<.01 ) .", "there \n were no significant associations with vaginal or urinary symptoms or exam \n findings .", "microbes isolated from the \n urine of women in the l. crispatus ctv-05 arm with pyuria included \n mixed gram - positive organisms ( 6 ) , lactobacillus ( 3 ) , e. coli ( 1 ) , enterococcus ( 1 ) , group b streptococcus ( 1 ) , and yeast \n ( 1 ) .", "one subject had a negative urine \n culture , and none had symptomatic infection . \n", "lactobacillus and mixed gram - positive rods were isolated at 10 - 10 cfu / ml from more women with pyuria ( 50% and 75% ) than without ( 19% and 46% , \n resp . ) at the 1-week visit ( p=.22 for lactobacillus , p=.60 for \n mixed gram - positive rods ) but not at the 4-week visit .", "none of the other isolated organisms was found \n at a significantly increased frequency in women randomized to l. crispatus ctv-05 versus placebo or \n in women with pyuria compared to those without pyuria at any visit .", "two women randomized to l. crispatus ctv-05 and one randomized to placebo had hematuria at \n the 4-week visit ( 30 red blood cells per mm ) .", "nine women in the l. crispatus ctv-05 arm and ten in the \n placebo arm completed the six - month follow - up telephone call .", "four women in the l. crispatus ctv-05 arm and one in the \n placebo arm reported one or more episodes of cystitis .", "28 of 30 women had vaginal \n colonization with h202-producing \n lactobacilli at baseline and all follow - up visits ( table 4 ) .", "two women \n randomized to placebo had no vaginal h202-producing \n lactobacilli detected at any clinic visit", ". \n one of these women had large quantities of gardnerella and bacteroides species morphotypes on gram stain .", "cultures \n from women with h202-producing \n lactobacilli yielded heavy ( 4 + ) growth .", "vaginal \n cultures from five women in each treatment arm yielded e. coli from one or more clinic visits .", "e. coli was isolated from vaginal cultures taken at the 1- and 4-week visits from 4 \n women randomized to l. crispatus ctv-05 and from one women randomized to placebo ( p=.33 ) .", "no inverse association between vaginal \n colonization with h202-producing \n lactobacilli and vaginal colonization with e. \n coli was detected .", "vaginal cultures \n frequently yielded candida at baseline \n as well as follow - up visits . using rep - pcr ,", "l. crispatus ctv-05 was detected in the vaginas of four women \n randomized to l. crispatus ctv-05 \n ( three women at each clinic visit ) .", "three \n women had a positive assay for l. \n crispatus ctv-05 detected at the baseline visit before administration of the \n study drug .", "no women randomized to \n placebo had a positive assay at any visit ( p = .22 each visit ) .", "we \n compared our rep - pcr results ( figure 3 ) \n to previously published rep - pcr patterns in order to identify the \n bacterial species . at baseline", ", 80% of \n subjects had l. crispatus , 37% had l. jensenii , 17% had l. gasseri , and 27% had other lactobacillus species detected in \n vaginal culture .", "the following \n combinations of lactobacillus species \n were found : l. crispatus ( 27% ) , l. crispatus and l. jensenii ( 20% ) , l. \n crispatus and l. gasseri ( 7% ) , l. crispatus and other lactobacillus species ( 13% ) , l. crispatus , l. jensenii , and l. gasseri ( 3% ) , l. crispatus , l. jensenii , and other lactobacillus species ( 7% ) , l. crispatus , l. gasseri , and other lactobacillus species ( 3% ) , l. jensenii ( 7% ) , l. gasseri ( 3% ) , other lactobacillus species ( 3% ) , and no lactobacillus ( 7% ) .", "the prevalences of individual species were \n relatively stable over time ( figure 4 ) .", "there were no significant differences in lactobacillus species prevalence between \n subjects in the two treatment arms with the exception of l. gasseri at the 1-week visit ( p = .02 ) ." ], [ "in phase i , placebo - controlled trial , use of a l. crispatus ctv-05 vaginal suppository , \n for the prevention of recurrent uti was well tolerated with minimal side effects .", "there were no serious adverse events , and \n despite the occurrence of abnormal vaginal discharge and external genital or \n vaginal irritation in several women , compliance was high .", "these mild to moderate symptoms appear to be \n secondary to the act of suppository use or a reaction to the preservation \n matrix rather than a consequence of l. \n crispatus ctv-05 as they occurred with similar frequency among women in \n each treatment arm .", "however , \n given the frequency of recurrent vaginal candidiasis experienced by subjects \n prior to enrollment and the prevalence of candida in baseline vaginal cultures , the relationship of this condition to study drug \n use is not compelling . ", "l. crispatus ctv-05 use was associated \n with pyuria ( detected by microscopy and urine leukocyte - esterase ) in seven women \n at either the 1- or 4-week visits ( p = .04 at 4 weeks ) .", "pyuria was not associated with urogenital \n symptoms , exam findings , or symptomatic uti . \n while lactobacillus was not isolated from the urine during every episode \n of pyuria , it is possible that some lactobacilli were misclassified as mixed gram - positive \n rods , as both were more frequently found in women with pyuria than in women \n without .", "our data suggest that vaginal \n instillation of l. crispatus ctv-05 induces \n a mild inflammatory response in the bladder or vaginal mucosa of some subjects \n without causing prolonged urogenital infection . \n", "the importance of this phenomenon with respect to safety or potential efficacy \n is unclear .", "it is possible that induction of an asymptomatic inflammatory \n response by lactobacilli protects against uropathogen colonization of the \n vagina or infection in the bladder .", "limited \n existing data from prior studies suggest that l. crispatus therapy is safe . \n", "women surveyed after participation in a clinical trial of a vaginal \n capsule formulation of l. crispatus ctv-05 \n for treatment of bacterial vaginosis rarely reported adverse effects , and those \n reported were largely related to a perceived difference in vaginal \n discharge . satisfaction with the vaginal \n capsule was high .", "no adverse effects were reported in a pilot study of l. crispatus ctv-05 given \n intravaginally to a small group of healthy women .", "nine women with a history of recurrent uti \n reported no adverse effects when given l. \n crispatus strain ga198332 as a vaginal suppository every other day for one \n year .", "studies of other probiotic \n strains of lactobacillus have \n indicated that they can be given to women safely with minimal or no side \n effects [ 1218 ] . to our knowledge , pyuria \n resulting from vaginal instillation of lactobacillus has not been previously reported , and should be evaluated further in future \n studies .", "we \n did not see an effect of l. crispatus ctv-05 use on vaginal lactobacillus growth . however , most study participants had heavy \n vaginal growth of h2o2-producing \n lactobacilli at baseline , making detection of subsequent changes difficult .", "data from a pilot study indicated that l. crispatus ctv-05 did not displace other \n endogenous vaginal lactobacilli .", "vaginal \n colonization was most successful in women lacking vaginal h2o2-producing \n lactobacilli at the outset , suggesting that a lactobacillus probiotic may be \n most effective at establishing vaginal colonization in women with abnormal \n flora , such as those with bacterial vaginosis or recurrent uti .", "to our knowledge , our study represents the first attempt to use rep - pcr to identify the genetic fingerprint of the probiotic strain l. crispatus ctv-05 in a larger - scale clinical study .", "although none of the placebo recipients were \n colonized with lactobacillus strains having the characteristic rep - pcr pattern of l. crispatus ctv-05 at any time , three \n women randomized to l. crispatus ctv-05 had at least one isolate in their vaginal culture at baseline that bore \n the characteristic fingerprint .", ". as there are no \n large - scale surveys of the prevalence and distribution of lactobacilli bearing this fingerprint , we do not know whether the rep - pcr technique failed to \n distinguish l. crispatus ctv-05 from \n other genetically closely related strains of l. crispatus naturally colonizing the vagina at baseline , or \n whether the identical strain was prevalent in our study population . of note ,", "subjects in our study had a much \n higher prevalence of vaginal h2o2-producing \n lactobacilli in baseline vaginal cultures than found in previous studies . by comparing our rep - pcr results", "to previous \n published rep - pcr patterns , we characterized lactobacillus isolates at the \n species level and found l. crispatus , l. jensenii , and l. gasseri to be the most prevalent species , with relatively stable \n prevalences over time .", "our prevalence \n data are consistent with what has been previously documented [ 20 , 21 ] .", "additional tests are under way to compare \n quantitative pcr results for l. crispatus in women before and after the introduction of active suppositories or placebo .", "our study was not \n designed nor statistically powered to evaluate the effect of l. crispatus ctv-05 on the rate of uti \n recurrence .", "therefore , it is difficult \n to interpret the report of cystitis in two women randomized to l. crispatus ctv-05 .", "other small studies have suggested that use of \n vaginally administered l. crispatus may \n be associated with a lower rate of uti recurrence [ 16 , 29 ] .", "the major strengths \n of our study include the placebo - controlled , randomized study design , excellent \n compliance , and extensive and complete followup .", "use of a study diary provided finer details \n of our subjects experiences that confirmed adverse events and symptoms \n reported from memory at clinic visits .", "weaknesses \n that should be mentioned include the inability to accurately document vaginal \n colonization with the probiotic strain , and the inherent lack of statistical \n power due to the small sample size typical of phase i studies .", "we are currently conducting a phase ii trial \n that will have more power to clarify some of the issues raised in this \n study . in \n conclusion ,", "l. crispatus ctv-05 is \n well tolerated when given as a vaginal suppository to healthy women with a \n history of recurrent uti .", "mild to \n moderate side effects related to suppository use occur but do not affect \n compliance .", "timing of administration and efficacy in \n preventing recurrent cystitis should be evaluated further in larger studies ." ] ]

[ "retinoblastoma is the most frequent childhood intraocular tumour with an approximately incidence of one in 15,00020,000 births in the world . in africa", "management of retinoblastoma has been changing during years with a survival rate of more than 95% in developed countries [ 5 , 6 ] .", "however , in developing countries these cures are less than 50% , primarily because of advanced disease at time of diagnosis [ 79 ] .", "previous studies had reported epidemiological and clinical characteristics of the disease [ 1012 ] . in the democratic republic of congo ( drc )", "in other previous study , retinoblastoma was the most common histologic form representing 31.7% of all malignant of the eye . however , there is still a paucity of information on clinical findings and on the outcomes of children with retinoblastoma .", "the last paper on presenting signs of retinoblastoma in congolese patients gave a five - year ophthalmology view .", "at the other side , it is necessary for health plan to have the main characteristics of all children with retinoblastoma followed in democratic republic of congo .", "this information will give the whole view of patients and may serve to rule out general public health and in - hospital cancer politics in poor resources settings . ", "the aim of this study is to present data over two decades about clinical presentation and outcome of children with retinoblastoma treated at the university hospital of kinshasa , which is a referral and tertiary care center in west of democratic republic of congo and to compare the results with those reported in the literature .", "the medical records of all patients with a diagnosis of retinoblastoma seen in the pediatrics oncology unit at the university teaching hospital of kinshasa , between january 1985 and december 2005 were reviewed retrospectively .", "we used hospital - based data from the records because there is no population - based cancer registry in democratic republic of congo .", "the diagnosis of retinoblastoma was based upon history , clinical examination at university hospital of kinshasa . clinical examination with complete ocular examination including dilated ophthalmoscopy under general anaesthesia and physical examination by a paediatrician", "ancillary studies consisted of ocular ultrasonography and computed tomography when affordable , which showed the presence of solid intraocular tumor with calcium deposits . the data collected included demographics features , reference , order among siblings , the initial sign noticed by the family , age at first symptom , clinical findings , age at diagnosis , laterality , different time lag , treatment modalities , treatment adherence , and outcome .", "age at first sign means the age of the child when families noticed for the first time the abnormality ( alarming sign ) .", "the clinical presentation report to what the physician had seen when examining the patient at our institution .", "different lag times include four latency periods : ( 1 ) the time from the first sign to a referral service , ( 2 ) the time from the reference to the pediatrics oncology unit , ( 3 ) the time from pediatrics consultation to the initiation of treatment , and ( 4 ) the observational time from treatment ( followup ) .", "we divided the study period in two parts considering the year 1998 as the limitation .", "chemoreduction regimen consisted in vincristine , adriamycin , cyclophosphamide for a maximum of six cycles given every 3 weeks before and after enucleation ) following the international conference on paediatric oncology in morocco in 1998 ( http://www.smhop.org.ma/ ) .", "statistical analysis was performed using the statistics software spss for windows ( 15.0 spss , chicago ) .", "data are represented as meanssd when the distribution was normal and median with range when the distribution was not normal .", "nine of them were excluded because of insufficient information , and data of 40 patients , including 28 males ( 70% ) and 12 females ( 30% ) were reviewed .", "our patients were referred from the ophthalmology unit of the hospital ( 33 cases 82.5% ) and other centres ( 7 cases 17.5% ) .", "their median order among siblings was 4 ( range 1 to 10 ) . during the study period , 31 cases ( 77.5% ) had unilateral disease [ 16 cases ( 40% ) left and 15 cases ( 37.5% ) right ] and 9 cases ( 22.5% ) had bilateral disease , resulting in 49 affected eyes . in unilateral retinoblastoma , there were 20 boys and 11 girls with sex ratio 2 : 1 . in bilateral disease , there were 8 boys and 1 girl with sex ratio 8 : 1 .", "the median age at the first sign noticed by families was 1.9 years ( range one month to six years ) for unilateral disease and 1.4 year ( range one month to three years and half ) for bilateral disease while the median age of the diagnosis in our institution was 2.8 years ( range six months to six years ) for unilateral disease and 2.4 years ( range nine months to four years ) for bilateral disease .", "leukokoria ( n = 27 ) and proptosis ( n = 6 ) were the most frequent initial signs noticed by families ( table 1 ) . at diagnosis in our institution ( table 2 ) , the most frequent clinical findings were proptosis ( n = 22 ) and leukokoria ( n = 10 ) .", "a median 9 months ( range , 1 to 79 months ) passed from detection of the first sign to referral ( lag time ) , 1 month ( range , 0 to 23 months ) from referral to paediatric oncology unit , 9.5 months ( range , 0 months to 3.3 years ) from paediatric consultation to treatment , and 2.3 months ( range , 0 to 18 moths ) from treatment initiation to last followup .", "an average of 15 months passed between the detection of the first sign and the beginning of therapy if applied . at diagnosis , 36 ( 90% ) patients had stage 4 of the reese - ellsworth classification .", "four patients had localized disease , twenty six had locoregional disease , and ten had metastatic disease .", "the sites of metastatic disease were brain ( n = 4 ) , lymph nodes ( n = 3 ) , bone ( n = 2 ) , and bone marrow ( n = 1 ) . from 1985 to 1998 , 23 patients ( 57.5% ) have been treated and 17 ( 42.5% ) from 1998 to the 2005 .", "the table 3 shows chemotherapy protocols to be used and those effectively applied to patients .", "the outcomes of our patients were as follows : 30 ( 75% ) children were lost from followup , 7 ( 17.5% ) died among which 5 had proptosis with an orbital extension , 2 had protruded mass development after enucleation ( characteristics presented in table 4 ) , one was referred abroad for treatment , and two were alive at the time of final data entry . assuming that cases lost to followup worsened and died ,", "our study is the first to look at the outcome and the limitations to management of retinoblastoma in democratic republic of congo .", "the study spanned 20 years and 49 cases of retinoblastoma were identified during that time . from mid 1998 to 2005 , 17 patients were identified .", "the calculation based on incidence of retinoblastoma in africa , data from index mundi available for 2005 ( http://www.indexmundi.com/g/g.aspx?c=cg&v=25/ ) , and estimate population in 1999 , we can give an estimate o incidence of retinoblastoma in democratic republic of congo approximately of 9761098 cases and the the total number of children in this study indicates that only a tiny fraction of congolese children with retinoblastoma benefited from treatment in our tertiary referral center .", "it is common for some children with retinoblastoma to be taken to herbalist or prayer houses instead of hospital .", "this situation is due to cultural beliefs , stigma , and ignorance [ 17 , 18 ] .", "many authors reported similar observations [ 14 , 16 , 19 , 20 ] , but in others the occurrence of retinoblastoma is almost the same in both sexes [ 11 , 21 , 22 ] .", "the fact of sex ratio in the present study had been reported to be related to environmental factors or to bias referral . ", "the main referral to paediatric oncology was the ophthalmology unit of our institution with 82.5% of patients . in a previous study from the ophthalmology unit of the university hospital of kinshasa ,", "this may be explained by some obstacles outside university hospital of kinshasa as lack of paediatrician and ophthalmologist . in - hospital", "reference from ophthalmologist to paediatrician could help in the early management because it was reported in argentina that the paediatrician underestimated the complaints in half patients while the diagnosis was suspected by the ophthalmologist on the first visit .", "this frequency varies from 10 to 50% in different studies [ 1 , 19 , 2426 ] . it might be that some bilateral cases are lost to followup before second eye involvement appears , leading to misclassification as unilateral in our context . ", "the age at diagnosis of retinoblastoma in developing countries varies from 24 months to 3 years [ 16 , 24 , 27 , 28 ] . in this study ,", "the age of our patients ranged from 6 months to 6.6 years and averaged 32 months .", "the mean age when first sign had noticed by parents was 20 , 4 months , meaning a year average lost of time before reaching a hospital due to lake of information , education , or poor socioeconomic level .", "different studies reported similar observations in developing countries [ 12 , 20 , 24 , 28 ] .", "leukokoria ( 67.5% ) had been the first sign noticed by parents . in medical consultation , proptosis and", "leukokoria were the most common clinical presentation , similar to that from other developing countries [ 4 , 20 , 23 , 26 , 28 , 29 ] .", "computer tomography is generally avoided in bilateral retinoblastoma because radiation may increase risk of second cancers .", "similar observations were reported in other studies in developing countries [ 20 , 23 , 26 , 30 ] .", "the predominance of proptosis is due to the delay in consultation and the development of the tumour because there is a link between the lag time and the expansion of the tumour .", "however , in other settings leukokoria still remains the presenting sign at the hospital [ 32 , 33 ] .", "another explanation rises as been reported from the use of self - treatments or traditional healers ' treatments before reaching a hospital or during the period between a health centre and the pediatrics oncology unit [ 34 , 35 ] .", "so , retinoblastoma is diagnosed late with extraocular dissemination , and there is tremendous scope for improvement in its managements as performed in developed countries .", "this should lead to set up an informational program for careful screening and referral of white pupil for early diagnosis and treatment as proposed elsewhere [ 36 , 37 ] . ", "the chemotherapy of retinoblastoma in kinshasa evolved from one drug to a 3 drugs combination therapy every 3 weeks during the study period .", "the chemotherapy regimen ( vincristine ; adriamycine cyclophosphamide ) had been systematically applied since 1998 .", "most of the parents of our patients are poor and because a free health care facility is not available in most developing countries , these drugs are not affordable to many of the patients .", "an additional factor is the unavailability and high cost of some drugs in democratic republic of congo .", "this situation contributes to lack of treatment adherence and patients being lost to followup . in our series ,", "this high treatment abandonment may be the result of late presentation as well as poor compliance with chemotherapy regimen .", "treatment abandonment as well as loss to followup has also been reported from other developing countries [ 20 , 24 , 30 ] .", "we think that it derives from complex factors such as ignorance , poverty without health insurance system , financial constraint regarding investigations and procurement of drugs , lack infrastructure resources , poor communication between medical teams and patients , and deep lack of financial and infrastructure resources . in our study ,", "limited management for above reasons and lack of an accurate tracing system are factors predicting a worse prognosis . during these 2 decades", ", retinoblastoma remains a life - threatening disease in democratic republic of congo characterized by advanced intraocular disease , late referral to a specialized unit , delay in treatment , important lost to followup , and affordability of chemotherapy .", "early educational detection program , prompt referral to specialized centers , use of current methods treatment , and an implementation of a health insurance system particularly for cancers will improve outcomes for children with retinoblastoma in our settings ." ]

[ "<S> \n background . to determine clinical profile and management of retinoblastoma among children at kinshasa in democratic republic of congo . </S>", "<S> patients and methods . the medical records of patients with a diagnosis of retinoblastoma seen at the university hospital of kinshasa from january 1985 till december 2005 were retrospectively reviewed . </S>", "<S> demographic profile , clinical data , modes of treatment and outcome were analysed . results . </S>", "<S> a total of 49 children , of whom 40 had adequate data on record were identified as retinoblastoma ( 28 males and 12 females ) . </S>", "<S> nine cases had bilateral disease . </S>", "<S> the median age at the first symptoms was 9 months ( range , 1 month to 6 years ) for unilateral retinoblastoma and 18 months ( range , 1 month to 3.5 years ) for bilateral retinoblastoma . </S>", "<S> the median age at the first oncology consultation was 2.4 years ( range , 6 months to 6 years ) for unilateral retinoblastoma and 2.4years ( range , 9 months to 4 years ) for bilateral disease . </S>", "<S> leukokoria was present in 67.5% of subjects . </S>", "<S> seventy - five percent abandoned the treatment . </S>", "<S> the mortality was 92.5% . </S>", "<S> conclusion . in democratic republic of congo , retinoblastoma remains a life threatening disease characterized by late referral to a specialized unit and affordability of chemotherapy ; all leading to an extension of the disease and high mortality . </S>" ]

[ "1. Introduction", "2. Methods", "3. Results", "4. Discussion" ]

[ [ "retinoblastoma is the most frequent childhood intraocular tumour with an approximately incidence of one in 15,00020,000 births in the world . in africa", "management of retinoblastoma has been changing during years with a survival rate of more than 95% in developed countries [ 5 , 6 ] .", "however , in developing countries these cures are less than 50% , primarily because of advanced disease at time of diagnosis [ 79 ] .", "previous studies had reported epidemiological and clinical characteristics of the disease [ 1012 ] . in the democratic republic of congo ( drc )", "in other previous study , retinoblastoma was the most common histologic form representing 31.7% of all malignant of the eye . however , there is still a paucity of information on clinical findings and on the outcomes of children with retinoblastoma .", "the last paper on presenting signs of retinoblastoma in congolese patients gave a five - year ophthalmology view .", "at the other side , it is necessary for health plan to have the main characteristics of all children with retinoblastoma followed in democratic republic of congo .", "this information will give the whole view of patients and may serve to rule out general public health and in - hospital cancer politics in poor resources settings . ", "the aim of this study is to present data over two decades about clinical presentation and outcome of children with retinoblastoma treated at the university hospital of kinshasa , which is a referral and tertiary care center in west of democratic republic of congo and to compare the results with those reported in the literature ." ], [ "the medical records of all patients with a diagnosis of retinoblastoma seen in the pediatrics oncology unit at the university teaching hospital of kinshasa , between january 1985 and december 2005 were reviewed retrospectively .", "we used hospital - based data from the records because there is no population - based cancer registry in democratic republic of congo .", "the diagnosis of retinoblastoma was based upon history , clinical examination at university hospital of kinshasa . clinical examination with complete ocular examination including dilated ophthalmoscopy under general anaesthesia and physical examination by a paediatrician", "ancillary studies consisted of ocular ultrasonography and computed tomography when affordable , which showed the presence of solid intraocular tumor with calcium deposits . the data collected included demographics features , reference , order among siblings , the initial sign noticed by the family , age at first symptom , clinical findings , age at diagnosis , laterality , different time lag , treatment modalities , treatment adherence , and outcome .", "age at first sign means the age of the child when families noticed for the first time the abnormality ( alarming sign ) .", "the clinical presentation report to what the physician had seen when examining the patient at our institution .", "different lag times include four latency periods : ( 1 ) the time from the first sign to a referral service , ( 2 ) the time from the reference to the pediatrics oncology unit , ( 3 ) the time from pediatrics consultation to the initiation of treatment , and ( 4 ) the observational time from treatment ( followup ) .", "we divided the study period in two parts considering the year 1998 as the limitation .", "chemoreduction regimen consisted in vincristine , adriamycin , cyclophosphamide for a maximum of six cycles given every 3 weeks before and after enucleation ) following the international conference on paediatric oncology in morocco in 1998 ( http://www.smhop.org.ma/ ) .", "statistical analysis was performed using the statistics software spss for windows ( 15.0 spss , chicago ) .", "data are represented as meanssd when the distribution was normal and median with range when the distribution was not normal ." ], [ "nine of them were excluded because of insufficient information , and data of 40 patients , including 28 males ( 70% ) and 12 females ( 30% ) were reviewed .", "our patients were referred from the ophthalmology unit of the hospital ( 33 cases 82.5% ) and other centres ( 7 cases 17.5% ) .", "their median order among siblings was 4 ( range 1 to 10 ) . during the study period , 31 cases ( 77.5% ) had unilateral disease [ 16 cases ( 40% ) left and 15 cases ( 37.5% ) right ] and 9 cases ( 22.5% ) had bilateral disease , resulting in 49 affected eyes . in unilateral retinoblastoma , there were 20 boys and 11 girls with sex ratio 2 : 1 . in bilateral disease , there were 8 boys and 1 girl with sex ratio 8 : 1 .", "the median age at the first sign noticed by families was 1.9 years ( range one month to six years ) for unilateral disease and 1.4 year ( range one month to three years and half ) for bilateral disease while the median age of the diagnosis in our institution was 2.8 years ( range six months to six years ) for unilateral disease and 2.4 years ( range nine months to four years ) for bilateral disease .", "leukokoria ( n = 27 ) and proptosis ( n = 6 ) were the most frequent initial signs noticed by families ( table 1 ) . at diagnosis in our institution ( table 2 ) , the most frequent clinical findings were proptosis ( n = 22 ) and leukokoria ( n = 10 ) .", "a median 9 months ( range , 1 to 79 months ) passed from detection of the first sign to referral ( lag time ) , 1 month ( range , 0 to 23 months ) from referral to paediatric oncology unit , 9.5 months ( range , 0 months to 3.3 years ) from paediatric consultation to treatment , and 2.3 months ( range , 0 to 18 moths ) from treatment initiation to last followup .", "an average of 15 months passed between the detection of the first sign and the beginning of therapy if applied . at diagnosis , 36 ( 90% ) patients had stage 4 of the reese - ellsworth classification .", "four patients had localized disease , twenty six had locoregional disease , and ten had metastatic disease .", "the sites of metastatic disease were brain ( n = 4 ) , lymph nodes ( n = 3 ) , bone ( n = 2 ) , and bone marrow ( n = 1 ) . from 1985 to 1998 , 23 patients ( 57.5% ) have been treated and 17 ( 42.5% ) from 1998 to the 2005 .", "the table 3 shows chemotherapy protocols to be used and those effectively applied to patients .", "the outcomes of our patients were as follows : 30 ( 75% ) children were lost from followup , 7 ( 17.5% ) died among which 5 had proptosis with an orbital extension , 2 had protruded mass development after enucleation ( characteristics presented in table 4 ) , one was referred abroad for treatment , and two were alive at the time of final data entry . assuming that cases lost to followup worsened and died ," ], [ "our study is the first to look at the outcome and the limitations to management of retinoblastoma in democratic republic of congo .", "the study spanned 20 years and 49 cases of retinoblastoma were identified during that time . from mid 1998 to 2005 , 17 patients were identified .", "the calculation based on incidence of retinoblastoma in africa , data from index mundi available for 2005 ( http://www.indexmundi.com/g/g.aspx?c=cg&v=25/ ) , and estimate population in 1999 , we can give an estimate o incidence of retinoblastoma in democratic republic of congo approximately of 9761098 cases and the the total number of children in this study indicates that only a tiny fraction of congolese children with retinoblastoma benefited from treatment in our tertiary referral center .", "it is common for some children with retinoblastoma to be taken to herbalist or prayer houses instead of hospital .", "this situation is due to cultural beliefs , stigma , and ignorance [ 17 , 18 ] .", "many authors reported similar observations [ 14 , 16 , 19 , 20 ] , but in others the occurrence of retinoblastoma is almost the same in both sexes [ 11 , 21 , 22 ] .", "the fact of sex ratio in the present study had been reported to be related to environmental factors or to bias referral . ", "the main referral to paediatric oncology was the ophthalmology unit of our institution with 82.5% of patients . in a previous study from the ophthalmology unit of the university hospital of kinshasa ,", "this may be explained by some obstacles outside university hospital of kinshasa as lack of paediatrician and ophthalmologist . in - hospital", "reference from ophthalmologist to paediatrician could help in the early management because it was reported in argentina that the paediatrician underestimated the complaints in half patients while the diagnosis was suspected by the ophthalmologist on the first visit .", "this frequency varies from 10 to 50% in different studies [ 1 , 19 , 2426 ] . it might be that some bilateral cases are lost to followup before second eye involvement appears , leading to misclassification as unilateral in our context . ", "the age at diagnosis of retinoblastoma in developing countries varies from 24 months to 3 years [ 16 , 24 , 27 , 28 ] . in this study ,", "the age of our patients ranged from 6 months to 6.6 years and averaged 32 months .", "the mean age when first sign had noticed by parents was 20 , 4 months , meaning a year average lost of time before reaching a hospital due to lake of information , education , or poor socioeconomic level .", "different studies reported similar observations in developing countries [ 12 , 20 , 24 , 28 ] .", "leukokoria ( 67.5% ) had been the first sign noticed by parents . in medical consultation , proptosis and", "leukokoria were the most common clinical presentation , similar to that from other developing countries [ 4 , 20 , 23 , 26 , 28 , 29 ] .", "computer tomography is generally avoided in bilateral retinoblastoma because radiation may increase risk of second cancers .", "similar observations were reported in other studies in developing countries [ 20 , 23 , 26 , 30 ] .", "the predominance of proptosis is due to the delay in consultation and the development of the tumour because there is a link between the lag time and the expansion of the tumour .", "however , in other settings leukokoria still remains the presenting sign at the hospital [ 32 , 33 ] .", "another explanation rises as been reported from the use of self - treatments or traditional healers ' treatments before reaching a hospital or during the period between a health centre and the pediatrics oncology unit [ 34 , 35 ] .", "so , retinoblastoma is diagnosed late with extraocular dissemination , and there is tremendous scope for improvement in its managements as performed in developed countries .", "this should lead to set up an informational program for careful screening and referral of white pupil for early diagnosis and treatment as proposed elsewhere [ 36 , 37 ] . ", "the chemotherapy of retinoblastoma in kinshasa evolved from one drug to a 3 drugs combination therapy every 3 weeks during the study period .", "the chemotherapy regimen ( vincristine ; adriamycine cyclophosphamide ) had been systematically applied since 1998 .", "most of the parents of our patients are poor and because a free health care facility is not available in most developing countries , these drugs are not affordable to many of the patients .", "an additional factor is the unavailability and high cost of some drugs in democratic republic of congo .", "this situation contributes to lack of treatment adherence and patients being lost to followup . in our series ,", "this high treatment abandonment may be the result of late presentation as well as poor compliance with chemotherapy regimen .", "treatment abandonment as well as loss to followup has also been reported from other developing countries [ 20 , 24 , 30 ] .", "we think that it derives from complex factors such as ignorance , poverty without health insurance system , financial constraint regarding investigations and procurement of drugs , lack infrastructure resources , poor communication between medical teams and patients , and deep lack of financial and infrastructure resources . in our study ,", "limited management for above reasons and lack of an accurate tracing system are factors predicting a worse prognosis . during these 2 decades", ", retinoblastoma remains a life - threatening disease in democratic republic of congo characterized by advanced intraocular disease , late referral to a specialized unit , delay in treatment , important lost to followup , and affordability of chemotherapy .", "early educational detection program , prompt referral to specialized centers , use of current methods treatment , and an implementation of a health insurance system particularly for cancers will improve outcomes for children with retinoblastoma in our settings ." ] ]

[ "temporary external ventricular drainage for refractory thoracolumbar cerebrospinal fluid ( csf ) leak is not reported in the literature .", "we present a young man who had multiple complex spinal surgeries for resection of innumerable metastatic ependymoma lesions .", "as a last resort , we decided to place an external ventricular catheter for proximal controlled csf drainage with great success .", "the 32-year - old man had a history of spinal myxopapillary ependymoma world health organization ( who ) grade i with local metastatic spinal disease . the patient first presented in 2005 with a 1-year history of severe low back pain . he was found to have an intradural lesion , and subsequently had a t12l3 laminectomy for resection of the tumor .", "small amounts of the tumor adherent to the nerve roots were purposefully left behind . each time an attempt was made to remove these residual small pieces of the tumor , significant amounts of neuromonitoring activity was noted .", "hence , the residual tumor was left behind so as to not cause a neurologic deficit .", "approximately 10 days after surgery , the patient fell at home and noticed significant amounts of a clear fluid consistent with csf leaking from his incision .", "he was taken to the operating room at that time and a dural defect was identified and closed primarily , then covered with gelfoam ( baxter healthcare , deerfield , il , united states ) and tisseel ( baxter healthcare ) . the patient again noted another csf leak from a different area of his incision 5 days later , and he was taken back to the operating room for another primary closure , as well as placement of a fat graft and more tisseel .", "adjuvant radiation treatment was undertaken after he recovered from the initial surgeries . in 2008 ,", "he was found to have a dorsally located intradural lesion at the t8t9 region , which was resected via a t7t10 laminectomy . in 2010 ,", "he was found to have growing intradural lesions at l4 and l5 . at that time", "one year later , our patient presented yet again with multiple thoracolumbar intradural lesions ( fig .", "he underwent redo decompressive laminectomies from t8s1 with a total of 13 intradural tumors being excised ( fig .", "2 ) . the dural closure proved to be quite complex , as there were several areas that were macerated and very difficult to reapproximate .", "fat grafts , duraform ( depuy synthes , west chester , pa , united states ) , and tisseel were all used on top of the attempted primary dural closure . \n", "preoperative sagittal t2-weighted magnetic resonance imaging ( mri ) ( a ) of the thoracic spine revealing multiple intradural tumors as well as an axial t2-weighted mri ( b ) revealing significant compression of the spinal cord . \n", "postoperative t2-weighted sagittal ( a ) and axial ( b ) magnetic resonance imaging revealing gross total resection of multiple intradural tumors .", "radiographic evidence of a refractory thoracolumbar cerebrospinal fluid leak was not readily evident on the immediate postoperative films . during his hospital course , the patient developed a spontaneous csf leak from his wound .", "it was initially treated by oversewing the skin , but the leak persisted over the next day .", "we decided to bring the patient back to the operating room for an exploration and an attempt at repairing the dural defects .", "multiple dural defects were noted . as a result of the significant dural maceration , another primary closure was not attempted .", "we placed more pieces of duraform , fat graft , and tisseel over the areas of csf leakage , and we again performed a tight multilayer closure of the muscle , fascia , fat , and subcutaneous tissue . despite our best efforts to stop the csf leak , the leak continued through the incisions from multiple areas .", "as a last resort , we decided to place a right frontal external ventricular drain ( evd ; integra life sciences , plainsboro , nj , united states ) after obtaining a preprocedure computed tomography scan of the head , to ensure the ventricular size could accommodate the catheter ( fig .", "he was placed on antibiotics for both the pneumonia as well as the csf leak and subsequent meningitis . on the day 8 of the evd in place", ", we continued to see cessation of the csf leak and decided to perform a 3-day clamping trial .", "no further csf leak was noted from the incision during the clamping trial . the evd was removed , and the patient was eventually discharged home without further complications . \n", "preprocedure computed tomography scan of the head to ensure the ventricular size could accommodate an external ventricular drain ( a ) .", "postprocedure scan showing successful placement of a right frontal external ventricular drain ( b ) .", "a watertight dural closure is essential in reducing the risk of csf leakage in spinal surgery .", "eismont et al recommended the meticulous closure of the dura at the time of surgery and reoperation and repair of dural tears upon the discovery of a persistent csf leak.1 conservative management may be initially attempted with bed rest for 3 days with subsequent slow advancement to ambulation .", "eighty - six of eighty - eight patients with dural tears studied by wang et al had resolution of csf leaks with primary dural closure at the time of surgery followed by bed rest.2 others have confirmed the efficacy of treating such leaks via csf diversion in the lumbar region.3 \n a review of the literature revealed scarce reports of csf leakage in more complex patients such as the patient in this case .", "this complexity arose from the multiple surgeries that were performed due to the recurrence of this intradural disease .", "they are benign , slow - growing , who grade i tumors that are amenable to surgical resection with optimal outcomes when gross total resection is achieved .", "although pathologically they may be low grade , they can be metastatic locally within the spine via the csf route .", "recurrence of the disease is usually in the same region and is substantiated by subtotal or piecemeal resection.4 \n spine surgeons occasionally encounter lumbar csf leak .", "routine iatrogenic csf leaks can usually be repaired primarily by suturing the dural defect , with the subsequent placement of a liquefied dural sealant such as tisseel .", "our patient 's dura was manipulated on multiple occasions over the span of half a decade . with each surgery to remove more metastatic lesions , the dura became more macerated .", "this damaged dura created multiple avenues for csf to leak , which was essentially refractory to all of our attempts at a repair . a case report by yeager demonstrated the use of an evd with subsequent conversion to a shunt for a persistent cervical csf leak after a schwannoma resection.5 however ,", "temporary external ventricular drainage for refractory lumbar spinal fluid leaks is not reported in the literature .", "lumbar drains have been utilized in the case of persistent cervical csf leaks.6 we feel that a lumbar drain in our particular case would not have been best suited for the patient we presented .", "we placed an evd in this situation as opposed to a lumbar drain because maximal csf diversion from the macerated dura would take place with a proximal avenue for controlled csf drainage , which would essentially decrease the pressure gradient of csf in an attempt to traverse the dural defects more distally .", "although the placement of external ventricular drains comes with associated risks , if they are placed safely after a preprocedure computed tomography scan is done , their usage can be a viable option for temporary proximal csf diversion for refractory thoracolumbar csf leaks ." ]

[ "<S> \n study design case report . \n </S>", "<S> objective temporary external ventricular drainage for refractory thoracolumbar cerebrospinal fluid ( csf ) leak is not reported in the literature . </S>", "<S> we describe a recent case that utilized this technique . \n </S>", "<S> methods retrospective review of the patient 's case notes was performed and the literature on this subject reviewed . \n </S>", "<S> results the patient underwent multiple complex spinal surgeries for resection of innumerable metastatic ependymoma lesions . a case of significant refractory csf leak developed and as a last resort a right frontal external ventricular drain was placed . </S>", "<S> the csf leak ceased , and the patient was eventually discharged home without further complication . \n </S>", "<S> conclusion external ventricular drainage can be a viable option for temporary proximal csf diversion to treat refractory thoracolumbar csf leaks . </S>" ]

[ "Introduction", "Case Report", "Discussion" ]

[ [ "temporary external ventricular drainage for refractory thoracolumbar cerebrospinal fluid ( csf ) leak is not reported in the literature .", "we present a young man who had multiple complex spinal surgeries for resection of innumerable metastatic ependymoma lesions .", "as a last resort , we decided to place an external ventricular catheter for proximal controlled csf drainage with great success ." ], [ "the 32-year - old man had a history of spinal myxopapillary ependymoma world health organization ( who ) grade i with local metastatic spinal disease . the patient first presented in 2005 with a 1-year history of severe low back pain . he was found to have an intradural lesion , and subsequently had a t12l3 laminectomy for resection of the tumor .", "small amounts of the tumor adherent to the nerve roots were purposefully left behind . each time an attempt was made to remove these residual small pieces of the tumor , significant amounts of neuromonitoring activity was noted .", "hence , the residual tumor was left behind so as to not cause a neurologic deficit .", "approximately 10 days after surgery , the patient fell at home and noticed significant amounts of a clear fluid consistent with csf leaking from his incision .", "he was taken to the operating room at that time and a dural defect was identified and closed primarily , then covered with gelfoam ( baxter healthcare , deerfield , il , united states ) and tisseel ( baxter healthcare ) . the patient again noted another csf leak from a different area of his incision 5 days later , and he was taken back to the operating room for another primary closure , as well as placement of a fat graft and more tisseel .", "adjuvant radiation treatment was undertaken after he recovered from the initial surgeries . in 2008 ,", "he was found to have a dorsally located intradural lesion at the t8t9 region , which was resected via a t7t10 laminectomy . in 2010 ,", "he was found to have growing intradural lesions at l4 and l5 . at that time", "one year later , our patient presented yet again with multiple thoracolumbar intradural lesions ( fig .", "he underwent redo decompressive laminectomies from t8s1 with a total of 13 intradural tumors being excised ( fig .", "2 ) . the dural closure proved to be quite complex , as there were several areas that were macerated and very difficult to reapproximate .", "fat grafts , duraform ( depuy synthes , west chester , pa , united states ) , and tisseel were all used on top of the attempted primary dural closure . \n", "preoperative sagittal t2-weighted magnetic resonance imaging ( mri ) ( a ) of the thoracic spine revealing multiple intradural tumors as well as an axial t2-weighted mri ( b ) revealing significant compression of the spinal cord . \n", "postoperative t2-weighted sagittal ( a ) and axial ( b ) magnetic resonance imaging revealing gross total resection of multiple intradural tumors .", "radiographic evidence of a refractory thoracolumbar cerebrospinal fluid leak was not readily evident on the immediate postoperative films . during his hospital course , the patient developed a spontaneous csf leak from his wound .", "it was initially treated by oversewing the skin , but the leak persisted over the next day .", "we decided to bring the patient back to the operating room for an exploration and an attempt at repairing the dural defects .", "multiple dural defects were noted . as a result of the significant dural maceration , another primary closure was not attempted .", "we placed more pieces of duraform , fat graft , and tisseel over the areas of csf leakage , and we again performed a tight multilayer closure of the muscle , fascia , fat , and subcutaneous tissue . despite our best efforts to stop the csf leak , the leak continued through the incisions from multiple areas .", "as a last resort , we decided to place a right frontal external ventricular drain ( evd ; integra life sciences , plainsboro , nj , united states ) after obtaining a preprocedure computed tomography scan of the head , to ensure the ventricular size could accommodate the catheter ( fig .", "he was placed on antibiotics for both the pneumonia as well as the csf leak and subsequent meningitis . on the day 8 of the evd in place", ", we continued to see cessation of the csf leak and decided to perform a 3-day clamping trial .", "no further csf leak was noted from the incision during the clamping trial . the evd was removed , and the patient was eventually discharged home without further complications . \n", "preprocedure computed tomography scan of the head to ensure the ventricular size could accommodate an external ventricular drain ( a ) .", "postprocedure scan showing successful placement of a right frontal external ventricular drain ( b ) ." ], [ "a watertight dural closure is essential in reducing the risk of csf leakage in spinal surgery .", "eismont et al recommended the meticulous closure of the dura at the time of surgery and reoperation and repair of dural tears upon the discovery of a persistent csf leak.1 conservative management may be initially attempted with bed rest for 3 days with subsequent slow advancement to ambulation .", "eighty - six of eighty - eight patients with dural tears studied by wang et al had resolution of csf leaks with primary dural closure at the time of surgery followed by bed rest.2 others have confirmed the efficacy of treating such leaks via csf diversion in the lumbar region.3 \n a review of the literature revealed scarce reports of csf leakage in more complex patients such as the patient in this case .", "this complexity arose from the multiple surgeries that were performed due to the recurrence of this intradural disease .", "they are benign , slow - growing , who grade i tumors that are amenable to surgical resection with optimal outcomes when gross total resection is achieved .", "although pathologically they may be low grade , they can be metastatic locally within the spine via the csf route .", "recurrence of the disease is usually in the same region and is substantiated by subtotal or piecemeal resection.4 \n spine surgeons occasionally encounter lumbar csf leak .", "routine iatrogenic csf leaks can usually be repaired primarily by suturing the dural defect , with the subsequent placement of a liquefied dural sealant such as tisseel .", "our patient 's dura was manipulated on multiple occasions over the span of half a decade . with each surgery to remove more metastatic lesions , the dura became more macerated .", "this damaged dura created multiple avenues for csf to leak , which was essentially refractory to all of our attempts at a repair . a case report by yeager demonstrated the use of an evd with subsequent conversion to a shunt for a persistent cervical csf leak after a schwannoma resection.5 however ,", "temporary external ventricular drainage for refractory lumbar spinal fluid leaks is not reported in the literature .", "lumbar drains have been utilized in the case of persistent cervical csf leaks.6 we feel that a lumbar drain in our particular case would not have been best suited for the patient we presented .", "we placed an evd in this situation as opposed to a lumbar drain because maximal csf diversion from the macerated dura would take place with a proximal avenue for controlled csf drainage , which would essentially decrease the pressure gradient of csf in an attempt to traverse the dural defects more distally .", "although the placement of external ventricular drains comes with associated risks , if they are placed safely after a preprocedure computed tomography scan is done , their usage can be a viable option for temporary proximal csf diversion for refractory thoracolumbar csf leaks ." ] ]

[ "ankylosing spondylitis ( as ) is a chronic inflammatory rheumatic disease that affects the axial skeleton , causing characteristics inflammatory back pain , which can lead to structural and functional impairments .", "asymmetric peripheral arthritis is present in about 2040% of patients with as . in europe ,", "estimates of prevalence of ankylosing spondylitis vary by tenfold from 0.08% to 0.86% [ 24 ] . the direct costs of as are substantial . in europe , the annual total direct costs per patient have been estimated at 1,800 to 2,800 .", "the introduction of antitumour necrosis factor alpha ( anti - tnf ) agents has increased the direct cost of as . ", "the first - line treatment of as is nonsteroidal anti - inflammatory drugs ( nsaids ) [ 79 ] .", "the nonselective nsaids ( nsnsaids ) have been associated with an increased risk of gastrointestinal ( gi ) adverse events such as ulcers and gi bleeding because of their inhibition of the gastroprotective cox-1 isoform .", "etoricoxib and celecoxib have been demonstrated to have a superior upper gastrointestinal ( gi ) safety profile [ 1114 ] . the medal ( multinational etoricoxib and diclofenac arthritis long term ) program demonstrated the risk of cardiovascular ( cv ) events with etoricoxib ( 60 mg / d and 90 mg / d ) comparable to diclofenac ( 75 mg bid and 50 mg tid ) . on the efficacy side , etoricoxib has been shown , at doses of 90 mg and 120 mg , to be superior compared to naproxen 1000 mg in the treatment of as .", "celecoxib ( 200 mg and 400 mg ) showed comparable efficacy to diclofenac ( 150 mg ) . ", "given the economic burden of as , a cost - effectiveness analysis of interventions for as is warranted .", "the objective of this study was to evaluate the cost - effectiveness of etoricoxib ( 90 mg ) compared to celecoxib ( 200 and 400 mg ) , diclofenac ( 150 mg ) , and naproxen ( 1000 mg ) in the treatment of patients with as in norway .", "in the present economic evaluation , a comprehensive decision bayesian modelling approach was used which integrates evidence synthesis and parameter estimation for efficacy and safety with cost - effectiveness modeling in a single unified framework .", "a previously published markov - state transition model was used to estimate the cost - effectiveness of etoricoxib versus celecoxib and nsnsaids in the treatment of as patients requiring daily nsaid treatment . the model consisted of eight health states reflecting treatment received : ( 1 ) initial nsaid ", "( etoricoxib , celecoxib , or nsnsaids , depending on intervention arm of the model ) , ( 2 ) initial nsaid with proton - pump inhibitor ( ppi ) , ( 3 ) alternative nsnsaids with ppi , ( 4 ) alternative nsnsaid with ppi and aspirin , ( 5 ) alternative nsnsaid , ( 6 ) anti - tnf treatment , ( 7 ) discontinued anti - tnf treatment , and ( 8) death .", "transitions from state to state were determined by lack of treatment efficacy , and the different types of events as presented in table 1 . ", "figure 1 presents the different types of cost generating gi , cv , and other events relevant to each markov cycle . for each health state", ", utilities were assigned based on the bath ankylosing spondylitis functional index ( basfi ) and the bath ankylosing spondylitis disease activity index ( basdai ) . over time , basfi will worsen thereby decreasing utilities .", "drug acquisition costs , and cost due to adverse events were taken into account . ", "the model followed individuals for a maximum of 30 cycles ( 30 years ) as by this time the majority of individuals had reached the absorbing state ( i.e. , death ) . ", "the efficacy of etoricoxib , celecoxib , diclofenac , or naproxen in as regarding basfi , basdai , and discontinuation was obtained from a previously performed systematic review and bayesian mixed treatment comparison ( mtc ) of randomized controlled trials using noninformative prior distributions [ 19 , 21 , 22 ] . in table 2", "the results of the mtc as used in the cost - effectiveness analysis are presented . for the model analysis , the expected change from baseline ( cfb ) estimates for basfi and basdai by treatment", "were subtracted from background basfi and basdai values which develop over time . over time , an increase in basfi of 0.5 ( scale 0100 ) per annum was assumed [ 6 , 20 ] . ", "it was assumed that background basdai scores remained stable over time [ 16 , 23 , 24 ] . for patients who continue responding to treatment", ", it is assumed that their treatment effect regarding basfi and basdai ( i.e. , the cfb scores ) remain constant over time .", "patients who switched to another nsnsaids were assumed to have the average treatment effect of diclofenac and naproxen as obtained from the mtc . for patients that switched to anti - tnf ,", "a treatment effect of 23 points and 19 points was used for basfi and basdai , respectively . ", "it was assumed that 10% withdraw from anti - tnf each year [ 20 , 25 ] . for patients who withdraw from anti - tnf treatment basdai , and", "basfi measurements revert back to baseline values as reported by ara et al . . ", "incidence rates of an upper gi perforation , ulcer , or bleeding ( pub ) for etoricoxib , celecoxib , and the nsnsaids were estimated with an indirect comparison of the relative incidence rates versus placebo as reported by ramey et al .", "incidence rates for suspected pubs were calculated by subtracting the pubs from all - investigator - reported pubs by ramey et al . .", "rates of minor gi symptoms were based on discontinuations due to clinical gi events in the medal programme . except for pubs ,", "the rates for other upper gi events with etoricoxib were also used for the celecoxib arm of the model .", "the probabilities of treatment of gi events were based on moore et al . . ", "the incidence of a thrombotic cv event with etoricoxib , diclofenac , and naproxen were obtained by performing an indirect comparison of the results from the medal programme by cannon et al .", "and the relative incidence rate of etoricoxib versus naproxen from a meta - analysis of thrombotic cv events in 12 phase ii - iv clinical trials [ 15 , 19 , 30 ] .", "the occurrence of edema , hypertension , coronary heart failure , hepatic adverse events , and renal events were obtained from the medal programme as well . for anti - tnf treatment , and treatment after anti - tnf", ", no adverse events were taken into consideration . the adverse event rate for second - line nsnsaid therapy", "was assumed to be equal to the average of those obtained for diclofenac and naproxen , with the exception of the incidence of an upper gi event in a patient receiving nsnsaid plus ppi therapy , which was assumed to be reduced by 40% .", "the gi and cv risk for a patient who switched to nsnsaid plus aspirin and ppi was assumed to be comparable to that for nsnsaid alone .", "the case - fatality of a ugi pub or lgi bleed was 3.6% [ 29 , 31 ] . ", "a 13% case - fatality for a cv event was used for etoricoxib and celecoxib , and 12.8% case - fatality for nsnsaids . for patients not experiencing gi or cv adverse events ,", "a norwegian annual age - dependent mortality was used , as obtained from the life tables for norway . for the evaluation ,", "an as population with an average age of 45 was assumed ( base - case scenario ) .", "utilities reflect the preference for a certain health state and are measured on 0 - 1 scale . ", "a value of 1 reflects perfect health and 0 represents death . by summarizing the utility value over time ,", "life years were transformed into qalys using a relation between utility ( eq-5d ) and basfi and basdai as derived by ara et al .", "utility loss associated with adverse events was obtained from the literature [ 29 , 3336 ] .", "annual drug acquisition costs were calculated based on the most commonly prescribed drug within a drug class and obtained from the norwegian medicines agency ( noma september 2007 ) . for anti - tnf treatment , annual costs of etanercept were used . for each type of gi event , numbers of units of health care resource use were assigned and respective unit costs applied to all healthcare resources to calculate the cost per event .", "the key cost items for gi events included costs of treatment ( drugs and dispensing ) , gp consultations , investigations , inpatient days , and surgery .", "cost for the other adverse events ( i.e. , edema , hypertension , hepatic , and renal ) were not taken into consideration .", "drug costs related to adverse events were obtained from noma ( september 2007 ) ; costs related to gp visits were obtained from the norwegian medical association ( july 2007 ) , and inpatient costs related to events were obtained from the drg price list .", "all costs were expressed in 2007 norwegian kroner ( nok ) . given the markov state - transition model structure , the source data were combined and translated into the following outcomes : quality adjusted life years , drug acquisition costs , costs of adverse events , total costs , and net monetary benefit ( nmb ) calculated as qalys multiplied with a willingness - to pay ratio ( wtp ) minus costs .", "wtp is the amount that decision makers are willing - to pay per additional qaly gained .", "since the model was fully probabilistic , outcomes were estimated with mcmc simulation using winbugs v 1.4 . for each iteration of the model , new parameter values", "were sampled from the estimated ( posterior ) or defined distributions for efficacy , safety , and costs ( see table 3 ) .", "the model was evaluated by averaging output values over many iterations ( i.e. , 10.000 ) , allowing uncertainty in model parameters to be accounted for .", "for each iteration , the qalys and cost accrued for each cycle were calculated for each of the treatments according to sonnenberg and beck . at the end of each iteration ,", "the cumulative qalys and costs over the cycles were obtained by summing the results over all cycles .", "next , the incremental cost , incremental qalys , and incremental cost - effectiveness ratio ( icer = incremental costs / incremental qalys ) of etoricoxib versus the other interventions were evaluated .", "the probability of cost - effectiveness was expressed with cost - effectiveness acceptability curves , calculated as the number of iterations out of the total number of iterations for which the nmb was greatest for a given treatment out of all 4 treatments .", "furthermore , analysis were performed to identify the impact of uncertainty in the source data on the uncertainty in the qalys , costs , and nmb estimates . in the base - case scenario ,", "etoricoxib ( 90 mg ) was compared with celecoxib ( 200 mg & 400 mg ) , diclofenac ( 150 mg ) , and naproxen ( 1000 mg ) . in alternative analyses ,", "the following scenarios were evaluated : ( 1 ) celecoxib 200 mg was used instead of celecoxib 200 mg/400 mg , ( 2 ) only gi events , ( 3 ) only cv events , ( 4 ) no adverse events , ( 5 ) no discounting on costs and effects , ( 6 ) 8% discounting on costs and effects , ( 7 ) stable basfi over time , and ( 8) assuming an age of 20 years , and ( 9 ) anti - tnf costs excluded . for each scenario uncertainty in input parameters", "in table 4 , the results of the base - case scenario are presented for 1 year , 5 years , and 30 years of follow - up .", "there was more than 98% probability that etoricoxib resulted in higher expected qalys than the other interventions of interest . ", "drug costs are expected to be the highest with celecoxib ( 200 & 400 mg ) followed by etoricoxib ( 90 mg ) .", "after 5 years , however , the lowest drug costs can be expected for the patients for whom treatment was initiated with etoricoxib due to the higher probability of staying on initial therapy and not switching to the far more expensive anti - tnf treatment .", "relative to a patient starting with nsnsaids , the costs due to gi events were lower for a patient starting with etoricoxib or celecoxib as a result of a reduced risk of treatment - requiring gi events .", "after 30 years , the gi - related costs with etoricoxib were higher than with celecoxib because this latter group of patients switched quicker to anti - tnf , which is not associated with gi events . until 5 years", ", costs related to thrombotic cv events were similar with etoricoxib , celecoxib , and diclofenac , and slightly higher than with naproxen .", "for the same reason as the gi - related costs , the cv - related costs with etoricoxib were higher than with celecoxib and diclofenac at 30 years of followup .", "overall , naproxen resulted in the lowest cost at 1 year ; however , at 5 years and beyond , etoricoxib resulted in the lowest direct costs of the four alternatives ; at 5 year , there is a > 96% probability that the lowest costs are obtained with etoricoxib .", "the difference in costs and qalys of etoricoxib relative to the other interventions is presented .", "given the more favourable outcomes regarding costs and qalys with etoricoxib after 5 years of followup , etoricoxib is considered an economically dominant intervention . at 1 year , etoricoxib is economically dominant over celecoxib . ", "the incremental cost - utility ratio ( icer ) of etoricoxib relative to diclofenac and naproxen at 1 year was 59,221 nok and 107,256 nok , respectively . in figure 2 ,", "the probability of cost - effectiveness for the different interventions at different willingness - to - pay ( wtp ) ratios are presented . for a wtp 200,000 nok per qaly there is an 85% probability that etoricoxib is the most cost - effective intervention at 1 year .", "this increased to more than 96% for wtp of 500,000 nok and higher . at 5 and 30 years", ", there is a more than 99% probability that etoricoxib is the most cost - effective intervention . ", "figure 3 provides an overview which of the parameters have the greatest impact on ( uncertainty ) in model outcomes .", "when celecoxib 200 mg was used as a comparator instead of celecoxib 200 mg & 400 mg combined , etoricoxib was no longer economically dominant at 1 year .", "given the qaly gain of 0.07 , this translates into a cost per qaly of 17,882 nok , a cost - effective result .", "scenarios where ( 1 ) only gi events were included as adverse events , ( 2 ) only cv events were included as adverse events , ( 3 ) no adverse events were included , ( 4 ) no discounting was applied , ( 5 ) 8% discounting was applied , ( 6 ) basfi was assumed to be stable over time , and ( 7 ) assuming an average age of 20 years provided comparable cost - effectiveness results as the base - case analysis . only for the scenario where anti - tnf costs were set to zero , etoricoxib was no longer dominant .", "however , etoricoxib can still be considered cost - effective , independent of the time horizon ( see table 6 ) .", "the economic evaluation demonstrated that etoricoxib ( 90 mg ) is an economically superior treatment of as to celecoxib ( 200 & 400 mg ) , diclofenac ( 150 mg ) , and naproxen ( 1000 mg ) for both qaly gains and cost savings for a time horizon longer than 5 years . ", "for a 1-year time horizon , etoricoxib is associated with greater costs than diclofenac ( 150 mg ) and naproxen ( 1000 mg ) , but can still be considered cost effective . ", "in addition to drug acquisition costs for the nsaids , also costs for anti - tnf treatment after failure on nsaids were taken into consideration .", "given the model structure we opted for , the average duration on any nsaid was estimated to be 11.2 years ( 95% cri 9.013.2 ) for the patients starting with etoricoxib , 7.8 ( 6.89.0 ) years with celecoxib , 8.3 ( 6.810.1 ) years with diclofenac , and 8.4 ( 6.910.2 ) years with naproxen .", "these differences explain the savings regarding drug acquisition observed when initiating treatment with etoricoxib over the other treatment strategies observed at the 5,- and 30-year time horizon . however , when the anti - tnf acquisition costs are taken out of the picture , etoricoxib is still economically more favorable than celecoxib , diclofenac , or naproxen ( see table 6 ) . for the current economic evaluation ,", "an indirect comparison of efficacy and safety estimates were integrated with cost - effectiveness analysis in a single framework .", "the advantage of this approach is that no assumptions were made regarding the uncertainty distributions used for sensitivity analysis ; the bayesian posterior uncertainty distributions of the treatment effect and gi and cv events as obtained from the mtc were directly propagated through the markov model .", "the most important factor in the cost - effectiveness analysis was the probability of discontinuation as estimated with the mtc ( see figure 3 ) the etoricoxib gi safety data as used in the analysis were obtained from clinical trials in oa , ra , as , and chronic low back pain patients ; the celecoxib gi safety data were obtained from the class study .", "these data were assumed applicable for as . it could be argued that as patients are likely younger than the average patient in the gi meta - analysis and , therefore , have a lower gi risk .", "however , as patients often receive higher nsnsaid doses than patients with other arthritic conditions , thereby increasing their risk for gi events .", "risk estimates of cv events were based on the medal programme comparing etoricoxib with diclofenac among oa and ra patients and a meta - analysis of 12 phase ii - iv clinical trials comparing etoricoxib with naproxen , among oa , ra , as , and chronic low back pain patients [ 15 , 30 ] . as", "as patients are on average younger than patients in the medal programme and the meta - analysis , it can be argued that the risk for cv events might have been overestimated in the model . in the model", ", the risk for a second cv event was set to be the same as before the cv event , assuming that the increased cv risk due to the history of a cv event was counterbalanced by adding aspirin to the nsaid .", "however , the history of a cv event might have a bigger impact than the protective effect by aspirin , which would imply an underestimation of the risk of cv events in the model , and , therefore , an underestimation of the costs due to cv events .", "the effect on the difference in costs , however , would be limited because the underestimation applies to both treatment initiated with etoricoxib and nsnsaids . ", "costs associated with severity of as ( i.e. , gp visits , specialist visits , paramedical visits , hospitalization , technical examinations , adaptations and aids ) were not included in the analysis . for the uk ,", "botteman et al . showed that each incremental change in one unit of basdai ( 0100 scale ) was estimated to be associated with a direct medical cost increase according to cost = 708.45 + 75.00*basdai .", "as a result , the cost savings of treatment of as have probably been underestimated , especially for etoricoxib .", "this evaluation was performed for the norwegian local situation . in general , it is difficult to transfer cost - effectiveness estimates obtained for one country to another , due to differences in treatment practices , resource use , and unit cost data , among other .", "although the cost - effectiveness findings in this study were primarily driven by differences in efficacy of the compared interventions , the cost - effectiveness of the different nsaids for the management of ankylosing spondylitis in other countries needs to be confirmed with country - specific analysis . in conclusion ,", "given the underlying assumptions and current evidence available , this economic evaluation demonstrated that etoricoxib is a cost - saving and qaly gaining therapy for as in norway from a health care perspective ." ]

[ "<S> \n objectives . to evaluate the cost - effectiveness of etoricoxib ( 90 mg ) relative to celecoxib ( 200/400 mg ) , and the nonselective nsaids naproxen ( 1000 mg ) and diclofenac ( 150 mg ) in the initial treatment of ankylosing spondylitis in norway . methods . </S>", "<S> a previously developed markov state - transition model was used to estimate costs and benefits associated with initiating treatment with the different competing nsaids . </S>", "<S> efficacy , gastrointestinal and cardiovascular safety , and resource use data were obtained from the literature . </S>", "<S> data from different studies were synthesized and translated into direct costs and quality adjusted life years by means of a bayesian comprehensive decision modeling approach . </S>", "<S> results . over a 30-year time horizon </S>", "<S> , etoricoxib is associated with about 0.4 more quality adjusted life years than the other interventions . at 1 year , naproxen is the most cost - saving strategy . </S>", "<S> however , etoricoxib is cost and quality adjusted life year saving relative to celecoxib , as well as diclofenac and naproxen after 5 years of follow - up . for a willingness - to - pay ceiling ratio of 200,000 norwegian krones per quality adjusted life year </S>", "<S> , there is a > 95% probability that etoricoxib is the most - cost - effective treatment when a time horizon of 5 or more years is considered . conclusions . </S>", "<S> etoricoxib is the most cost - effective nsaid for initiating treatment of ankylosing spondylitis in norway . </S>" ]

[ "1. Introduction", "2. Methods", "3. Results", "4. Discussion", " Funding", " Disclosure Statement" ]

[ [ "ankylosing spondylitis ( as ) is a chronic inflammatory rheumatic disease that affects the axial skeleton , causing characteristics inflammatory back pain , which can lead to structural and functional impairments .", "asymmetric peripheral arthritis is present in about 2040% of patients with as . in europe ,", "estimates of prevalence of ankylosing spondylitis vary by tenfold from 0.08% to 0.86% [ 24 ] . the direct costs of as are substantial . in europe , the annual total direct costs per patient have been estimated at 1,800 to 2,800 .", "the introduction of antitumour necrosis factor alpha ( anti - tnf ) agents has increased the direct cost of as . ", "the first - line treatment of as is nonsteroidal anti - inflammatory drugs ( nsaids ) [ 79 ] .", "the nonselective nsaids ( nsnsaids ) have been associated with an increased risk of gastrointestinal ( gi ) adverse events such as ulcers and gi bleeding because of their inhibition of the gastroprotective cox-1 isoform .", "etoricoxib and celecoxib have been demonstrated to have a superior upper gastrointestinal ( gi ) safety profile [ 1114 ] . the medal ( multinational etoricoxib and diclofenac arthritis long term ) program demonstrated the risk of cardiovascular ( cv ) events with etoricoxib ( 60 mg / d and 90 mg / d ) comparable to diclofenac ( 75 mg bid and 50 mg tid ) . on the efficacy side , etoricoxib has been shown , at doses of 90 mg and 120 mg , to be superior compared to naproxen 1000 mg in the treatment of as .", "celecoxib ( 200 mg and 400 mg ) showed comparable efficacy to diclofenac ( 150 mg ) . ", "given the economic burden of as , a cost - effectiveness analysis of interventions for as is warranted .", "the objective of this study was to evaluate the cost - effectiveness of etoricoxib ( 90 mg ) compared to celecoxib ( 200 and 400 mg ) , diclofenac ( 150 mg ) , and naproxen ( 1000 mg ) in the treatment of patients with as in norway ." ], [ "in the present economic evaluation , a comprehensive decision bayesian modelling approach was used which integrates evidence synthesis and parameter estimation for efficacy and safety with cost - effectiveness modeling in a single unified framework .", "a previously published markov - state transition model was used to estimate the cost - effectiveness of etoricoxib versus celecoxib and nsnsaids in the treatment of as patients requiring daily nsaid treatment . the model consisted of eight health states reflecting treatment received : ( 1 ) initial nsaid ", "( etoricoxib , celecoxib , or nsnsaids , depending on intervention arm of the model ) , ( 2 ) initial nsaid with proton - pump inhibitor ( ppi ) , ( 3 ) alternative nsnsaids with ppi , ( 4 ) alternative nsnsaid with ppi and aspirin , ( 5 ) alternative nsnsaid , ( 6 ) anti - tnf treatment , ( 7 ) discontinued anti - tnf treatment , and ( 8) death .", "transitions from state to state were determined by lack of treatment efficacy , and the different types of events as presented in table 1 . ", "figure 1 presents the different types of cost generating gi , cv , and other events relevant to each markov cycle . for each health state", ", utilities were assigned based on the bath ankylosing spondylitis functional index ( basfi ) and the bath ankylosing spondylitis disease activity index ( basdai ) . over time , basfi will worsen thereby decreasing utilities .", "drug acquisition costs , and cost due to adverse events were taken into account . ", "the model followed individuals for a maximum of 30 cycles ( 30 years ) as by this time the majority of individuals had reached the absorbing state ( i.e. , death ) . ", "the efficacy of etoricoxib , celecoxib , diclofenac , or naproxen in as regarding basfi , basdai , and discontinuation was obtained from a previously performed systematic review and bayesian mixed treatment comparison ( mtc ) of randomized controlled trials using noninformative prior distributions [ 19 , 21 , 22 ] . in table 2", "the results of the mtc as used in the cost - effectiveness analysis are presented . for the model analysis , the expected change from baseline ( cfb ) estimates for basfi and basdai by treatment", "were subtracted from background basfi and basdai values which develop over time . over time , an increase in basfi of 0.5 ( scale 0100 ) per annum was assumed [ 6 , 20 ] . ", "it was assumed that background basdai scores remained stable over time [ 16 , 23 , 24 ] . for patients who continue responding to treatment", ", it is assumed that their treatment effect regarding basfi and basdai ( i.e. , the cfb scores ) remain constant over time .", "patients who switched to another nsnsaids were assumed to have the average treatment effect of diclofenac and naproxen as obtained from the mtc . for patients that switched to anti - tnf ,", "a treatment effect of 23 points and 19 points was used for basfi and basdai , respectively . ", "it was assumed that 10% withdraw from anti - tnf each year [ 20 , 25 ] . for patients who withdraw from anti - tnf treatment basdai , and", "basfi measurements revert back to baseline values as reported by ara et al . . ", "incidence rates of an upper gi perforation , ulcer , or bleeding ( pub ) for etoricoxib , celecoxib , and the nsnsaids were estimated with an indirect comparison of the relative incidence rates versus placebo as reported by ramey et al .", "incidence rates for suspected pubs were calculated by subtracting the pubs from all - investigator - reported pubs by ramey et al . .", "rates of minor gi symptoms were based on discontinuations due to clinical gi events in the medal programme . except for pubs ,", "the rates for other upper gi events with etoricoxib were also used for the celecoxib arm of the model .", "the probabilities of treatment of gi events were based on moore et al . . ", "the incidence of a thrombotic cv event with etoricoxib , diclofenac , and naproxen were obtained by performing an indirect comparison of the results from the medal programme by cannon et al .", "and the relative incidence rate of etoricoxib versus naproxen from a meta - analysis of thrombotic cv events in 12 phase ii - iv clinical trials [ 15 , 19 , 30 ] .", "the occurrence of edema , hypertension , coronary heart failure , hepatic adverse events , and renal events were obtained from the medal programme as well . for anti - tnf treatment , and treatment after anti - tnf", ", no adverse events were taken into consideration . the adverse event rate for second - line nsnsaid therapy", "was assumed to be equal to the average of those obtained for diclofenac and naproxen , with the exception of the incidence of an upper gi event in a patient receiving nsnsaid plus ppi therapy , which was assumed to be reduced by 40% .", "the gi and cv risk for a patient who switched to nsnsaid plus aspirin and ppi was assumed to be comparable to that for nsnsaid alone .", "the case - fatality of a ugi pub or lgi bleed was 3.6% [ 29 , 31 ] . ", "a 13% case - fatality for a cv event was used for etoricoxib and celecoxib , and 12.8% case - fatality for nsnsaids . for patients not experiencing gi or cv adverse events ,", "a norwegian annual age - dependent mortality was used , as obtained from the life tables for norway . for the evaluation ,", "an as population with an average age of 45 was assumed ( base - case scenario ) .", "utilities reflect the preference for a certain health state and are measured on 0 - 1 scale . ", "a value of 1 reflects perfect health and 0 represents death . by summarizing the utility value over time ,", "life years were transformed into qalys using a relation between utility ( eq-5d ) and basfi and basdai as derived by ara et al .", "utility loss associated with adverse events was obtained from the literature [ 29 , 3336 ] .", "annual drug acquisition costs were calculated based on the most commonly prescribed drug within a drug class and obtained from the norwegian medicines agency ( noma september 2007 ) . for anti - tnf treatment , annual costs of etanercept were used . for each type of gi event , numbers of units of health care resource use were assigned and respective unit costs applied to all healthcare resources to calculate the cost per event .", "the key cost items for gi events included costs of treatment ( drugs and dispensing ) , gp consultations , investigations , inpatient days , and surgery .", "cost for the other adverse events ( i.e. , edema , hypertension , hepatic , and renal ) were not taken into consideration .", "drug costs related to adverse events were obtained from noma ( september 2007 ) ; costs related to gp visits were obtained from the norwegian medical association ( july 2007 ) , and inpatient costs related to events were obtained from the drg price list .", "all costs were expressed in 2007 norwegian kroner ( nok ) . given the markov state - transition model structure , the source data were combined and translated into the following outcomes : quality adjusted life years , drug acquisition costs , costs of adverse events , total costs , and net monetary benefit ( nmb ) calculated as qalys multiplied with a willingness - to pay ratio ( wtp ) minus costs .", "wtp is the amount that decision makers are willing - to pay per additional qaly gained .", "since the model was fully probabilistic , outcomes were estimated with mcmc simulation using winbugs v 1.4 . for each iteration of the model , new parameter values", "were sampled from the estimated ( posterior ) or defined distributions for efficacy , safety , and costs ( see table 3 ) .", "the model was evaluated by averaging output values over many iterations ( i.e. , 10.000 ) , allowing uncertainty in model parameters to be accounted for .", "for each iteration , the qalys and cost accrued for each cycle were calculated for each of the treatments according to sonnenberg and beck . at the end of each iteration ,", "the cumulative qalys and costs over the cycles were obtained by summing the results over all cycles .", "next , the incremental cost , incremental qalys , and incremental cost - effectiveness ratio ( icer = incremental costs / incremental qalys ) of etoricoxib versus the other interventions were evaluated .", "the probability of cost - effectiveness was expressed with cost - effectiveness acceptability curves , calculated as the number of iterations out of the total number of iterations for which the nmb was greatest for a given treatment out of all 4 treatments .", "furthermore , analysis were performed to identify the impact of uncertainty in the source data on the uncertainty in the qalys , costs , and nmb estimates . in the base - case scenario ,", "etoricoxib ( 90 mg ) was compared with celecoxib ( 200 mg & 400 mg ) , diclofenac ( 150 mg ) , and naproxen ( 1000 mg ) . in alternative analyses ,", "the following scenarios were evaluated : ( 1 ) celecoxib 200 mg was used instead of celecoxib 200 mg/400 mg , ( 2 ) only gi events , ( 3 ) only cv events , ( 4 ) no adverse events , ( 5 ) no discounting on costs and effects , ( 6 ) 8% discounting on costs and effects , ( 7 ) stable basfi over time , and ( 8) assuming an age of 20 years , and ( 9 ) anti - tnf costs excluded . for each scenario uncertainty in input parameters" ], [ "in table 4 , the results of the base - case scenario are presented for 1 year , 5 years , and 30 years of follow - up .", "there was more than 98% probability that etoricoxib resulted in higher expected qalys than the other interventions of interest . ", "drug costs are expected to be the highest with celecoxib ( 200 & 400 mg ) followed by etoricoxib ( 90 mg ) .", "after 5 years , however , the lowest drug costs can be expected for the patients for whom treatment was initiated with etoricoxib due to the higher probability of staying on initial therapy and not switching to the far more expensive anti - tnf treatment .", "relative to a patient starting with nsnsaids , the costs due to gi events were lower for a patient starting with etoricoxib or celecoxib as a result of a reduced risk of treatment - requiring gi events .", "after 30 years , the gi - related costs with etoricoxib were higher than with celecoxib because this latter group of patients switched quicker to anti - tnf , which is not associated with gi events . until 5 years", ", costs related to thrombotic cv events were similar with etoricoxib , celecoxib , and diclofenac , and slightly higher than with naproxen .", "for the same reason as the gi - related costs , the cv - related costs with etoricoxib were higher than with celecoxib and diclofenac at 30 years of followup .", "overall , naproxen resulted in the lowest cost at 1 year ; however , at 5 years and beyond , etoricoxib resulted in the lowest direct costs of the four alternatives ; at 5 year , there is a > 96% probability that the lowest costs are obtained with etoricoxib .", "the difference in costs and qalys of etoricoxib relative to the other interventions is presented .", "given the more favourable outcomes regarding costs and qalys with etoricoxib after 5 years of followup , etoricoxib is considered an economically dominant intervention . at 1 year , etoricoxib is economically dominant over celecoxib . ", "the incremental cost - utility ratio ( icer ) of etoricoxib relative to diclofenac and naproxen at 1 year was 59,221 nok and 107,256 nok , respectively . in figure 2 ,", "the probability of cost - effectiveness for the different interventions at different willingness - to - pay ( wtp ) ratios are presented . for a wtp 200,000 nok per qaly there is an 85% probability that etoricoxib is the most cost - effective intervention at 1 year .", "this increased to more than 96% for wtp of 500,000 nok and higher . at 5 and 30 years", ", there is a more than 99% probability that etoricoxib is the most cost - effective intervention . ", "figure 3 provides an overview which of the parameters have the greatest impact on ( uncertainty ) in model outcomes .", "when celecoxib 200 mg was used as a comparator instead of celecoxib 200 mg & 400 mg combined , etoricoxib was no longer economically dominant at 1 year .", "given the qaly gain of 0.07 , this translates into a cost per qaly of 17,882 nok , a cost - effective result .", "scenarios where ( 1 ) only gi events were included as adverse events , ( 2 ) only cv events were included as adverse events , ( 3 ) no adverse events were included , ( 4 ) no discounting was applied , ( 5 ) 8% discounting was applied , ( 6 ) basfi was assumed to be stable over time , and ( 7 ) assuming an average age of 20 years provided comparable cost - effectiveness results as the base - case analysis . only for the scenario where anti - tnf costs were set to zero , etoricoxib was no longer dominant .", "however , etoricoxib can still be considered cost - effective , independent of the time horizon ( see table 6 ) ." ], [ "the economic evaluation demonstrated that etoricoxib ( 90 mg ) is an economically superior treatment of as to celecoxib ( 200 & 400 mg ) , diclofenac ( 150 mg ) , and naproxen ( 1000 mg ) for both qaly gains and cost savings for a time horizon longer than 5 years . ", "for a 1-year time horizon , etoricoxib is associated with greater costs than diclofenac ( 150 mg ) and naproxen ( 1000 mg ) , but can still be considered cost effective . ", "in addition to drug acquisition costs for the nsaids , also costs for anti - tnf treatment after failure on nsaids were taken into consideration .", "given the model structure we opted for , the average duration on any nsaid was estimated to be 11.2 years ( 95% cri 9.013.2 ) for the patients starting with etoricoxib , 7.8 ( 6.89.0 ) years with celecoxib , 8.3 ( 6.810.1 ) years with diclofenac , and 8.4 ( 6.910.2 ) years with naproxen .", "these differences explain the savings regarding drug acquisition observed when initiating treatment with etoricoxib over the other treatment strategies observed at the 5,- and 30-year time horizon . however , when the anti - tnf acquisition costs are taken out of the picture , etoricoxib is still economically more favorable than celecoxib , diclofenac , or naproxen ( see table 6 ) . for the current economic evaluation ,", "an indirect comparison of efficacy and safety estimates were integrated with cost - effectiveness analysis in a single framework .", "the advantage of this approach is that no assumptions were made regarding the uncertainty distributions used for sensitivity analysis ; the bayesian posterior uncertainty distributions of the treatment effect and gi and cv events as obtained from the mtc were directly propagated through the markov model .", "the most important factor in the cost - effectiveness analysis was the probability of discontinuation as estimated with the mtc ( see figure 3 ) the etoricoxib gi safety data as used in the analysis were obtained from clinical trials in oa , ra , as , and chronic low back pain patients ; the celecoxib gi safety data were obtained from the class study .", "these data were assumed applicable for as . it could be argued that as patients are likely younger than the average patient in the gi meta - analysis and , therefore , have a lower gi risk .", "however , as patients often receive higher nsnsaid doses than patients with other arthritic conditions , thereby increasing their risk for gi events .", "risk estimates of cv events were based on the medal programme comparing etoricoxib with diclofenac among oa and ra patients and a meta - analysis of 12 phase ii - iv clinical trials comparing etoricoxib with naproxen , among oa , ra , as , and chronic low back pain patients [ 15 , 30 ] . as", "as patients are on average younger than patients in the medal programme and the meta - analysis , it can be argued that the risk for cv events might have been overestimated in the model . in the model", ", the risk for a second cv event was set to be the same as before the cv event , assuming that the increased cv risk due to the history of a cv event was counterbalanced by adding aspirin to the nsaid .", "however , the history of a cv event might have a bigger impact than the protective effect by aspirin , which would imply an underestimation of the risk of cv events in the model , and , therefore , an underestimation of the costs due to cv events .", "the effect on the difference in costs , however , would be limited because the underestimation applies to both treatment initiated with etoricoxib and nsnsaids . ", "costs associated with severity of as ( i.e. , gp visits , specialist visits , paramedical visits , hospitalization , technical examinations , adaptations and aids ) were not included in the analysis . for the uk ,", "botteman et al . showed that each incremental change in one unit of basdai ( 0100 scale ) was estimated to be associated with a direct medical cost increase according to cost = 708.45 + 75.00*basdai .", "as a result , the cost savings of treatment of as have probably been underestimated , especially for etoricoxib .", "this evaluation was performed for the norwegian local situation . in general , it is difficult to transfer cost - effectiveness estimates obtained for one country to another , due to differences in treatment practices , resource use , and unit cost data , among other .", "although the cost - effectiveness findings in this study were primarily driven by differences in efficacy of the compared interventions , the cost - effectiveness of the different nsaids for the management of ankylosing spondylitis in other countries needs to be confirmed with country - specific analysis . in conclusion ,", "given the underlying assumptions and current evidence available , this economic evaluation demonstrated that etoricoxib is a cost - saving and qaly gaining therapy for as in norway from a health care perspective ." ], [ "" ], [ "" ] ]

[ "an unprecedented outbreak of ebola virus disease ( evd ) is occurring in west africa .", "in addition to the morbidity and mortality effects on the population at large , there is a significant impact on healthcare providers in the region .", "although many healthcare worker exposures may occur prior to evd being suspected and appropriate isolation implemented , exposures can occur in spite of isolation procedures and ppe use .", "although there is anecdotal evidence regarding the efficacy of ppe , a review of literature reveals few controlled studies of ppe ensembles relevant to evd .", "field studies of ppe in the setting of evd are difficult because of ethical and safety concerns .", "it is thought a high level of training is required to safely don , use , and doff ppe .", "introducing a new ppe ensemble into the field could increase exposure risk of participants who are already well - trained on a current ppe ensemble . there may be some hesitancy among participants to try an unproven ppe set .", "finally , in a high disease prevalence area , with a relatively small number of events , a very large study population would be required to account for the possibility of evd acquisition from non - work exposures or activities ( i.e. non - medical contacts within the community ) .", "the possibility that exposures can occur in spite of ppe utilization is evident from recent disease acquisition by two healthcare workers in the united states .", "these regulatory standards are material standards so that testing of individual components ( gloves , gown , etc . ) result in a rating for the tested component .", "some ppe ensemble testing has been performed , so called man - in - suit - testing ( mist ) ) but this has primarily focused on radiation , thermal or chemical exposures where exposure risk is dose - dependent , or in aerosol scenarios .", "zamora et al . compared two personal protective systems ( ensembles ) for biological exposures in 2006 and found significant differences in contamination rates . in 2010", "the institute of medicine released a report regarding certification of personal protective technologies , noting that additional testing of ppe ensembles should be performed .", "given the large number and variations of individual ppe components , it would not be feasible to test all potential combinations of ppe commercially available .", "additionally , the recent surge in ppe purchasing in response to evd has caused significant shortages of some ppe components and manufacturers with a history of field use .", "some healthcare facilities have had to develop ppe protocols based on the best use of locally available components ( author , personal communication ) .", "ideally , a ppe ensemble would be evaluated at the local level by the staff anticipated to wear the equipment .", "doffing or removal of ppe after a patient encounter may be a particularly high risk activity .", "appropriate testing of ppe ensembles should include mist type testing to confirm product efficacy in real world scenarios .", "fluorescent agents have been used in training for hand - washing , and environmental decontamination , as well as ppe ensembles , .", "non - toxic and mixed with an appropriate carrier , these agents can potentially mimic the natural contamination which occurs with an infectious agent spread by contact route .", "the purpose of our study was to compare two ppe ensembles used by volunteers with minimal prior training , and to evaluate the feasibility of fluorescent markers as an assessment tool for ppe effectiveness .", "eight healthcare providers ( six registered nurses and two physicians ) volunteered for this preliminary study .", "the study protocol was approved by the texas tech university health science center institutional review board .", "the participants were randomized to one of two ppe ensembles that meet current ( as of 10/20/14 ) cdc recommendations for ppe .", "one ppe ensemble ( standard ) utilized commercial components that meet current cdc recommendations . the other ppe ensemble ( alternate ) was composed of components already available at local hospitals or retail stores .", "1 ) included a neck - to - ankle coverall with overlying water impermeable surgical gown , knee - length impermeable leggings , and stryker hood ( stryker , kalamazoo , mi , usa ) .", "2 ) ensemble included two plastic gowns ( worn over the front and back of the torso ) , rain - suit pants , a rain - suit hood cut from a rain jacket , ankle length shoe covers , and a plastic spark - shield commonly used for metal working to cover the face . both ppe ensembles utilized double gloving , with the outermost glove a forearm length surgical glove and n-95 masks .", "when complete , both ppe ensembles met the cdc recommendations for ppe and no skin was exposed in either group .", "subjects were then randomized to standard or high volume exposures ( hve ) to simulate fluid splash .", "subjects randomized to standard exposure came into contact with a training mannequin contaminated with fluorescent agents to simulate bodily fluids .", "subjects randomized to hve had standard exposure , but then also had an additional 100 ml of fluorescent agent splashed onto the front torso of their garment .", "the fluorescent agents used included fluorescent powder ( glogerm glogerm co , moab , ut , usa ) , liquid clothes detergent with bleach alternative ( tide proctor & gamble inc .", ", providence , ri , usa ) , and dissolvable fluorescent tablets ( bright dyes orange dye kingscote chemicals , miamisburg , oh , usa ) . a base mixture of 500 ml of liquid detergent , 500 ml of water and three fluorescent tablets was used to create body fluids .", "the base mixture was combined with oatmeal , chocolate powder and crushed cereal to simulate different bodily fluids .", "the testing area was divided into four areas , a ppe donning area ( staging area ) , patient encounter room , ppe doffing area , and a separate dark room for black light photography .", ", participants worked in pairs to perform a series of clinical tasks to care for the evd patient .", " participants were asked to clean the contaminated mannequin , change the mannequin gown , place an automated bp cuff , and check and record the temperature . after completing the tasks ,", "finally , the de - gowned participants were examined under black light for fluorescence indicative of possible contamination .", "an led black light panel , ( chauvet led shadow , model dmx-512 led uv , chauvet lighting , sunrise , florida ) was used to illuminate .", "one participant in each ppe ensemble arm had evidence of contamination under black light examination ( fig .", "the participant contaminated in the standard arm felt the exposure occurred because of a splash under her hood during hve .", "the trainer felt she may have inadvertently touched a contaminated portion of her leggings and subsequently touched her neck during ppe removal .", "the participant contaminated in the alternate group likely had a gap appear between her front and back gowns while placing a blood pressure cuff .", "this event was not noted at occurrence , but was noticed under black light examination .", "the contamination in the alternate ppe ensemble arm likely could have been prevented if a larger range of gown sizes was available .", "it may be helpful to consider the body habitus of evd responder when selecting ppe .", "clinical activities as part of the assessment process , as the contamination likely would not have occurred without the provider manipulating the mannequin to place the blood pressure cuff .", "it could be helpful to videotape exercises such as this to better identify the timing of contamination such as that which occurred in the standard arm .", "pin - pointing the conditions of contamination may allow the development of new mechanisms for prevention . in preparation for this exercise", ", we found it notable that taping gloves circumferentially is routinely shown in the lay press , but current guidelines do not make this recommendation . in our study ,", "circumferential taping did not result in contamination , but greatly impaired the ease of ppe removal . on the other hand ,", "a single strip of taped placed longitudinally along the sleeve and glove allowed the glove to be removed with the garment as a seamless piece .", "current recommendations do not comment on hair or jewelry , perhaps making the assumption that these will be taken care of as a matter of course . for our study", ", we did not specifically ask participants to pull back their hair . although we did not note any contamination specific to this ,", "long hair , rings and watches adversely affected the ease of ppe placement and removal and may be an additional risk factor for exposure .", "it may be useful for future recommendations to include stipulations about such accouterments . in this very small study ,", "ppe ensembles made from readily available components ( approximately $ 36 per outfit ) performed as well as the commercial ppe ensemble .", "additionally , it must be borne in mind that increased complexity of ppe may be self - defeating , as the complexity of ppe removal may offset gains made by complexity of ppe .", "it is difficult to assess the actual effectiveness of fluorescents as a proxy for ev exposure in this study .", "it is possible that small volume contamination occurred that was unable to be visualized , and the clinical relevance of such a small exposure is unknown .", "how an exposure on intact skin correlates with risk of subsequent evd development remains to be determined .", "the florescent mixtures used in this study were intended to mimic the mechanical effects of bodily fluids during a patient interaction . in spite of widespread use", ", it is unknown how these mixtures compare with actual bodily fluids considering the unique chemical and biochemical attributes of blood", ". the limitations of this type of testing prevent any assumptions of risk of evd development for a healthcare worker in the field .", "this type of testing could , however , allow comparison of different ppe ensembles and protocols under the assumption that more fluorescent exposures equates to higher risk of evd development .", "another use of this system could be to identify ppe ensembles with lower expense and equivalent testing attributes .", "finally , fluorescent markers can be used for training and proficiency testing of ppe ensembles for evd preparedness and even determine minimal standards of training repetition to achieve proficiency . as fluorescent agents and black lights are readily available for purchase in retail stores and over the internet , it is feasible for testing and training to be performed at a local level with ppe ensembles available at hand . beyond the current evd outbreak , this concept is applicable to training for other infectious diseases , such as methicillin - resistant staphylococcus aureus or multidrug resistant gram - negative bacteria , spread primarily by direct contact .", "the concept could also be used for some pathogens with a significant respiratory mode of transmission , such as pandemic influenza and novel pathogenic corona viruses , which have a recommended contact component of ppe ." ]

[ "<S> the recent ebola virus disease ( evd ) outbreak has created interest in personal protective equipment ( ppe ) content and usage . ppe testing has historically been done by individual component , rather than as a bundle for contact isolation . </S>", "<S> fluorescent agents are commonly used in training for infection control techniques . </S>", "<S> the purpose of our study was to compare 2 ppe bundles and to evaluate the feasibility of fluorescent markers as an assessment tool for ppe effectiveness . </S>", "<S> eight healthcare providers volunteered for this preliminary study . </S>", "<S> participants were randomized to 1 of 2 ppe bundles that meet current ( october 20 , 2014 ) cdc recommendations . </S>", "<S> one ppe bundle utilized commercial evd - recommended components . the other ppe bundle used components already available at local hospitals or retail stores . </S>", "<S> participants were also randomized to standard or high volume exposures ( hve ) to simulate fluid splash . </S>", "<S> each participant was assisted in ppe donning and doffing by an experienced trainer . </S>", "<S> a training mannequin was contaminated with fluorescent agents to simulate bodily fluids . </S>", "<S> participants were then given clinical tasks to care for the evd patient . </S>", "<S> de - gowned participants were examined under black light for fluorescence indicative of contamination . </S>", "<S> one participant in each ppe arm had evidence of contamination . </S>", "<S> one of the contamination events was suspected during the patient care exercise . </S>", "<S> the other contamination event was not suspected until black light examination . in spite of a large difference in cost of ppe , the two bundle arms performed similarly . </S>", "<S> bundle testing using fluorescent markers could help identify optimal ppe systems . </S>" ]

[ "Background", "Methodology", "Results", "Discussion" ]

[ [ "an unprecedented outbreak of ebola virus disease ( evd ) is occurring in west africa .", "in addition to the morbidity and mortality effects on the population at large , there is a significant impact on healthcare providers in the region .", "although many healthcare worker exposures may occur prior to evd being suspected and appropriate isolation implemented , exposures can occur in spite of isolation procedures and ppe use .", "although there is anecdotal evidence regarding the efficacy of ppe , a review of literature reveals few controlled studies of ppe ensembles relevant to evd .", "field studies of ppe in the setting of evd are difficult because of ethical and safety concerns .", "it is thought a high level of training is required to safely don , use , and doff ppe .", "introducing a new ppe ensemble into the field could increase exposure risk of participants who are already well - trained on a current ppe ensemble . there may be some hesitancy among participants to try an unproven ppe set .", "finally , in a high disease prevalence area , with a relatively small number of events , a very large study population would be required to account for the possibility of evd acquisition from non - work exposures or activities ( i.e. non - medical contacts within the community ) .", "the possibility that exposures can occur in spite of ppe utilization is evident from recent disease acquisition by two healthcare workers in the united states .", "these regulatory standards are material standards so that testing of individual components ( gloves , gown , etc . ) result in a rating for the tested component .", "some ppe ensemble testing has been performed , so called man - in - suit - testing ( mist ) ) but this has primarily focused on radiation , thermal or chemical exposures where exposure risk is dose - dependent , or in aerosol scenarios .", "zamora et al . compared two personal protective systems ( ensembles ) for biological exposures in 2006 and found significant differences in contamination rates . in 2010", "the institute of medicine released a report regarding certification of personal protective technologies , noting that additional testing of ppe ensembles should be performed .", "given the large number and variations of individual ppe components , it would not be feasible to test all potential combinations of ppe commercially available .", "additionally , the recent surge in ppe purchasing in response to evd has caused significant shortages of some ppe components and manufacturers with a history of field use .", "some healthcare facilities have had to develop ppe protocols based on the best use of locally available components ( author , personal communication ) .", "ideally , a ppe ensemble would be evaluated at the local level by the staff anticipated to wear the equipment .", "doffing or removal of ppe after a patient encounter may be a particularly high risk activity .", "appropriate testing of ppe ensembles should include mist type testing to confirm product efficacy in real world scenarios .", "fluorescent agents have been used in training for hand - washing , and environmental decontamination , as well as ppe ensembles , .", "non - toxic and mixed with an appropriate carrier , these agents can potentially mimic the natural contamination which occurs with an infectious agent spread by contact route .", "the purpose of our study was to compare two ppe ensembles used by volunteers with minimal prior training , and to evaluate the feasibility of fluorescent markers as an assessment tool for ppe effectiveness ." ], [ "eight healthcare providers ( six registered nurses and two physicians ) volunteered for this preliminary study .", "the study protocol was approved by the texas tech university health science center institutional review board .", "the participants were randomized to one of two ppe ensembles that meet current ( as of 10/20/14 ) cdc recommendations for ppe .", "one ppe ensemble ( standard ) utilized commercial components that meet current cdc recommendations . the other ppe ensemble ( alternate ) was composed of components already available at local hospitals or retail stores .", "1 ) included a neck - to - ankle coverall with overlying water impermeable surgical gown , knee - length impermeable leggings , and stryker hood ( stryker , kalamazoo , mi , usa ) .", "2 ) ensemble included two plastic gowns ( worn over the front and back of the torso ) , rain - suit pants , a rain - suit hood cut from a rain jacket , ankle length shoe covers , and a plastic spark - shield commonly used for metal working to cover the face . both ppe ensembles utilized double gloving , with the outermost glove a forearm length surgical glove and n-95 masks .", "when complete , both ppe ensembles met the cdc recommendations for ppe and no skin was exposed in either group .", "subjects were then randomized to standard or high volume exposures ( hve ) to simulate fluid splash .", "subjects randomized to standard exposure came into contact with a training mannequin contaminated with fluorescent agents to simulate bodily fluids .", "subjects randomized to hve had standard exposure , but then also had an additional 100 ml of fluorescent agent splashed onto the front torso of their garment .", "the fluorescent agents used included fluorescent powder ( glogerm glogerm co , moab , ut , usa ) , liquid clothes detergent with bleach alternative ( tide proctor & gamble inc .", ", providence , ri , usa ) , and dissolvable fluorescent tablets ( bright dyes orange dye kingscote chemicals , miamisburg , oh , usa ) . a base mixture of 500 ml of liquid detergent , 500 ml of water and three fluorescent tablets was used to create body fluids .", "the base mixture was combined with oatmeal , chocolate powder and crushed cereal to simulate different bodily fluids .", "the testing area was divided into four areas , a ppe donning area ( staging area ) , patient encounter room , ppe doffing area , and a separate dark room for black light photography .", ", participants worked in pairs to perform a series of clinical tasks to care for the evd patient .", " participants were asked to clean the contaminated mannequin , change the mannequin gown , place an automated bp cuff , and check and record the temperature . after completing the tasks ,", "finally , the de - gowned participants were examined under black light for fluorescence indicative of possible contamination .", "an led black light panel , ( chauvet led shadow , model dmx-512 led uv , chauvet lighting , sunrise , florida ) was used to illuminate ." ], [ "one participant in each ppe ensemble arm had evidence of contamination under black light examination ( fig .", "the participant contaminated in the standard arm felt the exposure occurred because of a splash under her hood during hve .", "the trainer felt she may have inadvertently touched a contaminated portion of her leggings and subsequently touched her neck during ppe removal .", "the participant contaminated in the alternate group likely had a gap appear between her front and back gowns while placing a blood pressure cuff .", "this event was not noted at occurrence , but was noticed under black light examination ." ], [ "the contamination in the alternate ppe ensemble arm likely could have been prevented if a larger range of gown sizes was available .", "it may be helpful to consider the body habitus of evd responder when selecting ppe .", "clinical activities as part of the assessment process , as the contamination likely would not have occurred without the provider manipulating the mannequin to place the blood pressure cuff .", "it could be helpful to videotape exercises such as this to better identify the timing of contamination such as that which occurred in the standard arm .", "pin - pointing the conditions of contamination may allow the development of new mechanisms for prevention . in preparation for this exercise", ", we found it notable that taping gloves circumferentially is routinely shown in the lay press , but current guidelines do not make this recommendation . in our study ,", "circumferential taping did not result in contamination , but greatly impaired the ease of ppe removal . on the other hand ,", "a single strip of taped placed longitudinally along the sleeve and glove allowed the glove to be removed with the garment as a seamless piece .", "current recommendations do not comment on hair or jewelry , perhaps making the assumption that these will be taken care of as a matter of course . for our study", ", we did not specifically ask participants to pull back their hair . although we did not note any contamination specific to this ,", "long hair , rings and watches adversely affected the ease of ppe placement and removal and may be an additional risk factor for exposure .", "it may be useful for future recommendations to include stipulations about such accouterments . in this very small study ,", "ppe ensembles made from readily available components ( approximately $ 36 per outfit ) performed as well as the commercial ppe ensemble .", "additionally , it must be borne in mind that increased complexity of ppe may be self - defeating , as the complexity of ppe removal may offset gains made by complexity of ppe .", "it is difficult to assess the actual effectiveness of fluorescents as a proxy for ev exposure in this study .", "it is possible that small volume contamination occurred that was unable to be visualized , and the clinical relevance of such a small exposure is unknown .", "how an exposure on intact skin correlates with risk of subsequent evd development remains to be determined .", "the florescent mixtures used in this study were intended to mimic the mechanical effects of bodily fluids during a patient interaction . in spite of widespread use", ", it is unknown how these mixtures compare with actual bodily fluids considering the unique chemical and biochemical attributes of blood", ". the limitations of this type of testing prevent any assumptions of risk of evd development for a healthcare worker in the field .", "this type of testing could , however , allow comparison of different ppe ensembles and protocols under the assumption that more fluorescent exposures equates to higher risk of evd development .", "another use of this system could be to identify ppe ensembles with lower expense and equivalent testing attributes .", "finally , fluorescent markers can be used for training and proficiency testing of ppe ensembles for evd preparedness and even determine minimal standards of training repetition to achieve proficiency . as fluorescent agents and black lights are readily available for purchase in retail stores and over the internet , it is feasible for testing and training to be performed at a local level with ppe ensembles available at hand . beyond the current evd outbreak , this concept is applicable to training for other infectious diseases , such as methicillin - resistant staphylococcus aureus or multidrug resistant gram - negative bacteria , spread primarily by direct contact .", "the concept could also be used for some pathogens with a significant respiratory mode of transmission , such as pandemic influenza and novel pathogenic corona viruses , which have a recommended contact component of ppe ." ] ]

[ "restoration of structure and function using autologous free fat grafts has always been a challenge .", "the advent and refinement of liposuction and lipoinjection techniques , abundant donor - tissue availability , and relative ease of harvesting have made autologous fat an attractive material for use as soft - tissue filler .", "fat is harvested using either ( 1 ) syringe aspiration or ( 2 ) liposuction aspiration,(1 ) performed with a liposuction pump ( byron medical , tucson , ariz . ) . a standard coleman aspirator cannula ( byron medical ) with a curettage tip", "( 500 mm mercury).(2 ) syringe aspiration is tedious if the amount of fat required is substantial .", "when the liposuction aspiration using a liposuction pump is performed the whole suction system , and the container needs to be kept sterile .", "( figure 1 ) we have devised a novel method of lipoaspiration to overcome these problems .", "the above picture helps us to compare the three modalities of lipoaspiration and also helps us to note the advantage of our technique", "materials required 20 - 50 cc disposable syringe , a piece of 2 2 cm polypropylene mesh , lipoaspiration suction pump ( figure 2a ) . ( a ) materials required for the procedure , ( b ) connected syringe mesh system , ( c ) close - up view of the mesh in liposuction aspiration , a suction pump is used . in our technique , a piece of polypropylene mesh is kept at one end of the lipoaspiration syringe , which is then connected to the suction pump .", "( figure 2b & 2c ) as one syringe fills , it is replaced by another one until the required amount of fat is obtained .", "these syringes are then centrifuged and the emulsified fat thus obtained is used for grafting ( video 1 ) . by using a polypropylene mesh in our technique , we are able to separate the transfusate from the harvested fat during harvesting itself", "in our experience , the fat graft obtained by this method is found to be concentrated and has fewer contaminants .", "this method also has the added advantage of having a short processing time , from harvesting to grafting .", "fat grafting remains unpredictable , and various anecdotal harvesting and preparation techniques have been advocated .", "our method is a more economical and an easier way to extract sterile emulsified fat .", "as it is a simple method , it can be practiced even in day care centers .", "hence , we recommend our technique as a reliable and routine method for extracting sterile emulsified fat in an economical way ." ]

[ "<S> background : successful restoration of structure and function using autologous free fat grafts has remained elusive . </S>", "<S> review of literature shows that various harvesting and preparation techniques have been suggested . </S>", "<S> the goal of these techniques is to obtain greater adipocyte cell survival and consequently more reliable clinical results.materials and methods : in our technique , a piece of mesh is kept at one end of the lipoaspiration syringe , which is then connected to the suction pump . </S>", "<S> as one syringe fills , it is replaced by another one until the required amount of fat is obtained.results:by using a polypropylene mesh in our technique , we can separate the transfusate from the harvested fat graft during harvesting itself . the fat graft thus obtained is dense and concentrated , with fewer impurities.conclusion:hence , we recommend our technique as a reliable method for extracting sterile emulsified fat in an economical way . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "CONCLUSION", "Video available on " ]

[ [ "restoration of structure and function using autologous free fat grafts has always been a challenge .", "the advent and refinement of liposuction and lipoinjection techniques , abundant donor - tissue availability , and relative ease of harvesting have made autologous fat an attractive material for use as soft - tissue filler .", "fat is harvested using either ( 1 ) syringe aspiration or ( 2 ) liposuction aspiration,(1 ) performed with a liposuction pump ( byron medical , tucson , ariz . ) . a standard coleman aspirator cannula ( byron medical ) with a curettage tip", "( 500 mm mercury).(2 ) syringe aspiration is tedious if the amount of fat required is substantial .", "when the liposuction aspiration using a liposuction pump is performed the whole suction system , and the container needs to be kept sterile .", "( figure 1 ) we have devised a novel method of lipoaspiration to overcome these problems .", "the above picture helps us to compare the three modalities of lipoaspiration and also helps us to note the advantage of our technique" ], [ "materials required 20 - 50 cc disposable syringe , a piece of 2 2 cm polypropylene mesh , lipoaspiration suction pump ( figure 2a ) . ( a ) materials required for the procedure , ( b ) connected syringe mesh system , ( c ) close - up view of the mesh in liposuction aspiration , a suction pump is used . in our technique , a piece of polypropylene mesh is kept at one end of the lipoaspiration syringe , which is then connected to the suction pump .", "( figure 2b & 2c ) as one syringe fills , it is replaced by another one until the required amount of fat is obtained .", "these syringes are then centrifuged and the emulsified fat thus obtained is used for grafting ( video 1 ) . by using a polypropylene mesh in our technique , we are able to separate the transfusate from the harvested fat during harvesting itself" ], [ "in our experience , the fat graft obtained by this method is found to be concentrated and has fewer contaminants .", "this method also has the added advantage of having a short processing time , from harvesting to grafting ." ], [ "fat grafting remains unpredictable , and various anecdotal harvesting and preparation techniques have been advocated .", "our method is a more economical and an easier way to extract sterile emulsified fat .", "as it is a simple method , it can be practiced even in day care centers .", "hence , we recommend our technique as a reliable and routine method for extracting sterile emulsified fat in an economical way ." ], [ "" ] ]

[ "turner syndrome ( ts ) is caused by partial or complete monosomy of the x chromosome .", "only a few cases of ts with ischemic stroke have been reported [ 2 , 3 , 4 , 5 ] . in previously reported cases , various arteriopathies affecting the cerebral arteries such as fibromuscular dysplasia , hypoplasia , moyamoya syndrome , and", ", we report a case of ts with multiple embolic infarcts caused by a cryptogenic embolism .", "a 28-year - old woman with ts was referred to our hospital because of abnormal findings on brain magnetic resonance imaging ( mri ) .", "she was diagnosed with ts [ karyotype : 45 , x/46 , x , + mar ] at the age of 16 years due to amenorrhea .", "she denied alcohol , tobacco , or drug use and had no family history of neurologic disorders .", "the patient was afebrile with a blood pressure of 180/130 mm hg and a heart rate of 89 beats / min .", "she was obese with a body mass index of 27.3 kg / m ( 160 cm height , 70 kg weight ) .", "she visited hospital due to sudden - onset severe headache with high blood pressure ( 240/140 mm hg ) .", "she underwent brain computed tomography and mri at the referring hospital for differential diagnosis of thunderclap headache including hypertensive intracranial hemorrhage , subarachnoid hemorrhage , arterial dissection , or reversible cerebral vasoconstriction syndrome .", "brain computed tomography was unremarkable , but diffusion - weighted imaging showed restricted diffusion in multiple lesions involving multiple vascular territories ( fig 1a ) .", "cerebral magnetic resonance angiography ( mra ) ( fig 1b ) and carotid sonography demonstrated no extracranial or intracranial arterial stenosis .", "laboratory tests revealed diabetes mellitus ( fasting plasma glucose 219 mg / dl , hemoglobin a1c 11.0% ) and hyperlipidemia ( low - density lipoprotein 189 mg / dl ) .", "transthoracic echocardiography revealed normal valvular structure and function with a normal left ventricular ejection fraction .", "patent foramen ovale , atrial septal aneurysm , or any aortic pathologies including aortic atheroma were not detected in transesophageal echocardiography .", "the results of the hypercoagulability panel , vasculitis panel , and cerebrospinal fluid examination were all negative .", "aspirin was administered , and medications for hypertension , hyperlipidemia , and diabetes were also started . within few hours after blood pressure control ,", "the patient was discharged from the hospital without any symptoms of stroke , and recurrence was not observed in the 2 years after discharge .", "cerebrovascular arteriopathies such as fibromuscular dysplasia , congenital hypoplasia , moyamoya syndrome , and premature atherosclerosis have been shown to be causes of ischemic stroke in previous case reports . in this case", "furthermore , our patient showed multiple lesions in different vascular territories that were strongly suggestive of embolic stroke .", "atrial fibrillation ( af ) could also be considered a potential source of embolic stroke in ts , as p - wave dispersion , a potential substrate for af , is increased in ts .", "the classical risk factors for developing af include hypertension , diabetes mellitus , and valvular disease , and these are more common in ts than in the general population .", "however , we could not completely exclude the possibility of paroxysmal af , as holter monitoring has a low yield for af detection ( 35% ) .", "other possible cardio - aortic sources of embolism include intracardiac shunts , thrombi , calcifications / vegetations in the mitral valve , or aortic atheroma plaque .", "we sought to identify possible cardio - aortic sources on transthoracic echocardiography , transesophageal echocardiography , and transcranial doppler shunt test ; however , all tests were negative .", "some case reports have described deep venous thrombosis and portal vein thrombosis in ts [ 11 , 12 ] .", "these factors include fibrinogen , d - dimer , factor viii , von willebrand factor , and proteins c and s .", "we also considered the possibility of hypercoagulability as a cause of stroke in this case ; however , levels of d - dimer , fibrinogen , protein c and s were normal .", "the embolic source of stroke in our patient remains unclear ; however , this is the first report of a ts patient with an embolic stroke pattern .", "our case suggests another possible mechanism of stroke in ts that is different from previous case reports .", "ischemic stroke in ts could be due to embolism as well as various cerebral arteriopathies .", "further investigation is needed to more fully understand the various mechanisms of ischemic stroke in ts .", "ischemic stroke in ts could be due to embolism as well as various cerebral arteriopathies , as documented in previous reports .", "further studies are needed to determine the extent of the underlying mechanisms of ischemic stroke in ts .", "this case report has been approved by the institutional review board of the inha university hospital ." ]

[ "<S> only a few cases of turner syndrome ( ts ) with ischemic stroke have been reported . </S>", "<S> various arteriopathies of the cerebral arteries , including fibromuscular dysplasia , congenital hypoplasia , moyamoya syndrome , and premature atherosclerosis have been assumed to be the cause of ischemic stroke in ts . </S>", "<S> there has been no case report of a ts patient presenting with an embolic stroke pattern without any cerebral arteriopathy . a 28-year - old woman with ts </S>", "<S> was referred to our hospital because of abnormal brain magnetic resonance imaging ( mri ) findings . </S>", "<S> she underwent brain mri at the referring hospital because she experienced sudden - onset diffuse headache . </S>", "<S> diffusion - weighted imaging revealed multiple acute embolic infarcts in different vascular territories . </S>", "<S> intracranial and extracranial arterial disease was not detected on cerebral magnetic resonance angiography and carotid sonography . </S>", "<S> embolic source workups , including transthoracic and transesophageal echocardiography , holter monitoring , and transcranial doppler shunt study , were all negative . </S>", "<S> hypercoagulability and vasculitis panels were also negative . </S>", "<S> our patient was diagnosed with cryptogenic embolic stroke . </S>", "<S> this is the first report of a ts patient with an embolic stroke pattern . </S>", "<S> our case shows that ischemic stroke in ts could be due to embolism as well as the various cerebral arteriopathies documented in previous reports . </S>" ]

[ "Introduction", "Case Presentation", "Discussion", "Conclusion", "Statement of Ethics", "Disclosure Statement" ]

[ [ "turner syndrome ( ts ) is caused by partial or complete monosomy of the x chromosome .", "only a few cases of ts with ischemic stroke have been reported [ 2 , 3 , 4 , 5 ] . in previously reported cases , various arteriopathies affecting the cerebral arteries such as fibromuscular dysplasia , hypoplasia , moyamoya syndrome , and", ", we report a case of ts with multiple embolic infarcts caused by a cryptogenic embolism ." ], [ "a 28-year - old woman with ts was referred to our hospital because of abnormal findings on brain magnetic resonance imaging ( mri ) .", "she was diagnosed with ts [ karyotype : 45 , x/46 , x , + mar ] at the age of 16 years due to amenorrhea .", "she denied alcohol , tobacco , or drug use and had no family history of neurologic disorders .", "the patient was afebrile with a blood pressure of 180/130 mm hg and a heart rate of 89 beats / min .", "she was obese with a body mass index of 27.3 kg / m ( 160 cm height , 70 kg weight ) .", "she visited hospital due to sudden - onset severe headache with high blood pressure ( 240/140 mm hg ) .", "she underwent brain computed tomography and mri at the referring hospital for differential diagnosis of thunderclap headache including hypertensive intracranial hemorrhage , subarachnoid hemorrhage , arterial dissection , or reversible cerebral vasoconstriction syndrome .", "brain computed tomography was unremarkable , but diffusion - weighted imaging showed restricted diffusion in multiple lesions involving multiple vascular territories ( fig 1a ) .", "cerebral magnetic resonance angiography ( mra ) ( fig 1b ) and carotid sonography demonstrated no extracranial or intracranial arterial stenosis .", "laboratory tests revealed diabetes mellitus ( fasting plasma glucose 219 mg / dl , hemoglobin a1c 11.0% ) and hyperlipidemia ( low - density lipoprotein 189 mg / dl ) .", "transthoracic echocardiography revealed normal valvular structure and function with a normal left ventricular ejection fraction .", "patent foramen ovale , atrial septal aneurysm , or any aortic pathologies including aortic atheroma were not detected in transesophageal echocardiography .", "the results of the hypercoagulability panel , vasculitis panel , and cerebrospinal fluid examination were all negative .", "aspirin was administered , and medications for hypertension , hyperlipidemia , and diabetes were also started . within few hours after blood pressure control ,", "the patient was discharged from the hospital without any symptoms of stroke , and recurrence was not observed in the 2 years after discharge ." ], [ "cerebrovascular arteriopathies such as fibromuscular dysplasia , congenital hypoplasia , moyamoya syndrome , and premature atherosclerosis have been shown to be causes of ischemic stroke in previous case reports . in this case", "furthermore , our patient showed multiple lesions in different vascular territories that were strongly suggestive of embolic stroke .", "atrial fibrillation ( af ) could also be considered a potential source of embolic stroke in ts , as p - wave dispersion , a potential substrate for af , is increased in ts .", "the classical risk factors for developing af include hypertension , diabetes mellitus , and valvular disease , and these are more common in ts than in the general population .", "however , we could not completely exclude the possibility of paroxysmal af , as holter monitoring has a low yield for af detection ( 35% ) .", "other possible cardio - aortic sources of embolism include intracardiac shunts , thrombi , calcifications / vegetations in the mitral valve , or aortic atheroma plaque .", "we sought to identify possible cardio - aortic sources on transthoracic echocardiography , transesophageal echocardiography , and transcranial doppler shunt test ; however , all tests were negative .", "some case reports have described deep venous thrombosis and portal vein thrombosis in ts [ 11 , 12 ] .", "these factors include fibrinogen , d - dimer , factor viii , von willebrand factor , and proteins c and s .", "we also considered the possibility of hypercoagulability as a cause of stroke in this case ; however , levels of d - dimer , fibrinogen , protein c and s were normal .", "the embolic source of stroke in our patient remains unclear ; however , this is the first report of a ts patient with an embolic stroke pattern .", "our case suggests another possible mechanism of stroke in ts that is different from previous case reports .", "ischemic stroke in ts could be due to embolism as well as various cerebral arteriopathies .", "further investigation is needed to more fully understand the various mechanisms of ischemic stroke in ts ." ], [ "ischemic stroke in ts could be due to embolism as well as various cerebral arteriopathies , as documented in previous reports .", "further studies are needed to determine the extent of the underlying mechanisms of ischemic stroke in ts ." ], [ "this case report has been approved by the institutional review board of the inha university hospital ." ], [ "" ] ]

[ "mating is random when two individuals in a population are just as likely as any other two individuals to mate .", "nonrandom mating results when individuals tend to choose mates with a specific phenotype and the associated genotype(s ) among compatible mates .", "dynamics of nonrandom mating have been studied in sexually dimorphic species in which size , sound , and color often describe the most preferred phenotype . among the microbial eukaryotes , mate - preference has been demonstrated in the yeast saccharomyces cerevisiae , in which the highest amount of pheromone produced defines the most preferred phenotype for the cells of either sex .", "it is largely unclear how mates are chosen in other unicellular organisms . often , unicellular species have more than two sexes , raising an obvious yet previously unanswered question : do more sexes mean more choice , thereby making evolution of mate preference among the sexes inevitable ?", "in other words , is selective mating observed when there is an opportunity to choose between many compatible sexes ?", "for instance , the multiple sexes of a species could form a hierarchy from the best phenotype ( the most preferred sex ) to the least preferred one . alternatively", ", the multiple sexes could be grouped such that sexes within a group mate more frequently with each other than those between groups , resulting in pronounced mating preferences between groups . ", "the ciliate tetrahymena thermophila has seven , self - incompatible mating types ( sexes ) .", "pair formation between cells of any two sexes initiates mating ( conjugation ) and subsequent genetic exchange . although each of the seven sexes identified in t. thermophila readily forms pairs with the other six sexes the frequency of pair formation between sexes , that is , the degree of selective mating , has not been quantified when more than two compatible sexes are simultaneously present in a population offering a choice of mate . in t. thermophila ,", "cells of different sexes engage in physical interactions ( costimulation ) , which last for up to 2 hours prior to pair formation .", "also , the extent of costimulation by one compatible sex does not affect the efficiency ( total amount and the speed ) of pairing with another compatible sex .", "although this shows that costimulation is not sex - specific , the exact molecular interactions that occur during costimulation are still a mystery . also , molecular differences between the seven sexes are unknown .", "current speculation is that a unique glycoprotein ligand - receptor pair may characterize each sex and that the interaction between a sex - specific ligand carried by one partner with its receptor displayed on the surface of the other partner may lead to mating pair formation in t. thermophila [ 68 ] . under this model , affinity between the ligand and receptor", "may determine how likely a sex is chosen as a mate , that is , pairs would more often be formed with the sex whose ligand shares the strongest affinity for the receptor .", "another way in which the molecular affinities may affect the pairing frequencies is through mating kinetics , which affect the rate of pair formation .", "a sex whose ligand and receptor share the strongest affinity may begin pairing with the other sexes early , resulting in nonrandom mating if limited time is available to find a partner ; however , given enough time , the initial differences in pairing frequencies may disappear .", "mating kinetics has been previously documented to contribute to nonrandom mating in yeast [ 10 , 11 ] . here , we tested the null hypothesis that the sexes of t. thermophila mate randomly with respect to sex of a partner .", "our experimental design allowed individuals a limited amount of time to choose between two compatible sexes ( or not to mate at all ) , and we recorded the number of times each compatible sex was chosen as a partner . under the conditions tested , we found that this species mates randomly with respect to sex .", "we obtained the strains cu427.4 , cu428.2 , and cu438.1 ( table 1 ) from the tetrahymena stock center ( cornell university ) .", "these strains have a sensitive phenotype in all drugs because their somatic nuclei contain the drug sensitive allele ( table 1 ) .", "all strains have the mat-2 allele at the sex determination locus [ 12 , 14 , 15 ] .", "we performed a genomic exclusion ( ge ) cross of each strain ( following the methods in [ 13 , 16 ] ) with the strain a*iii , which does not contribute its genome to the progeny .", "we obtained 4 progeny , with mating types ii , iv , v , and vii , from each parental strain ( figure 1(a ) , table 1 ) .", "each progeny inherits the mat-2 allele and expresses one of the seven sexes according to the sex determination pattern of mat-2 .", "the progeny of a strain carry the same drug resistance marker as their parent strain , but in contrast to the parental strain , the progeny express the drug resistant phenotype . ", "stocks of all strains were maintained frozen under liquid nitrogen for the entire duration of the study .", "frozen stocks were thawed , and cells were grown to log phase for 48 hours prior , to use in the experiments .", "we used 2% w / v proteose peptone ( pp ) to grow cells asexually .", "this medium , unlike 2% pp , buffers the pairs against osmotic shock , allowing completion of mating and subsequent asexual growth . to induce mating , all strains", "an overnight culture of klebsiella pneumoniae grown in 2% pp was diluted 1 : 50 with sterile water . in this medium , ciliates grow asexually by feeding on the bacteria and starve upon exhausting the bacteria in about 48 hours .", "we used 2% bp , instead of conventional starvation media ( e.g. , 10 mm tris ) , to mimic starvation in the natural environment . also , 2%bp is the least likely to modify the molecular interactions and influence mating propensities between different sexes .", "mating type ( mt ) tests are used to identify mating type ( sex ) of a new progeny cell produced as a result of a cross .", "self - incompatibility , which is the inability of cells to form pairs with other cells of the same sex , is a key property used in mt tests . when mixed separately with a culture of each of the seven sexes , a clonal culture forms pairs with all but one of the seven mating type tester strains .", "absence of pairing is interpreted as evidence that the progeny culture has the same sex as the tester strain .", "we used this protocol to determine the sex of each progeny strain generated by genomic exclusion ( table 1 ) . \n", "pmr is a dominant structural mutation in the coding region of small subunit of the rdna , and it confers resistance to paromomycin ( 30 ng/l ) .", "chx is a dominant mutation , which causes structural modification of large subunit of rdna , and confers resistance to cyclohexamide ( 15 ng/l ) [ 18 , 19 ] .", "the dominant mutant allele mpr is mapped to chromosome 2r , and confers resistance against 6-methylpurine(25 ng/l ) , which is a structural analog of adenine , and disrupts dna synthesis in sensitive cells . since all drugs are lethal at the respective concentrations , the sensitive phenotype manifests as the presence of dead cells .", "a resistant phenotype is indicated by the presence of log - phase cells after 72 hours of exposure to a single drug or 48 hours of exposure to two drugs applied simultaneously ( this study ) . ", "we verified the stability of drug resistance markers in the parental as well as the progeny strains listed in table 1 .", "parental strains obtained from the stock center carry resistance alleles in their germline nucleus , but sensitive alleles in the somatic nucleus . because alleles in the somatic nucleus determine phenotypes , each parental strain is expected to show sensitivity to all drugs , including the one for which they carry resistance alleles in the germline .", "each progeny strain is expected to be resistant to only one drug , characteristic of the resistance allele in the germline nucleus of its parental strain . from a clonal culture of each strain ( parental or progeny ) , we isolated 48 single cells and grew them asexually for 48 hours .", "each of the 48 cultures was exposed separately to the three drugs , and scored for resistant phenotype .", "this allowed us to determine the frequency with which cells spontaneously acquired or lost resistance to one or more of the drugs . ", "efficiency of a resistance marker is calculated as the frequency of observing a resistant phenotype when expected .", "we performed all pairwise crosses between the 12 progeny strains ( table 1 ) to construct strains heterozygous for every pairwise combination of the markers .", "we picked 48 pairs from each cross , let their cultures grow asexually for 72 hours , and identified which cultures are sexually immature .", "immaturity confirms that the pair mated successfully and exchanged the resistance markers ( see below ) .", "the extent of association between confirmed immaturity and the presence of dual drug resistance provides the efficiency of drug resistance markers in the presence of each other .", "we used mate choice assays to quantify pairing frequency between the sexes ii , iv , v , and vii .", "every assay was performed between three sexes , each sex carrying a different drug - resistant genotype ( figure 1(b ) ) . to create such a triplet of sexes", ", we used one progeny strain of each parent ( table 1 , figure 1 ) .", "thus , drug resistance allowed identification of morphologically indistinguishable sexes and quantification of the number of pairs formed between each of the three sexes .", ", we starved three sexes ( i.e. , the respective progeny strains ) separately and adjusted the density of each to 2 10 cells / ml . we mixed about equal numbers of the three sexes ( 6.6 10 cells per sex ) to a total density of 2 10 cells in 1 ml ( 0.33 ml per sex ) .", "a pair takes between 10 and 12 hours to complete mating and then separates . before separation ,", "the two sexes involved in a pair reciprocally exchange haploid genomes , including the drug - resistance markers . after separation , the partners ( now called progeny ) have dual drug - resistance , characteristic of the sexes involved in the pair . after 24 hours ( t24 ) , mostly single cells are observed , indicating separation of all pairs . for every triplet", ", the mate choice assay was performed under a strict competition regime by providing limited time for choosing a mate .", "we put each pair in an individual drop of 1% pp medium , and grew them asexually for 48 hours .", "we replicated each drop - culture into 2% pp medium containing each drug separately and into the pairwise combinations of the three drugs .", "if the pair mated successfully , its culture will have dual drug resistance characteristic of the two sexes in the pair and is expected to grow in the respective drug combination .", "if mating was unsuccessful , its culture will grow in the presence of two of the individual drugs , but not in any pairwise combination of drugs .", "for every triplet , we also repeated the mate choice assay giving the cells unlimited time to choose a mating partner by testing the dual - drug resistance of single cells isolated at t24 ( data not shown ) .", "in addition to the drug resistance markers , we used immaturity tests to verify whether the isolated pairs successfully completed mating .", "mating partners in a pair separate after about 10 to 12 hours . if genetic exchange was successful , the separated partners are now progeny cells , which are sexually immature and show no pairing with any of the seven mating types until they reach sexual maturity ( ~100 asexual divisions ) . in an unsuccessful mating ,", "the separated partners retain their sexual maturity . hence , soon after separation , they are able to form pairs with a compatible mating type .", "we tested cultures of each pair for immaturity by mating tests with the parental strains within 5 days after picking mating pairs .", "significant differences in viability of mating pairs between the progeny strains would skew our estimates of pairing frequencies .", "thus , we conducted a separate experiment to test how frequently mating pairs between progeny strains died before scoring for dual resistance .", "we made every pairwise cross ( total 36 crosses ) between the 12 progeny strains ( table 1 ) , and isolated 46 pairs at 6.5 hours after mixing the two progeny strains .", "we scored the survivorship as the proportion of viable pairs in 1% pp at 48 hours after - isolation .", "we used these viability data to correct the estimates of the pairing frequencies in mate choice trials .", "we divided the number of pairs observed between two sexes in a mate choice trial by the viability estimate for the corresponding pair of progeny strains ( table s1 available online at doi:10.1155/2012/201921 ) .", "we then normalized the corrected numbers to 96 : the total number of pairs isolated in each mate choice trial .", "this gave us an estimate of the actual number of pairs that likely formed between the two sexes .", "we used these normalized estimates of pairing frequencies in the analyses of mating biases . in every trial conducted on a triplet of sexes , three types of pairs", "can be formed between the three sexes ( figure 1(b ) ) . under random mating , we expect 1/3 of total pairs to be of each type .", "we used goodness - of - fit tests to determine if mating was random within a trial . also , we used mixed - effect anova to analyze if mating was random in a triplet ( i.e. , pooling the data for all trials conducted for a given triplet ) .", "the experimental design ( figure 1(b ) ) contained one fixed factor ( sex ) and one random factor ( drug resistant genotype ) .", "our aim was to investigate whether preferential mating occurs among sexes , when more than two sexes are present in a population .", "hence , we quantified biases in pair formation between four ( of seven ) sexes of t. thermophila .", "we created experimental populations containing three sexes each and used drug resistance markers to identify how often the various sexes formed mating pairs .", "we determined the reliability of the drug resistance alleles as genetic markers and found that they are stable against spontaneous mutations .", "the frequency of mutations conferring a loss or gain of resistance was below the limit of detection for all strains listed in table 1 ( data not shown ) , that is , we found no such spontaneous mutations .", "we also found the efficiency of the markers in conferring resistance to be 100% ( data not shown ) because only those strains that were immature showed dual drug resistance .", "thus , the three resistance alleles are reliable genetic markers in the genetic backgrounds used in our experiments .", "while we did not explicitly test the neutrality of these markers towards fitness ( growth rate ) of the strains , at no step in our mate choice assays was there an opportunity for fertility selection .", "we did not , however , observe any obvious differences between growth rates of the progeny strains with different markers . ", "we measured viability of mating pairs as the proportion of total pairs that survived and grew 48 hours after isolation in growth medium .", "we found that some mating pairs did die more frequently than others ( one - way anova , p < 2.2e 16 ) .", "this indicates that viability of the pairs is contingent upon the progeny strains forming the pairs ( table s1 ) .", "we could not associate the observed mortality with the presence of any particular resistance marker , but rather conclude that viability is a property of the specific genetic backgrounds of the strains . ", "each experimental trial ( figure 1(b ) ) contained three sexes in equal proportion , which ensures equal mating opportunity for each sex .", "thus , under random mating , the three types of pairs that could be formed among the three sexes should be represented at an equal frequency of ~0.33 .", "we tested this null hypothesis for every trial using a goodness - of - fit g - test and found evidence of nonrandom mating ( p < 0.05 ) in many trials but without a strong bias towards any particular sex ( data not shown ) .", "hence , the apparent deviations from random mating may not represent mating preference with respect to sex of an individual but instead reflect an effect of genetic backgrounds on pairing frequencies in a trial .", "the genetic backgrounds used in our experiments represent a randomly chosen subset of the available drug resistant backgrounds ; therefore , any effect of the genetic background on pairing frequencies may be specific to the strains we used , and must be isolated from the effect of the sexes . for every triplet of sexes ,", "we had conducted three trials in which the drug resistant , genetic backgrounds alternated among the three sexes ( figure 1(b ) ) .", "if sex of an individual was driving biases in pairing frequencies observed within a trial , we would expect a consistent bias towards a particular pair of sexes across the three trials irrespective of the associated genetic backgrounds . on the other hand , if the sexes exhibit no preferences , the pairing frequencies across the three trials will reflect random mating with respect to sex . to investigate any consistent effects of sex ( with alternating genetic backgrounds ) on pairing , we analyzed mating biases by pooling across the three trials conducted for a given triplet of sexes .", "a mixed effect anova indicated no significant effect of the sex on the dynamics of pairing in each triplet of the sexes ( table 2 , figure 2 ) .", "this suggests that mating was indeed random with respect to sex of an individual , and that the biases apparent in pairing frequencies in individual trials likely represent an effect of the genetic background rather than preferences among the sexes .", "nonpreferential mating , that is , random mating with respect to sex , was verified in assays conducted at t24 ( data not shown ) .", "nonrandom mating is pervasive in nature , occurring in various ways , and resulting in differing evolutionary consequences . in contrast", ", our results suggest that t. thermophila mate randomly among each other with respect to sex .", "this lack of mate preference was apparent even under strict competition for mates imposed by limited time to find a mating partner , and subsequently when time restriction was removed .", "sexes of t. thermophila are self - incompatible , that is , cells of the same sex never form mating pairs , but each of the seven sexes can pair with all the other ( i.e. , six ) sexes . thus , even though all sexes look morphologically alike , they are somehow able to distinguish self from nonself .", "our analysis suggests that beyond this basic distinction , most sexes of t. thermophila are unable to differentiate the various nonself sexes .", "thus , the choice in this multisex system is binary , that is , it is about whether or not to form a mating pair rather than which of the available sexes to form the pair with .", "our results are consistent with the previous finding that the sexes functionally substitute each other during costimulation the stage immediately prior to mating pair formation .", "although it used a similar experimental design , the previous study did not allow quantification of mating preference of the four sexes .", "our experimental design allowed us to verify that none of the sexes ii , iv , v , and vii show bias for mating among each other when presented simultaneously with a choice between two compatible sexes", ". this could be possible if the ligands characterizing various sexes diverged to be sex - specific but still share a common receptor , enabling the sexes to replace each other functionally .", "alternatively , random mating found in our study may reflect presence of a unique receptor for each sex - specific ligand and thus lack of competition between the sexes . ", "neither the molecular differences between the sexes nor the molecular interactions responsible for pairing are known in t. thermophila .", "interaction between a sex - specific ligand on the surface of one sex and the receptor on the surface of the other sex is hypothesized to be the underlying mechanism of pairing between sexes , but awaits empirical support .", "if pairing indeed results from such molecular interactions , then we suggest that affinities between the sex - specific ligands and receptor(s ) may be of equal strength , leading to random mating between the sexes .", "it is generally observed in other unicellular species that the affinities between sex - specific molecules rarely determine the intraspecific mating propensities .", "for instance , the unique pheromones secreted by the two mating types in the yeast saccharomyces cerevisiae bind to specific receptors displayed by the opposite mating type .", "variation in the amount of pheromone produced , but not in the affinity to the receptor was found to be the basis of nonrandom mating . in the ciliate euplotes raikovi , sex - specific pheromones and receptors characterize the multiple sexes , which show variable mating interactions among each other beyond self-/non - self - distinction .", "biased mating among the multiple sexes is a function of amount of pheromone produced such that higher secretion translates into higher mating success for any given sex .", "thus , factors other than molecular affinities largely determine mating propensities among sexes in a population of this species .", "also , affinities between sex - specific molecules play a minimal role in interspecific mating interactions . for instance , although recognition of opposite mating type occurs and interspecific hybrids sometimes form between the two closely related yeasts s. cerevisiae and s. paradoxus , mating takes place preferentially with conspecifics and is more efficient and frequent [ 10 , 23 ] . the overall genetic background rather than species - specific pheromone - receptor affinities are largely responsible for mating selectively within species .", "thus , unlike in anisogamous plants and animals , prezygotic reproductive barriers may rarely occur at the pheromone / receptor level in isogamous unicellular eukaryotes , allowing extensive diversification of mating types within species . ", "for example , pairing with the strain 5c always resulted in low viability ( table s1 ) for reasons that are yet unclear and may involve genetic incompatibilities , which could be investigated in future studies .", "although mating is random with respect to sex , the effect of genetic background on viability implies that some sexes may contribute to the gene pool more than the others , contingent upon the genetic background they are associated with .", "the effect of a sex and its genetic background on mating propensities could be explored in future studies using a full factorial design involving six ( instead of three ) trials for every triplet of sexes ( figure 1(b ) ) .", "natural populations of t. thermophila are likely patchy , thus due to self - incompatibility and the inability to switch sexes , finding a compatible mate may be difficult ( [ 2426 ] , p. doerder , pers .", ". random mating with respect to sex creates the highest possible opportunity for mating , contingent upon the sex ratio , the relative frequencies of multiple sexes , in the population .", "high frequency of sex has been documented in natural populations of t. thermophila [ 2527 ] .", "thus , when searching for mates is costly , random mating with respect to sex is likely to be advantageous by avoiding further delays in initiating mating .", "it is unclear , however , why self - incompatibility would be maintained under such a scenario , though it is possible that high levels of inbreeding depression select for the maintenance of self - incompatibility .", "transitions to self - compatibility in arabidopsis have been linked to mate - limiting conditions .", "mating between cells of the same sex and other selfing strategies , including autogamy , are also observed in many species of ciliates and may reflect ways to avoid the cost of finding a mate [ 7 , 29 ] .", "alternatively , presence of multiple sexes may compensate , at least partially , for the cost of self - incompatibility .", "very few data exist to test the correlation between the number of sexes and the capacity to transition to self - compatibility in ciliates .", "the demonstration of random mating with respect to sex verifies a key assumption of our model on sex ratio evolution in t. thermophila .", "although violating the assumption of random mating would not change the equilibria predicted in this model , it would change the approach ( time and the trajectory ) of the populations to those equilibria .", "we studied mating preferences of the four sexes specified by a single sex determining allele ( mat-2 ) .", "the analysis presented here does not rule out the possibility of finding nonrandom mating between sexes specified by different mat alleles , which may lead to assortative mating with respect to the genotypes at the mat locus in t. thermophila .", "discovery of mating bias among sexes in t. thermophila could have delivered insights into affinities between sexes at the molecular level and facilitated predictions about the structural differences between the sex - specific molecules , which are currently unknown .", "our results emphasize the need to decipher the molecular machinery that enables random mating among multiple sexes ." ]

[ "<S> ciliate mating systems are highly diversified , providing unique opportunities to study sexual differentiation and its implications for mating dynamics . many species of ciliates have multiple ( > 2 ) sexes . </S>", "<S> more sexes may mean more choice and an opportunity for evolution of preferential mating . </S>", "<S> we asked if the multiple sexes of the ciliate tetrahymena thermophila mate preferentially among each other . </S>", "<S> we quantified pairing frequencies among four sexes of t. thermophila using experiments that allowed the sexes to compete as mating partners . </S>", "<S> we found that all sexes mated equally frequently among each other , that is , we found no evidence of preferential mating with respect to sex . </S>", "<S> this suggests that the mate choice in this ciliate is binary , between whether to form a pair or not and , in this regard , sex facilitates only self-/non - self - distinction . </S>", "<S> thus , presence of multiple sexes does not necessarily result in the evolution of mating bias , which could decrease the maximum amount of mating that would otherwise be possible in a population . </S>", "<S> our result of random mating verifies a key assumption in the theoretical model of sex ratio evolution in t. thermophila . </S>", "<S> investigation into molecular differences between the sexes will be necessary to reveal the mechanistic basis of random mating among them . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion" ]

[ [ "mating is random when two individuals in a population are just as likely as any other two individuals to mate .", "nonrandom mating results when individuals tend to choose mates with a specific phenotype and the associated genotype(s ) among compatible mates .", "dynamics of nonrandom mating have been studied in sexually dimorphic species in which size , sound , and color often describe the most preferred phenotype . among the microbial eukaryotes , mate - preference has been demonstrated in the yeast saccharomyces cerevisiae , in which the highest amount of pheromone produced defines the most preferred phenotype for the cells of either sex .", "it is largely unclear how mates are chosen in other unicellular organisms . often , unicellular species have more than two sexes , raising an obvious yet previously unanswered question : do more sexes mean more choice , thereby making evolution of mate preference among the sexes inevitable ?", "in other words , is selective mating observed when there is an opportunity to choose between many compatible sexes ?", "for instance , the multiple sexes of a species could form a hierarchy from the best phenotype ( the most preferred sex ) to the least preferred one . alternatively", ", the multiple sexes could be grouped such that sexes within a group mate more frequently with each other than those between groups , resulting in pronounced mating preferences between groups . ", "the ciliate tetrahymena thermophila has seven , self - incompatible mating types ( sexes ) .", "pair formation between cells of any two sexes initiates mating ( conjugation ) and subsequent genetic exchange . although each of the seven sexes identified in t. thermophila readily forms pairs with the other six sexes the frequency of pair formation between sexes , that is , the degree of selective mating , has not been quantified when more than two compatible sexes are simultaneously present in a population offering a choice of mate . in t. thermophila ,", "cells of different sexes engage in physical interactions ( costimulation ) , which last for up to 2 hours prior to pair formation .", "also , the extent of costimulation by one compatible sex does not affect the efficiency ( total amount and the speed ) of pairing with another compatible sex .", "although this shows that costimulation is not sex - specific , the exact molecular interactions that occur during costimulation are still a mystery . also , molecular differences between the seven sexes are unknown .", "current speculation is that a unique glycoprotein ligand - receptor pair may characterize each sex and that the interaction between a sex - specific ligand carried by one partner with its receptor displayed on the surface of the other partner may lead to mating pair formation in t. thermophila [ 68 ] . under this model , affinity between the ligand and receptor", "may determine how likely a sex is chosen as a mate , that is , pairs would more often be formed with the sex whose ligand shares the strongest affinity for the receptor .", "another way in which the molecular affinities may affect the pairing frequencies is through mating kinetics , which affect the rate of pair formation .", "a sex whose ligand and receptor share the strongest affinity may begin pairing with the other sexes early , resulting in nonrandom mating if limited time is available to find a partner ; however , given enough time , the initial differences in pairing frequencies may disappear .", "mating kinetics has been previously documented to contribute to nonrandom mating in yeast [ 10 , 11 ] . here , we tested the null hypothesis that the sexes of t. thermophila mate randomly with respect to sex of a partner .", "our experimental design allowed individuals a limited amount of time to choose between two compatible sexes ( or not to mate at all ) , and we recorded the number of times each compatible sex was chosen as a partner . under the conditions tested , we found that this species mates randomly with respect to sex ." ], [ "we obtained the strains cu427.4 , cu428.2 , and cu438.1 ( table 1 ) from the tetrahymena stock center ( cornell university ) .", "these strains have a sensitive phenotype in all drugs because their somatic nuclei contain the drug sensitive allele ( table 1 ) .", "all strains have the mat-2 allele at the sex determination locus [ 12 , 14 , 15 ] .", "we performed a genomic exclusion ( ge ) cross of each strain ( following the methods in [ 13 , 16 ] ) with the strain a*iii , which does not contribute its genome to the progeny .", "we obtained 4 progeny , with mating types ii , iv , v , and vii , from each parental strain ( figure 1(a ) , table 1 ) .", "each progeny inherits the mat-2 allele and expresses one of the seven sexes according to the sex determination pattern of mat-2 .", "the progeny of a strain carry the same drug resistance marker as their parent strain , but in contrast to the parental strain , the progeny express the drug resistant phenotype . ", "stocks of all strains were maintained frozen under liquid nitrogen for the entire duration of the study .", "frozen stocks were thawed , and cells were grown to log phase for 48 hours prior , to use in the experiments .", "we used 2% w / v proteose peptone ( pp ) to grow cells asexually .", "this medium , unlike 2% pp , buffers the pairs against osmotic shock , allowing completion of mating and subsequent asexual growth . to induce mating , all strains", "an overnight culture of klebsiella pneumoniae grown in 2% pp was diluted 1 : 50 with sterile water . in this medium , ciliates grow asexually by feeding on the bacteria and starve upon exhausting the bacteria in about 48 hours .", "we used 2% bp , instead of conventional starvation media ( e.g. , 10 mm tris ) , to mimic starvation in the natural environment . also , 2%bp is the least likely to modify the molecular interactions and influence mating propensities between different sexes .", "mating type ( mt ) tests are used to identify mating type ( sex ) of a new progeny cell produced as a result of a cross .", "self - incompatibility , which is the inability of cells to form pairs with other cells of the same sex , is a key property used in mt tests . when mixed separately with a culture of each of the seven sexes , a clonal culture forms pairs with all but one of the seven mating type tester strains .", "absence of pairing is interpreted as evidence that the progeny culture has the same sex as the tester strain .", "we used this protocol to determine the sex of each progeny strain generated by genomic exclusion ( table 1 ) . \n", "pmr is a dominant structural mutation in the coding region of small subunit of the rdna , and it confers resistance to paromomycin ( 30 ng/l ) .", "chx is a dominant mutation , which causes structural modification of large subunit of rdna , and confers resistance to cyclohexamide ( 15 ng/l ) [ 18 , 19 ] .", "the dominant mutant allele mpr is mapped to chromosome 2r , and confers resistance against 6-methylpurine(25 ng/l ) , which is a structural analog of adenine , and disrupts dna synthesis in sensitive cells . since all drugs are lethal at the respective concentrations , the sensitive phenotype manifests as the presence of dead cells .", "a resistant phenotype is indicated by the presence of log - phase cells after 72 hours of exposure to a single drug or 48 hours of exposure to two drugs applied simultaneously ( this study ) . ", "we verified the stability of drug resistance markers in the parental as well as the progeny strains listed in table 1 .", "parental strains obtained from the stock center carry resistance alleles in their germline nucleus , but sensitive alleles in the somatic nucleus . because alleles in the somatic nucleus determine phenotypes , each parental strain is expected to show sensitivity to all drugs , including the one for which they carry resistance alleles in the germline .", "each progeny strain is expected to be resistant to only one drug , characteristic of the resistance allele in the germline nucleus of its parental strain . from a clonal culture of each strain ( parental or progeny ) , we isolated 48 single cells and grew them asexually for 48 hours .", "each of the 48 cultures was exposed separately to the three drugs , and scored for resistant phenotype .", "this allowed us to determine the frequency with which cells spontaneously acquired or lost resistance to one or more of the drugs . ", "efficiency of a resistance marker is calculated as the frequency of observing a resistant phenotype when expected .", "we performed all pairwise crosses between the 12 progeny strains ( table 1 ) to construct strains heterozygous for every pairwise combination of the markers .", "we picked 48 pairs from each cross , let their cultures grow asexually for 72 hours , and identified which cultures are sexually immature .", "immaturity confirms that the pair mated successfully and exchanged the resistance markers ( see below ) .", "the extent of association between confirmed immaturity and the presence of dual drug resistance provides the efficiency of drug resistance markers in the presence of each other .", "we used mate choice assays to quantify pairing frequency between the sexes ii , iv , v , and vii .", "every assay was performed between three sexes , each sex carrying a different drug - resistant genotype ( figure 1(b ) ) . to create such a triplet of sexes", ", we used one progeny strain of each parent ( table 1 , figure 1 ) .", "thus , drug resistance allowed identification of morphologically indistinguishable sexes and quantification of the number of pairs formed between each of the three sexes .", ", we starved three sexes ( i.e. , the respective progeny strains ) separately and adjusted the density of each to 2 10 cells / ml . we mixed about equal numbers of the three sexes ( 6.6 10 cells per sex ) to a total density of 2 10 cells in 1 ml ( 0.33 ml per sex ) .", "a pair takes between 10 and 12 hours to complete mating and then separates . before separation ,", "the two sexes involved in a pair reciprocally exchange haploid genomes , including the drug - resistance markers . after separation , the partners ( now called progeny ) have dual drug - resistance , characteristic of the sexes involved in the pair . after 24 hours ( t24 ) , mostly single cells are observed , indicating separation of all pairs . for every triplet", ", the mate choice assay was performed under a strict competition regime by providing limited time for choosing a mate .", "we put each pair in an individual drop of 1% pp medium , and grew them asexually for 48 hours .", "we replicated each drop - culture into 2% pp medium containing each drug separately and into the pairwise combinations of the three drugs .", "if the pair mated successfully , its culture will have dual drug resistance characteristic of the two sexes in the pair and is expected to grow in the respective drug combination .", "if mating was unsuccessful , its culture will grow in the presence of two of the individual drugs , but not in any pairwise combination of drugs .", "for every triplet , we also repeated the mate choice assay giving the cells unlimited time to choose a mating partner by testing the dual - drug resistance of single cells isolated at t24 ( data not shown ) .", "in addition to the drug resistance markers , we used immaturity tests to verify whether the isolated pairs successfully completed mating .", "mating partners in a pair separate after about 10 to 12 hours . if genetic exchange was successful , the separated partners are now progeny cells , which are sexually immature and show no pairing with any of the seven mating types until they reach sexual maturity ( ~100 asexual divisions ) . in an unsuccessful mating ,", "the separated partners retain their sexual maturity . hence , soon after separation , they are able to form pairs with a compatible mating type .", "we tested cultures of each pair for immaturity by mating tests with the parental strains within 5 days after picking mating pairs .", "significant differences in viability of mating pairs between the progeny strains would skew our estimates of pairing frequencies .", "thus , we conducted a separate experiment to test how frequently mating pairs between progeny strains died before scoring for dual resistance .", "we made every pairwise cross ( total 36 crosses ) between the 12 progeny strains ( table 1 ) , and isolated 46 pairs at 6.5 hours after mixing the two progeny strains .", "we scored the survivorship as the proportion of viable pairs in 1% pp at 48 hours after - isolation .", "we used these viability data to correct the estimates of the pairing frequencies in mate choice trials .", "we divided the number of pairs observed between two sexes in a mate choice trial by the viability estimate for the corresponding pair of progeny strains ( table s1 available online at doi:10.1155/2012/201921 ) .", "we then normalized the corrected numbers to 96 : the total number of pairs isolated in each mate choice trial .", "this gave us an estimate of the actual number of pairs that likely formed between the two sexes .", "we used these normalized estimates of pairing frequencies in the analyses of mating biases . in every trial conducted on a triplet of sexes , three types of pairs", "can be formed between the three sexes ( figure 1(b ) ) . under random mating , we expect 1/3 of total pairs to be of each type .", "we used goodness - of - fit tests to determine if mating was random within a trial . also , we used mixed - effect anova to analyze if mating was random in a triplet ( i.e. , pooling the data for all trials conducted for a given triplet ) .", "the experimental design ( figure 1(b ) ) contained one fixed factor ( sex ) and one random factor ( drug resistant genotype ) ." ], [ "our aim was to investigate whether preferential mating occurs among sexes , when more than two sexes are present in a population .", "hence , we quantified biases in pair formation between four ( of seven ) sexes of t. thermophila .", "we created experimental populations containing three sexes each and used drug resistance markers to identify how often the various sexes formed mating pairs .", "we determined the reliability of the drug resistance alleles as genetic markers and found that they are stable against spontaneous mutations .", "the frequency of mutations conferring a loss or gain of resistance was below the limit of detection for all strains listed in table 1 ( data not shown ) , that is , we found no such spontaneous mutations .", "we also found the efficiency of the markers in conferring resistance to be 100% ( data not shown ) because only those strains that were immature showed dual drug resistance .", "thus , the three resistance alleles are reliable genetic markers in the genetic backgrounds used in our experiments .", "while we did not explicitly test the neutrality of these markers towards fitness ( growth rate ) of the strains , at no step in our mate choice assays was there an opportunity for fertility selection .", "we did not , however , observe any obvious differences between growth rates of the progeny strains with different markers . ", "we measured viability of mating pairs as the proportion of total pairs that survived and grew 48 hours after isolation in growth medium .", "we found that some mating pairs did die more frequently than others ( one - way anova , p < 2.2e 16 ) .", "this indicates that viability of the pairs is contingent upon the progeny strains forming the pairs ( table s1 ) .", "we could not associate the observed mortality with the presence of any particular resistance marker , but rather conclude that viability is a property of the specific genetic backgrounds of the strains . ", "each experimental trial ( figure 1(b ) ) contained three sexes in equal proportion , which ensures equal mating opportunity for each sex .", "thus , under random mating , the three types of pairs that could be formed among the three sexes should be represented at an equal frequency of ~0.33 .", "we tested this null hypothesis for every trial using a goodness - of - fit g - test and found evidence of nonrandom mating ( p < 0.05 ) in many trials but without a strong bias towards any particular sex ( data not shown ) .", "hence , the apparent deviations from random mating may not represent mating preference with respect to sex of an individual but instead reflect an effect of genetic backgrounds on pairing frequencies in a trial .", "the genetic backgrounds used in our experiments represent a randomly chosen subset of the available drug resistant backgrounds ; therefore , any effect of the genetic background on pairing frequencies may be specific to the strains we used , and must be isolated from the effect of the sexes . for every triplet of sexes ,", "we had conducted three trials in which the drug resistant , genetic backgrounds alternated among the three sexes ( figure 1(b ) ) .", "if sex of an individual was driving biases in pairing frequencies observed within a trial , we would expect a consistent bias towards a particular pair of sexes across the three trials irrespective of the associated genetic backgrounds . on the other hand , if the sexes exhibit no preferences , the pairing frequencies across the three trials will reflect random mating with respect to sex . to investigate any consistent effects of sex ( with alternating genetic backgrounds ) on pairing , we analyzed mating biases by pooling across the three trials conducted for a given triplet of sexes .", "a mixed effect anova indicated no significant effect of the sex on the dynamics of pairing in each triplet of the sexes ( table 2 , figure 2 ) .", "this suggests that mating was indeed random with respect to sex of an individual , and that the biases apparent in pairing frequencies in individual trials likely represent an effect of the genetic background rather than preferences among the sexes .", "nonpreferential mating , that is , random mating with respect to sex , was verified in assays conducted at t24 ( data not shown ) ." ], [ "nonrandom mating is pervasive in nature , occurring in various ways , and resulting in differing evolutionary consequences . in contrast", ", our results suggest that t. thermophila mate randomly among each other with respect to sex .", "this lack of mate preference was apparent even under strict competition for mates imposed by limited time to find a mating partner , and subsequently when time restriction was removed .", "sexes of t. thermophila are self - incompatible , that is , cells of the same sex never form mating pairs , but each of the seven sexes can pair with all the other ( i.e. , six ) sexes . thus , even though all sexes look morphologically alike , they are somehow able to distinguish self from nonself .", "our analysis suggests that beyond this basic distinction , most sexes of t. thermophila are unable to differentiate the various nonself sexes .", "thus , the choice in this multisex system is binary , that is , it is about whether or not to form a mating pair rather than which of the available sexes to form the pair with .", "our results are consistent with the previous finding that the sexes functionally substitute each other during costimulation the stage immediately prior to mating pair formation .", "although it used a similar experimental design , the previous study did not allow quantification of mating preference of the four sexes .", "our experimental design allowed us to verify that none of the sexes ii , iv , v , and vii show bias for mating among each other when presented simultaneously with a choice between two compatible sexes", ". this could be possible if the ligands characterizing various sexes diverged to be sex - specific but still share a common receptor , enabling the sexes to replace each other functionally .", "alternatively , random mating found in our study may reflect presence of a unique receptor for each sex - specific ligand and thus lack of competition between the sexes . ", "neither the molecular differences between the sexes nor the molecular interactions responsible for pairing are known in t. thermophila .", "interaction between a sex - specific ligand on the surface of one sex and the receptor on the surface of the other sex is hypothesized to be the underlying mechanism of pairing between sexes , but awaits empirical support .", "if pairing indeed results from such molecular interactions , then we suggest that affinities between the sex - specific ligands and receptor(s ) may be of equal strength , leading to random mating between the sexes .", "it is generally observed in other unicellular species that the affinities between sex - specific molecules rarely determine the intraspecific mating propensities .", "for instance , the unique pheromones secreted by the two mating types in the yeast saccharomyces cerevisiae bind to specific receptors displayed by the opposite mating type .", "variation in the amount of pheromone produced , but not in the affinity to the receptor was found to be the basis of nonrandom mating . in the ciliate euplotes raikovi , sex - specific pheromones and receptors characterize the multiple sexes , which show variable mating interactions among each other beyond self-/non - self - distinction .", "biased mating among the multiple sexes is a function of amount of pheromone produced such that higher secretion translates into higher mating success for any given sex .", "thus , factors other than molecular affinities largely determine mating propensities among sexes in a population of this species .", "also , affinities between sex - specific molecules play a minimal role in interspecific mating interactions . for instance , although recognition of opposite mating type occurs and interspecific hybrids sometimes form between the two closely related yeasts s. cerevisiae and s. paradoxus , mating takes place preferentially with conspecifics and is more efficient and frequent [ 10 , 23 ] . the overall genetic background rather than species - specific pheromone - receptor affinities are largely responsible for mating selectively within species .", "thus , unlike in anisogamous plants and animals , prezygotic reproductive barriers may rarely occur at the pheromone / receptor level in isogamous unicellular eukaryotes , allowing extensive diversification of mating types within species . ", "for example , pairing with the strain 5c always resulted in low viability ( table s1 ) for reasons that are yet unclear and may involve genetic incompatibilities , which could be investigated in future studies .", "although mating is random with respect to sex , the effect of genetic background on viability implies that some sexes may contribute to the gene pool more than the others , contingent upon the genetic background they are associated with .", "the effect of a sex and its genetic background on mating propensities could be explored in future studies using a full factorial design involving six ( instead of three ) trials for every triplet of sexes ( figure 1(b ) ) .", "natural populations of t. thermophila are likely patchy , thus due to self - incompatibility and the inability to switch sexes , finding a compatible mate may be difficult ( [ 2426 ] , p. doerder , pers .", ". random mating with respect to sex creates the highest possible opportunity for mating , contingent upon the sex ratio , the relative frequencies of multiple sexes , in the population .", "high frequency of sex has been documented in natural populations of t. thermophila [ 2527 ] .", "thus , when searching for mates is costly , random mating with respect to sex is likely to be advantageous by avoiding further delays in initiating mating .", "it is unclear , however , why self - incompatibility would be maintained under such a scenario , though it is possible that high levels of inbreeding depression select for the maintenance of self - incompatibility .", "transitions to self - compatibility in arabidopsis have been linked to mate - limiting conditions .", "mating between cells of the same sex and other selfing strategies , including autogamy , are also observed in many species of ciliates and may reflect ways to avoid the cost of finding a mate [ 7 , 29 ] .", "alternatively , presence of multiple sexes may compensate , at least partially , for the cost of self - incompatibility .", "very few data exist to test the correlation between the number of sexes and the capacity to transition to self - compatibility in ciliates .", "the demonstration of random mating with respect to sex verifies a key assumption of our model on sex ratio evolution in t. thermophila .", "although violating the assumption of random mating would not change the equilibria predicted in this model , it would change the approach ( time and the trajectory ) of the populations to those equilibria .", "we studied mating preferences of the four sexes specified by a single sex determining allele ( mat-2 ) .", "the analysis presented here does not rule out the possibility of finding nonrandom mating between sexes specified by different mat alleles , which may lead to assortative mating with respect to the genotypes at the mat locus in t. thermophila .", "discovery of mating bias among sexes in t. thermophila could have delivered insights into affinities between sexes at the molecular level and facilitated predictions about the structural differences between the sex - specific molecules , which are currently unknown .", "our results emphasize the need to decipher the molecular machinery that enables random mating among multiple sexes ." ] ]

[ "stroke , with a high prevalence and long - term disabilities , is a major health problem in the \n world1 , 2 , and it was reported that about two million people suffer from \n stroke each year3 .", "impairments including \n loss of strength , sensation , and coordination abilities , which result in walking \n difficulties , balance disorders , and limb function disturbance , occur in 7080% of stroke \n patients4 .", "this affects the life quality \n of patients adversely and leads to a heavy economic burden on society .", "subsequently , \n effective rehabilitation interventions to optimize recovery in stroke patients have received \n high attention . in the treatment of balance and gait dysfunction ,", "exercise therapy is regarded as one of \n the commonly used methods that bring greater benefits in physical function for stroke \n patients5 .", "the effect of exercises on \n balance and gait dysfunction in stroke patients has been confirmed by many clinical \n studies3 , 6 , \n 7 .", "in addition , core stability exercises \n have been widely carried out in many fields , including medical rehabilitation , sports \n training , and medical care .", "core stability exercises were shown to improve dynamic standing \n balance , functional autonomy , static balance , flexibility , and stability8 . in recent years , core stability exercises have been reported to improve the rehabilitation \n effect in stroke patients . in a study of 20 stroke patients ,", "the control group underwent \n standard exercise therapy , while the experiment group underwent both the core \n stability - enhancing exercise and standard exercise therapy simultaneously .", "after 4 weeks , yu \n found that the mean trunk impairment scale score and muscle activity of the lower trunk \n increased in the experiment group significantly9 . in study by cabanas - valdes , 80 patients were randomly assigned to \n an experimental group and a control group , both groups underwent conventional therapy \n including exercises , and the experimental group performed core stability exercises .", "the \n results showed that core stability exercises improve the efficacy of conventional \n exercises10 .", "although these studies \n confirmed the effect of core stability exercises in the rehabilitation of stroke patients , \n few comparative studies have been carried out to compare the effect of core stability \n exercises and conventional exercises . which is better in improving the stability and balance \n of stroke patients , core stability or conventional exercises ?", "therefore , a prospective study including 40 participants was carried out in the department \n of neurology of yidu central hospital of weifang between january 2014 and february 2015 .", "the \n objective of the study was to determine which is better in the rehabilitation of stroke \n patients , core stability exercises or conventional exercises .", "participants with stroke - induced hemiplegia were recruited from the department of neurology \n of yidu central hospital of weifang between january 2014 and february 2015 .", "the diagnosis \n and lesion side of these participants were confirmed by history evaluation , physical \n examination , brain imaging , and examination of their medical records .", "the inclusion \n criteria9 were ability to walk more than \n 32 feet , duration of disorder > 6 months , no musculoskeletal problems , absence of any \n cardiac disorders , complete understanding of this research , and ability to communicate .", "the \n included participants were randomly divided into either an experimental or control group by \n a random computer - generated sequence .", "the randomization was managed by a physician who was \n not involved in the study .", "all the included patients \n signed an informed consent form , and the study was approved by the ethics committee of our \n hospital .", "the patients in the control group performed conventional exercises including limb \n stretching , passive mobilization of joints , walking between parallel bars , and occupational \n therapy11 .", "the patients in the \n experiment group performed core stability exercises including the plank , side plank , bridge , \n straight leg raise , and modified push - up12 . before the beginning of the study ,", "the patients were given \n individual instruction by the researchers . during the exercises , physicians provide \n necessary assistance to help the patients in executing the exercises .", "the exercises in both \n groups were performed for one hour per day , six times a week for six weeks . the modified barthel index ( mbi)13 and \n berg balance scale ( bbs)13 were used to \n assess the clinical outcomes of the two groups .", "the mbi , which demonstrates the ability of \n patients to deal with daily activities , consists of ten items including personal hygiene , \n taking a bath alone , eating , using the bathroom , ascending stairs , dressing , defecating , \n controlling urine , walking or using a wheelchair , and moving to a chair or bed .", "each item is \n rated on a scale of 5 ranging from independent conduct to impossible to conduct .", "the bbs , \n which can evaluate static and dynamic balance abilities of patients , consists of 14 items \n covering functional tasks in three domains , sitting , standing , and changing posture , that \n are scored on a scale of 04 .", "ibm spss statistics 21.0 ( ibm corp . , armonk , ny , usa ) was used for statistical analysis , \n and p < 0.05 was considered significant .", "the differences in the baseline data including \n age , gender , and disease course were compared using the t test or test . \n", "in the current study , 40 patients with stroke - induced hemiplegia were included and divided \n randomly into experimental and control groups , with 20 participants in each group at the \n beginning of the study .", "the baseline clinical data including age , gender , disease course , \n bbs , and mbi were recorded .", "there was no significant difference in baseline data between the \n two groups ( p>0.05 ) .", "the patients in the control group performed conventional exercises \n for six weeks and those in the experimental group carried out core stability exercises for \n six weeks .", "three patients in the experimental group and two in the control group withdrew from the \n study two weeks after treatment , and in total thirty - five patients completed the training . \n", "the final results showed that , compared with the baseline , the mean mbi scores of the \n experimental and control groups increased significantly from 53.5 and 55.1 to 66.8 and 61.2 \n ( p<0.05 ) , respectively , and that the mean bbs scores increased significantly from 30.2 \n and 31.9 to 38.6 and 35.7 ( p<0.05 ) , respectively .", "in addition , mbi scores were \n significantly lower ( p<0.05 ) in the control group than in the experimental group .", "the bbs \n scores in the control group were relatively lower than those in the experimental group , but \n there was no significant difference between the two groups ( p>0.05 ) .", "a prospective study was carried out to compare the effect of core stability exercises and \n conventional exercises on patients with stroke - induced hemiplegia . to the best of our \n knowledge", "the core , described as a box in the trunk with the \n abdominals , paraspinals , and gluteals ; diaphragm and pelvic floor ; and hip girdle \n musculature serving as the front wall , back wall , roof , and bottom , respectively , makes up \n the largest part of the body and plays an important role in stabilizing the body and \n controlling movement of daily activities9 . \n", "core stability is regarded as a prerequisite for maintaining proper posture of the lumbar \n and pelvic regions during sports activities9 . as a result ,", "core stability exercises have many advantages in the \n field of rehabilitation and can improve the activation and cooperative contractions of \n abdominal and multifidus muscles , facilitating the function and movement of the limbs9 , 12 .", "in the current study , the mbi and bbs scores after treatment were significantly increased \n in both the experimental and control groups when compared with those before treatment .", "this \n indicates that both core stability exercises and conventional exercises can improve the \n abilities of patients to deal with daily activities and control body balance , which confirms \n the viewpoints of many published studies9 , 10 .", "in addition , after treatment , the mbi scores were significantly lower in the control group \n than those in the experimental group , and the bbs scores in the control group were also \n lower than those in the experimental group .", "the outcomes demonstrate that compared with \n conventional exercises , core stability exercises can result in better effectiveness in \n patients with stroke - induced hemiplegia . however , in terms of comparison of the bbs scores \n at the end of treatment , although the mean bbs score in the experimental group was larger \n than that in the control group , there was no significant difference between the two groups . \n", "such a condition may be attributed to the relatively small sample size , and in a \n larger - scale study , the difference between the two groups may be significant .", "consequently , \n the outcomes in the present study can demonstrate that core stability exercises present with \n better effectiveness than conventional exercises .", "first , the sample size was small , and a large - scale \n clinical study may be better in a comparative study .", "second , the exercises results were \n evaluated using self - reported measures of function instead of an objective measurement , and \n this may have had some adverse influence on the final outcomes .", "third , there was a lack of \n long - term follow - up in the study , and it is unclear whether the effectiveness of core \n stability exercises will decrease in patients with hemiplegia with time ." ]

[ "<S> [ purpose ] the aim of this study was to determine which is better in the rehabilitation of \n stroke patients , core stability exercises or conventional exercises . [ subjects and \n methods ] forty participants with hemiplegia were recruited in the department of neurology \n of yidu central hospital of weifang between january 2014 and february 2015 and randomly \n divided into either an experimental or control group . </S>", "<S> the patients in the control group \n performed conventional exercises for six weeks , and those in the experiment group \n performed core stability exercises for six weeks . </S>", "<S> the outcomes were evaluated using \n modified barthel index and berg balance scale . </S>", "<S> [ results ] after treatment , the modified \n barthel index and berg balance scale were significantly increased in both groups when \n compared with the baseline . </S>", "<S> the modified barthel index was significantly lower in the \n control group compared with the experimental group . </S>", "<S> the berg balance scale scores in the \n control group were relatively lower than those in the experimental group , but there was no \n significant difference between the two groups . </S>", "<S> [ conclusion ] core stability exercises have \n a better effect on patients with hemiplegia than conventional exercises . </S>" ]

[ "INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "stroke , with a high prevalence and long - term disabilities , is a major health problem in the \n world1 , 2 , and it was reported that about two million people suffer from \n stroke each year3 .", "impairments including \n loss of strength , sensation , and coordination abilities , which result in walking \n difficulties , balance disorders , and limb function disturbance , occur in 7080% of stroke \n patients4 .", "this affects the life quality \n of patients adversely and leads to a heavy economic burden on society .", "subsequently , \n effective rehabilitation interventions to optimize recovery in stroke patients have received \n high attention . in the treatment of balance and gait dysfunction ,", "exercise therapy is regarded as one of \n the commonly used methods that bring greater benefits in physical function for stroke \n patients5 .", "the effect of exercises on \n balance and gait dysfunction in stroke patients has been confirmed by many clinical \n studies3 , 6 , \n 7 .", "in addition , core stability exercises \n have been widely carried out in many fields , including medical rehabilitation , sports \n training , and medical care .", "core stability exercises were shown to improve dynamic standing \n balance , functional autonomy , static balance , flexibility , and stability8 . in recent years , core stability exercises have been reported to improve the rehabilitation \n effect in stroke patients . in a study of 20 stroke patients ,", "the control group underwent \n standard exercise therapy , while the experiment group underwent both the core \n stability - enhancing exercise and standard exercise therapy simultaneously .", "after 4 weeks , yu \n found that the mean trunk impairment scale score and muscle activity of the lower trunk \n increased in the experiment group significantly9 . in study by cabanas - valdes , 80 patients were randomly assigned to \n an experimental group and a control group , both groups underwent conventional therapy \n including exercises , and the experimental group performed core stability exercises .", "the \n results showed that core stability exercises improve the efficacy of conventional \n exercises10 .", "although these studies \n confirmed the effect of core stability exercises in the rehabilitation of stroke patients , \n few comparative studies have been carried out to compare the effect of core stability \n exercises and conventional exercises . which is better in improving the stability and balance \n of stroke patients , core stability or conventional exercises ?", "therefore , a prospective study including 40 participants was carried out in the department \n of neurology of yidu central hospital of weifang between january 2014 and february 2015 .", "the \n objective of the study was to determine which is better in the rehabilitation of stroke \n patients , core stability exercises or conventional exercises ." ], [ "participants with stroke - induced hemiplegia were recruited from the department of neurology \n of yidu central hospital of weifang between january 2014 and february 2015 .", "the diagnosis \n and lesion side of these participants were confirmed by history evaluation , physical \n examination , brain imaging , and examination of their medical records .", "the inclusion \n criteria9 were ability to walk more than \n 32 feet , duration of disorder > 6 months , no musculoskeletal problems , absence of any \n cardiac disorders , complete understanding of this research , and ability to communicate .", "the \n included participants were randomly divided into either an experimental or control group by \n a random computer - generated sequence .", "the randomization was managed by a physician who was \n not involved in the study .", "all the included patients \n signed an informed consent form , and the study was approved by the ethics committee of our \n hospital .", "the patients in the control group performed conventional exercises including limb \n stretching , passive mobilization of joints , walking between parallel bars , and occupational \n therapy11 .", "the patients in the \n experiment group performed core stability exercises including the plank , side plank , bridge , \n straight leg raise , and modified push - up12 . before the beginning of the study ,", "the patients were given \n individual instruction by the researchers . during the exercises , physicians provide \n necessary assistance to help the patients in executing the exercises .", "the exercises in both \n groups were performed for one hour per day , six times a week for six weeks . the modified barthel index ( mbi)13 and \n berg balance scale ( bbs)13 were used to \n assess the clinical outcomes of the two groups .", "the mbi , which demonstrates the ability of \n patients to deal with daily activities , consists of ten items including personal hygiene , \n taking a bath alone , eating , using the bathroom , ascending stairs , dressing , defecating , \n controlling urine , walking or using a wheelchair , and moving to a chair or bed .", "each item is \n rated on a scale of 5 ranging from independent conduct to impossible to conduct .", "the bbs , \n which can evaluate static and dynamic balance abilities of patients , consists of 14 items \n covering functional tasks in three domains , sitting , standing , and changing posture , that \n are scored on a scale of 04 .", "ibm spss statistics 21.0 ( ibm corp . , armonk , ny , usa ) was used for statistical analysis , \n and p < 0.05 was considered significant .", "the differences in the baseline data including \n age , gender , and disease course were compared using the t test or test . \n" ], [ "in the current study , 40 patients with stroke - induced hemiplegia were included and divided \n randomly into experimental and control groups , with 20 participants in each group at the \n beginning of the study .", "the baseline clinical data including age , gender , disease course , \n bbs , and mbi were recorded .", "there was no significant difference in baseline data between the \n two groups ( p>0.05 ) .", "the patients in the control group performed conventional exercises \n for six weeks and those in the experimental group carried out core stability exercises for \n six weeks .", "three patients in the experimental group and two in the control group withdrew from the \n study two weeks after treatment , and in total thirty - five patients completed the training . \n", "the final results showed that , compared with the baseline , the mean mbi scores of the \n experimental and control groups increased significantly from 53.5 and 55.1 to 66.8 and 61.2 \n ( p<0.05 ) , respectively , and that the mean bbs scores increased significantly from 30.2 \n and 31.9 to 38.6 and 35.7 ( p<0.05 ) , respectively .", "in addition , mbi scores were \n significantly lower ( p<0.05 ) in the control group than in the experimental group .", "the bbs \n scores in the control group were relatively lower than those in the experimental group , but \n there was no significant difference between the two groups ( p>0.05 ) ." ], [ "a prospective study was carried out to compare the effect of core stability exercises and \n conventional exercises on patients with stroke - induced hemiplegia . to the best of our \n knowledge", "the core , described as a box in the trunk with the \n abdominals , paraspinals , and gluteals ; diaphragm and pelvic floor ; and hip girdle \n musculature serving as the front wall , back wall , roof , and bottom , respectively , makes up \n the largest part of the body and plays an important role in stabilizing the body and \n controlling movement of daily activities9 . \n", "core stability is regarded as a prerequisite for maintaining proper posture of the lumbar \n and pelvic regions during sports activities9 . as a result ,", "core stability exercises have many advantages in the \n field of rehabilitation and can improve the activation and cooperative contractions of \n abdominal and multifidus muscles , facilitating the function and movement of the limbs9 , 12 .", "in the current study , the mbi and bbs scores after treatment were significantly increased \n in both the experimental and control groups when compared with those before treatment .", "this \n indicates that both core stability exercises and conventional exercises can improve the \n abilities of patients to deal with daily activities and control body balance , which confirms \n the viewpoints of many published studies9 , 10 .", "in addition , after treatment , the mbi scores were significantly lower in the control group \n than those in the experimental group , and the bbs scores in the control group were also \n lower than those in the experimental group .", "the outcomes demonstrate that compared with \n conventional exercises , core stability exercises can result in better effectiveness in \n patients with stroke - induced hemiplegia . however , in terms of comparison of the bbs scores \n at the end of treatment , although the mean bbs score in the experimental group was larger \n than that in the control group , there was no significant difference between the two groups . \n", "such a condition may be attributed to the relatively small sample size , and in a \n larger - scale study , the difference between the two groups may be significant .", "consequently , \n the outcomes in the present study can demonstrate that core stability exercises present with \n better effectiveness than conventional exercises .", "first , the sample size was small , and a large - scale \n clinical study may be better in a comparative study .", "second , the exercises results were \n evaluated using self - reported measures of function instead of an objective measurement , and \n this may have had some adverse influence on the final outcomes .", "third , there was a lack of \n long - term follow - up in the study , and it is unclear whether the effectiveness of core \n stability exercises will decrease in patients with hemiplegia with time ." ] ]

[ "a 3-year - old male , neutered , domestic shorthair cat with a history of chronic regurgitation since being obtained as a kitten was presented for weight loss and regurgitation of all ingested food .", "the cat was in poor body condition and had a firm swelling in the ventral neck at the time of presentation .", "thoracic radiographs showed severe dilation of the entire cervical and cranial intrathoracic esophagus to the level of the heart base .", "computed tomographic angiography ( cta ) showed a persistent right aortic arch with an aberrant left subclavian artery and severe dilation of the cervical and intrathoracic esophagus cranial to the heart base .", "cta also showed a bicarotid trunk and kommerell s diverticulum to be present , which are rare vascular structures in the cat .", "removal of the obstructing trichobezoars resulted in resolution of clinical signs , and the cat was able to drink water and eat a canned food slurry without regurgitation .", "vascular ring anomaly ( vra ) should be considered in all cats with a history of regurgitation , regardless of their age at the time of presentation .", "cta is a valuable diagnostic imaging procedure that allows differentiation of a vra from other causes of esophageal obstruction and provides information about the vra that can be used to determine amenability to surgical correction .", "a 3 year - old male , neutered , domestic shorthair cat was presented with a 2 week history of severe regurgitation of all ingested food .", "the cat had a history of a voracious appetite and regurgitation of solid food since being acquired as a kitten , but was able to consume liquid meals without regurgitating until 2 weeks prior to presentation .", "the physical examination revealed a poor body condition score ( bcs 2/9 ) and a 4 cm firm tubular structure in the ventral neck , believed to be within or associated with the esophagus .", "as a result of the cat s fractious nature , it was necessary to induce general anesthesia for further evaluation .", "thoracic radiographs showed severe distention of the entire cervical and intrathoracic esophagus to the level of the heart base with heterogeneous soft tissue opaque material surrounded by a thin gas rim ( figure 1 ) .", "differentials for the radiographic findings were constriction due to a vascular ring anomaly ( vra ) , most commonly a persistent right aortic arch , a mural defect or benign esophageal stricture .", "the cervical and thoracic esophagus ( dashed lines ) is severely distended cranial to the carina , displacing the trachea ( dotted lines ) ventrally and to the right .", "note the marked leftward deviation of the trachea at the level of the heart base computed tomographic angiogram .", "structures of note include the persistent right aortic arch ( praa ) , bicarotid trunk ( bct ) , right subclavian artery ( rsa ) , aberrant left subclavian artery ( alsa ) , descending aorta ( dao ) , esophagus ( es ) , cranial vena cava ( crvc ) , pulmonary trunk ( pt ) and right auricle ( rau ) computed tomographic angiography ( cta ) was performed in order to confirm and classify the suspected vra and to determine if surgical correction would be possible .", "the esophagus cranial to the heart base was almost entirely filled with heterogeneous soft tissue , gas and mineral opaque material . at the level of the heart base ,", "the esophagus was focally and concentrically narrowed by the trachea and aorta on the right and the main pulmonary artery on the left .", "the site of constriction was apparent at the anatomic level of the ligamentum arteriosum ; however , the ligamentum arteriosum could not be specifically identified on cta .", "the aortic arch and proximal descending aorta were located just to the right of midline , with the distal descending aorta positioned immediately ventral to the thoracic spine .", "a bicarotid trunk and right subclavian artery were present , and branched from the aortic arch in close succession .", "additionally , the origin of the aberrant left subclavian artery from the aorta was markedly dilated and then abruptly narrowed distally to a normal diameter ; this finding is known as kommerell s diverticulum .", "although the aberrant left subclavian artery created a dorsal indentation in the intrathoracic esophagus , it did not appear to be the primary cause of the constriction .", "the esophagus remained dilated cranial to the level of the constriction and pulsation of the major vessels against the wall of the esophagus was observed at the level of the luminal narrowing ( see video in the supplementary material ) .", "evidence of mild - to - moderate esophagitis , presumably due to stagnation of trichobezoars and putrefaction of food , was observed cranial to the constriction .", "it was possible to advance the endoscope past the constriction and no gross abnormalities were noted in the stomach .", "following recovery from anesthesia , the cat was able to drink water and eat a canned food slurry without complication or regurgitation .", "surgical ligation and transection of the vascular ring anomaly with concurrent endoscopic balloon dilatation of the esophageal stricture was recommended as the treatment of choice .", "the cat was discharged with instructions to continue feeding a canned food slurry in an upright position .", "the owner was contacted prior to submission and reported that the cat was doing well with multiple small feedings of canned food and had not regurgitated since discharge .", "although vras are common in dogs , with several reports of diagnosis in adult dogs , they are uncommon in cats and infrequently diagnosed in adult cats .", "the most common feline vra is persistent right aortic arch ( praa ) with a left ligamentum arteriosum ; however , other described abnormalities include praa with coarctation of the aorta at the level of the left ligamentum arteriosum , double aortic arch , left aortic arch with right ligamentum arteriosum and praa with concurrent left cranial vena cava .", "recently , cta has become more widely available as a diagnostic modality and has increased the frequency and accuracy of diagnosis .", "cta proved valuable in this adult cat as it could confirm a congenital defect was present as the cause of the esophageal constriction and allow a surgical procedure to be planned . the vascular abnormality known as kommerell s diverticulum", "this vascular structure is classically defined as a dilation of the aorta at the insertion of either the left or right subclavian artery that is 1.5 times greater than the diameter of the distal subclavian artery .", "the presence of a bicarotid trunk branching directly from the aortic arch in this cat is also an unusual finding .", "the treatment of choice for vras in companion animals is surgical ligation and transection of the anomalous vessel(s ) . if possible", ", periesophageal fibrosis should be reduced , and the stricture site should be dilated intraluminally with a balloon dilatation catheter .", "this procedure has traditionally been performed via a thoracotomy , but thoracoscopy has also been used , and offers a less invasive treatment option .", "ligation and transection of the aberrant left subclavian artery can also be performed if the vessel contributes to the esophageal constriction . in an effort to decrease the severity of esophageal dilation and dysfunction ,", "although surgical correction is the treatment of choice , clinical signs may persist even after a successful procedure .", "esophageal dilation may cause irreversible myenteric nerve degeneration and esophageal hypomotility ; however , a 90% or greater recovery rate has been reported in dogs .", "nutritional management is critical in the postoperative period but is rarely successful as a primary treatment for vra .", "many of these patients require lifetime management with elevated feedings of canned or pured food in small meals , but it is possible to transition those with less severe obstructions to a more solid diet . in cases of severe esophageal dysfunction or malnutrition , a gastrostomy tube may be necessary to achieve adequate nutritional supplementation .", "congenital causes of esophageal obstruction should be considered in cats presented with a history of regurgitation , regardless of age at the time of presentation .", "cta confirmed the presence of an extraluminal constriction and provided valuable information about the vascular anomaly in the event that surgical correction would have been pursued .", "surgical removal of the extraluminal constriction with balloon dilatation of the esophageal stricture was recommended and is considered the treatment of choice .", "however , considering the guarded prognosis for return of normal esophageal function in an adult cat with chronic esophageal dilation , medical management provided a viable alternative for this cat ." ]

[ "<S> case summarya 3-year - old male , neutered , domestic shorthair cat with a history of chronic regurgitation since being obtained as a kitten was presented for weight loss and regurgitation of all ingested food . </S>", "<S> the cat was in poor body condition and had a firm swelling in the ventral neck at the time of presentation . </S>", "<S> thoracic radiographs showed severe dilation of the entire cervical and cranial intrathoracic esophagus to the level of the heart base . </S>", "<S> computed tomographic angiography ( cta ) showed a persistent right aortic arch with an aberrant left subclavian artery and severe dilation of the cervical and intrathoracic esophagus cranial to the heart base . </S>", "<S> cta also showed a bicarotid trunk and kommerell s diverticulum to be present , which are rare vascular structures in the cat . </S>", "<S> esophagoscopy showed esophageal dilation and multiple compact trichobezoars obstructing the esophagus . </S>", "<S> removal of the obstructing trichobezoars resulted in resolution of clinical signs , and the cat was able to drink water and eat a canned food slurry without regurgitation . </S>", "<S> surgical correction was not pursued.relevance and novel informationvascular ring anomaly ( vra ) should be considered in all cats with a history of regurgitation , regardless of their age at the time of presentation . </S>", "<S> cta is a valuable diagnostic imaging procedure that allows differentiation of a vra from other causes of esophageal obstruction and provides information about the vra that can be used to determine amenability to surgical correction . </S>" ]

[ "Case summary", "Relevance and novel information", "Case description", "Discussion", "Conclusions" ]

[ [ "a 3-year - old male , neutered , domestic shorthair cat with a history of chronic regurgitation since being obtained as a kitten was presented for weight loss and regurgitation of all ingested food .", "the cat was in poor body condition and had a firm swelling in the ventral neck at the time of presentation .", "thoracic radiographs showed severe dilation of the entire cervical and cranial intrathoracic esophagus to the level of the heart base .", "computed tomographic angiography ( cta ) showed a persistent right aortic arch with an aberrant left subclavian artery and severe dilation of the cervical and intrathoracic esophagus cranial to the heart base .", "cta also showed a bicarotid trunk and kommerell s diverticulum to be present , which are rare vascular structures in the cat .", "removal of the obstructing trichobezoars resulted in resolution of clinical signs , and the cat was able to drink water and eat a canned food slurry without regurgitation ." ], [ "vascular ring anomaly ( vra ) should be considered in all cats with a history of regurgitation , regardless of their age at the time of presentation .", "cta is a valuable diagnostic imaging procedure that allows differentiation of a vra from other causes of esophageal obstruction and provides information about the vra that can be used to determine amenability to surgical correction ." ], [ "a 3 year - old male , neutered , domestic shorthair cat was presented with a 2 week history of severe regurgitation of all ingested food .", "the cat had a history of a voracious appetite and regurgitation of solid food since being acquired as a kitten , but was able to consume liquid meals without regurgitating until 2 weeks prior to presentation .", "the physical examination revealed a poor body condition score ( bcs 2/9 ) and a 4 cm firm tubular structure in the ventral neck , believed to be within or associated with the esophagus .", "as a result of the cat s fractious nature , it was necessary to induce general anesthesia for further evaluation .", "thoracic radiographs showed severe distention of the entire cervical and intrathoracic esophagus to the level of the heart base with heterogeneous soft tissue opaque material surrounded by a thin gas rim ( figure 1 ) .", "differentials for the radiographic findings were constriction due to a vascular ring anomaly ( vra ) , most commonly a persistent right aortic arch , a mural defect or benign esophageal stricture .", "the cervical and thoracic esophagus ( dashed lines ) is severely distended cranial to the carina , displacing the trachea ( dotted lines ) ventrally and to the right .", "note the marked leftward deviation of the trachea at the level of the heart base computed tomographic angiogram .", "structures of note include the persistent right aortic arch ( praa ) , bicarotid trunk ( bct ) , right subclavian artery ( rsa ) , aberrant left subclavian artery ( alsa ) , descending aorta ( dao ) , esophagus ( es ) , cranial vena cava ( crvc ) , pulmonary trunk ( pt ) and right auricle ( rau ) computed tomographic angiography ( cta ) was performed in order to confirm and classify the suspected vra and to determine if surgical correction would be possible .", "the esophagus cranial to the heart base was almost entirely filled with heterogeneous soft tissue , gas and mineral opaque material . at the level of the heart base ,", "the esophagus was focally and concentrically narrowed by the trachea and aorta on the right and the main pulmonary artery on the left .", "the site of constriction was apparent at the anatomic level of the ligamentum arteriosum ; however , the ligamentum arteriosum could not be specifically identified on cta .", "the aortic arch and proximal descending aorta were located just to the right of midline , with the distal descending aorta positioned immediately ventral to the thoracic spine .", "a bicarotid trunk and right subclavian artery were present , and branched from the aortic arch in close succession .", "additionally , the origin of the aberrant left subclavian artery from the aorta was markedly dilated and then abruptly narrowed distally to a normal diameter ; this finding is known as kommerell s diverticulum .", "although the aberrant left subclavian artery created a dorsal indentation in the intrathoracic esophagus , it did not appear to be the primary cause of the constriction .", "the esophagus remained dilated cranial to the level of the constriction and pulsation of the major vessels against the wall of the esophagus was observed at the level of the luminal narrowing ( see video in the supplementary material ) .", "evidence of mild - to - moderate esophagitis , presumably due to stagnation of trichobezoars and putrefaction of food , was observed cranial to the constriction .", "it was possible to advance the endoscope past the constriction and no gross abnormalities were noted in the stomach .", "following recovery from anesthesia , the cat was able to drink water and eat a canned food slurry without complication or regurgitation .", "surgical ligation and transection of the vascular ring anomaly with concurrent endoscopic balloon dilatation of the esophageal stricture was recommended as the treatment of choice .", "the cat was discharged with instructions to continue feeding a canned food slurry in an upright position .", "the owner was contacted prior to submission and reported that the cat was doing well with multiple small feedings of canned food and had not regurgitated since discharge ." ], [ "although vras are common in dogs , with several reports of diagnosis in adult dogs , they are uncommon in cats and infrequently diagnosed in adult cats .", "the most common feline vra is persistent right aortic arch ( praa ) with a left ligamentum arteriosum ; however , other described abnormalities include praa with coarctation of the aorta at the level of the left ligamentum arteriosum , double aortic arch , left aortic arch with right ligamentum arteriosum and praa with concurrent left cranial vena cava .", "recently , cta has become more widely available as a diagnostic modality and has increased the frequency and accuracy of diagnosis .", "cta proved valuable in this adult cat as it could confirm a congenital defect was present as the cause of the esophageal constriction and allow a surgical procedure to be planned . the vascular abnormality known as kommerell s diverticulum", "this vascular structure is classically defined as a dilation of the aorta at the insertion of either the left or right subclavian artery that is 1.5 times greater than the diameter of the distal subclavian artery .", "the presence of a bicarotid trunk branching directly from the aortic arch in this cat is also an unusual finding .", "the treatment of choice for vras in companion animals is surgical ligation and transection of the anomalous vessel(s ) . if possible", ", periesophageal fibrosis should be reduced , and the stricture site should be dilated intraluminally with a balloon dilatation catheter .", "this procedure has traditionally been performed via a thoracotomy , but thoracoscopy has also been used , and offers a less invasive treatment option .", "ligation and transection of the aberrant left subclavian artery can also be performed if the vessel contributes to the esophageal constriction . in an effort to decrease the severity of esophageal dilation and dysfunction ,", "although surgical correction is the treatment of choice , clinical signs may persist even after a successful procedure .", "esophageal dilation may cause irreversible myenteric nerve degeneration and esophageal hypomotility ; however , a 90% or greater recovery rate has been reported in dogs .", "nutritional management is critical in the postoperative period but is rarely successful as a primary treatment for vra .", "many of these patients require lifetime management with elevated feedings of canned or pured food in small meals , but it is possible to transition those with less severe obstructions to a more solid diet . in cases of severe esophageal dysfunction or malnutrition , a gastrostomy tube may be necessary to achieve adequate nutritional supplementation ." ], [ "congenital causes of esophageal obstruction should be considered in cats presented with a history of regurgitation , regardless of age at the time of presentation .", "cta confirmed the presence of an extraluminal constriction and provided valuable information about the vascular anomaly in the event that surgical correction would have been pursued .", "surgical removal of the extraluminal constriction with balloon dilatation of the esophageal stricture was recommended and is considered the treatment of choice .", "however , considering the guarded prognosis for return of normal esophageal function in an adult cat with chronic esophageal dilation , medical management provided a viable alternative for this cat ." ] ]

[ "we wish to report some corrections to our study , none of which alters the interpretation of the data or the conclusions drawn . after publication", ", we noticed that one of the micro - array hybridizations ( on sample nb11 ) was performed on the same patient 's material as another hybridization ( sample nb4 ; see table 1 ; a corrected version of table 5 ) .", "as this error leads to an incorrect subclassification of the patients into the ' favourable ' and ' unfavourable ' neuroblastoma subgroups , we would like to exclude this data point from the differential expression analysis of favorable versus unfavorable neuroblastoma given under the heading ' differential expression analysis of favorable and unfavorable neuroblastoma ' in the results section of .", "careful reanalysis after exclusion of nb11 did not lead to important changes in the generated gene lists and conclusions ; the changes are given in the corrected paragraph and table 2 ( a corrected version of table 4 ) , and the additional data files 1 and 2 ( corrected versions of additional data files 2 and 3 ) available online with this article . we also noticed that sample nb1 is stage 1 instead of stage 4s and that sample nb2 was not localized to the adrenals ( see table 1 ) .", "so far , most published microarray studies on neuroblastomas mainly compared favorable with unfavorable neuroblastomas in order to identify prognostic markers or pathways that are involved in these clearly different neuroblastoma tumor types . in order to add value to such an analysis", ", we contrasted similar differentially expressed gene lists with the normal neuroblast expression profile ( additional data file 1 ) . in a first step , we compared the differentially expressed genes between these two tumor types with published prognostic gene lists .", "we found that 23 of the 193 genes on our list were previously reported , including the well established markers mycn , ntrk1 , and cd44 ( see nbgs analysis in additional data file 2 ) .", "subsequently , we looked for the corresponding gene expression levels of the differentially expressed genes in the normal counterpart cells , aiming to select neuroblastoma candidate genes .", "of the 100 genes that are more highly expressed in favorable tumors ( compared to unfavorable ) 41 also have a significant differential expression ( either higher or lower ) compared to neuroblasts , whereas 43 of the 93 genes that are more highly expressed in unfavorable tumors exhibit differential expression compared to the neuroblasts ( table 2 ) . from this analysis ,", "a few putative positional tumor suppressor candidates emerge : cdc42 on 1p36 , cacna2d3 on 3p21 and dlk1 on 14q .", "the latter two genes are of particular interest because they are highly expressed in neuroblasts and favorable neuroblastomas and their expression is significantly lower in unfavorable neuroblastomas . among the genes that are", "more highly expressed in unfavorable neurob - lastomas than in favorable ones and neuroblasts , the proven oncogenic transcription factor mycn emerges ( and putative downstream genes kifap3 , ophn1 , rgs7 , ascl1 , odc1 , twist1 and tyms , according to nbgs ) , as well as several other genes that have been identified or studied in the context of neuroblastoma such as alk and prame , and positional candidates on 17q including birc5 and rnu2 .", "so far , most published microarray studies on neuroblastomas mainly compared favorable with unfavorable neuroblastomas in order to identify prognostic markers or pathways that are involved in these clearly different neuroblastoma tumor types . in order to add value to such an analysis", ", we contrasted similar differentially expressed gene lists with the normal neuroblast expression profile ( additional data file 1 ) . in a first step , we compared the differentially expressed genes between these two tumor types with published prognostic gene lists .", "we found that 23 of the 193 genes on our list were previously reported , including the well established markers mycn , ntrk1 , and cd44 ( see nbgs analysis in additional data file 2 ) .", "subsequently , we looked for the corresponding gene expression levels of the differentially expressed genes in the normal counterpart cells , aiming to select neuroblastoma candidate genes .", "of the 100 genes that are more highly expressed in favorable tumors ( compared to unfavorable ) 41 also have a significant differential expression ( either higher or lower ) compared to neuroblasts , whereas 43 of the 93 genes that are more highly expressed in unfavorable tumors exhibit differential expression compared to the neuroblasts ( table 2 ) . from this analysis ,", "a few putative positional tumor suppressor candidates emerge : cdc42 on 1p36 , cacna2d3 on 3p21 and dlk1 on 14q .", "the latter two genes are of particular interest because they are highly expressed in neuroblasts and favorable neuroblastomas and their expression is significantly lower in unfavorable neuroblastomas . among the genes that are", "more highly expressed in unfavorable neurob - lastomas than in favorable ones and neuroblasts , the proven oncogenic transcription factor mycn emerges ( and putative downstream genes kifap3 , ophn1 , rgs7 , ascl1 , odc1 , twist1 and tyms , according to nbgs ) , as well as several other genes that have been identified or studied in the context of neuroblastoma such as alk and prame , and positional candidates on 17q including birc5 and rnu2 .", "additional data files 1 and 2 containing the corrected data available online with this article .", "corrected version of additional data file 2 . corrected version of additional data file 3 .", "clinical and genetic data of carefully selected neuroblastoma samples that were included in this study samples were subdivided into favorable or unfavorable type based on mycn amplification , ploidy and age at diagnosis .", "nd , not determined or unknown . genes that are differentially expressed in favorable vs unfavorable neuroblastoma genes that are differentially expressed compared with neuroblasts among the differentially expressed genes in favorable neuroblastoma ( nb ) vs unfavorable nb , with an indication of the number of neuroblastoma microarray studies in which these genes were found through nbgs analysis ." ]

[ "<S> a correction to human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes by k de preter , j vandesompele , p heimann , n yigit , s beckman , a schramm , a eggert , rl stallings , y benoit , m renard , a de paepe , g laureys , s phlman and f speleman . </S>", "<S> genome biology 2006 7:r84 </S>" ]

[ "None", "Results", "Differential expression analysis of favorable and unfavorable neuroblastoma", "Additional data files", "Supplementary Material", "Figures and Tables" ]

[ [ "we wish to report some corrections to our study , none of which alters the interpretation of the data or the conclusions drawn . after publication", ", we noticed that one of the micro - array hybridizations ( on sample nb11 ) was performed on the same patient 's material as another hybridization ( sample nb4 ; see table 1 ; a corrected version of table 5 ) .", "as this error leads to an incorrect subclassification of the patients into the ' favourable ' and ' unfavourable ' neuroblastoma subgroups , we would like to exclude this data point from the differential expression analysis of favorable versus unfavorable neuroblastoma given under the heading ' differential expression analysis of favorable and unfavorable neuroblastoma ' in the results section of .", "careful reanalysis after exclusion of nb11 did not lead to important changes in the generated gene lists and conclusions ; the changes are given in the corrected paragraph and table 2 ( a corrected version of table 4 ) , and the additional data files 1 and 2 ( corrected versions of additional data files 2 and 3 ) available online with this article . we also noticed that sample nb1 is stage 1 instead of stage 4s and that sample nb2 was not localized to the adrenals ( see table 1 ) ." ], [ "so far , most published microarray studies on neuroblastomas mainly compared favorable with unfavorable neuroblastomas in order to identify prognostic markers or pathways that are involved in these clearly different neuroblastoma tumor types . in order to add value to such an analysis", ", we contrasted similar differentially expressed gene lists with the normal neuroblast expression profile ( additional data file 1 ) . in a first step , we compared the differentially expressed genes between these two tumor types with published prognostic gene lists .", "we found that 23 of the 193 genes on our list were previously reported , including the well established markers mycn , ntrk1 , and cd44 ( see nbgs analysis in additional data file 2 ) .", "subsequently , we looked for the corresponding gene expression levels of the differentially expressed genes in the normal counterpart cells , aiming to select neuroblastoma candidate genes .", "of the 100 genes that are more highly expressed in favorable tumors ( compared to unfavorable ) 41 also have a significant differential expression ( either higher or lower ) compared to neuroblasts , whereas 43 of the 93 genes that are more highly expressed in unfavorable tumors exhibit differential expression compared to the neuroblasts ( table 2 ) . from this analysis ,", "a few putative positional tumor suppressor candidates emerge : cdc42 on 1p36 , cacna2d3 on 3p21 and dlk1 on 14q .", "the latter two genes are of particular interest because they are highly expressed in neuroblasts and favorable neuroblastomas and their expression is significantly lower in unfavorable neuroblastomas . among the genes that are", "more highly expressed in unfavorable neurob - lastomas than in favorable ones and neuroblasts , the proven oncogenic transcription factor mycn emerges ( and putative downstream genes kifap3 , ophn1 , rgs7 , ascl1 , odc1 , twist1 and tyms , according to nbgs ) , as well as several other genes that have been identified or studied in the context of neuroblastoma such as alk and prame , and positional candidates on 17q including birc5 and rnu2 ." ], [ "so far , most published microarray studies on neuroblastomas mainly compared favorable with unfavorable neuroblastomas in order to identify prognostic markers or pathways that are involved in these clearly different neuroblastoma tumor types . in order to add value to such an analysis", ", we contrasted similar differentially expressed gene lists with the normal neuroblast expression profile ( additional data file 1 ) . in a first step , we compared the differentially expressed genes between these two tumor types with published prognostic gene lists .", "we found that 23 of the 193 genes on our list were previously reported , including the well established markers mycn , ntrk1 , and cd44 ( see nbgs analysis in additional data file 2 ) .", "subsequently , we looked for the corresponding gene expression levels of the differentially expressed genes in the normal counterpart cells , aiming to select neuroblastoma candidate genes .", "of the 100 genes that are more highly expressed in favorable tumors ( compared to unfavorable ) 41 also have a significant differential expression ( either higher or lower ) compared to neuroblasts , whereas 43 of the 93 genes that are more highly expressed in unfavorable tumors exhibit differential expression compared to the neuroblasts ( table 2 ) . from this analysis ,", "a few putative positional tumor suppressor candidates emerge : cdc42 on 1p36 , cacna2d3 on 3p21 and dlk1 on 14q .", "the latter two genes are of particular interest because they are highly expressed in neuroblasts and favorable neuroblastomas and their expression is significantly lower in unfavorable neuroblastomas . among the genes that are", "more highly expressed in unfavorable neurob - lastomas than in favorable ones and neuroblasts , the proven oncogenic transcription factor mycn emerges ( and putative downstream genes kifap3 , ophn1 , rgs7 , ascl1 , odc1 , twist1 and tyms , according to nbgs ) , as well as several other genes that have been identified or studied in the context of neuroblastoma such as alk and prame , and positional candidates on 17q including birc5 and rnu2 ." ], [ "additional data files 1 and 2 containing the corrected data available online with this article ." ], [ "corrected version of additional data file 2 . corrected version of additional data file 3 ." ], [ "clinical and genetic data of carefully selected neuroblastoma samples that were included in this study samples were subdivided into favorable or unfavorable type based on mycn amplification , ploidy and age at diagnosis .", "nd , not determined or unknown . genes that are differentially expressed in favorable vs unfavorable neuroblastoma genes that are differentially expressed compared with neuroblasts among the differentially expressed genes in favorable neuroblastoma ( nb ) vs unfavorable nb , with an indication of the number of neuroblastoma microarray studies in which these genes were found through nbgs analysis ." ] ]

[ "the ultrasound ( us ) appearance of testicular microlithiasis ( tm ) was first described by doherty et al . in 1987 .", "the prevalence of tm in symptomatic korean men was found to be 6.0% with significant co - occurrence of tm , testicular cancer , and infertility by yee et al . ", "although the cause - and - effect relationships are unclear , tm has been seen in patients with cryptorchidism , varicoceles , infertility , testicular torsion , klinefelter syndrome , pulmonary alveolar microlithiasis , neurofibromatosis , acquired immunodeficiency syndrome , intratubular germ cell neoplasia , and most importantly , primary testicular neoplasms .", "reported that 74% of testes with tumors had associated ipsilateral tm on radiological inspection ; whereas , only 8% of testicular specimens with benign conditions had microcalcifications .", "although chen et al . reported that there was a significant difference in the rate of malignancy in males with tm compared with those without tm , the question remains whether tm independently increases the risk of testicular malignancy in taiwanese men .", ", after reporting an association between tm and testicular tumor specimens , suggested in 1982 that radiographic studies be incorporated into diagnosing tm because of the perceived risk for testicular cancer in testicles with microlithiasis", "performed us follow - up in 9 of 22 patients with tm for a mean of 32 months without any newly developing tumors being diagnosed .", "reported similar results ; therefore , a regular scrotal us is controversial in asymptomatic tm patients .", "the current recommendations , including those of the european association of urology , are that the presence of microlithiasis alone is not an indication for a regular scrotal us in the absence of other risk factors ( size<12 ml or atrophy , inhomogeneous parenchyma ) .", "however , there have been no reports describing the changes in calcification over time in pediatric patients with tm .", "the study protocol was approved by the institutional review board of the asan medical center .", "the medical records from july 1997 to august 2014 of the asan medical center , a tertiary referral center , were retrospectively screened for patients diagnosed with tm by scrotal us .", "twenty - three tm patients were included who had undergone scrotal us at least twice .", "we analyzed the patient characteristics , clinical manifestations , specific pathological features , and clinical outcomes .", "we measured the calcified area in maximum cross - sectional area , and we compared the calcific density in the initial diagnostic us and the final follow - up us .", "we defined the testis and the calcified area in terms of their maximal cross - sectional area and calculated those areas using image j software ( national institutes of health , bethesda , md , usa ) ( fig .", "patients that showed microlithiasis in three or more sections were defined as diffuse type , and patients with microlithiasis in less than three sections were defined as focal type ( fig .", "we classified the patients into three groups according to the change of microlithiasis : an increased group , 20% increase in microlithiasis ; a decreased group , 20% decrease in microlithiasis ; and a no change group , < 20% increase or decrease .", "differences were analyzed by a paired t - test , and crosstabs were used to assess dependent samples . in this study p - values < 0.05 were considered statistically significant .", "the mean age at presentation was 11.34.6 years , and the follow - up period ( interval of fist us and last us ) was 79.138.8 months ( range , 25.4 - 152.9 months ) .", "the mean age at last follow - up was 17.85.8 years ( range , 6.4 - 26.9 months ) .", "follow - up occurred for 19 of 23 patients over puberty ( defined as > 13 years old ) .", "of the 23 patients 20 had bilateral tm , 2 patients had unilateral tm , and 1 patient had an atrophic testis .", "less frequent comorbid conditions included testicular torsion ( 3 patients , 13.0% ) , epididymitis ( 3 patients , 13.0% ) , varicocele ( 2 patients , 8.7% ) , hydrocele ( 2 patients , 8.7% ) , and epididymal cyst ( 2 patients , 8.7% ) .", "calcific density was increased at the last follow - up us compared with the initial us ; however , this finding was not statistically different ( 3.74%6.0% vs. 3.06%4.38% , respectively , p=0.147 ) .", "we divided the subjects into two groups ( focal vs. diffuse ) based on the distribution of tm .", "we classified 23 testes as having focal tm and 20 testes as having diffuse tm .", "in focal tm calcific density decreased but not significantly ( 0.72%0.55% vs. 0.66%1.03% , p=0.813 ) . on the other hand ,", "the calcific density of diffuse tm show a trend toward increase ( 5.8%5.2% vs. 7.3%7.4% , p=0.457 ) ( table 2 ) ( fig .", "3 ) . to clarify the tendency of tm calcification toward increase vs. decrease we divided the subjects into three groups based on the calcific density change of tm ( increase : increased > 20% ; decrease : decreased > 20% ; no change : < 20% increase or decrease ) .", "we categorized 14 testes as increased , 18 testes as decreased , and 11 testes as no change .", "half of the patients with diffuse tm were assigned to the increased group , a proportion significantly higher than focal tm ( 10/20 [ 50% ] in diffuse tm , 4/23 [ 17.4% ] in focal tm , p=0.049 ) ( table 3 ) .", "in addition , 5 of 8 testes with cryptorchidism ( including 2 with bilateral cryptorchidism ) were categorized into the increased group .", "previous tm studies have focused only on the relationship between tm and testicular cancer , and not on the natural course of this disease progression . in our current study , we report the natural course of calcific density changes in pediatric tm during pubertal development .", "the mean age at presentation was 11.3 years and the mean follow - up period of approximately 5 years allowed follow - up of 23 patients over pubertal development . despite a follow - up interval that allowed potential pubertal changes to the testis , none of our patients developed testicular cancer or new abnormal symptoms . by contrast , there are some reported cases of patients with a known tm on us exam that eventually developed a primary testicular cancer .", "reported a yolk sac tumor developing in a 17-year - old boy being followed for bilateral tm that was originally detected because of an initial sonographic evaluation of unequal sized testes .", "reported the case of a man with tm seen on a sonograph performed due to bilateral testicular pain who presented three years later with a metastatic germ cell tumor of the left testicle .", "although , an association between tm and subsequent testicular tumors appears likely , whether there is a true cause - and - effect relationship remains unknown .", "( 7 patients , 45 months)-used us to follow patients with tm and , similarly to our present results , did not detect the appearance of new testicular tumors .", "compared with these prior studies , our current study incorporates more patients and a longer - term follow - up extending through puberty .", "an additional strength of our study over previous studies is in our investigation of changes in calcified density of the tm .", "one earlier study has reported nonquantitative changes in the prominence of tm on yearly us follow - up .", "the tm was less prominent in one patient , unchanged in four , and two patients were lost to follow - up . to our knowledge , our present study is the first report to provide a quantitative analysis of calcific density in tm .", "calcific density on us showed a nonstatistically significant trend toward increase over time in our study subjects .", "reported a relationship between the number of microliths and testicular cancer after subgrouping subjects into four ranges based on the number of microliths . in our present study , we divided tm into two nearly equal groups based on the distribution pattern of calcification ( focal type [ 23 testes ] vs. diffuse type [ 20 testes ] ) that appear to differ in terms of calcification trends .", "calcific density shows a trend toward a decrease in focal tm but toward an increase in 50% of the testes with diffuse tm .", "notably , the majority of testes ( 5/8 ) in our study series with cryptorchidism , including those with bilateral cryptorchidism , were categorized as being in the increased calcification group .", "during the follow - up through puberty of the microlithiasis in the 23 boys in our present study , we observed no significant changes in the density of their microliths and no development of testicular cancer ; however , we found that diffuse tm and cryptorchidism tend to increase calcific density .", "hence , close observation is recommended in cases of tm combined with cryptorchidism and diffuse microlithiasis ." ]

[ "<S> purposetesticular microlithiasis ( tm ) is a relatively rare clinical entity of controversial significance characterized by the existence of hydroxyapatite microliths located in the seminiferous tubules . </S>", "<S> the aim of this study was to observe the natural course of changes in the calcific density of pediatric tm.materials and methodswe included a total of 23 tm patients undergoing scrotal ultrasound ( us ) on at least two occasions from july 1997 to august 2014 . </S>", "<S> we retrospectively analyzed the patient characteristics , clinical manifestations , specific pathological features , and clinical outcomes . </S>", "<S> we measured the calcified area and compared the calcific density between the initial and final uss.resultsthe mean age at diagnosis was 11.34.6 years , and the follow - up period was 79.138.8 months ( range , 25.4 - 152.9 months ) . during the follow - up period , </S>", "<S> no patients developed testicular cancer . </S>", "<S> calcific density on us was increased in the last versus the initial us , but not to a statistically significant degree ( 3.74%6.0% vs. 3.06%4.38% , respectively , p=0.147 ) . </S>", "<S> when we defined groups with increased and decreased calcification , we found that diffuse tm was categorized into the increased group to a greater degree than focal tm ( 10/20 vs. 4/23 , respectively , p=0.049 ) . </S>", "<S> in addition , five of eight cases of cryptorchidism ( including two cases of bilateral cryptorchidism ) were categorized in the increased calcification group.conclusionsdiffuse tm and cryptorchidism tend to increase calcific density . </S>", "<S> close observation is therefore recommended for cases of tm combined with cryptorchidism and cases of diffuse tm . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS" ]

[ [ "the ultrasound ( us ) appearance of testicular microlithiasis ( tm ) was first described by doherty et al . in 1987 .", "the prevalence of tm in symptomatic korean men was found to be 6.0% with significant co - occurrence of tm , testicular cancer , and infertility by yee et al . ", "although the cause - and - effect relationships are unclear , tm has been seen in patients with cryptorchidism , varicoceles , infertility , testicular torsion , klinefelter syndrome , pulmonary alveolar microlithiasis , neurofibromatosis , acquired immunodeficiency syndrome , intratubular germ cell neoplasia , and most importantly , primary testicular neoplasms .", "reported that 74% of testes with tumors had associated ipsilateral tm on radiological inspection ; whereas , only 8% of testicular specimens with benign conditions had microcalcifications .", "although chen et al . reported that there was a significant difference in the rate of malignancy in males with tm compared with those without tm , the question remains whether tm independently increases the risk of testicular malignancy in taiwanese men .", ", after reporting an association between tm and testicular tumor specimens , suggested in 1982 that radiographic studies be incorporated into diagnosing tm because of the perceived risk for testicular cancer in testicles with microlithiasis", "performed us follow - up in 9 of 22 patients with tm for a mean of 32 months without any newly developing tumors being diagnosed .", "reported similar results ; therefore , a regular scrotal us is controversial in asymptomatic tm patients .", "the current recommendations , including those of the european association of urology , are that the presence of microlithiasis alone is not an indication for a regular scrotal us in the absence of other risk factors ( size<12 ml or atrophy , inhomogeneous parenchyma ) .", "however , there have been no reports describing the changes in calcification over time in pediatric patients with tm ." ], [ "the study protocol was approved by the institutional review board of the asan medical center .", "the medical records from july 1997 to august 2014 of the asan medical center , a tertiary referral center , were retrospectively screened for patients diagnosed with tm by scrotal us .", "twenty - three tm patients were included who had undergone scrotal us at least twice .", "we analyzed the patient characteristics , clinical manifestations , specific pathological features , and clinical outcomes .", "we measured the calcified area in maximum cross - sectional area , and we compared the calcific density in the initial diagnostic us and the final follow - up us .", "we defined the testis and the calcified area in terms of their maximal cross - sectional area and calculated those areas using image j software ( national institutes of health , bethesda , md , usa ) ( fig .", "patients that showed microlithiasis in three or more sections were defined as diffuse type , and patients with microlithiasis in less than three sections were defined as focal type ( fig .", "we classified the patients into three groups according to the change of microlithiasis : an increased group , 20% increase in microlithiasis ; a decreased group , 20% decrease in microlithiasis ; and a no change group , < 20% increase or decrease .", "differences were analyzed by a paired t - test , and crosstabs were used to assess dependent samples . in this study p - values < 0.05 were considered statistically significant ." ], [ "the mean age at presentation was 11.34.6 years , and the follow - up period ( interval of fist us and last us ) was 79.138.8 months ( range , 25.4 - 152.9 months ) .", "the mean age at last follow - up was 17.85.8 years ( range , 6.4 - 26.9 months ) .", "follow - up occurred for 19 of 23 patients over puberty ( defined as > 13 years old ) .", "of the 23 patients 20 had bilateral tm , 2 patients had unilateral tm , and 1 patient had an atrophic testis .", "less frequent comorbid conditions included testicular torsion ( 3 patients , 13.0% ) , epididymitis ( 3 patients , 13.0% ) , varicocele ( 2 patients , 8.7% ) , hydrocele ( 2 patients , 8.7% ) , and epididymal cyst ( 2 patients , 8.7% ) .", "calcific density was increased at the last follow - up us compared with the initial us ; however , this finding was not statistically different ( 3.74%6.0% vs. 3.06%4.38% , respectively , p=0.147 ) .", "we divided the subjects into two groups ( focal vs. diffuse ) based on the distribution of tm .", "we classified 23 testes as having focal tm and 20 testes as having diffuse tm .", "in focal tm calcific density decreased but not significantly ( 0.72%0.55% vs. 0.66%1.03% , p=0.813 ) . on the other hand ,", "the calcific density of diffuse tm show a trend toward increase ( 5.8%5.2% vs. 7.3%7.4% , p=0.457 ) ( table 2 ) ( fig .", "3 ) . to clarify the tendency of tm calcification toward increase vs. decrease we divided the subjects into three groups based on the calcific density change of tm ( increase : increased > 20% ; decrease : decreased > 20% ; no change : < 20% increase or decrease ) .", "we categorized 14 testes as increased , 18 testes as decreased , and 11 testes as no change .", "half of the patients with diffuse tm were assigned to the increased group , a proportion significantly higher than focal tm ( 10/20 [ 50% ] in diffuse tm , 4/23 [ 17.4% ] in focal tm , p=0.049 ) ( table 3 ) .", "in addition , 5 of 8 testes with cryptorchidism ( including 2 with bilateral cryptorchidism ) were categorized into the increased group ." ], [ "previous tm studies have focused only on the relationship between tm and testicular cancer , and not on the natural course of this disease progression . in our current study , we report the natural course of calcific density changes in pediatric tm during pubertal development .", "the mean age at presentation was 11.3 years and the mean follow - up period of approximately 5 years allowed follow - up of 23 patients over pubertal development . despite a follow - up interval that allowed potential pubertal changes to the testis , none of our patients developed testicular cancer or new abnormal symptoms . by contrast , there are some reported cases of patients with a known tm on us exam that eventually developed a primary testicular cancer .", "reported a yolk sac tumor developing in a 17-year - old boy being followed for bilateral tm that was originally detected because of an initial sonographic evaluation of unequal sized testes .", "reported the case of a man with tm seen on a sonograph performed due to bilateral testicular pain who presented three years later with a metastatic germ cell tumor of the left testicle .", "although , an association between tm and subsequent testicular tumors appears likely , whether there is a true cause - and - effect relationship remains unknown .", "( 7 patients , 45 months)-used us to follow patients with tm and , similarly to our present results , did not detect the appearance of new testicular tumors .", "compared with these prior studies , our current study incorporates more patients and a longer - term follow - up extending through puberty .", "an additional strength of our study over previous studies is in our investigation of changes in calcified density of the tm .", "one earlier study has reported nonquantitative changes in the prominence of tm on yearly us follow - up .", "the tm was less prominent in one patient , unchanged in four , and two patients were lost to follow - up . to our knowledge , our present study is the first report to provide a quantitative analysis of calcific density in tm .", "calcific density on us showed a nonstatistically significant trend toward increase over time in our study subjects .", "reported a relationship between the number of microliths and testicular cancer after subgrouping subjects into four ranges based on the number of microliths . in our present study , we divided tm into two nearly equal groups based on the distribution pattern of calcification ( focal type [ 23 testes ] vs. diffuse type [ 20 testes ] ) that appear to differ in terms of calcification trends .", "calcific density shows a trend toward a decrease in focal tm but toward an increase in 50% of the testes with diffuse tm .", "notably , the majority of testes ( 5/8 ) in our study series with cryptorchidism , including those with bilateral cryptorchidism , were categorized as being in the increased calcification group ." ], [ "during the follow - up through puberty of the microlithiasis in the 23 boys in our present study , we observed no significant changes in the density of their microliths and no development of testicular cancer ; however , we found that diffuse tm and cryptorchidism tend to increase calcific density .", "hence , close observation is recommended in cases of tm combined with cryptorchidism and diffuse microlithiasis ." ] ]

[ "bluetongue viruses , pathogenic only to domestic cattle and wild ruminants ( barratt - boyes and maclachlan , 1995 ; tsuboi and imada , 1997 ) , are non - pathogenic to humans ( xiao et al . , 2004 ; hu et al . , 2008 ) and thus , humans do not have pre - existing antibodies to btvs ( jeggo et al . , 1983 ; barratt - boyes and maclachlan , 1995 ; dong et al . , 1998 )", "not only can btv be grown easily in vitro ( jameson and grossberg , 1981 ; du , 1985 ; samal et al . , 1985 ; sundin and mecham , 1989 ; dong et al . , 1998 ; wechsler and mcholland , 1998 ; chen et al . , 1999 ; prasad et al .", ", 1999 ; xue , 2001 ; lei et al . , 2004 ; xiao et al . , 2004 ; liang et al . , 2006 ;", "hu et al . , 2008 ) , it has powerful oncolytic activity against many different in vitro cultured cancer cell lines ( chen et al .", "2008 ) , such as a498 human kidney cells ( atcc : htb-4 ) , hep - g2 human liver cells ( atcc : hb-8065 ) , a549 human lung epithelial cells ( atcc : ccl-185 ) , baby hamster kidney cells ( bhk-21 ) , monkey kidney ( vero ) cells ( ramig et al . , 1989 ) , mouse fibroblast cells ( nih 3t3 ) , spc - a-1 cells ( lei et al . , 2004 ) , ma 782 cells ( liang et al . , 2006 ) , and in mouse models .", "no normal human cells such as the primary human embryo lung fibroblast ( hel ) and primary murine embryos fibroblast ( mef ) have ever been successfully infected by btv ( dong et al . , 1998 ; chen et al . , 1999 ;", ", 2004 ; liang et al . , 2006 ; hu et al . , 2008 ) and thus , humans are normally sero - negative .", "madin darby canine kidney ( mdck ) which is sensitive to reovirus apoptosis also proved resistant to btv infection except in high moi ( unpublished data ) .", "btvs can also infect cultured insect cells ( king and alders , 1985 ; samal et al .", ", 1987 ; guirakhoo et al . , 1995 ; mullens et al . , 1995 ; mertens et al . , 1996", "; xu et al . , 1997 ; tan et al . , 2001 ) .", "in the last decade , our lab and collaborators have used both cell culture systems ( dong et al . , 1998 ;", "xiao et al . , 2004 ; liang et al . , 2006 ; hu et al . ,", "2008 ) and mice ( manuscript in preparation ) to investigate and determine the oncolytic activities and capacities of btvs and their selective degradation of human cancer cells as potential treatment of human cancers .", "human cancer cell lines such as hep-3b and hep - g2 , a549 , a498 , spc - a-1 , and ma 782 cells infected with btv 10 , 11 , 13 , 17 , and hbc3 at various mois exhibited the following easily detected characteristics 36 h post - infection ( pi ) but not in hel and mef cells in multiple determinations ( xiao et al . , 2004 ; hu et al . , 2008 ) .", "briefly : between 70 and 90% cytopathic effects ( cpe ) were readily observed and detected along with cellular apoptosis and necrosis in many human cancer cell lines .", "various degrees of morphological and cellular damage could be visualized by either light , confocal , or electron microscopy ( em ) .", "cellular dilated endoplasmic reticulum , nuclear chromatin condensation , and cytoplasmic shrinking were readily detected and easily observed in em .", "btv viral genomic ds - rna fragments and viral mrnas could be found in btv-10 , 13 , 17 , and hbc3 infected human cancer cells , but not in normal hel or mef cells by gel electrophoresis , elisa , rip , western blots , pcr , and qrt - pcr ( tiwari et al . , 2000 ) .", "the survival rate of several human cancer cell lines was low if high moi was used .", "primary hel and mef cells survived very well for 69 days independent of the moi used for testing .", "human cancer cells infected with btvs had more efficient viral replication and provided higher viral progeny yields .", "selective cell cycle arrest at sub - g1 peak in several human cancer cells infected with btv-10 ( hu et al . , 2008 ) and hbc3 similar to the reovirus - induced g2/m cell cycle arrest ( poggioli et al . , 2000", "after tumor cells were grown in culture and then injected as xenografts into mice ( eight mice / sample ) , tumors developed and all these mice died in less than 8 days ( preliminary data ) .", "when 0.1 ml of btvs with different mois in sterile phosphate - buffered saline ( pbs ) was injected at three different sites of each xenograft , the sizes of tumors in mice were reduced 6085% .", "most mice with the reduced tumors survived more than 35 days before they were sacrificed for tissue and organ samples ( kidney , lung , liver , heart . etc .", "thus , btvs selective cytotoxic effects to human tumor cells in vitro and partially in vivo have been clearly indicated and demonstrated . further in vivo research and clinical studies", "are now in progress to document and investigate this unique oncolyitc potential of btvs which will provide us better and safer application in humans either in monotherapy or combination therapy in the near future .", "bluetongue viruses administration has been shown to cause cell death and apoptosis in several cell lines as well as in several human tumor cell lines ( wechsler and mcholland , 1998 ; xiao et al . , 2004", "; liang et al . , 2006 ; hu et al . , 2008 ) .", "however , in experiments with cultured human cancer cells , the cells detached and died progressively throughout btv infection .", "all btv genes are transcribed and btv proteins are expressed during infection . in the analysis of secreted analyte expression , cytokine and chemokine levels from mock- and btv-17 infected cells", "were determined using multiplex immunoassay kits and reagents supplied from quansys biosciences ( logan , ut , usa ) , according to the supplier 's protocol .", "twenty - five analytes were tested and these included il-1a , il-1b , il-2 , il-4 , il-5 , il-6 , il-8 , il-10 , il-12p40 , il-12p70 , il-13 , ifn , tnf , tnf , tgf - b1 , mcp-1 , rantes , ang2 , hgf , timp , tpo , vegf , pdgf , fgf - basic , and crp ( manuscript in preparation ) .", "of the 25 analytes tested for during infection , only seven were found to be expressed from five cell lines : a498 human kidney cells , hep - g2 human liver cells , a549 human lung epithelial cells ( atcc : htb-4 , hb-8065 , ccl-185 , respectively ) , bhk-21 cells , or vero cells .", "of these seven analytes , five were found to be consistently elevated upon btv infection , with the expression pattern consistent among cell lines .", "these are il-6 , il-8 , mcp-1 , rantes , and fgf. the levels of vegf and timp were similar in control and btv - infected cells . however , a498 kidney cells responded to infection with the greatest secretion of cytokines , followed by a549 lung cells while hep - g2 liver cells secreted the least amount of cytokines .", "polyinosine : polycytosine ( poly i : c ) is a synthetic ds - rna that has been used to assess the role of ds - rna segments of many ds - rna viruses in vitro since it can induce differential interferon and cytokine production ( der et al . , 1998 ;", ", 2003 ; chiang et al . , 2006 ) . however , poly i : c alone did not cause cell death or have a significant impact on cytokine or chemokine expression in btv - infected human cancer cell lines . only il-6 and il-8 were found to be significantly elevated during poly i : c treatment .", "increases in il-6 and il-8 were observed only in kidney cells ( a498 and vero ) and were most prevalent in samples treated with 1 g / ml", "the other cytokines mcp-1 , rantes , and fgf were all at expression levels comparable to the placebo treated cells . with the identification of some of the antigenic epitopes of various btv proteins ( wang et al .", "gould et al . , 1988a , 1994 ; grieder and schultz , 1989 ; geysen , 1990 ; li and yang , 1990 , 1992 ; marshall and roy , 1990 ; mecham and jochim , 1990 ; eaton et al . , 1991 ; rossitto and maclachlan , 1992 ; du plessis et al . , 1994 , 1995 ;", ", 1997 ; nagesha et al . , 2001 ) , btv immunity has only been studied partially in the last two decades because of lack of supporting funding and reliable immune assays ( jeggo and wardley , 1982a , b ; gibbs et al . , 1983 ;", "jeggo et al . , 1983 ; campbell and grubman , 1985 ; stott et al . , 1985 ; fukusho et al .", ", 1987a ; marshall and roy , 1990 ; martyn et al . , 1991 ;", "; lin and zhou , 1996 ; wade - evans et al . , 1996 ; odeon et al . , 1999 ; prasad and minakshi , 1999 ; demaula et al . , 2000 ; roy , 2003 ) . with the recent development of more reliable immune assays and microarrays", ", we have found that btv can induce inflammatory cytokine expression in immortalized human kidney , lung , and liver cells and subsequently kill the infected cell with cell line - dependent severity .", "analysis of results from the cytokine profiles and expression levels in these five cell lines indicated that human tumor cells and tumor reduction might be a result of the combined effects of direct viral induced cell death and recruitment of t - lymphocytes ( jeggo and wardley , 1982a , b ; jones et al .", ", 1996 , 1997 ) to the tumor through elevated cytokine / chemokine production ( shmulevitz et al . , 2005 , 2010 ) .", "very high levels of pro - inflammatory cytokines were secreted from btv - infected cells and cytokine levels secreted from each of the seven cell types were directly proportional to the measured death of infected cells .", "thus , we hypothesize that the cell death and cytokine expression are related and may be caused by shared signaling pathways or events .", "if these same events occurred in human tumors , btv would be expected to reduce tumor size in infected patients by directly causing infected human cancer cell death and by inducing cytokine expression from the tumor that would lead to lymphocyte recruitment to the site of the tumor .", "thus , btv has the therapeutic potential in cancer because of its selective ability to cause preferential apoptosis of various human cancer cell lines and tumors .", "ideal oncolytic viruses should have essential survival data , systemic effect , resist immune system clearance , targeted delivery , and distribution , be highly mobile for intravenous spread , travel to and kill distant metastases , have a high rate of replication to stay ahead of immune system clearance , tolerable toxicity , and side effects , etc .", "systemic delivery makes sense in cancer where primary tumors and metastases are dispersed . however , systemic approach in cancer is a big problem because we do not have a good target specific delivery device that would facilitate uptake specifically for certain cells .", "if this can be achieved , then it would be better than the current and conventional therapies .", "however , it is very difficult to get enough control patients in clinical trials and to get biopsies from different tissues , organs and tumors after the oncolytic virus treatments to determine the efficacy or survival of most oncolytic viruses .", "oncovirologists consider oncolytic virus as a dark horse treatment but not a dead one since they know that many anti - cancer agents appear to be actively effective in lab cultures and animal models but failed in human trials .", "many oncolytic viruses ( norman and lee , 2005 ; alain et al . , 2006 , 2007 ; kim et al .", ", 2006 ; shen and nemunaitis , 2006 ; burroughs et al . , 2007 ; qi et al . , 2007", ", 2009 ; ou and yen , 2010 ) that are currently in phase i / ii / iii human clinical trial status include adenovirus , vaccinia virus , coxsackie virus , reovirus , newcastle disease virus , herpes simplex virus 1 , measles virus , and seneca valley virus ( burroughs et al . , 2007 ) . in 2007 , the only oncolytic virus approved for phase iii clinical trials in china was the adenovirus ( h101 ) with e1b deletion produced jointly by the sunway biotech of china and onyx pharmaceuticals of emeryville , ca , usa ( privilege communication ) .", "since mid-2005 to the end of 2010 , several other oncolytic viruses have also been approved for phase iii clinical trials primarily outside us as briefly described below .", "thus , cancer therapy with oncolytic viruses has survived and revived , and this unique approach is poised for a comeback ( aghi and martuza , 2005 ; norman and lee , 2005 ; alain et al .", "the results of the last 1015 years of investigation generated by researchers have increased interest in oncolytic viruses .", "investigators have generated and obtained some genetically modified viruses with greater oncolytic potentials ( aghi and martuza , 2005 ; norman and lee , 2005 ; alain et al .", "a significant effort is underway to understand the genetic and mechanistic basis of cancers , selective degradation of cancer cells by these oncolytic viruses , and their effects on downstream cellular events and pathways ( strong et al .", "vorburger et al . , 2004 ; aghi and martuza , 2005 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; liu , 2006 ; kim et al . , 2007 ; liu et al . , 2007 ;", "2009 ; hill and lee , 2010 ; ou and yen , 2010 ; thirukkumaran et al . , 2010 ) .", "increasing attention is being paid to protein families like proteases , caspases , and kinases which play critical roles in cancer .", "current therapeutic approaches for human cancer include the regimen of selective surgery , high dose but cytotoxic chemotherapy with two to three drugs and radiation .", "these approaches are not very effective to combat the increasing numbers of human cancers . using oncolytic viruses generates a paradigm shift from current cancer treatment using chemotherapy and radiation which typically destroy both normal and cancer tumor cells .", "as we learn more about cancer biology and oncolytic viruses , new strategies for treating cancers are rapidly evolving .", "uses of oncolytic virus alone or with synergistic anti - cancer drugs or irradiation in human clinical trials are currently under consideration or in progress .", "reovirus leads the field of oncolytic viruses and positive clinical trials are very encouraging ( strong et al . , 1998 ; norman et al . , 2004 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; kim et al .", "2009 ; hill and lee , 2010 ; thirukkumaran et al . , 2010 ) .", "other companies and institutes are not far behind . in mid - may 2010 , researchers of the university of helsinki and oncos therapeutics have treated 200 patients with their modified gmcsf armed oncolytic adenovirus which exhibited anti - tumor immunity that recruited natural killer cells and induced tumor - specific cytotoxic t - cells with strong efficacy and safety .", "the media report indicated that its clinical benefit is about 45% according to the recist criteria with no grade four to five side effects detected .", "this represents an extension of their initial investigation in 2008 . with these successful applications and those from other oncolytic viruses", ", we can now add the simple but oncolytic btv as a key component to the armamentarium of oncologists to combat and destroy the insidious cancer cells since btvs have similar characteristics and additional subtle advantages over reovirus and other oncolytic viruses .", "bluetongue viruses and reovirus are more advantageous than the genetically modified oncolytic dna viruses since the integration of viral ds - rna genome into the host cell genome have not been shown or reported . since humans are sero - negative to btvs , potential future use of btv to degrade human cancer by direct intratumoral injection to degrade the injected tumors will not be inhibited by pre - existing immune components within human cancer cells .", ". the current mechanisms of oncolytic reovirus toward human cancer cells have recently been summarized ( strong et al . , 1998 ; norman et al . , 2004 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; kim et al .", ", 2009 ; hill and lee , 2010 ; thirukkumaran et al . , 2010 ) .", "genetically modified reovirus serotype 3 , but not types 1 and 2 , is a proprietary product called reolysin produced by reolytics biotech , inc .", "data for cancer treatment with this agent from the last decade in animal models and lately in phase i / ii human clinical trials have shown encouraging indications of its potential oncolytic activities in cancer cells bearing an activated ras pathway .", "phase iii human clinical trials have been approved and initiated in 2009 . however , the pre - existing anti - reovirus sera in humans worldwide can trigger greater immune responses against this virus , reducing its efficacy and effectively shutting down its potential oncolytic treatment . uses of oncolytic btv have greater advantages over reovirus since no anti - btv serum is detected in most people globally .", "even though btv injected into tumors can activate dendritic cells for early detection of viral infection , it will take 1014 days before adaptive immune responses can be generated against btvs which can eradicate xenografted tumor cells in mice in less than 10 days ( preliminary data ) .", "oncolytics biotech at calgary , ab , canada showed the uses of reovirus ( reolysin ) plus cisplatin , a standard chemotherapy agent in both cultured melanoma cells and a mouse model .", "the combined uses of reolysin and cisplatin were much better than either of these two agents alone since they appeared to work synergistically ( abstract of the 4th international symposium on the treatment of cancers with oncolytic viruses at scottsdale , arizona march 1517 , 2007 ) .", "furthermore , they had also showed that simultaneous and combined uses of reolysin and gemcitabine could completely eradicate the transplanted colon cancer in four of the five mice ( abstract of the american association for cancer research ( aacr ) annual meeting in april 2007 ) . since mid - april , 2007", ", oncolytics biotech has demonstrated some success in multiple phase ii trials with several medical centers in the uk and us , administering intravenous reolysin to patients with sarcomas that have metastasized to the lung .", "multiple clinical trials for other tumors with the us national cancer institute ( nci ) were conducted in late 2007 .", "this will help to determine whether reolysin can migrate systemically to metastasized tumor sites and exhibit its oncolytic activity .", "oncolytics biotech has expended more than 65 million dollars and a decade of genetic modifications of reolysin before human clinical trials were approved .", "similar and greater expenditures have also been made by other companies and institutes in the last 1215 years to develop different oncolytic viruses with tremendous personnel efforts before some clinical trials are approved and conducted .", "we strongly believe that btv is the front runner of the next generation oncolytic viruses since it needs no genetic modifications and attenuation , and carries no payloads .", "it can grow fast and spread easily within many human cancer cells . since there is no pre - existing neutralizing antibody against btv in humans , they should be undetected nor immunologically inhibited in the initial injection alone or with anti - cancer agents .", "in addition to the comparison of the oncolysis of human cancer cells by reovirus and btv as shown in table 2 , btv has additional advantages .", "briefly : since btv does not infect normal human cells , we do not have to suppress the immune system before use", "btv is very stable since it can be stored at ph 8.28.4 at 4c for over 20 years with only 510% loss of infectivity ( unpublished data ) .", "thus , it can be shipped by air - mail globally without significant loss of its infectivity and oncolytic activities ( unpublished data ) .", "2-d gel electrophoresis and the 3-d assay ( lam et al . , 2007 ) for the measurement of btv - induced oncolysis are currently under optimization and further development and adaptation .", "btv infection of many cultured human tumor cells has led to the production of many inflammatory cytokines such as tnf - alpha and il-6 which strongly activate the kinase activity of p38 mapk and erk1/2 .", "these cytokines also assist the subsequent activation of p38-dependent mapk and pkr pathways which can bring about the necrosis and apoptosis of cancer cells .", "this strongly suggests that btv can prime and direct the immune system of cancer patients against certain solid tumors such as breast and prostate cancer .", "repeat screening of over 500 human cytokines using biotin label - based cytokine antibody array is currently in progress and data are under comparative analysis .", "construction and rescue of recombinant infectious btv ( boyce and roy , 2007 ) expressing the enhanced green fluorescent protein ( egfp ) , using a protocol similar to one recently published by our lab for human parainfluenza virus type 3 ( roth et al .", "comparison of oncolytic reovirus and bluetongue virus . * reovirus references : strong et al .", "an activated ras / ralgef / p38 pathway is potentially involved with the permissiveness of host cells to reovirus infection ( norman et al . , 2004 ; shmulevitz et al . , 2005 ; alain et al .", ", 2007 ; kim et al . , 2007 ; marcato et al . , 2007 ) .", "it has been shown that the c - k - ras gene inside the lung carcinoma cell line a549 we used has mutation at codon 12 ( valenzuela and groffen , 1986 ) .", "since the ras / ralgef / p38 signal pathway inside the a549 cell has been activated , this might allow btv to degrade human tumor cells efficiently similar to reovirus ( stott et al .", ", 1985 ; shmulevitz et al . , 2005 , 2010 ; kim et al . , 2007 ; marcato et al . , 2007", "we have further hypothesized that the genetic alterations / mutations of different human cancer cells , such as signals within a ras - activated pathway , play a key role in the susceptibility of different oncolytic viruses in addition to specific viral receptors which might be present in human cancer cells but not in normal human cells .", "identification of btv receptor(s ) on the surface of human cancer cells is also currently in progress .", "comparison of oncolytic potentials between reovirus and btv is summarized in table 2 . with regard to the potential cytotoxic effect mechanisms of btvs , the gp5 protein of btv identified by our lab ( li and yang , 1992 ; yang and li , 1992 , 1993 ; yang et al .", ", 1992a ) has recently been shown to induce or trigger apoptosis in mammalian cells ( mortola et al . , 2004 ) .", "furthermore , the non - structural btv protein , ns-3 , has been shown to be a ", "viroporin , the polymerization of which forms pores on the surface of btv - infected cells , leading to severe cellular leakage , and potential cell death ( han and harty , 2004 ) .", "we hypothesize that these two btv proteins and other(s ) are involved with the cytotoxic effect of human cancer cells after btv infection .", "individual btv gene silencing with rnai to determine the contribution of each of the btv gene products to onclysis of human cancer cells are also in progress .", "the protein toll ( means weird in german ) was discovered in the fruit fly drosophila melanogaster in 1980s .", "however , if mutations in toll occurred in the fruit fly , the infection by aspergillus fumigates ( a fungus ) is lethal .", "toll - related molecules involved with human innate immunity and coined as toll - like receptors ( tlrs ) were then discovered in mid-1990s by ruslan medzhitov and charles janeway ( medzhitov and janeway , 2002 ; norman et al . , 2004 ) .", "tlrs are molecular pattern recognition receptors and can recognize molecules that are broadly shared by many pathogens .", "thus , tlrs pathways play a key role in both innate immunity and normal immune physiology that can sense different pathogens and cancer cells .", "to determine whether mutated tlrs are involved with the recognition of btvs , we have initiated the silencing of each 1 of the 10 known human tlrs tlr- 110 using rnai .", "human cancer cell lines , a498 and hek 239 , are transfected with dna plasmids containing sirna sequences against human tlrs ( invivogen , ca , usa ) .", "preliminary data indicated that endosome - located tlr-3 which specifically recognizes ds - rna is potentially involved since cells transfected with this tlr-3 rnai plasmid produced substantially less btv 17 and 10 progeny .", "cells transfected with tlr-2 , 4 , 7 , 8 , and 9 rnai plasmids produced slightly less viral progeny in these two human cancer cell lines .", "experiments are now underway to determine what types of mutation are in the tlr-3 in these four human cancer cell lines and this might provide a potential explanation of why normal human cells are not infected by btv because the regular tlr-3 has not been modified nor mutated .", "several signal pathways related to tlrs are currently under screening using pathway screen arrays ( qiagen / sabiosciences ) .", "the protein toll ( means weird in german ) was discovered in the fruit fly drosophila melanogaster in 1980s .", "however , if mutations in toll occurred in the fruit fly , the infection by aspergillus fumigates ( a fungus ) is lethal .", "toll - related molecules involved with human innate immunity and coined as toll - like receptors ( tlrs ) were then discovered in mid-1990s by ruslan medzhitov and charles janeway ( medzhitov and janeway , 2002 ; norman et al . , 2004 ) .", "tlrs are molecular pattern recognition receptors and can recognize molecules that are broadly shared by many pathogens .", "thus , tlrs pathways play a key role in both innate immunity and normal immune physiology that can sense different pathogens and cancer cells .", "to determine whether mutated tlrs are involved with the recognition of btvs , we have initiated the silencing of each 1 of the 10 known human tlrs tlr- 110 using rnai .", "human cancer cell lines , a498 and hek 239 , are transfected with dna plasmids containing sirna sequences against human tlrs ( invivogen , ca , usa ) .", "preliminary data indicated that endosome - located tlr-3 which specifically recognizes ds - rna is potentially involved since cells transfected with this tlr-3 rnai plasmid produced substantially less btv 17 and 10 progeny .", "cells transfected with tlr-2 , 4 , 7 , 8 , and 9 rnai plasmids produced slightly less viral progeny in these two human cancer cell lines .", "experiments are now underway to determine what types of mutation are in the tlr-3 in these four human cancer cell lines and this might provide a potential explanation of why normal human cells are not infected by btv because the regular tlr-3 has not been modified nor mutated .", "several signal pathways related to tlrs are currently under screening using pathway screen arrays ( qiagen / sabiosciences ) .", "human cancers can be divided into two major types , 90 and 10% of which are solid ( carcinoma and sarcoma ) or liquid ( leukemia ) tumors , respectively .", "recent data and epidemiological analysis of many tumors reveal that most fda - approved cancer therapeutic drugs can only provide small incremental improvement and survival to cancer patients .", "companies that make many unrealistic claims for human cancer cures in scientific tabloid media do so strictly for financial reasons since these expensive cancer drugs can generate great revenues via physicians who make drug recommendation and to whom drug companies provide substantial and financial ", "however , the 90% of deaths of cancer patients are primarily caused by those cancer stem cells that have metastasized to other regions to form metastatic tumors .", "humans are sero - negative toward btvs since btvs do not infect normal human cells .", "infection and selective degradation of several human cancer cell lines , but not normal cells , in vitro by five us btv serotypes have been investigated by our lab for the last few years when btv oncolytic potential was discovered .", "we have also recently found that direct single injection of oncolytic btv into the human tumor xenografts in mice , have led to cytotoxic destruction and preferential lysis of cancer cells within the xenograft but not in normal healthy human or mouse cells ( preliminary data ) .", "thus , oncolytic btvs are safer and more effective than most current cancer treatments . however , we believe that oncolytic btv is not a stand - alone therapeutic and that simultaneous combination of btv with either radiation or chemotherapy can obliterate the tumors more rapidly and extensively .", "cancer patients should have fewer side effects since small dosage radiation and anti - cancer drugs are used for this combined treatment .", "related to the oncolytic btvs , the following questions have been frequently asked : is widely curing cancer by oncolytic viruses possible or just hype ?", "big is the gap between in vitro / in vivo studies and human application ?", "is oncolytic btv a vital alternative for cancer therapy ? what are the pathways btv used to carry out this task ?", "overall , we believe that the oncolytic ability of btv represents a significant potential to selectively degrade human cancer cells alone and would even be greater when combined with simultaneous anti - cancer drug and radiation treatment .", "our group and collaborators are currently engaged in several types of investigations with these goals : ( 1 ) study the mechanistic basis of lung , kidney , and liver cancer destruction by btvs , ( 2 ) examine and determine biological profiles of three types of solid tumors after btv infection , ( 3 ) correlate the responses of these human cancer cells to changes at the cellular dna , rna and protein level , and ( 4 ) construction and rescue of an infectious recombinant btv 17 expressing the enhanced egfp using the method that we have developed for the human parainfluenza virus type 3 ( roth et al . , 2010 ) with btv ds - rna fragments ( kowalik et al . , 1990b ; boyce and roy , 2007 ) , the success of which can further reveal the oncolytic pathways used by btvs .", "taking the next step in the investigation of btv oncolysis is very challenging since no one can individually excel in all the technical , scientific , and clinical avenues involved in this endeavor .", "our ongoing in vitro work with different human cancer cell lines will assist us in elucidating the potential mechanisms of the btv oncolytic activities .", "no cpe was detected nor viral progeny obtained when several primary human cell lines were infected by btv .", "when xenografts implanted in mice were injected directly with btv , no pathological damages were detected in any tissue or organs 721 days post - infection ( preliminary data ) .", "thus , the potential risks of btv oncotherapy is substantially lower than risks associated with other oncolytic viruses . however", ", this author sincerely hopes that expert advice and suggestions will be forthcoming from different champions after they have read this brief review .", "the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ." ]

[ "<S> humans are sero - negative toward bluetongue viruses ( btvs ) since btvs do not infect normal human cells . </S>", "<S> infection and selective degradation of several human cancer cell lines but not normal ones by five us btv serotypes have been investigated . </S>", "<S> we determined the susceptibilities of many normal and human cancer cells to btv infections and made comparative kinetic analyses of their cytopathic effects , survival rates , ultra - structural changes , cellular apoptosis and necrosis , cell cycle arrest , cytokine profiles , viral genome , mrnas , and progeny titers . </S>", "<S> the wild - type us btvs , without any genetic modifications , could preferentially infect and degrade several types of human cancer cells but not normal cells . </S>", "<S> their selective and preferential btv - degradation of human cancer cells is viral dose </S>", "<S> dependent , leading to effective viral replication , and induced apoptosis . </S>", "<S> xenograft tumors in mice were substantially reduced by a single intratumoral btv injection in initial in vivo experiments . </S>", "<S> thus , wild - type btvs , without genetic modifications , have oncolytic potentials . </S>", "<S> they represent an attractive , next generation of oncolytic viral approach for potential human cancer therapy combined with current anti - cancer agents and irradiation . </S>" ]

[ "Unique Novel Oncolytic activities of the BTV Virolomic System", "Inflammatory Mediators are Elevated During BTV Infection", "Progress and Perspectives of Oncolytic Viruses", "Comparative Viratherapy and Viratherapeutics of Reovirus (Strong et al., ", "Different Mechanisms by Which Oncolytic Reovirus and BTV Can Selectively Degrade Cancer Cells", "Toll-like receptor signaling and BTVs", "Conclusion", "Conflict of Interest Statement" ]

[ [ "bluetongue viruses , pathogenic only to domestic cattle and wild ruminants ( barratt - boyes and maclachlan , 1995 ; tsuboi and imada , 1997 ) , are non - pathogenic to humans ( xiao et al . , 2004 ; hu et al . , 2008 ) and thus , humans do not have pre - existing antibodies to btvs ( jeggo et al . , 1983 ; barratt - boyes and maclachlan , 1995 ; dong et al . , 1998 )", "not only can btv be grown easily in vitro ( jameson and grossberg , 1981 ; du , 1985 ; samal et al . , 1985 ; sundin and mecham , 1989 ; dong et al . , 1998 ; wechsler and mcholland , 1998 ; chen et al . , 1999 ; prasad et al .", ", 1999 ; xue , 2001 ; lei et al . , 2004 ; xiao et al . , 2004 ; liang et al . , 2006 ;", "hu et al . , 2008 ) , it has powerful oncolytic activity against many different in vitro cultured cancer cell lines ( chen et al .", "2008 ) , such as a498 human kidney cells ( atcc : htb-4 ) , hep - g2 human liver cells ( atcc : hb-8065 ) , a549 human lung epithelial cells ( atcc : ccl-185 ) , baby hamster kidney cells ( bhk-21 ) , monkey kidney ( vero ) cells ( ramig et al . , 1989 ) , mouse fibroblast cells ( nih 3t3 ) , spc - a-1 cells ( lei et al . , 2004 ) , ma 782 cells ( liang et al . , 2006 ) , and in mouse models .", "no normal human cells such as the primary human embryo lung fibroblast ( hel ) and primary murine embryos fibroblast ( mef ) have ever been successfully infected by btv ( dong et al . , 1998 ; chen et al . , 1999 ;", ", 2004 ; liang et al . , 2006 ; hu et al . , 2008 ) and thus , humans are normally sero - negative .", "madin darby canine kidney ( mdck ) which is sensitive to reovirus apoptosis also proved resistant to btv infection except in high moi ( unpublished data ) .", "btvs can also infect cultured insect cells ( king and alders , 1985 ; samal et al .", ", 1987 ; guirakhoo et al . , 1995 ; mullens et al . , 1995 ; mertens et al . , 1996", "; xu et al . , 1997 ; tan et al . , 2001 ) .", "in the last decade , our lab and collaborators have used both cell culture systems ( dong et al . , 1998 ;", "xiao et al . , 2004 ; liang et al . , 2006 ; hu et al . ,", "2008 ) and mice ( manuscript in preparation ) to investigate and determine the oncolytic activities and capacities of btvs and their selective degradation of human cancer cells as potential treatment of human cancers .", "human cancer cell lines such as hep-3b and hep - g2 , a549 , a498 , spc - a-1 , and ma 782 cells infected with btv 10 , 11 , 13 , 17 , and hbc3 at various mois exhibited the following easily detected characteristics 36 h post - infection ( pi ) but not in hel and mef cells in multiple determinations ( xiao et al . , 2004 ; hu et al . , 2008 ) .", "briefly : between 70 and 90% cytopathic effects ( cpe ) were readily observed and detected along with cellular apoptosis and necrosis in many human cancer cell lines .", "various degrees of morphological and cellular damage could be visualized by either light , confocal , or electron microscopy ( em ) .", "cellular dilated endoplasmic reticulum , nuclear chromatin condensation , and cytoplasmic shrinking were readily detected and easily observed in em .", "btv viral genomic ds - rna fragments and viral mrnas could be found in btv-10 , 13 , 17 , and hbc3 infected human cancer cells , but not in normal hel or mef cells by gel electrophoresis , elisa , rip , western blots , pcr , and qrt - pcr ( tiwari et al . , 2000 ) .", "the survival rate of several human cancer cell lines was low if high moi was used .", "primary hel and mef cells survived very well for 69 days independent of the moi used for testing .", "human cancer cells infected with btvs had more efficient viral replication and provided higher viral progeny yields .", "selective cell cycle arrest at sub - g1 peak in several human cancer cells infected with btv-10 ( hu et al . , 2008 ) and hbc3 similar to the reovirus - induced g2/m cell cycle arrest ( poggioli et al . , 2000", "after tumor cells were grown in culture and then injected as xenografts into mice ( eight mice / sample ) , tumors developed and all these mice died in less than 8 days ( preliminary data ) .", "when 0.1 ml of btvs with different mois in sterile phosphate - buffered saline ( pbs ) was injected at three different sites of each xenograft , the sizes of tumors in mice were reduced 6085% .", "most mice with the reduced tumors survived more than 35 days before they were sacrificed for tissue and organ samples ( kidney , lung , liver , heart . etc .", "thus , btvs selective cytotoxic effects to human tumor cells in vitro and partially in vivo have been clearly indicated and demonstrated . further in vivo research and clinical studies", "are now in progress to document and investigate this unique oncolyitc potential of btvs which will provide us better and safer application in humans either in monotherapy or combination therapy in the near future ." ], [ "bluetongue viruses administration has been shown to cause cell death and apoptosis in several cell lines as well as in several human tumor cell lines ( wechsler and mcholland , 1998 ; xiao et al . , 2004", "; liang et al . , 2006 ; hu et al . , 2008 ) .", "however , in experiments with cultured human cancer cells , the cells detached and died progressively throughout btv infection .", "all btv genes are transcribed and btv proteins are expressed during infection . in the analysis of secreted analyte expression , cytokine and chemokine levels from mock- and btv-17 infected cells", "were determined using multiplex immunoassay kits and reagents supplied from quansys biosciences ( logan , ut , usa ) , according to the supplier 's protocol .", "twenty - five analytes were tested and these included il-1a , il-1b , il-2 , il-4 , il-5 , il-6 , il-8 , il-10 , il-12p40 , il-12p70 , il-13 , ifn , tnf , tnf , tgf - b1 , mcp-1 , rantes , ang2 , hgf , timp , tpo , vegf , pdgf , fgf - basic , and crp ( manuscript in preparation ) .", "of the 25 analytes tested for during infection , only seven were found to be expressed from five cell lines : a498 human kidney cells , hep - g2 human liver cells , a549 human lung epithelial cells ( atcc : htb-4 , hb-8065 , ccl-185 , respectively ) , bhk-21 cells , or vero cells .", "of these seven analytes , five were found to be consistently elevated upon btv infection , with the expression pattern consistent among cell lines .", "these are il-6 , il-8 , mcp-1 , rantes , and fgf. the levels of vegf and timp were similar in control and btv - infected cells . however , a498 kidney cells responded to infection with the greatest secretion of cytokines , followed by a549 lung cells while hep - g2 liver cells secreted the least amount of cytokines .", "polyinosine : polycytosine ( poly i : c ) is a synthetic ds - rna that has been used to assess the role of ds - rna segments of many ds - rna viruses in vitro since it can induce differential interferon and cytokine production ( der et al . , 1998 ;", ", 2003 ; chiang et al . , 2006 ) . however , poly i : c alone did not cause cell death or have a significant impact on cytokine or chemokine expression in btv - infected human cancer cell lines . only il-6 and il-8 were found to be significantly elevated during poly i : c treatment .", "increases in il-6 and il-8 were observed only in kidney cells ( a498 and vero ) and were most prevalent in samples treated with 1 g / ml", "the other cytokines mcp-1 , rantes , and fgf were all at expression levels comparable to the placebo treated cells . with the identification of some of the antigenic epitopes of various btv proteins ( wang et al .", "gould et al . , 1988a , 1994 ; grieder and schultz , 1989 ; geysen , 1990 ; li and yang , 1990 , 1992 ; marshall and roy , 1990 ; mecham and jochim , 1990 ; eaton et al . , 1991 ; rossitto and maclachlan , 1992 ; du plessis et al . , 1994 , 1995 ;", ", 1997 ; nagesha et al . , 2001 ) , btv immunity has only been studied partially in the last two decades because of lack of supporting funding and reliable immune assays ( jeggo and wardley , 1982a , b ; gibbs et al . , 1983 ;", "jeggo et al . , 1983 ; campbell and grubman , 1985 ; stott et al . , 1985 ; fukusho et al .", ", 1987a ; marshall and roy , 1990 ; martyn et al . , 1991 ;", "; lin and zhou , 1996 ; wade - evans et al . , 1996 ; odeon et al . , 1999 ; prasad and minakshi , 1999 ; demaula et al . , 2000 ; roy , 2003 ) . with the recent development of more reliable immune assays and microarrays", ", we have found that btv can induce inflammatory cytokine expression in immortalized human kidney , lung , and liver cells and subsequently kill the infected cell with cell line - dependent severity .", "analysis of results from the cytokine profiles and expression levels in these five cell lines indicated that human tumor cells and tumor reduction might be a result of the combined effects of direct viral induced cell death and recruitment of t - lymphocytes ( jeggo and wardley , 1982a , b ; jones et al .", ", 1996 , 1997 ) to the tumor through elevated cytokine / chemokine production ( shmulevitz et al . , 2005 , 2010 ) .", "very high levels of pro - inflammatory cytokines were secreted from btv - infected cells and cytokine levels secreted from each of the seven cell types were directly proportional to the measured death of infected cells .", "thus , we hypothesize that the cell death and cytokine expression are related and may be caused by shared signaling pathways or events .", "if these same events occurred in human tumors , btv would be expected to reduce tumor size in infected patients by directly causing infected human cancer cell death and by inducing cytokine expression from the tumor that would lead to lymphocyte recruitment to the site of the tumor .", "thus , btv has the therapeutic potential in cancer because of its selective ability to cause preferential apoptosis of various human cancer cell lines and tumors ." ], [ "ideal oncolytic viruses should have essential survival data , systemic effect , resist immune system clearance , targeted delivery , and distribution , be highly mobile for intravenous spread , travel to and kill distant metastases , have a high rate of replication to stay ahead of immune system clearance , tolerable toxicity , and side effects , etc .", "systemic delivery makes sense in cancer where primary tumors and metastases are dispersed . however , systemic approach in cancer is a big problem because we do not have a good target specific delivery device that would facilitate uptake specifically for certain cells .", "if this can be achieved , then it would be better than the current and conventional therapies .", "however , it is very difficult to get enough control patients in clinical trials and to get biopsies from different tissues , organs and tumors after the oncolytic virus treatments to determine the efficacy or survival of most oncolytic viruses .", "oncovirologists consider oncolytic virus as a dark horse treatment but not a dead one since they know that many anti - cancer agents appear to be actively effective in lab cultures and animal models but failed in human trials .", "many oncolytic viruses ( norman and lee , 2005 ; alain et al . , 2006 , 2007 ; kim et al .", ", 2006 ; shen and nemunaitis , 2006 ; burroughs et al . , 2007 ; qi et al . , 2007", ", 2009 ; ou and yen , 2010 ) that are currently in phase i / ii / iii human clinical trial status include adenovirus , vaccinia virus , coxsackie virus , reovirus , newcastle disease virus , herpes simplex virus 1 , measles virus , and seneca valley virus ( burroughs et al . , 2007 ) . in 2007 , the only oncolytic virus approved for phase iii clinical trials in china was the adenovirus ( h101 ) with e1b deletion produced jointly by the sunway biotech of china and onyx pharmaceuticals of emeryville , ca , usa ( privilege communication ) .", "since mid-2005 to the end of 2010 , several other oncolytic viruses have also been approved for phase iii clinical trials primarily outside us as briefly described below .", "thus , cancer therapy with oncolytic viruses has survived and revived , and this unique approach is poised for a comeback ( aghi and martuza , 2005 ; norman and lee , 2005 ; alain et al .", "the results of the last 1015 years of investigation generated by researchers have increased interest in oncolytic viruses .", "investigators have generated and obtained some genetically modified viruses with greater oncolytic potentials ( aghi and martuza , 2005 ; norman and lee , 2005 ; alain et al .", "a significant effort is underway to understand the genetic and mechanistic basis of cancers , selective degradation of cancer cells by these oncolytic viruses , and their effects on downstream cellular events and pathways ( strong et al .", "vorburger et al . , 2004 ; aghi and martuza , 2005 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; liu , 2006 ; kim et al . , 2007 ; liu et al . , 2007 ;", "2009 ; hill and lee , 2010 ; ou and yen , 2010 ; thirukkumaran et al . , 2010 ) .", "increasing attention is being paid to protein families like proteases , caspases , and kinases which play critical roles in cancer .", "current therapeutic approaches for human cancer include the regimen of selective surgery , high dose but cytotoxic chemotherapy with two to three drugs and radiation .", "these approaches are not very effective to combat the increasing numbers of human cancers . using oncolytic viruses generates a paradigm shift from current cancer treatment using chemotherapy and radiation which typically destroy both normal and cancer tumor cells .", "as we learn more about cancer biology and oncolytic viruses , new strategies for treating cancers are rapidly evolving .", "uses of oncolytic virus alone or with synergistic anti - cancer drugs or irradiation in human clinical trials are currently under consideration or in progress .", "reovirus leads the field of oncolytic viruses and positive clinical trials are very encouraging ( strong et al . , 1998 ; norman et al . , 2004 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; kim et al .", "2009 ; hill and lee , 2010 ; thirukkumaran et al . , 2010 ) .", "other companies and institutes are not far behind . in mid - may 2010 , researchers of the university of helsinki and oncos therapeutics have treated 200 patients with their modified gmcsf armed oncolytic adenovirus which exhibited anti - tumor immunity that recruited natural killer cells and induced tumor - specific cytotoxic t - cells with strong efficacy and safety .", "the media report indicated that its clinical benefit is about 45% according to the recist criteria with no grade four to five side effects detected .", "this represents an extension of their initial investigation in 2008 . with these successful applications and those from other oncolytic viruses", ", we can now add the simple but oncolytic btv as a key component to the armamentarium of oncologists to combat and destroy the insidious cancer cells since btvs have similar characteristics and additional subtle advantages over reovirus and other oncolytic viruses ." ], [ "bluetongue viruses and reovirus are more advantageous than the genetically modified oncolytic dna viruses since the integration of viral ds - rna genome into the host cell genome have not been shown or reported . since humans are sero - negative to btvs , potential future use of btv to degrade human cancer by direct intratumoral injection to degrade the injected tumors will not be inhibited by pre - existing immune components within human cancer cells .", ". the current mechanisms of oncolytic reovirus toward human cancer cells have recently been summarized ( strong et al . , 1998 ; norman et al . , 2004 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; kim et al .", ", 2009 ; hill and lee , 2010 ; thirukkumaran et al . , 2010 ) .", "genetically modified reovirus serotype 3 , but not types 1 and 2 , is a proprietary product called reolysin produced by reolytics biotech , inc .", "data for cancer treatment with this agent from the last decade in animal models and lately in phase i / ii human clinical trials have shown encouraging indications of its potential oncolytic activities in cancer cells bearing an activated ras pathway .", "phase iii human clinical trials have been approved and initiated in 2009 . however , the pre - existing anti - reovirus sera in humans worldwide can trigger greater immune responses against this virus , reducing its efficacy and effectively shutting down its potential oncolytic treatment . uses of oncolytic btv have greater advantages over reovirus since no anti - btv serum is detected in most people globally .", "even though btv injected into tumors can activate dendritic cells for early detection of viral infection , it will take 1014 days before adaptive immune responses can be generated against btvs which can eradicate xenografted tumor cells in mice in less than 10 days ( preliminary data ) .", "oncolytics biotech at calgary , ab , canada showed the uses of reovirus ( reolysin ) plus cisplatin , a standard chemotherapy agent in both cultured melanoma cells and a mouse model .", "the combined uses of reolysin and cisplatin were much better than either of these two agents alone since they appeared to work synergistically ( abstract of the 4th international symposium on the treatment of cancers with oncolytic viruses at scottsdale , arizona march 1517 , 2007 ) .", "furthermore , they had also showed that simultaneous and combined uses of reolysin and gemcitabine could completely eradicate the transplanted colon cancer in four of the five mice ( abstract of the american association for cancer research ( aacr ) annual meeting in april 2007 ) . since mid - april , 2007", ", oncolytics biotech has demonstrated some success in multiple phase ii trials with several medical centers in the uk and us , administering intravenous reolysin to patients with sarcomas that have metastasized to the lung .", "multiple clinical trials for other tumors with the us national cancer institute ( nci ) were conducted in late 2007 .", "this will help to determine whether reolysin can migrate systemically to metastasized tumor sites and exhibit its oncolytic activity .", "oncolytics biotech has expended more than 65 million dollars and a decade of genetic modifications of reolysin before human clinical trials were approved .", "similar and greater expenditures have also been made by other companies and institutes in the last 1215 years to develop different oncolytic viruses with tremendous personnel efforts before some clinical trials are approved and conducted .", "we strongly believe that btv is the front runner of the next generation oncolytic viruses since it needs no genetic modifications and attenuation , and carries no payloads .", "it can grow fast and spread easily within many human cancer cells . since there is no pre - existing neutralizing antibody against btv in humans , they should be undetected nor immunologically inhibited in the initial injection alone or with anti - cancer agents .", "in addition to the comparison of the oncolysis of human cancer cells by reovirus and btv as shown in table 2 , btv has additional advantages .", "briefly : since btv does not infect normal human cells , we do not have to suppress the immune system before use", "btv is very stable since it can be stored at ph 8.28.4 at 4c for over 20 years with only 510% loss of infectivity ( unpublished data ) .", "thus , it can be shipped by air - mail globally without significant loss of its infectivity and oncolytic activities ( unpublished data ) .", "2-d gel electrophoresis and the 3-d assay ( lam et al . , 2007 ) for the measurement of btv - induced oncolysis are currently under optimization and further development and adaptation .", "btv infection of many cultured human tumor cells has led to the production of many inflammatory cytokines such as tnf - alpha and il-6 which strongly activate the kinase activity of p38 mapk and erk1/2 .", "these cytokines also assist the subsequent activation of p38-dependent mapk and pkr pathways which can bring about the necrosis and apoptosis of cancer cells .", "this strongly suggests that btv can prime and direct the immune system of cancer patients against certain solid tumors such as breast and prostate cancer .", "repeat screening of over 500 human cytokines using biotin label - based cytokine antibody array is currently in progress and data are under comparative analysis .", "construction and rescue of recombinant infectious btv ( boyce and roy , 2007 ) expressing the enhanced green fluorescent protein ( egfp ) , using a protocol similar to one recently published by our lab for human parainfluenza virus type 3 ( roth et al .", "comparison of oncolytic reovirus and bluetongue virus . * reovirus references : strong et al ." ], [ "an activated ras / ralgef / p38 pathway is potentially involved with the permissiveness of host cells to reovirus infection ( norman et al . , 2004 ; shmulevitz et al . , 2005 ; alain et al .", ", 2007 ; kim et al . , 2007 ; marcato et al . , 2007 ) .", "it has been shown that the c - k - ras gene inside the lung carcinoma cell line a549 we used has mutation at codon 12 ( valenzuela and groffen , 1986 ) .", "since the ras / ralgef / p38 signal pathway inside the a549 cell has been activated , this might allow btv to degrade human tumor cells efficiently similar to reovirus ( stott et al .", ", 1985 ; shmulevitz et al . , 2005 , 2010 ; kim et al . , 2007 ; marcato et al . , 2007", "we have further hypothesized that the genetic alterations / mutations of different human cancer cells , such as signals within a ras - activated pathway , play a key role in the susceptibility of different oncolytic viruses in addition to specific viral receptors which might be present in human cancer cells but not in normal human cells .", "identification of btv receptor(s ) on the surface of human cancer cells is also currently in progress .", "comparison of oncolytic potentials between reovirus and btv is summarized in table 2 . with regard to the potential cytotoxic effect mechanisms of btvs , the gp5 protein of btv identified by our lab ( li and yang , 1992 ; yang and li , 1992 , 1993 ; yang et al .", ", 1992a ) has recently been shown to induce or trigger apoptosis in mammalian cells ( mortola et al . , 2004 ) .", "furthermore , the non - structural btv protein , ns-3 , has been shown to be a ", "viroporin , the polymerization of which forms pores on the surface of btv - infected cells , leading to severe cellular leakage , and potential cell death ( han and harty , 2004 ) .", "we hypothesize that these two btv proteins and other(s ) are involved with the cytotoxic effect of human cancer cells after btv infection .", "individual btv gene silencing with rnai to determine the contribution of each of the btv gene products to onclysis of human cancer cells are also in progress .", "the protein toll ( means weird in german ) was discovered in the fruit fly drosophila melanogaster in 1980s .", "however , if mutations in toll occurred in the fruit fly , the infection by aspergillus fumigates ( a fungus ) is lethal .", "toll - related molecules involved with human innate immunity and coined as toll - like receptors ( tlrs ) were then discovered in mid-1990s by ruslan medzhitov and charles janeway ( medzhitov and janeway , 2002 ; norman et al . , 2004 ) .", "tlrs are molecular pattern recognition receptors and can recognize molecules that are broadly shared by many pathogens .", "thus , tlrs pathways play a key role in both innate immunity and normal immune physiology that can sense different pathogens and cancer cells .", "to determine whether mutated tlrs are involved with the recognition of btvs , we have initiated the silencing of each 1 of the 10 known human tlrs tlr- 110 using rnai .", "human cancer cell lines , a498 and hek 239 , are transfected with dna plasmids containing sirna sequences against human tlrs ( invivogen , ca , usa ) .", "preliminary data indicated that endosome - located tlr-3 which specifically recognizes ds - rna is potentially involved since cells transfected with this tlr-3 rnai plasmid produced substantially less btv 17 and 10 progeny .", "cells transfected with tlr-2 , 4 , 7 , 8 , and 9 rnai plasmids produced slightly less viral progeny in these two human cancer cell lines .", "experiments are now underway to determine what types of mutation are in the tlr-3 in these four human cancer cell lines and this might provide a potential explanation of why normal human cells are not infected by btv because the regular tlr-3 has not been modified nor mutated .", "several signal pathways related to tlrs are currently under screening using pathway screen arrays ( qiagen / sabiosciences ) ." ], [ "the protein toll ( means weird in german ) was discovered in the fruit fly drosophila melanogaster in 1980s .", "however , if mutations in toll occurred in the fruit fly , the infection by aspergillus fumigates ( a fungus ) is lethal .", "toll - related molecules involved with human innate immunity and coined as toll - like receptors ( tlrs ) were then discovered in mid-1990s by ruslan medzhitov and charles janeway ( medzhitov and janeway , 2002 ; norman et al . , 2004 ) .", "tlrs are molecular pattern recognition receptors and can recognize molecules that are broadly shared by many pathogens .", "thus , tlrs pathways play a key role in both innate immunity and normal immune physiology that can sense different pathogens and cancer cells .", "to determine whether mutated tlrs are involved with the recognition of btvs , we have initiated the silencing of each 1 of the 10 known human tlrs tlr- 110 using rnai .", "human cancer cell lines , a498 and hek 239 , are transfected with dna plasmids containing sirna sequences against human tlrs ( invivogen , ca , usa ) .", "preliminary data indicated that endosome - located tlr-3 which specifically recognizes ds - rna is potentially involved since cells transfected with this tlr-3 rnai plasmid produced substantially less btv 17 and 10 progeny .", "cells transfected with tlr-2 , 4 , 7 , 8 , and 9 rnai plasmids produced slightly less viral progeny in these two human cancer cell lines .", "experiments are now underway to determine what types of mutation are in the tlr-3 in these four human cancer cell lines and this might provide a potential explanation of why normal human cells are not infected by btv because the regular tlr-3 has not been modified nor mutated .", "several signal pathways related to tlrs are currently under screening using pathway screen arrays ( qiagen / sabiosciences ) ." ], [ "human cancers can be divided into two major types , 90 and 10% of which are solid ( carcinoma and sarcoma ) or liquid ( leukemia ) tumors , respectively .", "recent data and epidemiological analysis of many tumors reveal that most fda - approved cancer therapeutic drugs can only provide small incremental improvement and survival to cancer patients .", "companies that make many unrealistic claims for human cancer cures in scientific tabloid media do so strictly for financial reasons since these expensive cancer drugs can generate great revenues via physicians who make drug recommendation and to whom drug companies provide substantial and financial ", "however , the 90% of deaths of cancer patients are primarily caused by those cancer stem cells that have metastasized to other regions to form metastatic tumors .", "humans are sero - negative toward btvs since btvs do not infect normal human cells .", "infection and selective degradation of several human cancer cell lines , but not normal cells , in vitro by five us btv serotypes have been investigated by our lab for the last few years when btv oncolytic potential was discovered .", "we have also recently found that direct single injection of oncolytic btv into the human tumor xenografts in mice , have led to cytotoxic destruction and preferential lysis of cancer cells within the xenograft but not in normal healthy human or mouse cells ( preliminary data ) .", "thus , oncolytic btvs are safer and more effective than most current cancer treatments . however , we believe that oncolytic btv is not a stand - alone therapeutic and that simultaneous combination of btv with either radiation or chemotherapy can obliterate the tumors more rapidly and extensively .", "cancer patients should have fewer side effects since small dosage radiation and anti - cancer drugs are used for this combined treatment .", "related to the oncolytic btvs , the following questions have been frequently asked : is widely curing cancer by oncolytic viruses possible or just hype ?", "big is the gap between in vitro / in vivo studies and human application ?", "is oncolytic btv a vital alternative for cancer therapy ? what are the pathways btv used to carry out this task ?", "overall , we believe that the oncolytic ability of btv represents a significant potential to selectively degrade human cancer cells alone and would even be greater when combined with simultaneous anti - cancer drug and radiation treatment .", "our group and collaborators are currently engaged in several types of investigations with these goals : ( 1 ) study the mechanistic basis of lung , kidney , and liver cancer destruction by btvs , ( 2 ) examine and determine biological profiles of three types of solid tumors after btv infection , ( 3 ) correlate the responses of these human cancer cells to changes at the cellular dna , rna and protein level , and ( 4 ) construction and rescue of an infectious recombinant btv 17 expressing the enhanced egfp using the method that we have developed for the human parainfluenza virus type 3 ( roth et al . , 2010 ) with btv ds - rna fragments ( kowalik et al . , 1990b ; boyce and roy , 2007 ) , the success of which can further reveal the oncolytic pathways used by btvs .", "taking the next step in the investigation of btv oncolysis is very challenging since no one can individually excel in all the technical , scientific , and clinical avenues involved in this endeavor .", "our ongoing in vitro work with different human cancer cell lines will assist us in elucidating the potential mechanisms of the btv oncolytic activities .", "no cpe was detected nor viral progeny obtained when several primary human cell lines were infected by btv .", "when xenografts implanted in mice were injected directly with btv , no pathological damages were detected in any tissue or organs 721 days post - infection ( preliminary data ) .", "thus , the potential risks of btv oncotherapy is substantially lower than risks associated with other oncolytic viruses . however", ", this author sincerely hopes that expert advice and suggestions will be forthcoming from different champions after they have read this brief review ." ], [ "the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ." ] ]

[ "in the last few years , improvements in the endourological armamentarium together with the downsizing of flexible ureteroscopes , the advent of digital technology , and the increasing number of requests for minimally invasive procedures have made the use of flexible ureteroscopy ( furs ) for renal calculi increasingly attractive , even for stones larger than 2 cm in diameter including the most difficult clinical scenarios .", "recently , roboflex avicenna , a new robotic system , has been introduced for assisting furs , showing good outcomes in terms of stone free rate ( sfr ) and safety , with significant improvements in ergonomics . to date", ", no information exists about the learning curve of roboflex avicenna compared to furs without the robotic device .", "it has been demonstrated that surgeons can become familiar with a specific surgical procedure and improve their skills through simulators by repetitive training in a stress - free environment .", "recently , it has been showed that a new simulator for ureteroscopy , the kidney - box ( k - box , porgs - coloplast ) , represents a valid training model for initiate medical students to flexible ureteroscopy .", "the aim of this study was to evaluate the acquisition of basic ureteroscopy skills with and without roboflex avicenna by subjects with no prior surgical training , using the k - box simulator .", "ten 5-year medical students were voluntarily recruited from pierre and marie curie university of paris .", "subjects were randomized in block fashion to include 5 medical students in each group ; group 1 was trained with avicenna roboflex and group 2 with a standard flexible ureteroscope .", "the flexible ureteroscope used in both groups was urf - v2 ( olympus ) and the simulator for training the students was the k - box ( porgs - coloplast ) .", "all the exercises were assessed with the boxes closed ( non - transparent simulators ) . under supervision of a trained endourological instructor , all students received didactic teaching for 10 lessons in 10 days , at 10 minutes for each session .", "the instructor was responsible for correcting students mistakes , answering and explaining the participants questions and doubts .", "all participants were evaluated by an experienced surgeon ( ot ) , blinded to the randomization table , through two different exercises in the k - box ( figures 1 , 2 ) , both aimed to discover the small spheres present in the simulator .", "while the exercise was done with real time endoscopic vision transmission , the evaluation was performed simultaneously in a separate room in order not to be influenced by the device used .", "study s flow diagram . using an objective structured assessment of technical skills ( osats ) according to previous evaluations in a simulator model performed by hu et al . and chou et al . ", "all participants were scored on their global ability to perform , or not , the exercises recording their times analyzing the knowledge of the procedure , the instrument handling and the trainees competence .", "in addition , the participants were evaluated on the quality of their performance , using a 5-point likert rating scale from 1 ( poor ) to 5 ( excellent ) exclusively made for endoscopic evaluations .", "the scale contains the following parameters : respect of the surrounding environment , flow of the operation , orientation , centered vision and stability ( table 1 ) .", "time to complete the exercise was measured ; the students had a 2 minute timeframe to perform each exercise .", "the rating scale used to assess the quality of students performance statistical analysis was performed with spss version 20.0 ( statistical package for the social sciences , spss inc , chicago , il ) for mac os x. data extracted from our database was automatically converted into spss .", "normality testing ( dagostino and pearson test ) was performed to determine whether data were sampled from a gaussian distribution .", "chi - square and fisher s exact test were used to analyze proportions , as appropriate .", "student s t test and the mann - whitney u - test were performed to compare continuous parametric and non - parametric variables , respectively .", "all the tests were 2-sided and statistically significant difference was considered for p < 0.05 .", "all ten students completed the training . according to the osats evaluation , the global quality of performance was correct in all students .", "the first exercise was completed only by three out of five of students in group 1 ( robotic training ) and by four out of five students in group 2 ( furs alone ) .", "stability with the scope was significantly more accurate in the first group compared with the second , 4.6 + /-", "0.7 ( p = 0.02 ) respectively . there were no differences in timing , flow or orientation between groups .", "although not significant , a tendency of respecting the surrounding tissue and maintaining centered vision was observed more in the first group .", "as for the second exercise , there were no differences between groups in regard of orientation , flow , respecting the surrounding tissue , stability or the ability of maintaining centered vision .", "although not significant , the second group had a tendency of performing the exercise faster .", "nowadays , the technological advances combined with healthcare and ethic law standards have made urologists explore the use of surgical simulators to overtake the first steps in the learning curve of certain procedures [ 10 , 11 ] . it is known that untrained medical students , residents and skilled surgeons ( even tutors ) can become familiar with a specific surgical procedure and efficiently improve their skills through simulation technology by repetitive training in a stress - free environment .", "endourology simulators such as virtual reality , cadaveric , artificial models and animal models [ 11 , 12 ] are being widely and effectively used to master the learning curve of an evolving technology such as furs .", "it has been published and estimated that a surgeon must perform at least 50 furs to achieve surgical competence .", "simulators can actively improve surgeons skills and may possibly accelerate this path . when comparing simulation vs non - simulation furs training , hu et al .", "showed in a cohort of 36 medical students , that those participants with previous training experience in transparent and non - transparent models performed objectively better than those without it .", "the analysis also showed an even better performance in the group with transparent simulators , with all differences being statistically significant .", "a comparison between virtual reality and animal models reported that after a two month training period , all of their 36 participants evaluated by an objective qualitative scale , acquired adequate skills to perform a basic furs independently of the simulator used . in this study", "we used the k - box simulator ( porgs - coloplast ) which is an artificial model made of four different boxes that reproduces all anatomic variability of the upper urinary tract .", "this is a non - transparent model that permits the trainee to handle the actual ureteroscope while looking at a monitor with real time vision transmission .", "the k - box has proven to be a valid simulator to learn furs in a prospectively randomized study that compared a non - trained control group with trainees that underwent a ten day practice experience ( five days to learn the flexible ureteroscope handling and five more days to practice how to catch and release stones with a nitinol basket ) .", "the comparison between groups made by an experienced endourologist with the objective endoscopic skill scale used in this paper ( table 1 ) , revealed that all scores obtained by trained students were significantly higher compared to non - trained students . also , trained students performed the exercises significantly faster than the control group .", "as for the recent introduction of robotic furs , there is no evidence in the literature about the learning curve of this technique . also there is no evidence about simulation of neither the newly robotic furs nor comparisons between skills gained comparing robotic and standard furs simulations .", "we conducted the first prospective randomized trial in the literature comparing the simulation between robotic and standard furs . in our study , ten medical students after ten training sessions were able to acquire good ureteroscopic skills in a simulator model both in robotic and standard furs .", "the skills were evaluated according the validation tool : objective structured assessment of technical skills for surgical residents ( osats ) that analyzes the knowledge of the procedure , the instrument handling and the trainees competence .", "the results demonstrate the rapid acquisition of ureteroscopic skills for trainees with no prior surgical training , and therefore the efficacy of training models in furs .", "moreover , no major significant differences were seen between the groups , except for the stability item that was better in the robotic group for the first exercise , although it took the same group longer time to perform the second exercise .", "these data potentially suggest similar learning curves for robotic and standard furs in a training model that may later maximize the surgeons skills and support the trainers proficiency and excellence .", "nevertheless , these results should be considered with caution because our study certainly presents some limitations due to low numbers of students in each group and , consequently , a lack of adequate statistical power ; but to our best knowledge , this is the first study that compares the acquisition of endourological skills with and without roboflex avicenna in a simulator model .", "further investigation with a wider cohort of participants is needed to establish the real lack of statistical differences in the acquisition of basic ureteroscopic skills with and without robotic furs .", "according to these preliminary results , the acquisition of basic ureteroscopic skills with and without robotic furs in the k - box simulator , by subjects with no prior surgical training , is similar .", "traxer o : consultant for coloplast , rocamed , olympus , lumenis , boston scientific , biohealth , ems .", "giusti g : consultant for coloplast , rocamed , olympus , lumenis , boston scientific , karl storz , cook medical ." ]

[ "<S> introductionthe aim of this study was to evaluate the acquisition of basic ureteroscopic skills with and without roboflex avicenna by subjects with no prior surgical training.material and methodsten medical students were divided in two groups : group 1 was trained with roboflex avicenna and group 2 with flexible ureteroscope alone , using the k - box simulator model . </S>", "<S> participants were scored on their ability to perform or not two exercises , recording the time . </S>", "<S> in addition , the participants were evaluated on the quality of their performance for the following parameters : respect of the surrounding environment , flow of the operation , orientation , vision centering and stability.resultsthe first exercise was completed only by three and four out of five of students in group 1 and group 2 , respectively . </S>", "<S> stability with the scope was significantly more accurate in the first group compared with the second ( p = 0.02 ) . </S>", "<S> there were no differences in timing , flow or orientation between groups . </S>", "<S> although not significant , a tendency of respecting the surrounding tissue and maintaining centered vision was perceived more in the first group . as for the second exercise , there were no differences between groups in regard of orientation , flow , respecting the surrounding tissue , stability or the ability of maintaining centered vision . </S>", "<S> although not significant , the second group had a tendency of performing the exercise faster.conclusionsaccording to these preliminary results , the acquisition of basic ureteroscopic skills with and without robotic furs in the k - box simulator , by subjects with no prior surgical training , is similar . </S>" ]

[ "INTRODUCTION", "MATERIAL AND METHODS", "RESULTS", "DISCUSSIONS", "CONCLUSIONS", "CONFLICTS OF INTEREST" ]

[ [ "in the last few years , improvements in the endourological armamentarium together with the downsizing of flexible ureteroscopes , the advent of digital technology , and the increasing number of requests for minimally invasive procedures have made the use of flexible ureteroscopy ( furs ) for renal calculi increasingly attractive , even for stones larger than 2 cm in diameter including the most difficult clinical scenarios .", "recently , roboflex avicenna , a new robotic system , has been introduced for assisting furs , showing good outcomes in terms of stone free rate ( sfr ) and safety , with significant improvements in ergonomics . to date", ", no information exists about the learning curve of roboflex avicenna compared to furs without the robotic device .", "it has been demonstrated that surgeons can become familiar with a specific surgical procedure and improve their skills through simulators by repetitive training in a stress - free environment .", "recently , it has been showed that a new simulator for ureteroscopy , the kidney - box ( k - box , porgs - coloplast ) , represents a valid training model for initiate medical students to flexible ureteroscopy .", "the aim of this study was to evaluate the acquisition of basic ureteroscopy skills with and without roboflex avicenna by subjects with no prior surgical training , using the k - box simulator ." ], [ "ten 5-year medical students were voluntarily recruited from pierre and marie curie university of paris .", "subjects were randomized in block fashion to include 5 medical students in each group ; group 1 was trained with avicenna roboflex and group 2 with a standard flexible ureteroscope .", "the flexible ureteroscope used in both groups was urf - v2 ( olympus ) and the simulator for training the students was the k - box ( porgs - coloplast ) .", "all the exercises were assessed with the boxes closed ( non - transparent simulators ) . under supervision of a trained endourological instructor , all students received didactic teaching for 10 lessons in 10 days , at 10 minutes for each session .", "the instructor was responsible for correcting students mistakes , answering and explaining the participants questions and doubts .", "all participants were evaluated by an experienced surgeon ( ot ) , blinded to the randomization table , through two different exercises in the k - box ( figures 1 , 2 ) , both aimed to discover the small spheres present in the simulator .", "while the exercise was done with real time endoscopic vision transmission , the evaluation was performed simultaneously in a separate room in order not to be influenced by the device used .", "study s flow diagram . using an objective structured assessment of technical skills ( osats ) according to previous evaluations in a simulator model performed by hu et al . and chou et al . ", "all participants were scored on their global ability to perform , or not , the exercises recording their times analyzing the knowledge of the procedure , the instrument handling and the trainees competence .", "in addition , the participants were evaluated on the quality of their performance , using a 5-point likert rating scale from 1 ( poor ) to 5 ( excellent ) exclusively made for endoscopic evaluations .", "the scale contains the following parameters : respect of the surrounding environment , flow of the operation , orientation , centered vision and stability ( table 1 ) .", "time to complete the exercise was measured ; the students had a 2 minute timeframe to perform each exercise .", "the rating scale used to assess the quality of students performance statistical analysis was performed with spss version 20.0 ( statistical package for the social sciences , spss inc , chicago , il ) for mac os x. data extracted from our database was automatically converted into spss .", "normality testing ( dagostino and pearson test ) was performed to determine whether data were sampled from a gaussian distribution .", "chi - square and fisher s exact test were used to analyze proportions , as appropriate .", "student s t test and the mann - whitney u - test were performed to compare continuous parametric and non - parametric variables , respectively .", "all the tests were 2-sided and statistically significant difference was considered for p < 0.05 ." ], [ "all ten students completed the training . according to the osats evaluation , the global quality of performance was correct in all students .", "the first exercise was completed only by three out of five of students in group 1 ( robotic training ) and by four out of five students in group 2 ( furs alone ) .", "stability with the scope was significantly more accurate in the first group compared with the second , 4.6 + /-", "0.7 ( p = 0.02 ) respectively . there were no differences in timing , flow or orientation between groups .", "although not significant , a tendency of respecting the surrounding tissue and maintaining centered vision was observed more in the first group .", "as for the second exercise , there were no differences between groups in regard of orientation , flow , respecting the surrounding tissue , stability or the ability of maintaining centered vision .", "although not significant , the second group had a tendency of performing the exercise faster ." ], [ "nowadays , the technological advances combined with healthcare and ethic law standards have made urologists explore the use of surgical simulators to overtake the first steps in the learning curve of certain procedures [ 10 , 11 ] . it is known that untrained medical students , residents and skilled surgeons ( even tutors ) can become familiar with a specific surgical procedure and efficiently improve their skills through simulation technology by repetitive training in a stress - free environment .", "endourology simulators such as virtual reality , cadaveric , artificial models and animal models [ 11 , 12 ] are being widely and effectively used to master the learning curve of an evolving technology such as furs .", "it has been published and estimated that a surgeon must perform at least 50 furs to achieve surgical competence .", "simulators can actively improve surgeons skills and may possibly accelerate this path . when comparing simulation vs non - simulation furs training , hu et al .", "showed in a cohort of 36 medical students , that those participants with previous training experience in transparent and non - transparent models performed objectively better than those without it .", "the analysis also showed an even better performance in the group with transparent simulators , with all differences being statistically significant .", "a comparison between virtual reality and animal models reported that after a two month training period , all of their 36 participants evaluated by an objective qualitative scale , acquired adequate skills to perform a basic furs independently of the simulator used . in this study", "we used the k - box simulator ( porgs - coloplast ) which is an artificial model made of four different boxes that reproduces all anatomic variability of the upper urinary tract .", "this is a non - transparent model that permits the trainee to handle the actual ureteroscope while looking at a monitor with real time vision transmission .", "the k - box has proven to be a valid simulator to learn furs in a prospectively randomized study that compared a non - trained control group with trainees that underwent a ten day practice experience ( five days to learn the flexible ureteroscope handling and five more days to practice how to catch and release stones with a nitinol basket ) .", "the comparison between groups made by an experienced endourologist with the objective endoscopic skill scale used in this paper ( table 1 ) , revealed that all scores obtained by trained students were significantly higher compared to non - trained students . also , trained students performed the exercises significantly faster than the control group .", "as for the recent introduction of robotic furs , there is no evidence in the literature about the learning curve of this technique . also there is no evidence about simulation of neither the newly robotic furs nor comparisons between skills gained comparing robotic and standard furs simulations .", "we conducted the first prospective randomized trial in the literature comparing the simulation between robotic and standard furs . in our study , ten medical students after ten training sessions were able to acquire good ureteroscopic skills in a simulator model both in robotic and standard furs .", "the skills were evaluated according the validation tool : objective structured assessment of technical skills for surgical residents ( osats ) that analyzes the knowledge of the procedure , the instrument handling and the trainees competence .", "the results demonstrate the rapid acquisition of ureteroscopic skills for trainees with no prior surgical training , and therefore the efficacy of training models in furs .", "moreover , no major significant differences were seen between the groups , except for the stability item that was better in the robotic group for the first exercise , although it took the same group longer time to perform the second exercise .", "these data potentially suggest similar learning curves for robotic and standard furs in a training model that may later maximize the surgeons skills and support the trainers proficiency and excellence .", "nevertheless , these results should be considered with caution because our study certainly presents some limitations due to low numbers of students in each group and , consequently , a lack of adequate statistical power ; but to our best knowledge , this is the first study that compares the acquisition of endourological skills with and without roboflex avicenna in a simulator model .", "further investigation with a wider cohort of participants is needed to establish the real lack of statistical differences in the acquisition of basic ureteroscopic skills with and without robotic furs ." ], [ "according to these preliminary results , the acquisition of basic ureteroscopic skills with and without robotic furs in the k - box simulator , by subjects with no prior surgical training , is similar ." ], [ "traxer o : consultant for coloplast , rocamed , olympus , lumenis , boston scientific , biohealth , ems .", "giusti g : consultant for coloplast , rocamed , olympus , lumenis , boston scientific , karl storz , cook medical ." ] ]

[ "a 35-year - old male farm - worker presented to the emergency room with alleged history of horse bite to the right forearm about 2 hours prior to presentation while feeding the horse .", "there was deformity of the forearm with multiple puncture wounds , deep abrasions and small lacerations on the distal - third of the forearm .", "copious irrigation with normal saline was done and he was administered anti - tetanus and postexposure rabies prophylaxis .", "he underwent emergency wound debridement , and the ulna was stabilised with an intra - medullary square nail .", "he had an uneventful recovery and at three - month follow - up , the fractures had healed radiographically in anatomic alignment . at two - year follow - up , he is doing well , is pain free and has a normal range of motion compared to the contralateral side .", "horse bites behave as compound fractures however rabies prophylaxis will be needed and careful observation is needed .", "early radical debridement , preliminary skeletal stabilisation , re - debridement and conversion osteosynthesis to plate , and antibiotic prophylaxis were the key to the successful management of our patient .", "falls and kicks are common mechanism of injuries in people handling horses [ 1 - 3 ] .", "we present a case of forearm open fracture due to horse bite and its management .", "a 35-year - old male farm - worker presented to the emergency room with alleged history of horse bite to the right forearm about 2 hours prior to presenting to us .", "he had recently bought the horse for renting it for marriages and other social and religious functions . on examination , there was deformity of the forearm with multiple puncture wounds , deep abrasions and small lacerations on the distal - third of the forearm .", "1(a ) and ( b ) ) front and dorsal view of the forearm on presentation showing the bite wounds .", "he was given 0.5 ml tetanus toxoid and 500 iu tetglob ( human tetanus immunoglobulin bp ) , and received post - exposure rabies prophylaxis with vaxirab ( purified duck embryo vaccine for rabies ip ) .", "radiographs revealed fracture of radius and ulna in the mid - shaft region ( fig .", "he was taken to the operating room ( or ) where careful debridement of the wounds was done under loupe - magnification .", "the ulna was stabilised with an intra - medullary talwalkar square nail ( inor , mumbai , india ) .", "seventy - two hours later , he was taken to the or for re - debridement .", "the nail in ulna was removed and conversion osteosynthesis using dynamic compression plate and screws for radius and ulna was done ( fig .", "he was changed to oral cephalexin and ciprofloxacin on day 6 which were given till suture removal .", "he had an uneventful recovery and was discharged from the hospital on the 10th day from injury .", "the patient , however , sold the horse as he was too scared to keep it . at three - months", "three - month follow - up radiographs showing fracture healing in anatomic alignment . at two - year follow - up", ", he is doing well , is pain free and has a normal range of motion compared to the contralateral side .", "he is able to use his hand for all routine work and has returned to his work in the farm .", "in horse - related accidents , the majority of injuries occur when the person falls from a horse [ 1 - 3 ] .", "blow by a horse , getting struck by an object while riding a horse , being kicked by a horse , the horse falling on the patient , being entangled by reins , and being bitten by a horse are other less common horse - related accidents .", "most people bitten by horses do not seek medical advice as most bite injuries are minor and self - treated or do not require admission to hospital .", "thomas et al in their study estimated that 102,900 people are treated yearly in emergency rooms in the us due to nonfatal horse related injuries , and about 1800 patients are treated each year after horse bites .", "our patient was too frightened to keep the horse and risk another bite , and hence , sold it not caring for the implications of financial loss .", "domestic animals at home differ between societies , and therefore , local traditions affect the epidemiology of animal - related injuries . comparing animal bite wounds , cat bites result in punctured deep wounds , dog bites cause rather superficial abrasion and laceration type wounds ; because of the great deal of force exerted by an equine in closing its jaws , in horse and donkey bites , the severity of injuries may range from mild superficial pressure trauma , cutaneous breaks of the skin , deep lacerations with loss of tissue , to amputations of digits and even the nose .", "peel et al reported a case of fracture of the forearm bones following horse bite that was treated with open reduction and internal fixation primarily .", "he sustained repeated infections with purulent wound discharge from which mixed cultures of bacteria , including staphylococcus aureus , prevotella melaninogenica , escherichia coli , and pasteurella multocida were isolated .", "more than 3 months after the initial attack by the horse actinobacillus suis was isolated by bone biopsy specimen . though the acute trauma is readily apparent from a biting event ,", "there is also the risk of exposure to various microbes in the oral secretions of equines .", "transmission of such agents with zoonotic potential can also occur from the non - bite exposures to the oral and respiratory secretions .", "because of the large number of bacteria in the mouth , animal bite wounds are generally contaminated and their treatment is difficult , particularly in extensive lesions .", "the occurrence of bacterial infection after animal bites depends on several factors , such as species of animal aggressors ( humans would be associated with a higher infection risk ) , type and site of the injuries ( wounds located in hands have a higher infection risk ) , care given to the wound , inherent factors in the individual ( greater risk in elderly , those with diabetes mellitus , vascular disease , etc ) . in relation to wound type , puncture wounds", "have been reported to have a higher infection rate after animal bites , possibly due to the deposition of bacteria deep in the skin .", "there is evidence that the use of antibiotic prophylactic after bites of the hand reduces infection .", "horse bites most commonly lead to infections with burkholderia , streptococcus , staphylococcus , rhodococcus , actinobacillus , yersinia , and pasteurella , escherichia , neisseria , prevotella , pseudomonas , listeria , hendra virus , vesicular stomatitis virus species .", "bites , kicks or strikes , animal contact and cutting or scratching were the most frequent mechanisms of injury reported .", "nearly 20% of reported horse - related injuries , 14% of cattle and cats and 11% of dog - related injuries resulted in admission .", "brouwer et al reported a case of streptococcus equi meningitis complicated by brain abscesses , as a result of a horse bite .", "kse et al performed a retrospective evaluation of 24 patients presenting with animal bites ( 19 horse and 5 donkey bites ) .", "the head and neck were the most frequent bite sites ( 14 cases ) , followed by the extremities ( eight cases ) and the trunk ( two cases ) .", "our primary aim was damage - control to aggressively debride and at the same time give stability to the limb for wound dressings .", "preliminary stabilisation of the forearm by an intramedullary talwalkar square nail ( inor , mumbai , india ) in the ulna was done following debridement of the wounds and excision of devitalised tissues . fixing the ulna with a nail also helped in maintaining forearm length .", "re - debridement at 72 hours , showed no features of infection and decision of internal fixation with plate and screws was taken .", "we also wish to highlight to the journal reader community that putting in implants when there is any doubt of contamination is risking long term bone infection and other associated complications of delay - in - union and non - union with compromised hand function .", "intra - medullary nail was preferred over external fixator in this case , though in fractures with severe soft tissue damage the use of external fixator is indicated .", "the problems of using external fixation in the forearm include : pin - track - infection , nerve damage due to insufficient anatomical exposure and a relatively high rate of non - union .", "conversion osteosynthesis to internal fixation after primary external fixation is associated with high rates of osteomyelitis .", "plating is established as the standard method in the operative treatment of forearm fractures in adults .", "we successfully converted the fixation from intra - medullary nail to plating with no incidence of infection or delay - in - union .", "therefore , it makes sense to debride wounds which have necrotic tissue early rather than later .", "considering the most common complication of zoonotic infection related to animal bite injuries , our successful surgical treatment without complication appears to have definite clinical relevance .", "the systematic treatment protocol of early radical debridement , preliminary skeletal stabilisation , re - debridement and conversion osteosynthesis to plate , and antibiotic prophylaxis were the key to the successful management of our patient .", "horse or other animal bites are uncommon injuries . the management of these cases should be staged with the principles of damage control and infection control taking precedence . through this publication", "we wish to highlight these rare injuries in the orthopaedic community and the journal readers ." ]

[ "<S> introduction : fractures have been described mainly following falling accidents in horse - related injuries . </S>", "<S> horse bites are uncommon accidents . </S>", "<S> we present a case of open fracture of the forearm due to horse bite.case report : a 35-year - old male farm - worker presented to the emergency room with alleged history of horse bite to the right forearm about 2 hours prior to presentation while feeding the horse . </S>", "<S> there was deformity of the forearm with multiple puncture wounds , deep abrasions and small lacerations on the distal - third of the forearm . copious irrigation with normal saline was done and he was administered anti - tetanus and postexposure rabies prophylaxis . </S>", "<S> prophylactic antibiotic therapy was commenced . </S>", "<S> radiographs revealed fracture of radius and ulna in the mid - shaft region . </S>", "<S> he underwent emergency wound debridement , and the ulna was stabilised with an intra - medullary square nail . </S>", "<S> seventy - two hours later , he underwent re - debridement and conversion osteosynthesis . </S>", "<S> he had an uneventful recovery and at three - month follow - up , the fractures had healed radiographically in anatomic alignment . at two - year follow - up , he is doing well , is pain free and has a normal range of motion compared to the contralateral side.conclusion:horse bites behave as compound fractures however rabies prophylaxis will be needed and careful observation is needed . </S>", "<S> early radical debridement , preliminary skeletal stabilisation , re - debridement and conversion osteosynthesis to plate , and antibiotic prophylaxis were the key to the successful management of our patient . </S>" ]

[ "Introduction:", "Case Report:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion" ]

[ [ "" ], [ "a 35-year - old male farm - worker presented to the emergency room with alleged history of horse bite to the right forearm about 2 hours prior to presentation while feeding the horse .", "there was deformity of the forearm with multiple puncture wounds , deep abrasions and small lacerations on the distal - third of the forearm .", "copious irrigation with normal saline was done and he was administered anti - tetanus and postexposure rabies prophylaxis .", "he underwent emergency wound debridement , and the ulna was stabilised with an intra - medullary square nail .", "he had an uneventful recovery and at three - month follow - up , the fractures had healed radiographically in anatomic alignment . at two - year follow - up , he is doing well , is pain free and has a normal range of motion compared to the contralateral side ." ], [ "horse bites behave as compound fractures however rabies prophylaxis will be needed and careful observation is needed .", "early radical debridement , preliminary skeletal stabilisation , re - debridement and conversion osteosynthesis to plate , and antibiotic prophylaxis were the key to the successful management of our patient ." ], [ "falls and kicks are common mechanism of injuries in people handling horses [ 1 - 3 ] .", "we present a case of forearm open fracture due to horse bite and its management ." ], [ "a 35-year - old male farm - worker presented to the emergency room with alleged history of horse bite to the right forearm about 2 hours prior to presenting to us .", "he had recently bought the horse for renting it for marriages and other social and religious functions . on examination , there was deformity of the forearm with multiple puncture wounds , deep abrasions and small lacerations on the distal - third of the forearm .", "1(a ) and ( b ) ) front and dorsal view of the forearm on presentation showing the bite wounds .", "he was given 0.5 ml tetanus toxoid and 500 iu tetglob ( human tetanus immunoglobulin bp ) , and received post - exposure rabies prophylaxis with vaxirab ( purified duck embryo vaccine for rabies ip ) .", "radiographs revealed fracture of radius and ulna in the mid - shaft region ( fig .", "he was taken to the operating room ( or ) where careful debridement of the wounds was done under loupe - magnification .", "the ulna was stabilised with an intra - medullary talwalkar square nail ( inor , mumbai , india ) .", "seventy - two hours later , he was taken to the or for re - debridement .", "the nail in ulna was removed and conversion osteosynthesis using dynamic compression plate and screws for radius and ulna was done ( fig .", "he was changed to oral cephalexin and ciprofloxacin on day 6 which were given till suture removal .", "he had an uneventful recovery and was discharged from the hospital on the 10th day from injury .", "the patient , however , sold the horse as he was too scared to keep it . at three - months", "three - month follow - up radiographs showing fracture healing in anatomic alignment . at two - year follow - up", ", he is doing well , is pain free and has a normal range of motion compared to the contralateral side .", "he is able to use his hand for all routine work and has returned to his work in the farm ." ], [ "in horse - related accidents , the majority of injuries occur when the person falls from a horse [ 1 - 3 ] .", "blow by a horse , getting struck by an object while riding a horse , being kicked by a horse , the horse falling on the patient , being entangled by reins , and being bitten by a horse are other less common horse - related accidents .", "most people bitten by horses do not seek medical advice as most bite injuries are minor and self - treated or do not require admission to hospital .", "thomas et al in their study estimated that 102,900 people are treated yearly in emergency rooms in the us due to nonfatal horse related injuries , and about 1800 patients are treated each year after horse bites .", "our patient was too frightened to keep the horse and risk another bite , and hence , sold it not caring for the implications of financial loss .", "domestic animals at home differ between societies , and therefore , local traditions affect the epidemiology of animal - related injuries . comparing animal bite wounds , cat bites result in punctured deep wounds , dog bites cause rather superficial abrasion and laceration type wounds ; because of the great deal of force exerted by an equine in closing its jaws , in horse and donkey bites , the severity of injuries may range from mild superficial pressure trauma , cutaneous breaks of the skin , deep lacerations with loss of tissue , to amputations of digits and even the nose .", "peel et al reported a case of fracture of the forearm bones following horse bite that was treated with open reduction and internal fixation primarily .", "he sustained repeated infections with purulent wound discharge from which mixed cultures of bacteria , including staphylococcus aureus , prevotella melaninogenica , escherichia coli , and pasteurella multocida were isolated .", "more than 3 months after the initial attack by the horse actinobacillus suis was isolated by bone biopsy specimen . though the acute trauma is readily apparent from a biting event ,", "there is also the risk of exposure to various microbes in the oral secretions of equines .", "transmission of such agents with zoonotic potential can also occur from the non - bite exposures to the oral and respiratory secretions .", "because of the large number of bacteria in the mouth , animal bite wounds are generally contaminated and their treatment is difficult , particularly in extensive lesions .", "the occurrence of bacterial infection after animal bites depends on several factors , such as species of animal aggressors ( humans would be associated with a higher infection risk ) , type and site of the injuries ( wounds located in hands have a higher infection risk ) , care given to the wound , inherent factors in the individual ( greater risk in elderly , those with diabetes mellitus , vascular disease , etc ) . in relation to wound type , puncture wounds", "have been reported to have a higher infection rate after animal bites , possibly due to the deposition of bacteria deep in the skin .", "there is evidence that the use of antibiotic prophylactic after bites of the hand reduces infection .", "horse bites most commonly lead to infections with burkholderia , streptococcus , staphylococcus , rhodococcus , actinobacillus , yersinia , and pasteurella , escherichia , neisseria , prevotella , pseudomonas , listeria , hendra virus , vesicular stomatitis virus species .", "bites , kicks or strikes , animal contact and cutting or scratching were the most frequent mechanisms of injury reported .", "nearly 20% of reported horse - related injuries , 14% of cattle and cats and 11% of dog - related injuries resulted in admission .", "brouwer et al reported a case of streptococcus equi meningitis complicated by brain abscesses , as a result of a horse bite .", "kse et al performed a retrospective evaluation of 24 patients presenting with animal bites ( 19 horse and 5 donkey bites ) .", "the head and neck were the most frequent bite sites ( 14 cases ) , followed by the extremities ( eight cases ) and the trunk ( two cases ) .", "our primary aim was damage - control to aggressively debride and at the same time give stability to the limb for wound dressings .", "preliminary stabilisation of the forearm by an intramedullary talwalkar square nail ( inor , mumbai , india ) in the ulna was done following debridement of the wounds and excision of devitalised tissues . fixing the ulna with a nail also helped in maintaining forearm length .", "re - debridement at 72 hours , showed no features of infection and decision of internal fixation with plate and screws was taken .", "we also wish to highlight to the journal reader community that putting in implants when there is any doubt of contamination is risking long term bone infection and other associated complications of delay - in - union and non - union with compromised hand function .", "intra - medullary nail was preferred over external fixator in this case , though in fractures with severe soft tissue damage the use of external fixator is indicated .", "the problems of using external fixation in the forearm include : pin - track - infection , nerve damage due to insufficient anatomical exposure and a relatively high rate of non - union .", "conversion osteosynthesis to internal fixation after primary external fixation is associated with high rates of osteomyelitis .", "plating is established as the standard method in the operative treatment of forearm fractures in adults .", "we successfully converted the fixation from intra - medullary nail to plating with no incidence of infection or delay - in - union .", "therefore , it makes sense to debride wounds which have necrotic tissue early rather than later ." ], [ "considering the most common complication of zoonotic infection related to animal bite injuries , our successful surgical treatment without complication appears to have definite clinical relevance .", "the systematic treatment protocol of early radical debridement , preliminary skeletal stabilisation , re - debridement and conversion osteosynthesis to plate , and antibiotic prophylaxis were the key to the successful management of our patient .", "horse or other animal bites are uncommon injuries . the management of these cases should be staged with the principles of damage control and infection control taking precedence . through this publication", "we wish to highlight these rare injuries in the orthopaedic community and the journal readers ." ] ]

[ "other exclusion criteria were history or clinical evidence of coronary or valvular heart disease , congestive heart failure , hyperlipidemia , peripheral vascular disease , chronic gastrointestinal diseases associated with malabsorption , chronic pancreatitis , history of any malignant disease , history of alcohol or drug abuse , liver or kidney failure , and treatments able to modify glucose metabolism .", "after 12-h fasting , a 75-g ogtt was performed with 0- , 30- , 60- , 90- , and 120-min sampling for plasma glucose and insulin .", "glucose tolerance status was defined on the basis of ogtt using the world health organization ( who ) criteria .", "insulin sensitivity was evaluated using the matsuda index ( insulin sensitivity index [ isi ] ) , calculated as follows : 10,000/square root of [ fasting glucose ( millimoles per liter ) fasting insulin ( milliunits per liter ) ] [ mean glucose mean insulin during ogtt ] . the matsuda index is strongly related to euglycemic - hyperinsulinemic clamp , which represents the gold standard test for measuring insulin sensitivity ( 13 ) .", "the ethics committee approved the protocol , and informed written consent was obtained from all participants .", "all of the investigations were performed in accordance with the principles of the declaration of helsinki .", "readings of clinic blood pressure were obtained in the left arm of the supine patients , after 5 min of quiet rest , with a mercury sphygmomanometer .", "a minimum of three blood pressure readings were taken on three separate occasions at least 2 weeks apart .", "systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were recorded at the first appearance ( phase i ) and the disappearance ( phase v ) of korotkoff sounds .", "baseline blood pressure values were the average of the last two of the three consecutive measurements obtained at intervals of 3 min .", "patients with a clinic sbp > 140 mmhg and/or dbp > 90 mmhg were defined as hypertensive .", "plasma glucose was measured by the glucose oxidation method ( beckman glucose analyzer ii ; beckman instruments , milan , italy ) .", "triglyceride and total , ldl , and hdl cholesterol concentrations were measured by enzymatic methods ( roche diagnostics , mannheim , germany ) .", "plasma insulin concentration was determined by a chemiluminescence - based assay ( roche diagnostics ) .", "tracings were taken with the patient in a partial left decubitus position , using a vivid 7 pro ultrasound machine ( ge technologies , milwaukee , wi ) with an annular phased array 2.5-mhz transducer .", "echocardiographic readings were made in random order by the investigator , who had no knowledge of patients blood pressure or other clinical data .", "the mean values from at least five measurements of each parameter for each patient were computed .", "having the same experienced sonographer perform all studies in a dimly lit and quiet room optimized the reproducibility of measurements .", "subjects with a left ventricular ejection fraction < 50% were excluded from this study . in our laboratory ,", "the intraobserver coefficients of variation were 3.85% for posterior wall thickness , 3.7% for interventricular septal thickness , 1.5% for left ventricular internal diameter , and 5.1% for left ventricular mass ( lvm ) .", "tracings were recorded under two - dimensional guidance , and m - mode measurements were taken at the tip of the mitral valve or just below .", "measurements of interventricular septal thickness , posterior wall thickness , and left ventricular internal diameter were made at end diastole and end systole , as recommended by the american society of echocardiography ( 14 ) .", "lvm was calculated using the devereux formula and normalized by body surface area ( lvmi ) .", "left ventricular diastolic function was evaluated according to diagnostic criteria proposed by the american society of echocardiography ( 15 ) .", "evaluation of left atrium volume was obtained using the apical four - chamber and two - chamber views .", "pulsed doppler transmitral flow velocity profile was obtained from the apical four - chamber view , and the sample volume was positioned at the tip of the mitral valve leaflets .", "the following parameters were evaluated for diastolic function : peak transvalvular flow velocity in early diastole ( e wave ) , peak transvalvular flow velocity in late diastole ( a wave ) , e - to - a ratio , deceleration time ( dt ) ( time measured between peak e velocity and the point where the deceleration slope of the e velocity crosses the zero baseline ) , and isovolumic relaxation time ( ivrt ) ( time elapsed between aortic valve closure and mitral valve opening ) .", "pulsed wave tissue doppler imaging ( tdi ) was performed at the junction of the septal and lateral mitral annulus .", "early diastolic ( septal e and lateral e ) and late diastolic ( septal a and lateral a ) velocities were recorded ; ratio of e - to - e ( average ) was also calculated .", "the intraobserver coefficients of variation for measurement of diastolic parameters ranged from 1.8 to 4.1% .", "anova for clinical and biological data was performed to test the differences among groups , and the bonferroni post hoc test for multiple comparisons was further performed .", "linear regression analysis was performed to relate parameters of diastolic function with the following covariates : age , bmi , sbp , dbp , fasting and 1- and 2-h postload plasma glucose levels , fasting and 1- and 2-h postload insulin , matsuda index , and lvmi .", "subsequently , variables reaching statistical significance and sex , as dichotomic value , were inserted in a stepwise multivariate linear regression model to determine the independent predictors of diastolic dysfunction .", "correlational analysis was performed for the whole study population and according to different groups of glucose tolerance .", "all comparisons were performed using the statistical package spss 16.0 for windows ( spss , chicago , il ) .", "readings of clinic blood pressure were obtained in the left arm of the supine patients , after 5 min of quiet rest , with a mercury sphygmomanometer .", "a minimum of three blood pressure readings were taken on three separate occasions at least 2 weeks apart .", "systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were recorded at the first appearance ( phase i ) and the disappearance ( phase v ) of korotkoff sounds .", "baseline blood pressure values were the average of the last two of the three consecutive measurements obtained at intervals of 3 min .", "patients with a clinic sbp > 140 mmhg and/or dbp > 90 mmhg were defined as hypertensive .", "plasma glucose was measured by the glucose oxidation method ( beckman glucose analyzer ii ; beckman instruments , milan , italy ) .", "triglyceride and total , ldl , and hdl cholesterol concentrations were measured by enzymatic methods ( roche diagnostics , mannheim , germany ) .", "plasma insulin concentration was determined by a chemiluminescence - based assay ( roche diagnostics ) .", "tracings were taken with the patient in a partial left decubitus position , using a vivid 7 pro ultrasound machine ( ge technologies , milwaukee , wi ) with an annular phased array 2.5-mhz transducer .", "echocardiographic readings were made in random order by the investigator , who had no knowledge of patients blood pressure or other clinical data .", "the mean values from at least five measurements of each parameter for each patient were computed .", "having the same experienced sonographer perform all studies in a dimly lit and quiet room optimized the reproducibility of measurements .", "subjects with a left ventricular ejection fraction < 50% were excluded from this study . in our laboratory ,", "the intraobserver coefficients of variation were 3.85% for posterior wall thickness , 3.7% for interventricular septal thickness , 1.5% for left ventricular internal diameter , and 5.1% for left ventricular mass ( lvm ) .", "tracings were recorded under two - dimensional guidance , and m - mode measurements were taken at the tip of the mitral valve or just below .", "measurements of interventricular septal thickness , posterior wall thickness , and left ventricular internal diameter were made at end diastole and end systole , as recommended by the american society of echocardiography ( 14 ) .", "lvm was calculated using the devereux formula and normalized by body surface area ( lvmi ) .", "left ventricular diastolic function was evaluated according to diagnostic criteria proposed by the american society of echocardiography ( 15 ) .", "evaluation of left atrium volume was obtained using the apical four - chamber and two - chamber views .", "pulsed doppler transmitral flow velocity profile was obtained from the apical four - chamber view , and the sample volume was positioned at the tip of the mitral valve leaflets .", "the following parameters were evaluated for diastolic function : peak transvalvular flow velocity in early diastole ( e wave ) , peak transvalvular flow velocity in late diastole ( a wave ) , e - to - a ratio , deceleration time ( dt ) ( time measured between peak e velocity and the point where the deceleration slope of the e velocity crosses the zero baseline ) , and isovolumic relaxation time ( ivrt ) ( time elapsed between aortic valve closure and mitral valve opening ) .", "pulsed wave tissue doppler imaging ( tdi ) was performed at the junction of the septal and lateral mitral annulus .", "early diastolic ( septal e and lateral e ) and late diastolic ( septal a and lateral a ) velocities were recorded ; ratio of e - to - e ( average ) was also calculated .", "the intraobserver coefficients of variation for measurement of diastolic parameters ranged from 1.8 to 4.1% .", "tracings were recorded under two - dimensional guidance , and m - mode measurements were taken at the tip of the mitral valve or just below .", "measurements of interventricular septal thickness , posterior wall thickness , and left ventricular internal diameter were made at end diastole and end systole , as recommended by the american society of echocardiography ( 14 ) .", "lvm was calculated using the devereux formula and normalized by body surface area ( lvmi ) .", "left ventricular diastolic function was evaluated according to diagnostic criteria proposed by the american society of echocardiography ( 15 ) .", "evaluation of left atrium volume was obtained using the apical four - chamber and two - chamber views .", "pulsed doppler transmitral flow velocity profile was obtained from the apical four - chamber view , and the sample volume was positioned at the tip of the mitral valve leaflets .", "the following parameters were evaluated for diastolic function : peak transvalvular flow velocity in early diastole ( e wave ) , peak transvalvular flow velocity in late diastole ( a wave ) , e - to - a ratio , deceleration time ( dt ) ( time measured between peak e velocity and the point where the deceleration slope of the e velocity crosses the zero baseline ) , and isovolumic relaxation time ( ivrt ) ( time elapsed between aortic valve closure and mitral valve opening ) .", "e - to - a ratio between 1 and 2 was defined as normal . pulsed wave tissue doppler imaging ( tdi ) was performed at the junction of the septal and lateral mitral annulus .", "early diastolic ( septal e and lateral e ) and late diastolic ( septal a and lateral a ) velocities were recorded ; ratio of e - to - e ( average ) was also calculated .", "the intraobserver coefficients of variation for measurement of diastolic parameters ranged from 1.8 to 4.1% .", "anova for clinical and biological data was performed to test the differences among groups , and the bonferroni post hoc test for multiple comparisons was further performed .", "linear regression analysis was performed to relate parameters of diastolic function with the following covariates : age , bmi , sbp , dbp , fasting and 1- and 2-h postload plasma glucose levels , fasting and 1- and 2-h postload insulin , matsuda index , and lvmi .", "subsequently , variables reaching statistical significance and sex , as dichotomic value , were inserted in a stepwise multivariate linear regression model to determine the independent predictors of diastolic dysfunction .", "correlational analysis was performed for the whole study population and according to different groups of glucose tolerance .", "all comparisons were performed using the statistical package spss 16.0 for windows ( spss , chicago , il ) .", "of 161 patients examined by ogtt , 120 had normal glucose tolerance ( ngt ) , 26 had igt , and 15 had newly diagnosed t2d .", "a 1-h postload plasma glucose cutoff point of 155 mg / dl during ogtt was used to stratify ngt subjects into two groups : 90 patients with 1-h postload plasma glucose < 155 mg / dl ( ngt < 155 ) and 30 individuals with 1-h postload plasma glucose 155 mg / dl ( ngt 155 ) .", "table 1 shows the demographic , clinical , and biochemical characteristics of the four study groups .", "anthropometric , hemodynamic , and biochemical characteristics of the study population according to glucose tolerance data are means sd unless otherwise indicated .", "there were no significant differences among groups for sex , age , bmi , sbp , dbp , and total cholesterol .", "from the first to the fourth group , there was a significant increase of triglyceride ( p = 0.002 ) and a significant reduction of hdl cholesterol ( p = 0.034 ) . obviously , a progressive increase of fasting and 1-h and 2-h postload glucose parallels the worsening of glucose tolerance ( p < 0.0001 ) .", "fasting and postload insulin values were higher in the ngt 155 group and igt subjects in comparison with those in the ngt<155 group and diabetic patients , respectively .", "echocardiographic parameters for the study population , according to glucose tolerance groups , are reported in table 2 .", "t2d patients had the highest lvmi value ( p = 0.020 ) , and clinically relevant , ngt 155 subjects showed an lvmi value not significantly different from igt patients ( p = 0.691 ) but significantly higher than ngt < 155 subjects ( p = 0.042 ) .", "lvmi and echocardiographic parameters of left ventricular diastolic function according to glucose tolerance data are means sd unless otherwise indicated .", "it is interesting to note that the left atrium volume and ivrt values significantly increased from the first to the fourth group ( p < 0.0001 ) and that ngt 155 subjects showed both parameters significantly higher than ngt < 155 subjects ( p < 0.0001 for left atrium and p = 0.037 for ivrt ) .", "moreover , left atrium dimensions in ngt 155 subjects were similar to those of the igt group and t2d patients ; ngt 155 subjects showed an ivrt value similar to that of igt subjects ( p = 0.614 ) and t2d patients ( p = 0.120 ) .", "by contrast , e - to - a ratio significantly decreased from the first to the fourth group ( p < 0.0001 ) , and it was significantly lower in ngt 155 than in ngt<155 subjects .", "no significant differences among groups were observed for the dt duration ( p = 0.391 ) .", "e - to - e ratio significantly increased from the first to the fourth group ( p < 0.044 ) and in ngt 155 subjects was significantly higher than in ngt < 155 subjects ( p < 0.036 ) and similar to that of diabetic patients .", "all remaining tissue doppler parameters were significantly decreased from the first to the fourth group , confirming the progressive impairment of left ventricular diastolic dysfunction from the first to the fourth group of glucose tolerance .", "it is important to remark that ngt 155 subjects had the same characteristics observed in igt and t2d patients .", "a linear regression analysis was performed to test the correlation between echocardiographic parameters and different covariates ( table 3 ) .", "one - hour postload glucose was linearly correlated with ivrt ( r = 0.426 ; p < 0.0001 ) and left atrium volume ( r = 0.366 ; p = 0.0001 ) and inversely correlated with septal e-to - a ratio ( r = 0.502 ; p < 0.0001 ) , lateral e ( r = 0.427 ; p < 0.0001 ) , septal e ( r = 0.389 ; p < 0.0001 ) , lateral e-to - a ratio ( r = 0.384 ; p < 0.0001 ) , e - to - e ratio ( r = 0.161 ; p = 0.020 ) , and e - to - a ratio ( r = 0.238 ; p = 0.001 ) . linear regression analysis", "( r / p ) between diastolic function parameters and different covariates bsa , body surface area ; dt , deceleration time .", "thus , variables reaching statistical significance and sex , as dichotomic value , were inserted in a stepwise multivariate linear regression model to determine the independent predictors of left ventricular diastolic function parameters ( table 4 ) . in the study population ,", "1-h postload glucose was the first predictor of left atrium volume , ivrt , and dt , explaining 25.6 ( p < 0.0001 ) , 18.2 ( p < 0.0001 ) , and 2.9% ( p = 0.031 ) of its variation , respectively .", "age , sex , and dbp added another 5.6 ( p = 0.001 ) , 3.1 ( p = 0.013 ) , and 2.8% ( p = 0.032 ) to explaining left atrium volume , ivrt , and dt , respectively .", "as for the tissue doppler parameters , we observed that 1-h postload glucose was retained as the first correlated of septal e , septal e-to - aratio , lateral e , and lateral e-to - a ratio , explaining 15.2 , 25.2 , 18.3 , and 14.8% of their variations , respectively .", "age and sbp were retained as the independent predictors of e - to - e ratio , explaining 8.3% of its variation .", "stepwise multiple regression analysis on diastolic function parameters as dependent variables in the whole study population bsa , body surface area .", "echocardiographic parameters for the study population , according to glucose tolerance groups , are reported in table 2 .", "t2d patients had the highest lvmi value ( p = 0.020 ) , and clinically relevant , ngt 155 subjects showed an lvmi value not significantly different from igt patients ( p = 0.691 ) but significantly higher than ngt < 155 subjects ( p = 0.042 ) .", "lvmi and echocardiographic parameters of left ventricular diastolic function according to glucose tolerance data are means sd unless otherwise indicated .", "it is interesting to note that the left atrium volume and ivrt values significantly increased from the first to the fourth group ( p < 0.0001 ) and that ngt 155 subjects showed both parameters significantly higher than ngt < 155 subjects ( p < 0.0001 for left atrium and p = 0.037 for ivrt ) .", "moreover , left atrium dimensions in ngt 155 subjects were similar to those of the igt group and t2d patients ; ngt 155 subjects showed an ivrt value similar to that of igt subjects ( p = 0.614 ) and t2d patients ( p = 0.120 ) .", "by contrast , e - to - a ratio significantly decreased from the first to the fourth group ( p < 0.0001 ) , and it was significantly lower in ngt 155 than in ngt<155 subjects .", "no significant differences among groups were observed for the dt duration ( p = 0.391 ) .", "e - to - e ratio significantly increased from the first to the fourth group ( p < 0.044 ) and in ngt 155 subjects was significantly higher than in ngt < 155 subjects ( p < 0.036 ) and similar to that of diabetic patients .", "all remaining tissue doppler parameters were significantly decreased from the first to the fourth group , confirming the progressive impairment of left ventricular diastolic dysfunction from the first to the fourth group of glucose tolerance .", "it is important to remark that ngt 155 subjects had the same characteristics observed in igt and t2d patients .", "a linear regression analysis was performed to test the correlation between echocardiographic parameters and different covariates ( table 3 ) .", "one - hour postload glucose was linearly correlated with ivrt ( r = 0.426 ; p < 0.0001 ) and left atrium volume ( r = 0.366 ; p = 0.0001 ) and inversely correlated with septal e-to - a ratio ( r = 0.502 ; p < 0.0001 ) , lateral e ( r = 0.427 ; p < 0.0001 ) , septal e ( r = 0.389 ; p < 0.0001 ) , lateral e-to - a ratio ( r = 0.384 ; p < 0.0001 ) , e - to - e ratio ( r = 0.161 ; p = 0.020 ) , and e - to - a ratio ( r = 0.238 ; p = 0.001 ) .", "linear regression analysis ( r / p ) between diastolic function parameters and different covariates bsa , body surface area ; dt , deceleration time .", "thus , variables reaching statistical significance and sex , as dichotomic value , were inserted in a stepwise multivariate linear regression model to determine the independent predictors of left ventricular diastolic function parameters ( table 4 ) . in the study population ,", "1-h postload glucose was the first predictor of left atrium volume , ivrt , and dt , explaining 25.6 ( p < 0.0001 ) , 18.2 ( p < 0.0001 ) , and 2.9% ( p = 0.031 ) of its variation , respectively .", "age , sex , and dbp added another 5.6 ( p = 0.001 ) , 3.1 ( p = 0.013 ) , and 2.8% ( p = 0.032 ) to explaining left atrium volume , ivrt , and dt , respectively .", "as for the tissue doppler parameters , we observed that 1-h postload glucose was retained as the first correlated of septal e , septal e-to - aratio , lateral e , and lateral e-to - a ratio , explaining 15.2 , 25.2 , 18.3 , and 14.8% of their variations , respectively .", "age and sbp were retained as the independent predictors of e - to - e ratio , explaining 8.3% of its variation .", "stepwise multiple regression analysis on diastolic function parameters as dependent variables in the whole study population bsa , body surface area .", "this study , conducted in a cohort of never - treated and well - characterized hypertensive patients , showed that the worsening of glucose tolerance was associated with an impairment of left ventricular diastolic function .", "the main finding of this study is that 1-h postload plasma glucose in ngt subjects is associated with left ventricular diastolic dysfunction .", "this result persists after adjustment for all significant covariates reported in table 3 . of interest and clinically relevant , ngt 155 subjects had significantly worse diastolic function compared with ngt<155 subjects and similar compared with igt and t2d patients . to our knowledge , this is the first study that demonstrates this association , confirming the usefulness of early diagnosis in the stratification of overall cardiovascular risk ; in addition , it highlights the links between early alterations of glucose tolerance and early alterations in cardiac function .", "there is a consistent body of evidences demonstrating the association between t2d or insulin resistance and diastolic dysfunction , which is recognized as the first stage of diabetic cardiomyopathy that may lead to chronic heart failure , independent of other cardiovascular heart diseases such as hypertension or atherosclerotic ischemic coronary disease ( 16,17 ) .", "the development of diabetic cardiomyopathy is likely multifactorial involving several mechanisms including metabolic disturbances , endothelial dysfunction , coronary microvascular impairment , modification in the extracellular matrix , and sympathetic hyperactivity ( 16,17 ) .", "all of these factors contribute to the increase of ventricular stiffness , promoting cardiac structure abnormalities such as left ventricular remodeling or hypertrophy .", "cardiac fibrosis causes an imbalance between extracellular matrix deposition and degradation within the heart resulting in excessive fibroblast proliferation .", "in addition , ventricular fibrosis causes progressive stiffening of the ventricular wall resulting in ventricular dysfunction , increase in end diastolic pressure , and atrial dilatation . some of these effects could be related to chronic hyperglycemia that induces , in diabetic patients , nonenzymatic glycation of circulating and cellular membrane proteins , leading to the formation of advanced glycation end products ( ages ) and , through protein kinase c activation , to reactive oxygen species production with increased oxidative stress ( 18 ) .", "ages accumulation , in the myocardium and arterial wall , makes irreversible and stable links with collagen polymers , leading to fibrosis development with reduction of ventricular compliance and increase of lvm , as observed in animal models of igt ( 19 ) .", "moreover , under chronic hyperglycemia condition , there is an increased turnover of free fatty acids , with a shift of myocardial metabolism toward the oxidation of the latter , with intracellular accumulation of intermediate products that lead , via increased oxidative stress , to deleterious effects ( 20 ) . in keeping with this ,", "our results , obtained in ngt 155 subjects , clearly indicate that these modifications begin early , at a clinically silent phase , and support reconsideration of the notion that ngt subjects are a homogeneous group with a low cardiovascular risk profile .", "the activation of both the renin - angiotensin - aldosterone system and sympathetic nervous system is another important mechanism potentially involved in activation of cardiac fibroblasts and collagen production ( 21 ) , leading to fibrosis and likely subsequent to the development of diastolic dysfunction .", "finally , we should not ignore the role of coronary microcirculation abnormalities that may lead to myocardial cell injury and reactive fibrosis / hypertrophy . of interest", ", the impairment of coronary microcirculation , occurring without obstructive atherosclerotic lesions on epicardial coronary arteries , induces a reduction of coronary flow reserve , as demonstrated in type 1 diabetic patients ( 22 ) and hypertensive subjects ( 23 ) .", "the reduction of coronary flow reserve seems to be a direct consequence of elevated glycemia ( 24 ) .", "( 25 ) showing that postprandial hyperglycemia induces myocardial perfusion defects in t2d patients , secondary to deterioration in microvascular function causing a decrease in myocardial blood flow .", "the most clinically relevant information from this study , is that there is a statistically significant and direct correlation between 1-h postload plasma glucose and diastolic dysfunction in ngt hypertensive patients .", "our data have allowed us to identify a new early predictor of subclinical organ damage and emphasize the importance of performing an ogtt in all subjects affected by essential hypertension , paying attention not only to 2- h but also to 1-h postload plasma glucose values , which are more strongly associated with diastolic dysfunction , in order to better stratify the global cardiovascular risk in hypertensive patients ." ]

[ "<S> objectiveto address whether glucose tolerance status , and in particular 1-h postload plasma glucose levels , may affect diastolic function in 161 never - treated hypertensive white subjects . </S>", "<S> impaired left ventricular relaxation , an early sign of diastolic dysfunction , represents the first manifestation of myocardial involvement in diabetic cardiomyopathy . </S>", "<S> a plasma glucose value 155 mg / dl for the 1-h postload plasma glucose during an oral glucose tolerance test ( ogtt ) is able to identify subjects with normal glucose tolerance ( ngt ) at high risk for type 2 diabetes and with subclinical organ damage.research design and methodssubjects underwent ogtt and standard echocardiography . </S>", "<S> diastolic function was assessed by pulsed doppler transmitral flow velocity and tissue doppler imaging . </S>", "<S> insulin sensitivity was assessed by matsuda index.resultsamong the participants , 120 had ngt , 26 had impaired glucose tolerance ( igt ) , and 15 had type 2 diabetes . according to the 1-h postload plasma glucose cutoff point of 155 mg / dl , we divided ngt subjects as follows : ngt < 155 mg / dl ( n = 90 ) and ngt 155 mg / dl ( n = 30 ) . those with ngt 155 mg / dl </S>", "<S> had higher left atrium dimensions ( p < 0.0001 ) and isovolumetric relaxation time ( ivrt ) ( p = 0.037 ) than those with ngt < 155 mg / dl . by contrast , early / late transmitral flow velocity and all tissue doppler parameters were significantly lower in those with ngt 155 mg / dl than in those with ngt<155 mg / dl . at multiple regression analysis , </S>", "<S> 1-h glucose was the major determinant of left atrium area , ivrt , septal e , septal e-to - a ratio , lateral e , and lateral e-to - a ratio.conclusionsthe main finding of this study is that 1-h postload plasma glucose is associated with left ventricular diastolic dysfunction . </S>", "<S> subjects with ngt 155 mg / dl had significantly worse diastolic function than those with ngt<155 mg / dl . </S>" ]

[ "RESEARCH DESIGN AND METHODS", "Blood pressure measurements", "Laboratory determinations", "Echocardiograms", "M-mode measurements.", "Diastolic function.", "Statistical analysis", "RESULTS", "Echocardiographic parameters and glucose tolerance", "Correlational analysis", "CONCLUSIONS" ]

[ [ "the study group consisted of 161 outpatients with uncomplicated hypertension , 101 men and 60 women aged 3865 years ( mean sd 43.7 11.7 years ) , participating in the catanzaro metabolic risk factors study ( catameris ) .", "other exclusion criteria were history or clinical evidence of coronary or valvular heart disease , congestive heart failure , hyperlipidemia , peripheral vascular disease , chronic gastrointestinal diseases associated with malabsorption , chronic pancreatitis , history of any malignant disease , history of alcohol or drug abuse , liver or kidney failure , and treatments able to modify glucose metabolism .", "after 12-h fasting , a 75-g ogtt was performed with 0- , 30- , 60- , 90- , and 120-min sampling for plasma glucose and insulin .", "glucose tolerance status was defined on the basis of ogtt using the world health organization ( who ) criteria .", "insulin sensitivity was evaluated using the matsuda index ( insulin sensitivity index [ isi ] ) , calculated as follows : 10,000/square root of [ fasting glucose ( millimoles per liter ) fasting insulin ( milliunits per liter ) ] [ mean glucose mean insulin during ogtt ] . the matsuda index is strongly related to euglycemic - hyperinsulinemic clamp , which represents the gold standard test for measuring insulin sensitivity ( 13 ) .", "the ethics committee approved the protocol , and informed written consent was obtained from all participants .", "all of the investigations were performed in accordance with the principles of the declaration of helsinki .", "readings of clinic blood pressure were obtained in the left arm of the supine patients , after 5 min of quiet rest , with a mercury sphygmomanometer .", "a minimum of three blood pressure readings were taken on three separate occasions at least 2 weeks apart .", "systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were recorded at the first appearance ( phase i ) and the disappearance ( phase v ) of korotkoff sounds .", "baseline blood pressure values were the average of the last two of the three consecutive measurements obtained at intervals of 3 min .", "patients with a clinic sbp > 140 mmhg and/or dbp > 90 mmhg were defined as hypertensive .", "plasma glucose was measured by the glucose oxidation method ( beckman glucose analyzer ii ; beckman instruments , milan , italy ) .", "triglyceride and total , ldl , and hdl cholesterol concentrations were measured by enzymatic methods ( roche diagnostics , mannheim , germany ) .", "plasma insulin concentration was determined by a chemiluminescence - based assay ( roche diagnostics ) .", "tracings were taken with the patient in a partial left decubitus position , using a vivid 7 pro ultrasound machine ( ge technologies , milwaukee , wi ) with an annular phased array 2.5-mhz transducer .", "echocardiographic readings were made in random order by the investigator , who had no knowledge of patients blood pressure or other clinical data .", "the mean values from at least five measurements of each parameter for each patient were computed .", "having the same experienced sonographer perform all studies in a dimly lit and quiet room optimized the reproducibility of measurements .", "subjects with a left ventricular ejection fraction < 50% were excluded from this study . in our laboratory ,", "the intraobserver coefficients of variation were 3.85% for posterior wall thickness , 3.7% for interventricular septal thickness , 1.5% for left ventricular internal diameter , and 5.1% for left ventricular mass ( lvm ) .", "tracings were recorded under two - dimensional guidance , and m - mode measurements were taken at the tip of the mitral valve or just below .", "measurements of interventricular septal thickness , posterior wall thickness , and left ventricular internal diameter were made at end diastole and end systole , as recommended by the american society of echocardiography ( 14 ) .", "lvm was calculated using the devereux formula and normalized by body surface area ( lvmi ) .", "left ventricular diastolic function was evaluated according to diagnostic criteria proposed by the american society of echocardiography ( 15 ) .", "evaluation of left atrium volume was obtained using the apical four - chamber and two - chamber views .", "pulsed doppler transmitral flow velocity profile was obtained from the apical four - chamber view , and the sample volume was positioned at the tip of the mitral valve leaflets .", "the following parameters were evaluated for diastolic function : peak transvalvular flow velocity in early diastole ( e wave ) , peak transvalvular flow velocity in late diastole ( a wave ) , e - to - a ratio , deceleration time ( dt ) ( time measured between peak e velocity and the point where the deceleration slope of the e velocity crosses the zero baseline ) , and isovolumic relaxation time ( ivrt ) ( time elapsed between aortic valve closure and mitral valve opening ) .", "pulsed wave tissue doppler imaging ( tdi ) was performed at the junction of the septal and lateral mitral annulus .", "early diastolic ( septal e and lateral e ) and late diastolic ( septal a and lateral a ) velocities were recorded ; ratio of e - to - e ( average ) was also calculated .", "the intraobserver coefficients of variation for measurement of diastolic parameters ranged from 1.8 to 4.1% .", "anova for clinical and biological data was performed to test the differences among groups , and the bonferroni post hoc test for multiple comparisons was further performed .", "linear regression analysis was performed to relate parameters of diastolic function with the following covariates : age , bmi , sbp , dbp , fasting and 1- and 2-h postload plasma glucose levels , fasting and 1- and 2-h postload insulin , matsuda index , and lvmi .", "subsequently , variables reaching statistical significance and sex , as dichotomic value , were inserted in a stepwise multivariate linear regression model to determine the independent predictors of diastolic dysfunction .", "correlational analysis was performed for the whole study population and according to different groups of glucose tolerance .", "all comparisons were performed using the statistical package spss 16.0 for windows ( spss , chicago , il ) ." ], [ "readings of clinic blood pressure were obtained in the left arm of the supine patients , after 5 min of quiet rest , with a mercury sphygmomanometer .", "a minimum of three blood pressure readings were taken on three separate occasions at least 2 weeks apart .", "systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were recorded at the first appearance ( phase i ) and the disappearance ( phase v ) of korotkoff sounds .", "baseline blood pressure values were the average of the last two of the three consecutive measurements obtained at intervals of 3 min .", "patients with a clinic sbp > 140 mmhg and/or dbp > 90 mmhg were defined as hypertensive ." ], [ "plasma glucose was measured by the glucose oxidation method ( beckman glucose analyzer ii ; beckman instruments , milan , italy ) .", "triglyceride and total , ldl , and hdl cholesterol concentrations were measured by enzymatic methods ( roche diagnostics , mannheim , germany ) .", "plasma insulin concentration was determined by a chemiluminescence - based assay ( roche diagnostics ) ." ], [ "tracings were taken with the patient in a partial left decubitus position , using a vivid 7 pro ultrasound machine ( ge technologies , milwaukee , wi ) with an annular phased array 2.5-mhz transducer .", "echocardiographic readings were made in random order by the investigator , who had no knowledge of patients blood pressure or other clinical data .", "the mean values from at least five measurements of each parameter for each patient were computed .", "having the same experienced sonographer perform all studies in a dimly lit and quiet room optimized the reproducibility of measurements .", "subjects with a left ventricular ejection fraction < 50% were excluded from this study . in our laboratory ,", "the intraobserver coefficients of variation were 3.85% for posterior wall thickness , 3.7% for interventricular septal thickness , 1.5% for left ventricular internal diameter , and 5.1% for left ventricular mass ( lvm ) .", "tracings were recorded under two - dimensional guidance , and m - mode measurements were taken at the tip of the mitral valve or just below .", "measurements of interventricular septal thickness , posterior wall thickness , and left ventricular internal diameter were made at end diastole and end systole , as recommended by the american society of echocardiography ( 14 ) .", "lvm was calculated using the devereux formula and normalized by body surface area ( lvmi ) .", "left ventricular diastolic function was evaluated according to diagnostic criteria proposed by the american society of echocardiography ( 15 ) .", "evaluation of left atrium volume was obtained using the apical four - chamber and two - chamber views .", "pulsed doppler transmitral flow velocity profile was obtained from the apical four - chamber view , and the sample volume was positioned at the tip of the mitral valve leaflets .", "the following parameters were evaluated for diastolic function : peak transvalvular flow velocity in early diastole ( e wave ) , peak transvalvular flow velocity in late diastole ( a wave ) , e - to - a ratio , deceleration time ( dt ) ( time measured between peak e velocity and the point where the deceleration slope of the e velocity crosses the zero baseline ) , and isovolumic relaxation time ( ivrt ) ( time elapsed between aortic valve closure and mitral valve opening ) .", "pulsed wave tissue doppler imaging ( tdi ) was performed at the junction of the septal and lateral mitral annulus .", "early diastolic ( septal e and lateral e ) and late diastolic ( septal a and lateral a ) velocities were recorded ; ratio of e - to - e ( average ) was also calculated .", "the intraobserver coefficients of variation for measurement of diastolic parameters ranged from 1.8 to 4.1% ." ], [ "tracings were recorded under two - dimensional guidance , and m - mode measurements were taken at the tip of the mitral valve or just below .", "measurements of interventricular septal thickness , posterior wall thickness , and left ventricular internal diameter were made at end diastole and end systole , as recommended by the american society of echocardiography ( 14 ) .", "lvm was calculated using the devereux formula and normalized by body surface area ( lvmi ) ." ], [ "left ventricular diastolic function was evaluated according to diagnostic criteria proposed by the american society of echocardiography ( 15 ) .", "evaluation of left atrium volume was obtained using the apical four - chamber and two - chamber views .", "pulsed doppler transmitral flow velocity profile was obtained from the apical four - chamber view , and the sample volume was positioned at the tip of the mitral valve leaflets .", "the following parameters were evaluated for diastolic function : peak transvalvular flow velocity in early diastole ( e wave ) , peak transvalvular flow velocity in late diastole ( a wave ) , e - to - a ratio , deceleration time ( dt ) ( time measured between peak e velocity and the point where the deceleration slope of the e velocity crosses the zero baseline ) , and isovolumic relaxation time ( ivrt ) ( time elapsed between aortic valve closure and mitral valve opening ) .", "e - to - a ratio between 1 and 2 was defined as normal . pulsed wave tissue doppler imaging ( tdi ) was performed at the junction of the septal and lateral mitral annulus .", "early diastolic ( septal e and lateral e ) and late diastolic ( septal a and lateral a ) velocities were recorded ; ratio of e - to - e ( average ) was also calculated .", "the intraobserver coefficients of variation for measurement of diastolic parameters ranged from 1.8 to 4.1% ." ], [ "anova for clinical and biological data was performed to test the differences among groups , and the bonferroni post hoc test for multiple comparisons was further performed .", "linear regression analysis was performed to relate parameters of diastolic function with the following covariates : age , bmi , sbp , dbp , fasting and 1- and 2-h postload plasma glucose levels , fasting and 1- and 2-h postload insulin , matsuda index , and lvmi .", "subsequently , variables reaching statistical significance and sex , as dichotomic value , were inserted in a stepwise multivariate linear regression model to determine the independent predictors of diastolic dysfunction .", "correlational analysis was performed for the whole study population and according to different groups of glucose tolerance .", "all comparisons were performed using the statistical package spss 16.0 for windows ( spss , chicago , il ) ." ], [ "of 161 patients examined by ogtt , 120 had normal glucose tolerance ( ngt ) , 26 had igt , and 15 had newly diagnosed t2d .", "a 1-h postload plasma glucose cutoff point of 155 mg / dl during ogtt was used to stratify ngt subjects into two groups : 90 patients with 1-h postload plasma glucose < 155 mg / dl ( ngt < 155 ) and 30 individuals with 1-h postload plasma glucose 155 mg / dl ( ngt 155 ) .", "table 1 shows the demographic , clinical , and biochemical characteristics of the four study groups .", "anthropometric , hemodynamic , and biochemical characteristics of the study population according to glucose tolerance data are means sd unless otherwise indicated .", "there were no significant differences among groups for sex , age , bmi , sbp , dbp , and total cholesterol .", "from the first to the fourth group , there was a significant increase of triglyceride ( p = 0.002 ) and a significant reduction of hdl cholesterol ( p = 0.034 ) . obviously , a progressive increase of fasting and 1-h and 2-h postload glucose parallels the worsening of glucose tolerance ( p < 0.0001 ) .", "fasting and postload insulin values were higher in the ngt 155 group and igt subjects in comparison with those in the ngt<155 group and diabetic patients , respectively .", "echocardiographic parameters for the study population , according to glucose tolerance groups , are reported in table 2 .", "t2d patients had the highest lvmi value ( p = 0.020 ) , and clinically relevant , ngt 155 subjects showed an lvmi value not significantly different from igt patients ( p = 0.691 ) but significantly higher than ngt < 155 subjects ( p = 0.042 ) .", "lvmi and echocardiographic parameters of left ventricular diastolic function according to glucose tolerance data are means sd unless otherwise indicated .", "it is interesting to note that the left atrium volume and ivrt values significantly increased from the first to the fourth group ( p < 0.0001 ) and that ngt 155 subjects showed both parameters significantly higher than ngt < 155 subjects ( p < 0.0001 for left atrium and p = 0.037 for ivrt ) .", "moreover , left atrium dimensions in ngt 155 subjects were similar to those of the igt group and t2d patients ; ngt 155 subjects showed an ivrt value similar to that of igt subjects ( p = 0.614 ) and t2d patients ( p = 0.120 ) .", "by contrast , e - to - a ratio significantly decreased from the first to the fourth group ( p < 0.0001 ) , and it was significantly lower in ngt 155 than in ngt<155 subjects .", "no significant differences among groups were observed for the dt duration ( p = 0.391 ) .", "e - to - e ratio significantly increased from the first to the fourth group ( p < 0.044 ) and in ngt 155 subjects was significantly higher than in ngt < 155 subjects ( p < 0.036 ) and similar to that of diabetic patients .", "all remaining tissue doppler parameters were significantly decreased from the first to the fourth group , confirming the progressive impairment of left ventricular diastolic dysfunction from the first to the fourth group of glucose tolerance .", "it is important to remark that ngt 155 subjects had the same characteristics observed in igt and t2d patients .", "a linear regression analysis was performed to test the correlation between echocardiographic parameters and different covariates ( table 3 ) .", "one - hour postload glucose was linearly correlated with ivrt ( r = 0.426 ; p < 0.0001 ) and left atrium volume ( r = 0.366 ; p = 0.0001 ) and inversely correlated with septal e-to - a ratio ( r = 0.502 ; p < 0.0001 ) , lateral e ( r = 0.427 ; p < 0.0001 ) , septal e ( r = 0.389 ; p < 0.0001 ) , lateral e-to - a ratio ( r = 0.384 ; p < 0.0001 ) , e - to - e ratio ( r = 0.161 ; p = 0.020 ) , and e - to - a ratio ( r = 0.238 ; p = 0.001 ) . linear regression analysis", "( r / p ) between diastolic function parameters and different covariates bsa , body surface area ; dt , deceleration time .", "thus , variables reaching statistical significance and sex , as dichotomic value , were inserted in a stepwise multivariate linear regression model to determine the independent predictors of left ventricular diastolic function parameters ( table 4 ) . in the study population ,", "1-h postload glucose was the first predictor of left atrium volume , ivrt , and dt , explaining 25.6 ( p < 0.0001 ) , 18.2 ( p < 0.0001 ) , and 2.9% ( p = 0.031 ) of its variation , respectively .", "age , sex , and dbp added another 5.6 ( p = 0.001 ) , 3.1 ( p = 0.013 ) , and 2.8% ( p = 0.032 ) to explaining left atrium volume , ivrt , and dt , respectively .", "as for the tissue doppler parameters , we observed that 1-h postload glucose was retained as the first correlated of septal e , septal e-to - aratio , lateral e , and lateral e-to - a ratio , explaining 15.2 , 25.2 , 18.3 , and 14.8% of their variations , respectively .", "age and sbp were retained as the independent predictors of e - to - e ratio , explaining 8.3% of its variation .", "stepwise multiple regression analysis on diastolic function parameters as dependent variables in the whole study population bsa , body surface area ." ], [ "echocardiographic parameters for the study population , according to glucose tolerance groups , are reported in table 2 .", "t2d patients had the highest lvmi value ( p = 0.020 ) , and clinically relevant , ngt 155 subjects showed an lvmi value not significantly different from igt patients ( p = 0.691 ) but significantly higher than ngt < 155 subjects ( p = 0.042 ) .", "lvmi and echocardiographic parameters of left ventricular diastolic function according to glucose tolerance data are means sd unless otherwise indicated .", "it is interesting to note that the left atrium volume and ivrt values significantly increased from the first to the fourth group ( p < 0.0001 ) and that ngt 155 subjects showed both parameters significantly higher than ngt < 155 subjects ( p < 0.0001 for left atrium and p = 0.037 for ivrt ) .", "moreover , left atrium dimensions in ngt 155 subjects were similar to those of the igt group and t2d patients ; ngt 155 subjects showed an ivrt value similar to that of igt subjects ( p = 0.614 ) and t2d patients ( p = 0.120 ) .", "by contrast , e - to - a ratio significantly decreased from the first to the fourth group ( p < 0.0001 ) , and it was significantly lower in ngt 155 than in ngt<155 subjects .", "no significant differences among groups were observed for the dt duration ( p = 0.391 ) .", "e - to - e ratio significantly increased from the first to the fourth group ( p < 0.044 ) and in ngt 155 subjects was significantly higher than in ngt < 155 subjects ( p < 0.036 ) and similar to that of diabetic patients .", "all remaining tissue doppler parameters were significantly decreased from the first to the fourth group , confirming the progressive impairment of left ventricular diastolic dysfunction from the first to the fourth group of glucose tolerance .", "it is important to remark that ngt 155 subjects had the same characteristics observed in igt and t2d patients ." ], [ "a linear regression analysis was performed to test the correlation between echocardiographic parameters and different covariates ( table 3 ) .", "one - hour postload glucose was linearly correlated with ivrt ( r = 0.426 ; p < 0.0001 ) and left atrium volume ( r = 0.366 ; p = 0.0001 ) and inversely correlated with septal e-to - a ratio ( r = 0.502 ; p < 0.0001 ) , lateral e ( r = 0.427 ; p < 0.0001 ) , septal e ( r = 0.389 ; p < 0.0001 ) , lateral e-to - a ratio ( r = 0.384 ; p < 0.0001 ) , e - to - e ratio ( r = 0.161 ; p = 0.020 ) , and e - to - a ratio ( r = 0.238 ; p = 0.001 ) .", "linear regression analysis ( r / p ) between diastolic function parameters and different covariates bsa , body surface area ; dt , deceleration time .", "thus , variables reaching statistical significance and sex , as dichotomic value , were inserted in a stepwise multivariate linear regression model to determine the independent predictors of left ventricular diastolic function parameters ( table 4 ) . in the study population ,", "1-h postload glucose was the first predictor of left atrium volume , ivrt , and dt , explaining 25.6 ( p < 0.0001 ) , 18.2 ( p < 0.0001 ) , and 2.9% ( p = 0.031 ) of its variation , respectively .", "age , sex , and dbp added another 5.6 ( p = 0.001 ) , 3.1 ( p = 0.013 ) , and 2.8% ( p = 0.032 ) to explaining left atrium volume , ivrt , and dt , respectively .", "as for the tissue doppler parameters , we observed that 1-h postload glucose was retained as the first correlated of septal e , septal e-to - aratio , lateral e , and lateral e-to - a ratio , explaining 15.2 , 25.2 , 18.3 , and 14.8% of their variations , respectively .", "age and sbp were retained as the independent predictors of e - to - e ratio , explaining 8.3% of its variation .", "stepwise multiple regression analysis on diastolic function parameters as dependent variables in the whole study population bsa , body surface area ." ], [ "this study , conducted in a cohort of never - treated and well - characterized hypertensive patients , showed that the worsening of glucose tolerance was associated with an impairment of left ventricular diastolic function .", "the main finding of this study is that 1-h postload plasma glucose in ngt subjects is associated with left ventricular diastolic dysfunction .", "this result persists after adjustment for all significant covariates reported in table 3 . of interest and clinically relevant , ngt 155 subjects had significantly worse diastolic function compared with ngt<155 subjects and similar compared with igt and t2d patients . to our knowledge , this is the first study that demonstrates this association , confirming the usefulness of early diagnosis in the stratification of overall cardiovascular risk ; in addition , it highlights the links between early alterations of glucose tolerance and early alterations in cardiac function .", "there is a consistent body of evidences demonstrating the association between t2d or insulin resistance and diastolic dysfunction , which is recognized as the first stage of diabetic cardiomyopathy that may lead to chronic heart failure , independent of other cardiovascular heart diseases such as hypertension or atherosclerotic ischemic coronary disease ( 16,17 ) .", "the development of diabetic cardiomyopathy is likely multifactorial involving several mechanisms including metabolic disturbances , endothelial dysfunction , coronary microvascular impairment , modification in the extracellular matrix , and sympathetic hyperactivity ( 16,17 ) .", "all of these factors contribute to the increase of ventricular stiffness , promoting cardiac structure abnormalities such as left ventricular remodeling or hypertrophy .", "cardiac fibrosis causes an imbalance between extracellular matrix deposition and degradation within the heart resulting in excessive fibroblast proliferation .", "in addition , ventricular fibrosis causes progressive stiffening of the ventricular wall resulting in ventricular dysfunction , increase in end diastolic pressure , and atrial dilatation . some of these effects could be related to chronic hyperglycemia that induces , in diabetic patients , nonenzymatic glycation of circulating and cellular membrane proteins , leading to the formation of advanced glycation end products ( ages ) and , through protein kinase c activation , to reactive oxygen species production with increased oxidative stress ( 18 ) .", "ages accumulation , in the myocardium and arterial wall , makes irreversible and stable links with collagen polymers , leading to fibrosis development with reduction of ventricular compliance and increase of lvm , as observed in animal models of igt ( 19 ) .", "moreover , under chronic hyperglycemia condition , there is an increased turnover of free fatty acids , with a shift of myocardial metabolism toward the oxidation of the latter , with intracellular accumulation of intermediate products that lead , via increased oxidative stress , to deleterious effects ( 20 ) . in keeping with this ,", "our results , obtained in ngt 155 subjects , clearly indicate that these modifications begin early , at a clinically silent phase , and support reconsideration of the notion that ngt subjects are a homogeneous group with a low cardiovascular risk profile .", "the activation of both the renin - angiotensin - aldosterone system and sympathetic nervous system is another important mechanism potentially involved in activation of cardiac fibroblasts and collagen production ( 21 ) , leading to fibrosis and likely subsequent to the development of diastolic dysfunction .", "finally , we should not ignore the role of coronary microcirculation abnormalities that may lead to myocardial cell injury and reactive fibrosis / hypertrophy . of interest", ", the impairment of coronary microcirculation , occurring without obstructive atherosclerotic lesions on epicardial coronary arteries , induces a reduction of coronary flow reserve , as demonstrated in type 1 diabetic patients ( 22 ) and hypertensive subjects ( 23 ) .", "the reduction of coronary flow reserve seems to be a direct consequence of elevated glycemia ( 24 ) .", "( 25 ) showing that postprandial hyperglycemia induces myocardial perfusion defects in t2d patients , secondary to deterioration in microvascular function causing a decrease in myocardial blood flow .", "the most clinically relevant information from this study , is that there is a statistically significant and direct correlation between 1-h postload plasma glucose and diastolic dysfunction in ngt hypertensive patients .", "our data have allowed us to identify a new early predictor of subclinical organ damage and emphasize the importance of performing an ogtt in all subjects affected by essential hypertension , paying attention not only to 2- h but also to 1-h postload plasma glucose values , which are more strongly associated with diastolic dysfunction , in order to better stratify the global cardiovascular risk in hypertensive patients ." ] ]

[ "malignant salivary gland neoplasms account for < 0.5% of all malignancies and approximately 35% of all head and neck cancers .", "progress in understanding the cell biology of salivary gland carcinomas ( sgcs ) and detecting vulnerable molecular pathways may lead to the development of new targeted therapy options in these rare cancers with poor prognosis .", "anti - egfr agents include ( i ) monoclonal antibodies ( cetuximab or erbitux , panitumumab ) which block the binding of natural egfr ligands like egf or tgf- resulting in inhibition of downstream signal - transduction pathways and ( ii ) small molecule tyrosine kinase inhibitors ( tkis ) which act by binding the atp pocket within the kinase domain of the egfr and impairing its catalytic activity ( gefitinib , erlotinib , lapatinib ) . downstream signaling pathways triggered by egfr include the ras - raf - extracellular signal - regulated kinase / mitogen activated protein kinase ( mek / mapk ) pathway , which is mainly correlated to cell proliferation , and the p13k - pten - akt axis .", "recently , we were able to demonstrate that frequent egfr overexpression and the absence of drug - resistance egfr mutations in sgc plead in favor of further therapeutic trials with egfr - targeting monoclonal antibodies .", "one of the signaling effectors downstream of egfr , kras , was shown by us to be rarely mutated in sgc [ 2 , 3 ] .", "wildtype kras is one of the clinically proven prerequisites for a successful anti - egfr therapy and therefore anti - egfr monoclonal antibodies are approved only for metastatic colorectal cancer patients whose tumors display wildtype kras . in the absence of kras mutations , resistance to anti - egfr treatments could be caused by alterations of other members of the ras - raf - mapk pathway .", "braf ( v - raf murine sarcoma viral oncogene homolog b1 ) , a serine / threonine kinase , is the downstream effector of kras in the ras - raf - mapk signaling pathway . a somatic mutation ( v600e ) in exon 15 of braf has been identified in multiple human cancers with a mutation rate of 66% in malignant melanomas and at lower frequency in other human carcinomas .", "recently , it was demonstrated that wildtype braf is required for the response of patients with metastatic colorectal cancer to cetuximab and panitumumab .", "the aim of this study was to determine the braf v600e mutation frequency in a large cohort of sgcs of the main histopathological types and to design an allele - specific pcr as an effective screening method .", "surgically removed , formalin - fixed tumor samples were obtained from 65 patients ( 35 males and 30 females with a median age at diagnosis of 55 years ) treated with the histopathological diagnosis of an sgc according to the who classification .", "egfr - targeted therapy was not applied . the study cohort consisted of adenoid cystic carcinoma ( n = 25 ) mucoepidermoid carcinoma ( n = 10 ) , myoepithelial carcinoma ( n = 8) , acinic cell carcinoma ( n = 12 ) and adenocarcinoma ex pleomorphic adenoma ( n = 10 ) . genomic dna was extracted and pooled from oral mucosa samples of five healthy individuals .", "this pooled dna was used as a normal dna control for the development of the pcr assay .", "heterozygous mutant control dna was extracted from cells of the colorectal cancer cell line ht 29 which contains the heterozygous braf v600e mutation .", "the basis for discrimination using allele - specific pcr is that a pcr primer mismatched at its 3 end with the dna template will react less efficiently than one that is entirely complementary .", "our allele - specific multiplex pcr was designed with one common forward ( bf ) and two separate reverse primers ( br and bmu ) : bf : 5-ctcttcataatgcttgctctgatagg-3 , br : 5-agttgagaccttcaatgactttctagt-3 , bmu : 5-cccactccatcgagatttct-3. the forward primer bf and the reverse primer br amplify a 273 bp fragment of both mutant and wildtype alleles and thus serve as amplification control .", "the second reverse primer ( bmu ) is specific for the mutated allele at the 3 end .", "this primer together with bf generates an 143 bp product only in the presence of the v600e ( gtg > gag ) mutation ( figure 1(a ) ) . a series of annealing temperatures ( 52c60c ) , primer concentrations ( 0.10.4 mol / l ) , and mg concentrations ( 1.53.5", "reactions consisted of : 80100 ng genomic dna ; 200 mol / l dntp ; 0.1 mol / l of primers ; 1.5 mmol / l mgcl2 ; 0.5 u taq polymerase ( roche diagnostics , penzberg , germany ) .", "final cycling conditions were as follows : 5 minutes of denaturing at 94c and 30 cycles of 94c for 30 seconds , annealing 55c for 45 seconds and 72c for 60 seconds .", "a volume of 10 l of the pcr products was electrophoresed on a standard 2.5% agarose gel stained with sybr - green i for visualization under uv light .", "the allele - specific pcr for the detection of the braf v600e mutation demonstrated high specificity ( i.e. , detection of only the normal or only the mutant allele ) , high sensitivity ( i.e. , no spurious pcr fragments ) , and acceptable yield . all 65 sgc in this cohort ( 100% ) presented the braf wildtype ( 95% exact confidence limit 00.07 ) .", "the 273 bp pcr fragment was always amplified confirming the integrity of the isolated dna from clinical tissue samples ( figure 1(a ) ) . to test the sensitivity of the mutation - specific pcr", ", we made a serial dilution of ht 29 dna ( which contains the heterozygous braf v600e mutation ) , in control dna with wildtype braf .", "no braf v600e mutation was detected in additionally screened dna samples from microdissected normal tissue adjacent to the tumor cells ( 5 cases ) .", "the activation of the egfr - ras - raf signaling cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules .", "egfr transmits signals to the nuclei instructing cancer cells to proliferate and metastasize , and kras and braf are those downstream signaling molecules .", "anti - egfr therapies interrupt the cancer - triggering signaling cascade , however , if the kras or the braf gene is mutated , their proteins are locked into an active conformation , regardless of whether the egfr is therapeutically blocked . ", "cetuximab ( erbitux ) has already been tested in two phase ii studies in patients with recurrent and/or metastatic sgc including mainly acc , a cancer with generally poor outcome . in 50% of patients , clinical benefit ( i.e. , response or stable disease for 6 month )", "gefitinib was associated with a 53% stable disease rate ( 10/19 ) in acc , but had no effects on patients with salivary duct tumors and mucoepidermoid cancer .", "lapatinib , inhibiting erbb1 and erbb2 tyrosine kinases , was studied in a phase ii trial and stabilized disease for greater than 6 months in 47% of acc patients . ", "key molecules of the egfr - ras - raf signaling cascade and predictive markers of treatment outcome under anti - egfr therapies have not been comprehensively examined in sgc .", "investigations of the mutation status of proteins in the cascade downstream of egfr identified markers for egfr - targeted therapy in colorectal cancer . in recent studies , wildtype braf as well as wildtype kras and intact pten pik3ca", "were found to be required for the response of colorectal cancer patients treated with cetuximab or panitumumab [ 5 , 10 , 11 ] .", "we were able to demonstrate in this and in a former study that kras and braf mutations seem to be extremely rare in sgc .", "these findings imply that salivary gland carcinomas which rarely acquire mutations that result in constitutive activation of the signaling cascade downstream of egfr may be good candidates for anti - egfr therapies .", "molecular analyses of alternative members of the egfr signaling cascade , such as akt-1 and mek-1 , may further contribute to elucidating predictive markers of treatment outcome under anti - egfr therapies in sgc . because of its universal availability as a standard methodology in molecular medicine , we designed a braf mutation screening assay based on pcr .", "allele - specific pcr , also known as amplification refractory mutation system ( arms ) , is a well - established method for discriminating between different alleles at specific loci resulting from single base mutations [ 12 , 13 ] .", "we used the methodology to establish an assay with three pcr primers which allows the discrimination of allele - specific pcr fragments by agarose gel electrophoresis without the need of capillary electrophoresis devices . with our assay", "interpretation of the results can be made by simple visual inspection of the stained gel to determine whether or not a specific primer pair amplified a fragment with the template dna .", "because microdissected tumor areas were used for the allele - specific pcr , and a control amplification was incorporated into the pcr reaction to ensure dna integrity , we can exclude false negative results .", "so far , genomic screening for braf mutations has been based mainly on direct sequencing .", "our protocol provides an alternative rapid , sensitive , and cost - effective braf screening method ." ]

[ "<S> braf is the main effector of kras in the ras - raf - mapk axis , a signaling pathway downstream of egfr . </S>", "<S> the activation of this cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules . </S>", "<S> recent studies in metastatic colorectal cancer found that an oncogenic activation of braf by a point mutation in exon 15 ( v600e ) could bypass the egfr - initiated signaling cascade with the effect that patients bearing the mutant braf allele are not likely to benefit from egfr - targeted therapies . </S>", "<S> we designed an allele - specific pcr and screened 65 salivary gland carcinoma ( sgc ) of the main histopathological types for the braf v600e mutation . </S>", "<S> all 65 sgc in this cohort ( 100% ) presented the braf wildtype . in a previous study </S>", "<S> , we found a kras wildtype in 98.5% of sgc . \n </S>", "<S> these findings imply that sgc rarely acquires mutations that result in a constitutive activation of the signaling cascade downstream of egfr and this pleads in favor of further therapeutic trials with egfr - targeting monoclonal \n antibodies . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion and Conclusions" ]

[ [ "malignant salivary gland neoplasms account for < 0.5% of all malignancies and approximately 35% of all head and neck cancers .", "progress in understanding the cell biology of salivary gland carcinomas ( sgcs ) and detecting vulnerable molecular pathways may lead to the development of new targeted therapy options in these rare cancers with poor prognosis .", "anti - egfr agents include ( i ) monoclonal antibodies ( cetuximab or erbitux , panitumumab ) which block the binding of natural egfr ligands like egf or tgf- resulting in inhibition of downstream signal - transduction pathways and ( ii ) small molecule tyrosine kinase inhibitors ( tkis ) which act by binding the atp pocket within the kinase domain of the egfr and impairing its catalytic activity ( gefitinib , erlotinib , lapatinib ) . downstream signaling pathways triggered by egfr include the ras - raf - extracellular signal - regulated kinase / mitogen activated protein kinase ( mek / mapk ) pathway , which is mainly correlated to cell proliferation , and the p13k - pten - akt axis .", "recently , we were able to demonstrate that frequent egfr overexpression and the absence of drug - resistance egfr mutations in sgc plead in favor of further therapeutic trials with egfr - targeting monoclonal antibodies .", "one of the signaling effectors downstream of egfr , kras , was shown by us to be rarely mutated in sgc [ 2 , 3 ] .", "wildtype kras is one of the clinically proven prerequisites for a successful anti - egfr therapy and therefore anti - egfr monoclonal antibodies are approved only for metastatic colorectal cancer patients whose tumors display wildtype kras . in the absence of kras mutations , resistance to anti - egfr treatments could be caused by alterations of other members of the ras - raf - mapk pathway .", "braf ( v - raf murine sarcoma viral oncogene homolog b1 ) , a serine / threonine kinase , is the downstream effector of kras in the ras - raf - mapk signaling pathway . a somatic mutation ( v600e ) in exon 15 of braf has been identified in multiple human cancers with a mutation rate of 66% in malignant melanomas and at lower frequency in other human carcinomas .", "recently , it was demonstrated that wildtype braf is required for the response of patients with metastatic colorectal cancer to cetuximab and panitumumab .", "the aim of this study was to determine the braf v600e mutation frequency in a large cohort of sgcs of the main histopathological types and to design an allele - specific pcr as an effective screening method ." ], [ "surgically removed , formalin - fixed tumor samples were obtained from 65 patients ( 35 males and 30 females with a median age at diagnosis of 55 years ) treated with the histopathological diagnosis of an sgc according to the who classification .", "egfr - targeted therapy was not applied . the study cohort consisted of adenoid cystic carcinoma ( n = 25 ) mucoepidermoid carcinoma ( n = 10 ) , myoepithelial carcinoma ( n = 8) , acinic cell carcinoma ( n = 12 ) and adenocarcinoma ex pleomorphic adenoma ( n = 10 ) . genomic dna was extracted and pooled from oral mucosa samples of five healthy individuals .", "this pooled dna was used as a normal dna control for the development of the pcr assay .", "heterozygous mutant control dna was extracted from cells of the colorectal cancer cell line ht 29 which contains the heterozygous braf v600e mutation .", "the basis for discrimination using allele - specific pcr is that a pcr primer mismatched at its 3 end with the dna template will react less efficiently than one that is entirely complementary .", "our allele - specific multiplex pcr was designed with one common forward ( bf ) and two separate reverse primers ( br and bmu ) : bf : 5-ctcttcataatgcttgctctgatagg-3 , br : 5-agttgagaccttcaatgactttctagt-3 , bmu : 5-cccactccatcgagatttct-3. the forward primer bf and the reverse primer br amplify a 273 bp fragment of both mutant and wildtype alleles and thus serve as amplification control .", "the second reverse primer ( bmu ) is specific for the mutated allele at the 3 end .", "this primer together with bf generates an 143 bp product only in the presence of the v600e ( gtg > gag ) mutation ( figure 1(a ) ) . a series of annealing temperatures ( 52c60c ) , primer concentrations ( 0.10.4 mol / l ) , and mg concentrations ( 1.53.5", "reactions consisted of : 80100 ng genomic dna ; 200 mol / l dntp ; 0.1 mol / l of primers ; 1.5 mmol / l mgcl2 ; 0.5 u taq polymerase ( roche diagnostics , penzberg , germany ) .", "final cycling conditions were as follows : 5 minutes of denaturing at 94c and 30 cycles of 94c for 30 seconds , annealing 55c for 45 seconds and 72c for 60 seconds .", "a volume of 10 l of the pcr products was electrophoresed on a standard 2.5% agarose gel stained with sybr - green i for visualization under uv light ." ], [ "the allele - specific pcr for the detection of the braf v600e mutation demonstrated high specificity ( i.e. , detection of only the normal or only the mutant allele ) , high sensitivity ( i.e. , no spurious pcr fragments ) , and acceptable yield . all 65 sgc in this cohort ( 100% ) presented the braf wildtype ( 95% exact confidence limit 00.07 ) .", "the 273 bp pcr fragment was always amplified confirming the integrity of the isolated dna from clinical tissue samples ( figure 1(a ) ) . to test the sensitivity of the mutation - specific pcr", ", we made a serial dilution of ht 29 dna ( which contains the heterozygous braf v600e mutation ) , in control dna with wildtype braf .", "no braf v600e mutation was detected in additionally screened dna samples from microdissected normal tissue adjacent to the tumor cells ( 5 cases ) ." ], [ "the activation of the egfr - ras - raf signaling cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules .", "egfr transmits signals to the nuclei instructing cancer cells to proliferate and metastasize , and kras and braf are those downstream signaling molecules .", "anti - egfr therapies interrupt the cancer - triggering signaling cascade , however , if the kras or the braf gene is mutated , their proteins are locked into an active conformation , regardless of whether the egfr is therapeutically blocked . ", "cetuximab ( erbitux ) has already been tested in two phase ii studies in patients with recurrent and/or metastatic sgc including mainly acc , a cancer with generally poor outcome . in 50% of patients , clinical benefit ( i.e. , response or stable disease for 6 month )", "gefitinib was associated with a 53% stable disease rate ( 10/19 ) in acc , but had no effects on patients with salivary duct tumors and mucoepidermoid cancer .", "lapatinib , inhibiting erbb1 and erbb2 tyrosine kinases , was studied in a phase ii trial and stabilized disease for greater than 6 months in 47% of acc patients . ", "key molecules of the egfr - ras - raf signaling cascade and predictive markers of treatment outcome under anti - egfr therapies have not been comprehensively examined in sgc .", "investigations of the mutation status of proteins in the cascade downstream of egfr identified markers for egfr - targeted therapy in colorectal cancer . in recent studies , wildtype braf as well as wildtype kras and intact pten pik3ca", "were found to be required for the response of colorectal cancer patients treated with cetuximab or panitumumab [ 5 , 10 , 11 ] .", "we were able to demonstrate in this and in a former study that kras and braf mutations seem to be extremely rare in sgc .", "these findings imply that salivary gland carcinomas which rarely acquire mutations that result in constitutive activation of the signaling cascade downstream of egfr may be good candidates for anti - egfr therapies .", "molecular analyses of alternative members of the egfr signaling cascade , such as akt-1 and mek-1 , may further contribute to elucidating predictive markers of treatment outcome under anti - egfr therapies in sgc . because of its universal availability as a standard methodology in molecular medicine , we designed a braf mutation screening assay based on pcr .", "allele - specific pcr , also known as amplification refractory mutation system ( arms ) , is a well - established method for discriminating between different alleles at specific loci resulting from single base mutations [ 12 , 13 ] .", "we used the methodology to establish an assay with three pcr primers which allows the discrimination of allele - specific pcr fragments by agarose gel electrophoresis without the need of capillary electrophoresis devices . with our assay", "interpretation of the results can be made by simple visual inspection of the stained gel to determine whether or not a specific primer pair amplified a fragment with the template dna .", "because microdissected tumor areas were used for the allele - specific pcr , and a control amplification was incorporated into the pcr reaction to ensure dna integrity , we can exclude false negative results .", "so far , genomic screening for braf mutations has been based mainly on direct sequencing .", "our protocol provides an alternative rapid , sensitive , and cost - effective braf screening method ." ] ]

[ "wiskott aldrich syndrome ( was ) is a monogenic x - linked primary immunodeficiency characterized by the classic triad of microthrombocytopenia , eczema and recurrent infections .", "multiple autoimmune manifestations and tumors are serious complications and the life expectancy of was patients is severely reduced , unless they are successfully cured by hematopoietic stem cell transplantation .", "was is caused by mutations in the was gene impairing the expression and/or function of the was protein ( wasp ) , a hematopoietic - specific regulator of cytoskeletal reorganization also involved in signal transduction of cells .", "the absence or reduction of wasp compromises multiple processes of different immune cell types involved in innate and adaptive responses , resulting in a variable and progressive immunodeficiency .", "susceptibility of was patients to develop autoimmune diseases has been mainly attributed to the breakdown of self tolerance sustained by dysfunction of both natural t regulatory and effector t cells ( reviewed in ref . )", ". however , b - cell intrinsic defects have been demonstrated to critically contribute to was - associated autoimmunity in was mouse model . in humans , the contribution of b - cell defects in the pathogenesis of was has been partially investigated .", "b cells from patients exhibit lower motility , migratory and adhesive capacities , likely due to defective f - actin nucleation . in contrast , despite the role of wasp in b - cell receptor ( bcr ) signaling , abnormalities in b - cell activation still remain controversial . a skewed distribution of serum immunoglobulin ( ig ) classes and the inability to mount a proper antibody response , particularly to t - cell independent ( ti ) antigens , suggest defects in b - cell effector function .", "previous findings in was patients show phenotypical b - cell perturbations in the periphery . in order to evaluate whether an abnormal b - cell development might generate a b - cell repertoire unable to unsure full protection against pathogens and tolerance against self - antigens ,", "to this end , we have combined a detailed phenotypical analysis of b - cell maturation stages , from the bone marrow ( bm ) to the periphery , with a molecular study of ig repertoire and in vivo b - cell maturation processes in a large cohort of was pediatric patients .", "our data show that wasp - deficiency affects critical stages of central and peripheral b - cell differentiation contributing to abnormalities in humoral immunity and b - cell tolerance in humans .", "the diagnoses were clinically defined and confirmed by genetic analysis . a description of all patients is reported in supplementary table 1 .", "human samples were obtained according to the code of ethics of the world medical association ( declaration of helsinki ) with the approval of the local medical ethical committees of the erasmus mc and the san raffaele scientific institute internal review board ( tiget02 ) .", "all results obtained from samples of was patients were compared to age and sex matched healthy donors ( hds ) .", "the composition of the precursor b - cell compartment was analyzed by flow cytometric immunophenotyping as described in the supplementary material .", "for the analysis of replication history and somatic hypermutation , four b - cell subsets were isolated from thawed peripheral blood mononuclear cells ( pbmcs ) using a facs diva cell sorter ( bd biosciences ) .", "gating on cd19 cells , transitional ( cd27cd24cd38 ) , mature nave ( cd27igdcd24cd38 ) , natural effector ( cd27igd ) and memory ( cd27igd ) b - cell subsets were sorted with a purity of > 95% for all fractions . for intracytoplasmic detection of human wasp ,", "cells were fixed and permeabilized using a cytofix / cytoperm kit ( bd pharmingen , oregon , usa ) .", "the anti - wasp antibody 503 ( a kind gift from prof h. d. ochs , seattle , wa , and l. d. notarangelo , boston , ma ) was used , followed by detection with pacific blue - labeled anti - rabbit igg secondary antibody ( invitrogen , san diego , usa ) .", "cd20 positive cells were purified from pbmcs of pediatric was patients and age - matched hds by immunomagnetic beads ( miltenyi biotec , germany ) or facs sorting .", "the purity of the isolated cells were analyzed by facs and ranged from 84% to 98% . ", "after isolation , cells were left overnight at 37 c in culture medium composed of rpmi-1640 , 10% fbs , 2 mm glutamine , 100 iu / ml penicillin and 100 g / ml streptomycin ( lonza , basel , switzerland ) . in vitro chemotaxis assay", "was performed using 5 m pore - size transwell inserts ( costar corporation , corning , ny , us ) in 24-well plates .", "filters were prewet 30 min at 37 c in presence of 600 l of medium supplemented with 250 ng / ml of recombinant human stromal cell - derived factor ( sdf)-1 ( cxcl12 ; peprotech , rocky hill , us ) .", "fifty thousand cd20 positive or negative cells were resuspended in 100 l of culture medium , seeded in the upper chamber and incubated at 37 c for 3 h. transmigrated cells , collected in the lower chamber , were counted for viable cells and stained with anti - cd19 , anti - cd24 , anti - cd38 , anti - cd27 and anti - cd3 ( bd biosciences ) for the phenotypical analysis by facs .", "migration frequency was estimated as the [ ( cell n at the lower chamber / the initial b cell input in the upper chamber ) 100 ] .", "levels of b - cell activating factor ( baff ) were measured in duplicate in plasma samples of was patients and hds using a quantikine human baff / blys / tnfsf13b immunoassay kit ( r&d systems , minneapolis , usa ) .", "the assay was performed according to manufacturer 's instructions and the od was determined using a microplate reader set to 450 nm .", "ig and igh gene rearrangements were amplified and analyzed as described in the supplementary material .", "dna was extracted from sorted b - cell subsets with the genelute mammalian total dna miniprep kit ( sigma aldrich ) and used to perform the ig-deleting recombination excision circles ( krec ) assay .", "the amounts of coding and signal joints of the ig-deleting rearrangement were measured by real - time quantitative pcr on an abi prism 7000 sequence detection system ( applied biosystems ) .", "ig restriction enzyme - based hot - spot mutation assay ( igrehma ) was performed on genomic dna isolated from sorted b - cell subsets .", "briefly , v3 - 20-j rearrangements were pcr - amplified and the pcr products ( 500 bp ) were first digested using the restriction enzyme fnu4hi and then kpni .", "the unmutated gene products can be visualized as 244 or 247 bp hex - coupled fragments and the mutated as 262 bp hex - coupled fragment .", "comparisons between proportions were calculated by using the chi - square test ( test ) ( with continuity correction ) .", "until now , the composition of the b - cell compartment in the bm of was patients has not been reported . to evaluate the effect of wasp - deficiency on b - cell development in the bm", ", we characterized five stages of b - cell differentiation in three was patients .", "we observed a decreased frequency of immature b lymphocytes in was patients ( fig . ", "this observation was further assessed in four additional patients , focusing on late stages of b - cell differentiation based on the expression of cd10 and cd20 ( gating strategy is shown in suppl . ", "we confirmed a significant decrease in the frequency of immature b cells compared with age - matched hds ( fig . ", "in addition , at the earliest differentiation stages , we observed a significant increase of small pre - b - ii cells in was patients ; while no statistical difference was detected in large pre - b - ii cell frequency ( fig . ", "these findings might reflect a partial block in the bm b - cell differentiation between the small pre - b - ii and immature b - cell stages in was , or an early egress of immature b cells into the periphery .", "next we examined the distribution of b - cell subsets in the peripheral blood of was patients and age - matched hds .", "when available , we evaluated the absolute count of total b cells , which was found significantly reduced in patients less than three years old ( < 3y ) , while no differences were observed in the older was group ( fig . ", "four b - cell subsets were identified according to the expression of cd24 , cd38 , igd and cd27 : transitional , mature nave , memory and plasmablasts ( fig . ", "transitional b cells ( cd24cd38 ) are the most immature peripheral b - cell population that were significantly increased in both was age groups as frequency ( fig . 2c ) and absolute number ( suppl . ", "the next maturation stage , represented by mature nave b cells ( cd27igdcd24cd38 ) , was significantly decreased only in was patients less than 3 years old both in frequency ( fig . ", "finally , the frequency of plasmablasts ( cd24cd38 ) was significantly increased in the older group of patients ( fig . ", "2c ) , while the absolute number of both memory b cells ( cd27igdcd24cd38 ) and plasmablasts were significantly decreased in the younger group of was patients ( suppl . ", "altogether , these findings demonstrate that was patients have a perturbed peripheral b - cell distribution . the overrepresentation of transitional b cells in the blood of was patients led us to hypothesize that an early egress of immature b cells from bm could occur .", "cxcr4 , the chemokine receptor for sdf-1 , has an important role in retaining b - cell precursors within the bm in order to prevent premature migration to the periphery [ 2224 ] .", "we tested the chemotactic response to sdf-1 of b lymphocytes of was patients and hds by transwell migration assay .", "as shown in fig . 3 , both transitional and mature peripheral b cells were less responsive to sdf-1 in the absence of wasp ( fig . ", "this defect was specific for b lymphocytes since cd3 positive cells of was patients showed a normal migratory capacity to sdf-1 ( suppl . ", "thus , we hypothesize that a diminished retention signal mediated by cxcr4/sdf-1 could explain the premature egress of immature b cells from the bm leading to an increase of transitional b cells in periphery . to further characterize the peripheral b - cell distribution , we focused our analysis on b - cell subsets found expanded in autoimmune diseases and potentially autoreactive [ 2528 ] .", "we found a significant increase of cd19cd21cd35 subset in both was age groups ( fig . ", "4a ) , confirming data previously reported by park and colleagues . we also analyzed the frequency of cd19cd21cd38 cells ( referred to as cd21 ) described to be expanded in autoimmune diseases and immunodeficiencies \n .", "interestingly , we observed that the frequency of this unusual population was markedly increased in was patients , both in the younger and older groups ( fig . ", "in addition , higher levels of soluble baff were found in the plasma of was patients as compared to pediatric hds ( fig . ", "this finding was also associated with a decreased expression of its receptor , baffr , in transitional b cells of was patients ( fig . ", "because of the role of baff in b - cell homeostasis and peripheral b - cell tolerance , high baff levels may affect the stringency of peripheral b - cell selection thus favoring the survival of b - cell subsets containing potentially autoreactive clones . in order to evaluate the effect of wasp - deficiency in an antigen dependent context , we analyzed in detail the memory b - cell compartment of was patients .", "we identified six distinct memory b - cell subsets ( gating strategy is shown in suppl . ", "igm - only ) and cd27igg deriving from primary germinal center ( gc ) responses ; igg cd27 and iga cd27 switched memory b cells originating from secondary gc responses ; cd27igd ( named natural effector ) and cd27iga generated in a ti manner in the gastrointestinal tract or in the splenic marginal zone area , respectively .", "we did not observe differences in memory b - cell populations deriving from both primary and secondary gc responses between was patients and hds ( fig . ", "a marked reduction in natural effector b cells was found in both age groups of was patients both as frequency ( fig . ", "the frequency of cd27iga b cells was decreased in 312 years old patients compared with hds ( fig . ", ", the absence of wasp mainly affects ti responses of b cells , whereas only mildly impairs the generation of early memory b cells in gc without influencing the class - switched memory b - cell compartment .", "we determined the in vivo b - cell proliferation history by krec assay and in parallel shm by igrehma in sorted transitional , mature nave , natural effector and memory b cells .", "as expected , transitional b lymphocytes did not undergo cell divisions and mature nave b cells had a limited number of cell divisions in hds ( fig . ", "both b - cell subsets were characterized by the absence of shm ( fig . ", "6b ) and we did not find differences in transitional and mature nave b cells of was subjects ( fig . ", "in contrast , the memory compartment showed less proliferation in was memory b cells in association with a reduced shm level ( fig . ", "interestingly , the defect in maturation was even more pronounced in natural effector was b lymphocytes . to study shm also in the ig heavy chain ( igh )", ", we analyzed the frequency of mutated nucleotides in rearranged variable region of igh ( ighv ) genes by cloning and sequencing the most frequent ighv subgroups , the ighv3 and ighv4 gene families , of both - and -chain of constant regions ( c and c ) .", "the mutational frequency of was b cells was significantly lower for both c and c transcripts in all domains of v region ( fig . ", "thus , the decreased in vivo proliferation and shm levels of was memory b cells could be responsible of a lower protection against infections and persistence of pathogens that finally lead to autoimmunity .", "was b cells showed an increased frequency of ighv3 - 30 in both ig classes ( fig . ", "7a ) and the absence of ighv3 - 48 genes in c sequences ( suppl . ", "7a ) . in ighv4 transcripts , we noticed a dominant usage of the ighv4 - 34 genes in was c ( fig . ", "moreover , the antibody repertoire of was b cells was devoid of ighv4 - 59 , a gene commonly used in b cells from hds .", "the distribution of d families was only slightly altered in was patients ( suppl . ", "finally , we evaluated the csr process in total b cells by analyzing the frequency of ig subclasses in sequenced igh transcripts .", "a preferential usage of -chain c region 3 ( c3 ) and c1 , igh - proximal genes ( the igh locus is schematized in fig . ", "7d ) , accompanied by a reduction in the expression of distal c2 and c4 , were detected in was patients ( fig . ", "the analysis of iga transcripts showed no significant difference in the subclass usage ( fig . ", "7e ) . in conclusion was patients show a skewed ig gene usage suggesting an altered selection of the antibody repertoire during b - cell development .", "we reported herein that wasp - deficiency affects many aspects of b - cell development , first in the bm and then in the periphery .", "indeed , precursor b - cell as well as memory b - cell development and selection appear affected by the absence of functional wasp . in the bm", "we have observed a decreased frequency of immature b cells , while in the periphery an overrepresentation of transitional b cells likely due to a reduced retention in the bm and/or a decreased migration to peripheral lymphoid tissues .", "the interaction of the chemokine receptor cxcr4 with its ligand sdf-1 is required for the retention of developing b cells in the bm .", "wasp is actively involved in cxcr4 signaling [ 3739 ] and was murine b cells show defective migratory response to sdf-1 .", "consistently , here we demonstrate in was patients that b cells migrate less to sdf-1. thus , was immature b cells could be unable to properly sense retention signals derived from cxcr4 in the bm leading to a premature release in the periphery .", "the increase of transitional b cells could also reflect an altered migration to the peripheral lymphoid organs resulting in a prolonged persistence in the circulation .", "consistently , our data allow us to exclude that the expansion of transitional b cells is due to homeostatic proliferation since we demonstrated the absence of replication history in this subset .", "transitional b cells are found expanded in immunodeficient conditions or autoimmune diseases and represent a reservoir of autoreactive b cells .", "the cohort of patients analyzed here did not show any overt sign of autoimmunity , probably due to their young age .", "however , the analysis of serum autoantibodies performed in four patients showed positivity for anti - nuclear antibodies in three patients .", "one of them also showed the presence of anti - platelet antibodies ( data not shown ) .", "these cells are enriched in anergic autoreactive clones and expanded in systemic lupus erythematosus ( sle ) , rheumatoid arthritis and in common variable immunodeficiency groupia patients developing autoimmune syndromes .", "baff levels and signals through baffr coordinate the maintenance of the primary b - cell pool and the fate of self - reactive b cells .", "herein , we report for the first time increased baff levels and decreased baffr expression in was patients .", "elevated baff serum levels are often present in immunodeficiencies , autoimmune diseases and viral infections and may lower the thresholds for the survival of autoreactive b cell clones .", "the enrichment of cd21 b cells and the alterations in baff levels and expression of its receptor suggest that the mechanisms of b - cell selection could be altered in was patients .", "our data also suggest the presence of a defective selection of was b cells producing high - affinity antibodies .", "indeed , we noticed a restricted or null presence of ighv3 - 48 and ighv4 - 59 gene families and a preferential usage of ighv3 - 30 and ighv4 - 34 .", "in particular , ighv3 - 48 gene is selectively used against polysaccharide antigens and its decrease in was could account for an inefficient antibody response .", "in contrast , ighv3 - 30 is highly represented among anti - platelet autoantibodies from patients with idiopathic thrombocytopenic purpura and in sle patients . additionally , ighv4 - 34 encoded antibodies are intrinsically autoreactive when unmutated , as observed in our patients .", "this peculiar antibody repertoire reflects an altered selection of both protective and autoreactive ig gene families .", "the perturbation of b - cell homeostasis present in was patients supports the administration of anti - cd20 mab in the conditioning regimen of gene therapy to deplete b cells . in the memory b - cell compartment , the frequency of isotype - switched igg b cells", "we observed a normal frequency of memory b cells and of switched cd27igg and cd27iga b cells in our cohort of was patients .", "this suggests that t - cell dependent antigen response is induced normally in was patients .", "in contrast , the frequency and the shm level of ti memory b - cell subsets were reduced , likely due to their reduced in vivo proliferation . the marked reduction in the natural effector b - cell subset , resembling marginal zone b cells in the spleen , mirrors the decreased number of splenic marginal zone b cells and the histological defects in the marginal zone area of the spleen already described in was mice and patients . in addition , both the reduction in natural effector and igacd27 b cells , which are generated during ti responses , provide evidence of a defect in b - cell function independent from the cross - talk with t lymphocytes .", "the mutational status of both heavy and light ig chains is reduced in was in presence of a diminished rate of proliferation in the total memory b - cell pool .", "in addition , the analysis of ig gene selection showed a preferential use of igh - proximal genes ( ighg1 and ighg3 ) accompanied by reduced shm suggesting that was b cells have undergone less gc reactions .", "reduced igg2 switching in was patients could account for the poor response to ti antigens which is typical of the syndrome .", "the cytokine profile of was patients shows an impairment in the production of th1 cytokines that could explain their reduced switching to igg2 subclass .", "moreover , the isotype - switching outcome is also influenced by two additional factors : cell division and antibody affinity to antigens .", "the defective proliferation of memory and natural effector b cells and the reduced affinity maturation found in was might contribute to these alterations in class switching .", "our results in was patients show an early egress of immature b cells from bm leading to an overrepresentation of transitional b cells in the periphery .", "the memory compartment is characterized by a reduced maturation status that could affect the b - cell effector functions contributing to a lower clearance of pathogens and leading to chronic inflammation that can break tolerance . in conclusion ,", "our results add novel immunological features of was b - cell phenotype that can complement the evaluation of the efficacy of various treatment approaches ." ]

[ "<S> wiskott aldrich syndrome protein ( wasp ) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the wiskott </S>", "<S> aldrich syndrome ( was ) , a primary immunodeficiency with microthrombocytopenia , eczema and a higher susceptibility to develop tumors . </S>", "<S> autoimmune manifestations , frequently observed in was patients , are associated with an increased risk of mortality and still represent an unsolved aspect of the disease . </S>", "<S> b cells play a crucial role both in immune competence and self - tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity . we performed a phenotypical and molecular analysis of central and peripheral b - cell compartments in was pediatric patients . </S>", "<S> we found a decreased proportion of immature b cells in the bone marrow correlating with an increased presence of transitional b cells in the periphery . </S>", "<S> these results could be explained by the defective migratory response of was b cells to sdf-1 , essential for the retention of immature b cells in the bm . in the periphery </S>", "<S> , we observed an unusual expansion of cd21low b - cell population and increased plasma baff levels that may contribute to the high susceptibility to develop autoimmune manifestations in was patients . </S>", "<S> was memory b cells were characterized by a reduced in vivo proliferation , decreased somatic hypermutation and preferential usage of ighv4 - 34 , an immunoglobulin gene commonly found in autoreactive b cells.in conclusion , our findings demonstrate that wasp - deficiency perturbs b - cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in was patients . </S>" ]

[ "Introduction", "Material and methods", "Results", "Discussion and conclusions", "Conflict of interest" ]

[ [ "wiskott aldrich syndrome ( was ) is a monogenic x - linked primary immunodeficiency characterized by the classic triad of microthrombocytopenia , eczema and recurrent infections .", "multiple autoimmune manifestations and tumors are serious complications and the life expectancy of was patients is severely reduced , unless they are successfully cured by hematopoietic stem cell transplantation .", "was is caused by mutations in the was gene impairing the expression and/or function of the was protein ( wasp ) , a hematopoietic - specific regulator of cytoskeletal reorganization also involved in signal transduction of cells .", "the absence or reduction of wasp compromises multiple processes of different immune cell types involved in innate and adaptive responses , resulting in a variable and progressive immunodeficiency .", "susceptibility of was patients to develop autoimmune diseases has been mainly attributed to the breakdown of self tolerance sustained by dysfunction of both natural t regulatory and effector t cells ( reviewed in ref . )", ". however , b - cell intrinsic defects have been demonstrated to critically contribute to was - associated autoimmunity in was mouse model . in humans , the contribution of b - cell defects in the pathogenesis of was has been partially investigated .", "b cells from patients exhibit lower motility , migratory and adhesive capacities , likely due to defective f - actin nucleation . in contrast , despite the role of wasp in b - cell receptor ( bcr ) signaling , abnormalities in b - cell activation still remain controversial . a skewed distribution of serum immunoglobulin ( ig ) classes and the inability to mount a proper antibody response , particularly to t - cell independent ( ti ) antigens , suggest defects in b - cell effector function .", "previous findings in was patients show phenotypical b - cell perturbations in the periphery . in order to evaluate whether an abnormal b - cell development might generate a b - cell repertoire unable to unsure full protection against pathogens and tolerance against self - antigens ,", "to this end , we have combined a detailed phenotypical analysis of b - cell maturation stages , from the bone marrow ( bm ) to the periphery , with a molecular study of ig repertoire and in vivo b - cell maturation processes in a large cohort of was pediatric patients .", "our data show that wasp - deficiency affects critical stages of central and peripheral b - cell differentiation contributing to abnormalities in humoral immunity and b - cell tolerance in humans ." ], [ "the diagnoses were clinically defined and confirmed by genetic analysis . a description of all patients is reported in supplementary table 1 .", "human samples were obtained according to the code of ethics of the world medical association ( declaration of helsinki ) with the approval of the local medical ethical committees of the erasmus mc and the san raffaele scientific institute internal review board ( tiget02 ) .", "all results obtained from samples of was patients were compared to age and sex matched healthy donors ( hds ) .", "the composition of the precursor b - cell compartment was analyzed by flow cytometric immunophenotyping as described in the supplementary material .", "for the analysis of replication history and somatic hypermutation , four b - cell subsets were isolated from thawed peripheral blood mononuclear cells ( pbmcs ) using a facs diva cell sorter ( bd biosciences ) .", "gating on cd19 cells , transitional ( cd27cd24cd38 ) , mature nave ( cd27igdcd24cd38 ) , natural effector ( cd27igd ) and memory ( cd27igd ) b - cell subsets were sorted with a purity of > 95% for all fractions . for intracytoplasmic detection of human wasp ,", "cells were fixed and permeabilized using a cytofix / cytoperm kit ( bd pharmingen , oregon , usa ) .", "the anti - wasp antibody 503 ( a kind gift from prof h. d. ochs , seattle , wa , and l. d. notarangelo , boston , ma ) was used , followed by detection with pacific blue - labeled anti - rabbit igg secondary antibody ( invitrogen , san diego , usa ) .", "cd20 positive cells were purified from pbmcs of pediatric was patients and age - matched hds by immunomagnetic beads ( miltenyi biotec , germany ) or facs sorting .", "the purity of the isolated cells were analyzed by facs and ranged from 84% to 98% . ", "after isolation , cells were left overnight at 37 c in culture medium composed of rpmi-1640 , 10% fbs , 2 mm glutamine , 100 iu / ml penicillin and 100 g / ml streptomycin ( lonza , basel , switzerland ) . in vitro chemotaxis assay", "was performed using 5 m pore - size transwell inserts ( costar corporation , corning , ny , us ) in 24-well plates .", "filters were prewet 30 min at 37 c in presence of 600 l of medium supplemented with 250 ng / ml of recombinant human stromal cell - derived factor ( sdf)-1 ( cxcl12 ; peprotech , rocky hill , us ) .", "fifty thousand cd20 positive or negative cells were resuspended in 100 l of culture medium , seeded in the upper chamber and incubated at 37 c for 3 h. transmigrated cells , collected in the lower chamber , were counted for viable cells and stained with anti - cd19 , anti - cd24 , anti - cd38 , anti - cd27 and anti - cd3 ( bd biosciences ) for the phenotypical analysis by facs .", "migration frequency was estimated as the [ ( cell n at the lower chamber / the initial b cell input in the upper chamber ) 100 ] .", "levels of b - cell activating factor ( baff ) were measured in duplicate in plasma samples of was patients and hds using a quantikine human baff / blys / tnfsf13b immunoassay kit ( r&d systems , minneapolis , usa ) .", "the assay was performed according to manufacturer 's instructions and the od was determined using a microplate reader set to 450 nm .", "ig and igh gene rearrangements were amplified and analyzed as described in the supplementary material .", "dna was extracted from sorted b - cell subsets with the genelute mammalian total dna miniprep kit ( sigma aldrich ) and used to perform the ig-deleting recombination excision circles ( krec ) assay .", "the amounts of coding and signal joints of the ig-deleting rearrangement were measured by real - time quantitative pcr on an abi prism 7000 sequence detection system ( applied biosystems ) .", "ig restriction enzyme - based hot - spot mutation assay ( igrehma ) was performed on genomic dna isolated from sorted b - cell subsets .", "briefly , v3 - 20-j rearrangements were pcr - amplified and the pcr products ( 500 bp ) were first digested using the restriction enzyme fnu4hi and then kpni .", "the unmutated gene products can be visualized as 244 or 247 bp hex - coupled fragments and the mutated as 262 bp hex - coupled fragment .", "comparisons between proportions were calculated by using the chi - square test ( test ) ( with continuity correction ) ." ], [ "until now , the composition of the b - cell compartment in the bm of was patients has not been reported . to evaluate the effect of wasp - deficiency on b - cell development in the bm", ", we characterized five stages of b - cell differentiation in three was patients .", "we observed a decreased frequency of immature b lymphocytes in was patients ( fig . ", "this observation was further assessed in four additional patients , focusing on late stages of b - cell differentiation based on the expression of cd10 and cd20 ( gating strategy is shown in suppl . ", "we confirmed a significant decrease in the frequency of immature b cells compared with age - matched hds ( fig . ", "in addition , at the earliest differentiation stages , we observed a significant increase of small pre - b - ii cells in was patients ; while no statistical difference was detected in large pre - b - ii cell frequency ( fig . ", "these findings might reflect a partial block in the bm b - cell differentiation between the small pre - b - ii and immature b - cell stages in was , or an early egress of immature b cells into the periphery .", "next we examined the distribution of b - cell subsets in the peripheral blood of was patients and age - matched hds .", "when available , we evaluated the absolute count of total b cells , which was found significantly reduced in patients less than three years old ( < 3y ) , while no differences were observed in the older was group ( fig . ", "four b - cell subsets were identified according to the expression of cd24 , cd38 , igd and cd27 : transitional , mature nave , memory and plasmablasts ( fig . ", "transitional b cells ( cd24cd38 ) are the most immature peripheral b - cell population that were significantly increased in both was age groups as frequency ( fig . 2c ) and absolute number ( suppl . ", "the next maturation stage , represented by mature nave b cells ( cd27igdcd24cd38 ) , was significantly decreased only in was patients less than 3 years old both in frequency ( fig . ", "finally , the frequency of plasmablasts ( cd24cd38 ) was significantly increased in the older group of patients ( fig . ", "2c ) , while the absolute number of both memory b cells ( cd27igdcd24cd38 ) and plasmablasts were significantly decreased in the younger group of was patients ( suppl . ", "altogether , these findings demonstrate that was patients have a perturbed peripheral b - cell distribution . the overrepresentation of transitional b cells in the blood of was patients led us to hypothesize that an early egress of immature b cells from bm could occur .", "cxcr4 , the chemokine receptor for sdf-1 , has an important role in retaining b - cell precursors within the bm in order to prevent premature migration to the periphery [ 2224 ] .", "we tested the chemotactic response to sdf-1 of b lymphocytes of was patients and hds by transwell migration assay .", "as shown in fig . 3 , both transitional and mature peripheral b cells were less responsive to sdf-1 in the absence of wasp ( fig . ", "this defect was specific for b lymphocytes since cd3 positive cells of was patients showed a normal migratory capacity to sdf-1 ( suppl . ", "thus , we hypothesize that a diminished retention signal mediated by cxcr4/sdf-1 could explain the premature egress of immature b cells from the bm leading to an increase of transitional b cells in periphery . to further characterize the peripheral b - cell distribution , we focused our analysis on b - cell subsets found expanded in autoimmune diseases and potentially autoreactive [ 2528 ] .", "we found a significant increase of cd19cd21cd35 subset in both was age groups ( fig . ", "4a ) , confirming data previously reported by park and colleagues . we also analyzed the frequency of cd19cd21cd38 cells ( referred to as cd21 ) described to be expanded in autoimmune diseases and immunodeficiencies \n .", "interestingly , we observed that the frequency of this unusual population was markedly increased in was patients , both in the younger and older groups ( fig . ", "in addition , higher levels of soluble baff were found in the plasma of was patients as compared to pediatric hds ( fig . ", "this finding was also associated with a decreased expression of its receptor , baffr , in transitional b cells of was patients ( fig . ", "because of the role of baff in b - cell homeostasis and peripheral b - cell tolerance , high baff levels may affect the stringency of peripheral b - cell selection thus favoring the survival of b - cell subsets containing potentially autoreactive clones . in order to evaluate the effect of wasp - deficiency in an antigen dependent context , we analyzed in detail the memory b - cell compartment of was patients .", "we identified six distinct memory b - cell subsets ( gating strategy is shown in suppl . ", "igm - only ) and cd27igg deriving from primary germinal center ( gc ) responses ; igg cd27 and iga cd27 switched memory b cells originating from secondary gc responses ; cd27igd ( named natural effector ) and cd27iga generated in a ti manner in the gastrointestinal tract or in the splenic marginal zone area , respectively .", "we did not observe differences in memory b - cell populations deriving from both primary and secondary gc responses between was patients and hds ( fig . ", "a marked reduction in natural effector b cells was found in both age groups of was patients both as frequency ( fig . ", "the frequency of cd27iga b cells was decreased in 312 years old patients compared with hds ( fig . ", ", the absence of wasp mainly affects ti responses of b cells , whereas only mildly impairs the generation of early memory b cells in gc without influencing the class - switched memory b - cell compartment .", "we determined the in vivo b - cell proliferation history by krec assay and in parallel shm by igrehma in sorted transitional , mature nave , natural effector and memory b cells .", "as expected , transitional b lymphocytes did not undergo cell divisions and mature nave b cells had a limited number of cell divisions in hds ( fig . ", "both b - cell subsets were characterized by the absence of shm ( fig . ", "6b ) and we did not find differences in transitional and mature nave b cells of was subjects ( fig . ", "in contrast , the memory compartment showed less proliferation in was memory b cells in association with a reduced shm level ( fig . ", "interestingly , the defect in maturation was even more pronounced in natural effector was b lymphocytes . to study shm also in the ig heavy chain ( igh )", ", we analyzed the frequency of mutated nucleotides in rearranged variable region of igh ( ighv ) genes by cloning and sequencing the most frequent ighv subgroups , the ighv3 and ighv4 gene families , of both - and -chain of constant regions ( c and c ) .", "the mutational frequency of was b cells was significantly lower for both c and c transcripts in all domains of v region ( fig . ", "thus , the decreased in vivo proliferation and shm levels of was memory b cells could be responsible of a lower protection against infections and persistence of pathogens that finally lead to autoimmunity .", "was b cells showed an increased frequency of ighv3 - 30 in both ig classes ( fig . ", "7a ) and the absence of ighv3 - 48 genes in c sequences ( suppl . ", "7a ) . in ighv4 transcripts , we noticed a dominant usage of the ighv4 - 34 genes in was c ( fig . ", "moreover , the antibody repertoire of was b cells was devoid of ighv4 - 59 , a gene commonly used in b cells from hds .", "the distribution of d families was only slightly altered in was patients ( suppl . ", "finally , we evaluated the csr process in total b cells by analyzing the frequency of ig subclasses in sequenced igh transcripts .", "a preferential usage of -chain c region 3 ( c3 ) and c1 , igh - proximal genes ( the igh locus is schematized in fig . ", "7d ) , accompanied by a reduction in the expression of distal c2 and c4 , were detected in was patients ( fig . ", "the analysis of iga transcripts showed no significant difference in the subclass usage ( fig . ", "7e ) . in conclusion was patients show a skewed ig gene usage suggesting an altered selection of the antibody repertoire during b - cell development ." ], [ "we reported herein that wasp - deficiency affects many aspects of b - cell development , first in the bm and then in the periphery .", "indeed , precursor b - cell as well as memory b - cell development and selection appear affected by the absence of functional wasp . in the bm", "we have observed a decreased frequency of immature b cells , while in the periphery an overrepresentation of transitional b cells likely due to a reduced retention in the bm and/or a decreased migration to peripheral lymphoid tissues .", "the interaction of the chemokine receptor cxcr4 with its ligand sdf-1 is required for the retention of developing b cells in the bm .", "wasp is actively involved in cxcr4 signaling [ 3739 ] and was murine b cells show defective migratory response to sdf-1 .", "consistently , here we demonstrate in was patients that b cells migrate less to sdf-1. thus , was immature b cells could be unable to properly sense retention signals derived from cxcr4 in the bm leading to a premature release in the periphery .", "the increase of transitional b cells could also reflect an altered migration to the peripheral lymphoid organs resulting in a prolonged persistence in the circulation .", "consistently , our data allow us to exclude that the expansion of transitional b cells is due to homeostatic proliferation since we demonstrated the absence of replication history in this subset .", "transitional b cells are found expanded in immunodeficient conditions or autoimmune diseases and represent a reservoir of autoreactive b cells .", "the cohort of patients analyzed here did not show any overt sign of autoimmunity , probably due to their young age .", "however , the analysis of serum autoantibodies performed in four patients showed positivity for anti - nuclear antibodies in three patients .", "one of them also showed the presence of anti - platelet antibodies ( data not shown ) .", "these cells are enriched in anergic autoreactive clones and expanded in systemic lupus erythematosus ( sle ) , rheumatoid arthritis and in common variable immunodeficiency groupia patients developing autoimmune syndromes .", "baff levels and signals through baffr coordinate the maintenance of the primary b - cell pool and the fate of self - reactive b cells .", "herein , we report for the first time increased baff levels and decreased baffr expression in was patients .", "elevated baff serum levels are often present in immunodeficiencies , autoimmune diseases and viral infections and may lower the thresholds for the survival of autoreactive b cell clones .", "the enrichment of cd21 b cells and the alterations in baff levels and expression of its receptor suggest that the mechanisms of b - cell selection could be altered in was patients .", "our data also suggest the presence of a defective selection of was b cells producing high - affinity antibodies .", "indeed , we noticed a restricted or null presence of ighv3 - 48 and ighv4 - 59 gene families and a preferential usage of ighv3 - 30 and ighv4 - 34 .", "in particular , ighv3 - 48 gene is selectively used against polysaccharide antigens and its decrease in was could account for an inefficient antibody response .", "in contrast , ighv3 - 30 is highly represented among anti - platelet autoantibodies from patients with idiopathic thrombocytopenic purpura and in sle patients . additionally , ighv4 - 34 encoded antibodies are intrinsically autoreactive when unmutated , as observed in our patients .", "this peculiar antibody repertoire reflects an altered selection of both protective and autoreactive ig gene families .", "the perturbation of b - cell homeostasis present in was patients supports the administration of anti - cd20 mab in the conditioning regimen of gene therapy to deplete b cells . in the memory b - cell compartment , the frequency of isotype - switched igg b cells", "we observed a normal frequency of memory b cells and of switched cd27igg and cd27iga b cells in our cohort of was patients .", "this suggests that t - cell dependent antigen response is induced normally in was patients .", "in contrast , the frequency and the shm level of ti memory b - cell subsets were reduced , likely due to their reduced in vivo proliferation . the marked reduction in the natural effector b - cell subset , resembling marginal zone b cells in the spleen , mirrors the decreased number of splenic marginal zone b cells and the histological defects in the marginal zone area of the spleen already described in was mice and patients . in addition , both the reduction in natural effector and igacd27 b cells , which are generated during ti responses , provide evidence of a defect in b - cell function independent from the cross - talk with t lymphocytes .", "the mutational status of both heavy and light ig chains is reduced in was in presence of a diminished rate of proliferation in the total memory b - cell pool .", "in addition , the analysis of ig gene selection showed a preferential use of igh - proximal genes ( ighg1 and ighg3 ) accompanied by reduced shm suggesting that was b cells have undergone less gc reactions .", "reduced igg2 switching in was patients could account for the poor response to ti antigens which is typical of the syndrome .", "the cytokine profile of was patients shows an impairment in the production of th1 cytokines that could explain their reduced switching to igg2 subclass .", "moreover , the isotype - switching outcome is also influenced by two additional factors : cell division and antibody affinity to antigens .", "the defective proliferation of memory and natural effector b cells and the reduced affinity maturation found in was might contribute to these alterations in class switching .", "our results in was patients show an early egress of immature b cells from bm leading to an overrepresentation of transitional b cells in the periphery .", "the memory compartment is characterized by a reduced maturation status that could affect the b - cell effector functions contributing to a lower clearance of pathogens and leading to chronic inflammation that can break tolerance . in conclusion ,", "our results add novel immunological features of was b - cell phenotype that can complement the evaluation of the efficacy of various treatment approaches ." ], [ "" ] ]

[ "the direct nucleophilic substitution of alcohols is of high interest as it provides access to a wide variety of derivatives , with the formation of water as the only by - product .", "indeed , the acs green chemistry institute pharmaceutical roundtable identified oh activation for nucleophilic substitutions as a priority area currently used in the preparation of pharmaceutical intermediates that would greatly benefit from the development of better methods.1 unarguably , propargylic substitutions have progressed substantially since the pioneering work of nicholas on octacarbonyldicobalt - stabilised propargylic cations.2 the versatility of the propargylic moiety as a synthon in organic chemistry as well as its occurrence in natural products and synthetic pharmaceuticals have been the main driving forces for these advances .", "furthermore , propargylic alcohols are easily prepared from the corresponding aldehydes or ketones by addition of an alkynyl anion .", "diverse transition metals,3 such as ruthenium , palladium , gold or silver,4 have been successfully used in this context .", "however , the cost of the catalyst , together with its selectivity ( metal allenylidene vs. metal propargylic intermediates ) remain important issues to solve .", "the direct displacement of activated alcohols such as benzylic , allylic , and propargylic alcohols can also be achieved using brnsted or lewis acids by simple sn1 reactions.5 important advantages of brnsted acids over lewis acids often include lower catalytic loadings and easier handling as they are generally more stable towards oxygen and water .", "sulfonic acids are the most commonly used brnsted acids for the nucleophilic substitution of propargylic alcohols as described in extensive work by sanz and co - workers with p - toluenesulfonic acid.6,7 inorganic acids , such as phosphomolybdic acid on silica , have also been studied with c- , n- and o - nucleophiles.8 depending on the substrates , the reactions required either 10 mol-% of acid at room temperature , or 1 mol-% in refluxing toluene .", "an additional asset of these inorganic acids is their straightforward separation from the organic products through a simple basic workup .", "a common feature for all these catalytic systems is their compatibility with air and reagent - grade solvents , although they are mostly undesirable ones ( toxic , costly to dispose of , such as meno2 ) .", "hbf4 is a common acid in academic and industrial laboratories that has found diverse applications in synthesis , either as a reagent ( nucleophilic fluorination,9 synthesis of vinylidene metal complexes10 ) , or catalyst ( amidation of olefins,11 biginelli reaction,12 acylation of aldehydes13 ) .", "crafts alkylation of benzylic alcohols in the presence of an excess of hbf4oet2 solution at 78 c has been reported.14 even though high diastereoselectivities could be achieved with this methodology , the excess of acid and low reaction temperatures represent important drawbacks . herein , we report the use of hbf4 as a highly efficient catalyst for sn1 reactions of propargylic alcohols with different nucleophiles under mild , simple reaction conditions .", "in a first step , the effect of different solvents was tested on the reaction of propargylic alcohol 1a with meoh to give 2a with 1 mol-% of hbf4 ( table1 ) .", "of meoh and a commercially available 48 wt.-% solution of hbf4 in water as catalyst .", "no attempts were made to optimise the reaction times . whereas sluggish reactions were observed in thf or in water ( table1 , entries 1 and 2 ) ,", "high conversions were obtained in dcm , acetonitrile , and acetone ( table1 , entries 46 ) .", "overall , acetone was chosen as our preferred solvent because of its greener profile.15 it is important to note that all tested solvents were technical grade , and in particular , the acetone employed in these reactions was standard laboratory washing acetone .", "schuster rearrangement16 was not observed either in the model reaction or during the study of the scope of the reaction .", "the use of different o - nucleophiles was first explored ( scheme 1 ) .", "propargylic alcohol 1a was treated with different primary and secondary alcohols to form the expected ethers 2a h in high yields under our standard conditions .", "when chiral alcohols were used , the corresponding ethers 2 g and 2h were formed as a mixture of inseparable diastereoisomers . a tertiary alcohol , tbuoh , only led to low yields of the ether 2i , and the major product of that reaction ( 51 % conversion ) formed from dimerisation of the starting propargylic alcohol ( 3a ; vide infra for further details ) . on the other hand , an ortho - disubstituted aryl group was not detrimental to the reactivity of the propargylic alcohol , as exemplified with the formation of 2k . also ,", "unlike most transition - metal - based methodologies,3 the reaction is not limited to terminal alkynes , and alkyl ( 2j , 2k ) , aryl ( 2l ) or silyl groups ( 2 m , 2n ) at the acetylenic position did not have any major effect on the outcome of the reaction ( scheme 1 ) .", "also , several functional groups ( ketone , halogen or sulfone ) were shown to be compatible with the reaction conditions . in the absence of any other nucleophile , the starting propargylic alcohol dimerised to form the symmetrical ether as a mixture of diastereoisomers ( scheme 2 ) .", "dr = diastereoisomeric ratio . [ a ] isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % . [ b ] 51 % h nmr conversion into dimer 3a .", "[ a ] isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % .", "on the other hand , when r on the starting propargylic alcohol was not an electron - rich aryl group , no reaction was observed at room temperature . in most cases , however , the formation of the desired ethers was possible by increasing the reaction temperature and/or the acid loading ( table2 ) .", "good yields could be then obtained , except for a nitro - substituted substrate ( table2 , entry 4 ) .", "it is important to note that , since no decomposition or undesired reactions were observed at room temperature , this difference in reactivity could be used to selectively functionalise a more complex molecule with two electronically dissimilar propargylic alcohols ( vide infra ) .", "isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % ; n.r . =", "the inherent basicity of most amines is an obvious potential limitation of any brnsted acid catalysed reaction as they might simply neutralise the catalyst .", "our conditions , however , could be successfully applied to different carbamates and sulfonamides , as well as weakly basic anilines ( scheme 3 ) , and the expected products 4 were prepared in good yields at room temperature , with the exception of 4e .", "[ a ] isolated yields , h nmr conversions are provided in parentheses when lower than 95 % . [ b ] reaction carried out with 5 mol-% of hbf4 at 60 c .", "carbon nucleophiles were also investigated , and diketones as well as electron - rich arenes reacted to form the expected products 5 in good to excellent yields ( scheme 4 ) .", "very similar results were obtained with pentane-2,4-dione and a variety of substituted propargylic alcohols . for the formation of 5d , with an electron - neutral aryl group at the propargylic position , a higher catalyst loading and an elevated temperature were required in order to obtain high conversions .", "phenol also reacted efficiently in a friedel crafts - type reaction,17 to give para - substituted derivatives 5e g exclusively .", "when using 2-phenylphenol as the nucleophile , the hydroxy group had a stronger directing power than the arene , as expected ( scheme 4 , compound 5h ) .", "we then tested a phenol with a second strongly activating group at the para position ( 4-methoxyphenol , for the formation of 5i ) . in this case , only the product derived from reaction at the ortho position to the phenol was isolated .", "a ] isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % .", "[ b ] reaction carried out with 5 mol-% of hbf4 in mecn at 80 c .", "surprisingly , allyltrimethylsilane proved to be a very poor reaction partner for the substitution of propargylic alcohols with hbf4 as the catalyst .", "low conversions were obtained in either acetone or hot toluene , even when higher catalyst or nucleophile loadings were used ( table3 ) .", "overall , the best results were obtained in mecn at 80 c , and still product 5j could only be isolated in 50 % yield .", "it is important to note that alcohol 1a was stable under the studied conditions , and besides the expected product 5j , only 1a and ether 3a were evidenced in the h nmr spectra .", "hence , no amide formation , which could potentially take place by a ritter reaction,18,19 was observed under these conditions .", "when furan was used as nucleophile , the h nmr spectrum of the crude reaction mixture showed the formation of a complex mixture of products .", "prompt purification allowed the isolation of 5k in moderate yield , but it is important to note that 5k still decomposed rapidly after purification .", "these observations were perhaps not surprising as many furan derivatives are well - known to be acid - sensitive .", "this is also the case for some indoles , but as it can be seen in table4 , better yields were obtained with this important family of heterocyclic nucleophiles.20 hbf4-catalysed propargylation reactions of heterocycles .", "isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % .", "reaction carried out with 5 mol-% of hbf4 in toluene at 80 c . in order to investigate the influence of substitution on the nucleophile ,", "different indole derivatives were treated with the same propargylic alcohol 1a . 1-methyl- and 1h - indoles reacted regioselectively at c-3 , as expected , leading to the formation of 5l and 5 m , respectively , in good yields .", "this might be due to a higher instability under acidic conditions or its low solubility either in acetone or dcm . even under more forcing conditions ( 5 mol-% of acid at 80 c ) , no evidence for reaction of the hydroxy group could be detected .", "gratifyingly , products 5o q , derived from a friedel crafts reaction at c-2 could be prepared under very simple reaction conditions . to the best of our knowledge ,", "this is the first example of the synthesis of 2,3-disubstituted indoles by brnsted acid catalysed propargylation reactions.21 some of the reactions in table4 were carried out in dcm , instead of acetone , to avoid the formation of undesired by - products .", "it has previously been reported that indoles can react with ketones or aldehydes as electrophiles under acidic conditions.22 indeed , when 1-methylindole was treated with propargylic alcohol 1a under our standard conditions in acetone , the expected product 5l was formed preferentially , but it was contaminated with bis(indole ) 6 ( scheme 5a ) .", "the high yield obtained of 5l indicates that the indole reacts preferentially with the propargylic cation formed from 1a and that the reaction between the excess of indole and acetone is quite sluggish .", "the formation of 6 remains undesirable , and hence acetone was avoided as a solvent for these reactions .", "more problematic was the reaction of 1a with tryptophol as nucleophile ( scheme 5b ) . in this case the expected product could only be isolated in 40 % yield because of a competitive oxa - pictet ", "spengler acid - catalysed cyclocondensation of tryptophol with the solvent,23 which consumed 70 % of the available nucleophile .", "next , two competition experiments were performed to exploit the particular activity of hbf4 in propargylation reactions ( scheme 6 ) .", "firstly , relatively electron - rich alcohol 1a reacted selectively in the presence of 1 g , bearing a chloro substituent at the para position of the phenyl ring ( scheme 6a ) .", "also , the unexpected diminished reactivity of allylsilanes as nucleophiles was exploited when 1a was treated with 2 equiv . of benzyl alcohol and 2 equiv .", "only 2b , derived from the reaction with benzyl alcohol , was formed under these conditions .", "importantly , these chemoselectivities are not possible when using other brnsted acids reported for this transformation , such as p - toluenesulfonic acid,6a or phosphomolybdic acid.8a competition experiments with hbf4 .", "finally , a gram - scale reaction was performed to further demonstrate the applicability of this reaction , and compound 5r was isolated in high yield when using our optimised conditions ( scheme 7 ) .", "the scope and limitations of hbf4 as a practical catalyst for propargylation reactions have been explored . in general ,", "good to excellent yields for the formation of c o , c n and c ", "c bonds were obtained under exceptionally simple reaction conditions . challenging substrates such as electron - poor propargylic alcohols , or acid - sensitive indoles could be used with this methodology , even if slightly more forcing conditions were sometimes required .", "all reactions were carried out in air and in technical solvents , and the acid used was a commercially available aqueous solution .", "all the reactions were also completely regioselective , and no allene products were observed in any case .", "furthermore , many of the reactions were extremely clean , and the desired products could be isolated analytically pure without the need for further purification after a simple aqueous workup ( i.e. , 2a , 2j , 2l o , 5b c ) .", "overall , this is a convenient and powerful methodology that does not employ a costly metal catalyst .", "for instance , we were pleased to see that an allylic alcohol also reacted with meoh at room temperature in very high yields ( scheme 8) .", "general procedure for the nucleophilic substitution of propargylic alcohols : in a vial fitted with a screw cap and a stirring bar , the propargylic alcohol 1 ( 1 mmol ) , nucleophile ( 2 mmol ) and technical acetone ( 2 ml ) were introduced .", "an aqueous solution of hbf4 ( 48 wt.-% , 1.2 l,1 mol-% ) was then added , and the reaction mixture was stirred at room temperature for 18 h. the reaction was quenched with a saturated aqueous solution of nahco3 and the mixture extracted with ethyl acetate .", "the combined organic extracts were washed with brine , dried with anhydrous mgso4 , filtered , and concentrated under reduced pressure to give the title compound .", "as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors" ]

[ "<S> the activity of hbf4 ( aqueous solution ) as a catalyst in propargylation reactions is presented . </S>", "<S> diverse types of nucleophiles were employed in order to form new c o , c n and c </S>", "<S> c bonds in technical acetone and in air . </S>", "<S> good to excellent yields and good chemoselectivities were obtained using low acid loading ( typically 1 mol-% ) under simple reaction conditions . </S>" ]

[ "Introduction", "Results and Discussion", "Conclusions", "Experimental Section", "Supporting Information" ]

[ [ "the direct nucleophilic substitution of alcohols is of high interest as it provides access to a wide variety of derivatives , with the formation of water as the only by - product .", "indeed , the acs green chemistry institute pharmaceutical roundtable identified oh activation for nucleophilic substitutions as a priority area currently used in the preparation of pharmaceutical intermediates that would greatly benefit from the development of better methods.1 unarguably , propargylic substitutions have progressed substantially since the pioneering work of nicholas on octacarbonyldicobalt - stabilised propargylic cations.2 the versatility of the propargylic moiety as a synthon in organic chemistry as well as its occurrence in natural products and synthetic pharmaceuticals have been the main driving forces for these advances .", "furthermore , propargylic alcohols are easily prepared from the corresponding aldehydes or ketones by addition of an alkynyl anion .", "diverse transition metals,3 such as ruthenium , palladium , gold or silver,4 have been successfully used in this context .", "however , the cost of the catalyst , together with its selectivity ( metal allenylidene vs. metal propargylic intermediates ) remain important issues to solve .", "the direct displacement of activated alcohols such as benzylic , allylic , and propargylic alcohols can also be achieved using brnsted or lewis acids by simple sn1 reactions.5 important advantages of brnsted acids over lewis acids often include lower catalytic loadings and easier handling as they are generally more stable towards oxygen and water .", "sulfonic acids are the most commonly used brnsted acids for the nucleophilic substitution of propargylic alcohols as described in extensive work by sanz and co - workers with p - toluenesulfonic acid.6,7 inorganic acids , such as phosphomolybdic acid on silica , have also been studied with c- , n- and o - nucleophiles.8 depending on the substrates , the reactions required either 10 mol-% of acid at room temperature , or 1 mol-% in refluxing toluene .", "an additional asset of these inorganic acids is their straightforward separation from the organic products through a simple basic workup .", "a common feature for all these catalytic systems is their compatibility with air and reagent - grade solvents , although they are mostly undesirable ones ( toxic , costly to dispose of , such as meno2 ) .", "hbf4 is a common acid in academic and industrial laboratories that has found diverse applications in synthesis , either as a reagent ( nucleophilic fluorination,9 synthesis of vinylidene metal complexes10 ) , or catalyst ( amidation of olefins,11 biginelli reaction,12 acylation of aldehydes13 ) .", "crafts alkylation of benzylic alcohols in the presence of an excess of hbf4oet2 solution at 78 c has been reported.14 even though high diastereoselectivities could be achieved with this methodology , the excess of acid and low reaction temperatures represent important drawbacks . herein , we report the use of hbf4 as a highly efficient catalyst for sn1 reactions of propargylic alcohols with different nucleophiles under mild , simple reaction conditions ." ], [ "in a first step , the effect of different solvents was tested on the reaction of propargylic alcohol 1a with meoh to give 2a with 1 mol-% of hbf4 ( table1 ) .", "of meoh and a commercially available 48 wt.-% solution of hbf4 in water as catalyst .", "no attempts were made to optimise the reaction times . whereas sluggish reactions were observed in thf or in water ( table1 , entries 1 and 2 ) ,", "high conversions were obtained in dcm , acetonitrile , and acetone ( table1 , entries 46 ) .", "overall , acetone was chosen as our preferred solvent because of its greener profile.15 it is important to note that all tested solvents were technical grade , and in particular , the acetone employed in these reactions was standard laboratory washing acetone .", "schuster rearrangement16 was not observed either in the model reaction or during the study of the scope of the reaction .", "the use of different o - nucleophiles was first explored ( scheme 1 ) .", "propargylic alcohol 1a was treated with different primary and secondary alcohols to form the expected ethers 2a h in high yields under our standard conditions .", "when chiral alcohols were used , the corresponding ethers 2 g and 2h were formed as a mixture of inseparable diastereoisomers . a tertiary alcohol , tbuoh , only led to low yields of the ether 2i , and the major product of that reaction ( 51 % conversion ) formed from dimerisation of the starting propargylic alcohol ( 3a ; vide infra for further details ) . on the other hand , an ortho - disubstituted aryl group was not detrimental to the reactivity of the propargylic alcohol , as exemplified with the formation of 2k . also ,", "unlike most transition - metal - based methodologies,3 the reaction is not limited to terminal alkynes , and alkyl ( 2j , 2k ) , aryl ( 2l ) or silyl groups ( 2 m , 2n ) at the acetylenic position did not have any major effect on the outcome of the reaction ( scheme 1 ) .", "also , several functional groups ( ketone , halogen or sulfone ) were shown to be compatible with the reaction conditions . in the absence of any other nucleophile , the starting propargylic alcohol dimerised to form the symmetrical ether as a mixture of diastereoisomers ( scheme 2 ) .", "dr = diastereoisomeric ratio . [ a ] isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % . [ b ] 51 % h nmr conversion into dimer 3a .", "[ a ] isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % .", "on the other hand , when r on the starting propargylic alcohol was not an electron - rich aryl group , no reaction was observed at room temperature . in most cases , however , the formation of the desired ethers was possible by increasing the reaction temperature and/or the acid loading ( table2 ) .", "good yields could be then obtained , except for a nitro - substituted substrate ( table2 , entry 4 ) .", "it is important to note that , since no decomposition or undesired reactions were observed at room temperature , this difference in reactivity could be used to selectively functionalise a more complex molecule with two electronically dissimilar propargylic alcohols ( vide infra ) .", "isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % ; n.r . =", "the inherent basicity of most amines is an obvious potential limitation of any brnsted acid catalysed reaction as they might simply neutralise the catalyst .", "our conditions , however , could be successfully applied to different carbamates and sulfonamides , as well as weakly basic anilines ( scheme 3 ) , and the expected products 4 were prepared in good yields at room temperature , with the exception of 4e .", "[ a ] isolated yields , h nmr conversions are provided in parentheses when lower than 95 % . [ b ] reaction carried out with 5 mol-% of hbf4 at 60 c .", "carbon nucleophiles were also investigated , and diketones as well as electron - rich arenes reacted to form the expected products 5 in good to excellent yields ( scheme 4 ) .", "very similar results were obtained with pentane-2,4-dione and a variety of substituted propargylic alcohols . for the formation of 5d , with an electron - neutral aryl group at the propargylic position , a higher catalyst loading and an elevated temperature were required in order to obtain high conversions .", "phenol also reacted efficiently in a friedel crafts - type reaction,17 to give para - substituted derivatives 5e g exclusively .", "when using 2-phenylphenol as the nucleophile , the hydroxy group had a stronger directing power than the arene , as expected ( scheme 4 , compound 5h ) .", "we then tested a phenol with a second strongly activating group at the para position ( 4-methoxyphenol , for the formation of 5i ) . in this case , only the product derived from reaction at the ortho position to the phenol was isolated .", "a ] isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % .", "[ b ] reaction carried out with 5 mol-% of hbf4 in mecn at 80 c .", "surprisingly , allyltrimethylsilane proved to be a very poor reaction partner for the substitution of propargylic alcohols with hbf4 as the catalyst .", "low conversions were obtained in either acetone or hot toluene , even when higher catalyst or nucleophile loadings were used ( table3 ) .", "overall , the best results were obtained in mecn at 80 c , and still product 5j could only be isolated in 50 % yield .", "it is important to note that alcohol 1a was stable under the studied conditions , and besides the expected product 5j , only 1a and ether 3a were evidenced in the h nmr spectra .", "hence , no amide formation , which could potentially take place by a ritter reaction,18,19 was observed under these conditions .", "when furan was used as nucleophile , the h nmr spectrum of the crude reaction mixture showed the formation of a complex mixture of products .", "prompt purification allowed the isolation of 5k in moderate yield , but it is important to note that 5k still decomposed rapidly after purification .", "these observations were perhaps not surprising as many furan derivatives are well - known to be acid - sensitive .", "this is also the case for some indoles , but as it can be seen in table4 , better yields were obtained with this important family of heterocyclic nucleophiles.20 hbf4-catalysed propargylation reactions of heterocycles .", "isolated yields ; h nmr conversions are provided in parentheses when lower than 95 % .", "reaction carried out with 5 mol-% of hbf4 in toluene at 80 c . in order to investigate the influence of substitution on the nucleophile ,", "different indole derivatives were treated with the same propargylic alcohol 1a . 1-methyl- and 1h - indoles reacted regioselectively at c-3 , as expected , leading to the formation of 5l and 5 m , respectively , in good yields .", "this might be due to a higher instability under acidic conditions or its low solubility either in acetone or dcm . even under more forcing conditions ( 5 mol-% of acid at 80 c ) , no evidence for reaction of the hydroxy group could be detected .", "gratifyingly , products 5o q , derived from a friedel crafts reaction at c-2 could be prepared under very simple reaction conditions . to the best of our knowledge ,", "this is the first example of the synthesis of 2,3-disubstituted indoles by brnsted acid catalysed propargylation reactions.21 some of the reactions in table4 were carried out in dcm , instead of acetone , to avoid the formation of undesired by - products .", "it has previously been reported that indoles can react with ketones or aldehydes as electrophiles under acidic conditions.22 indeed , when 1-methylindole was treated with propargylic alcohol 1a under our standard conditions in acetone , the expected product 5l was formed preferentially , but it was contaminated with bis(indole ) 6 ( scheme 5a ) .", "the high yield obtained of 5l indicates that the indole reacts preferentially with the propargylic cation formed from 1a and that the reaction between the excess of indole and acetone is quite sluggish .", "the formation of 6 remains undesirable , and hence acetone was avoided as a solvent for these reactions .", "more problematic was the reaction of 1a with tryptophol as nucleophile ( scheme 5b ) . in this case the expected product could only be isolated in 40 % yield because of a competitive oxa - pictet ", "spengler acid - catalysed cyclocondensation of tryptophol with the solvent,23 which consumed 70 % of the available nucleophile .", "next , two competition experiments were performed to exploit the particular activity of hbf4 in propargylation reactions ( scheme 6 ) .", "firstly , relatively electron - rich alcohol 1a reacted selectively in the presence of 1 g , bearing a chloro substituent at the para position of the phenyl ring ( scheme 6a ) .", "also , the unexpected diminished reactivity of allylsilanes as nucleophiles was exploited when 1a was treated with 2 equiv . of benzyl alcohol and 2 equiv .", "only 2b , derived from the reaction with benzyl alcohol , was formed under these conditions .", "importantly , these chemoselectivities are not possible when using other brnsted acids reported for this transformation , such as p - toluenesulfonic acid,6a or phosphomolybdic acid.8a competition experiments with hbf4 .", "finally , a gram - scale reaction was performed to further demonstrate the applicability of this reaction , and compound 5r was isolated in high yield when using our optimised conditions ( scheme 7 ) ." ], [ "the scope and limitations of hbf4 as a practical catalyst for propargylation reactions have been explored . in general ,", "good to excellent yields for the formation of c o , c n and c ", "c bonds were obtained under exceptionally simple reaction conditions . challenging substrates such as electron - poor propargylic alcohols , or acid - sensitive indoles could be used with this methodology , even if slightly more forcing conditions were sometimes required .", "all reactions were carried out in air and in technical solvents , and the acid used was a commercially available aqueous solution .", "all the reactions were also completely regioselective , and no allene products were observed in any case .", "furthermore , many of the reactions were extremely clean , and the desired products could be isolated analytically pure without the need for further purification after a simple aqueous workup ( i.e. , 2a , 2j , 2l o , 5b c ) .", "overall , this is a convenient and powerful methodology that does not employ a costly metal catalyst .", "for instance , we were pleased to see that an allylic alcohol also reacted with meoh at room temperature in very high yields ( scheme 8) ." ], [ "general procedure for the nucleophilic substitution of propargylic alcohols : in a vial fitted with a screw cap and a stirring bar , the propargylic alcohol 1 ( 1 mmol ) , nucleophile ( 2 mmol ) and technical acetone ( 2 ml ) were introduced .", "an aqueous solution of hbf4 ( 48 wt.-% , 1.2 l,1 mol-% ) was then added , and the reaction mixture was stirred at room temperature for 18 h. the reaction was quenched with a saturated aqueous solution of nahco3 and the mixture extracted with ethyl acetate .", "the combined organic extracts were washed with brine , dried with anhydrous mgso4 , filtered , and concentrated under reduced pressure to give the title compound ." ], [ "as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors" ] ]

[ "notch proteins are evolutionarily conserved cell - surface receptors for transmembrane ligands of the dsl family ( named after the delta and serrate ligands of drosophila ) . signaling by notch regulates a broad range of cell - fate decisions during development and various human diseases , including cancers ,", "the extracellular part of notch contains 36 epidermal growth factor - like ( egf ) repeats carrying the ligand - binding region and three lin12/notch repeats ( lnrs ) that limit proteolytic cleavage of the receptor at the s2 site , and hence limit its activation .", "the egf repeats are modified by two types of o - linked glycosylation , o - glucosylation and o - fucosylation ( figure 1a ) ; both are important for notch activity [ 3 - 6 ] ( figure 1a ) .", "( a ) schematic representation of the drosophila notch receptor . the extracellular domain ( necd )", "is composed of three lin12/notch repeats ( lnrs ) ( black ) and 36 egf - like repeats ; the egf repeats are predicted to be either o - glucosylated ( cyan ) , o - fucosylated ( yellow ) or doubly modified ( green ) adapted from .", "the s2 cleavage site is indicated by an arrow . the intracellular domain ( nicd )", "contains various elements involved in transcriptional activation : a ram domain , seven ankyrin repeats ( ank ) , a transactivation domain ( tad ) and a pest domain ( p ) .", "( b , c ) two models for the roles of ofut1 in notch trafficking .", "each model is illustrated in a schematic cell that is shown as wild type on the left and ofut1 mutant on the right , gray - shaded side .", "( b ) model 1 : in the er , newly synthesized , unfolded notch ( with the necd in red and the nicd in white ) becomes properly folded ( necd in green ) and is o - fucosylated by ofut1 ( yellow diamond ) .", "it is then transported to the plasma membrane and adherens junctions ( ajs , cyan ) through the golgi . in the absence of ofut1 , notch remains unfolded and is retained in the er . ( c ) model 2 : o - fucosylated notch is first transported via the golgi to the apical membrane and then transported to ajs by a transcytosis mechanism . in ofut1 mutant cells , notch goes to the membrane , is internalized and accumulates in an uncharacterized endocytic compartment .", "o - fucosylation of notch is catalyzed by an o - fucosyltransferase ( pofut1 in mammals and ofut1 in drosophila ) that uses gdp - fucose as a substrate . the o - fucose residue added by this o - fucosyltransferase", "can be further elongated by the addition of an n - acetylglucosamine by fringe , an egf - o - fucose 1,3 n - acetylglucosamyltransferase .", "in drosophila , the activity of fringe is required for notch signaling events involved in boundary formation , in which it reduces the affinity of notch for serrate while enhancing that for delta .", "given that ofut1 is thought to be the sole enzyme that o - fucosylates notch , the activity of fringe is predicted to require the o - fucosylation activity of ofut1 .", "however , ofut1 has additional functions , and other catalytic and non - catalytic activities have been proposed [ 8,11 - 16 ] .", "these include roles in the folding of notch in the endoplasmic reticulum ( er ) , in notch - ligand binding and in endocytic trafficking of notch .", "there are several observations that support each of these models , and there has been controversy about which one is correct . a recent paper in bmc biology by kenneth irvine and colleagues ( okajima et al . )", "the first model proposes that ofut1 acts in the er , where it performs two separable functions : to o - fucosylate notch , thereby modulating notch - ligand interaction , and to promote the correct folding of the extracellular domain of notch ( a non - catalytic function ) .", "it partially co - localizes with er markers and acts , at least in part , in the er : the carboxy - terminal extremity of ofut1 contains a lys - asp - glu - leu ( kdel)-like motif that is dispensable for its catalytic activity but is required for both er retention and function .", "the notch protein accumulates in intracellular compartments marked by er markers , and knockdown of ofut1 using double - stranded rna in cultured cells inhibits the secretion of a soluble version of the notch extracellular domain ( necd ) .", "this activity of ofut1 does not require its o - fucosylation activity because expression of a mutant version of ofut1 demonstrated to be catalytically dead ( ofut1 ) mostly rescues the notch localization defects .", "in addition , ofut1 and ofut1both bind the necd , and expression of ofut1 in cultured cells can increase both the amount and the ligand - binding activity of secreted necd .", "consistent with this proposed o - fucosylation - independent activity , notch localizes at the cell cortex in gdp - mannose 4,6-dehydratase ( gmd ) mutants , which contain no gdp - fucose , indicating that non - o - fucosylated notch exits the er . together , these results led to a simple model ( model 1 , figure 1b ) in which ofut1 acts as an er chaperone to promote the proper folding of the egf repeats of notch , thereby ensuring its correct cell - surface localization and ligand - binding activity .", "the second model proposes that ofut1 acts at two post - exocytosis steps in the trafficking of notch : it regulates both the endocytosis of notch and its transcytosis from the apical plasma membrane to the adherens junctions ( ajs ) .", "first , ofut1 is required for the proper distribution of notch at ajs . in ofut1 mutant cells", ", notch does not accumulate at ajs but instead accumulates into intracellular ' dots ' that co - localize with er markers only poorly .", "in addition , surface - staining experiments indicate that a low level of notch is also present at the surface of ofut1 mutant cells .", "these data were interpreted to suggest that ofut1 is not required for the er exit and cell - surface delivery of notch .", "second , ofut1 is required to regulate the endocytic trafficking of notch , as monitored by antibody uptake .", "anti - notch antibodies are internalized in ofut1 mutant cells , albeit at a lower level than in wild - type cells , but fail to accumulate in endosomes .", "it is therefore proposed that notch accumulates in an uncharacterized endocytic compartment in ofut1 mutant cells . finally , the second model is supported by the observation that a lower level of intracellular notch is detected in gmd mutant cells than in ofut1 single - mutant or ofut1 , gmd double - mutant cells .", "this suggests that the endocytic activity of ofut1 does not depend on its catalytic activity .", "in addition , the failure of notch to localize at ajs in gmd mutant cells is interpreted in the light of a speculative model of the transcytosis of notch to suggest that ofut1 acts in a catalysis - dependent manner to regulate the transcytosis of notch .", "we feel that this transcytosis model needs further experimental validation , so we will not detail the possible role of ofut1 in this process further .", "we note , however , that this proposed role of ofut1 in the transcytosis of notch suggests that the o - fucosylation activity of ofut1 is required for fringe - independent signaling events .", "together , these observations suggest a model ( model 2 , figure 1c ) in which ofut1 regulates the endocytic trafficking of notch at a post - internalization step so that notch accumulates in an undefined endocytic compartment in the absence of ofut1 activity .", "thus , these two models offer two different interpretations of the cellular basis of the ofut1 mutant phenotype : in model 1 , the reason notch does not signal is because it is trapped in the er , whereas in model 2 , it is because notch accumulates in an undefined endocytic compartment with a low level of ( inactive ) notch at the surface .", "one implication of model 2 is that a fraction of ofut1 must escape er retention and act in the endocytic pathway .", "consistent with this possibility , experiments using cultured cells indicate that a fraction of ofut1 is secreted , interacts with the extracellular part of notch at the cell surface , and is internalized in a notch - dependent manner .", "in addition , the notch localization defects seen in cells treated with ofut1 double - stranded rna can be rescued by adding ofut1-containing conditioned medium .", "whether this happens in vivo is not clear . indeed , ofut1 acts in a cell - autonomous manner to regulate the localization of notch , arguing that secretion is not important in this regulation .", "moreover , the observed rescue of the ofut1 knockdown phenotype by extracellular ofut1 could also be consistent with model 1 , given that endocytosed ofut1 may be transported back into the er .", "so , how can we distinguish between the two models ? clearly , answering the following questions would help .", "does notch reach the cell surface in the absence of ofut1 activity ? where does notch accumulate in ofut1 mutant cells ? is the o - fucosylation activity of ofut1 required for proper notch localization and activity ? or is it only required for fringe - dependent signaling ?", "okajima et al . have addressed these issues and provide compelling evidence in favor of model 1 .", "okajima et al . have shown that the o - fucosylation activity of ofut1 is required for fringe - dependent but not for fringe - independent signaling events .", "they found that embryos lacking both maternal and zygotic contributions of gmd develop into larvae , as fringe mutant embryos do .", "the complete loss of ofut1 activity results in a strong phenotype mimicking a loss of notch activity that is rescued to larval viability by the expression of ofut1 .", "these data show that the o - fucosylation activity of ofut1 is dispensable for notch signaling in the embryo .", "this conclusion is entirely consistent with the observation that the activity of fringe is dispensable in the embryo .", "it is therefore clear that non - fucosylated notch can reach the cell surface and can signal .", "the role of the o - fucosylation activity of ofut1 during wing imaginal disc development led to identical conclusions .", "although the loss of ofut1 activity in clones leads to phenotypes mimicking a loss of notch activity , expression of ofut1rescued notch receptor activity in these ofut1 mutant cells and led to phenotypes mimicking a loss of fringe activity .", "this confirms that non - fucosylated notch can signal , and further indicates that the fucosylation activity of ofut1 is required only for fringe - dependent signaling events .", "this therefore implies that the transcytosis step of model 2 , which was proposed to be dependent on ofut1 catalytic activity , is not essential for notch signaling . because the controversy over the exact role of ofut1 is in part due to methodological differences between the various studies and also to technical limitations in subcellular localization analysis , okajima et al . ", "also re - examined the localization of notch in ofut1 mutant cells using a detergent - free cell - surface staining protocol .", "a striking difference in surface staining was observed between wild - type and ofut1 mutant cells .", "this convincingly shows that notch is not present at detectable levels at the surface of mutant cells .", "this contradicts the results obtained by sasaki et al . , who used a different protocol to assay the presence of notch at the cell surface of ofut1 mutant cells .", "whether the difference in protocols accounts for these opposite conclusions remains to be addressed experimentally .", "the second piece of evidence in support of a cell surface accumulation of notch in ofut1 mutant cells comes from antibody uptake experiments showing that anti - notch antibodies can be internalized by ofut1 mutant cells .", "however , as discussed by okijama et al . , the low level of antibody uptake can probably be accounted for by the fluid - phase uptake of anti - notch antibodies by live ofut1 mutant cells , followed by the specific retention of internalized antibodies by notch accumulating intracellularly .", "together , the published data are best interpreted as concluding that notch does not reach the cell surface in ofut1 mutant cells .", "notch was shown to partially co - localize with four different er markers that , in fact , show only partial co - localization among themselves .", "thus , the only partially overlapping distribution of er markers may explain the poor co - localization of notch with the two er markers seen by sasamura et al . .", "therefore propose that notch accumulates in ofut1 mutant cells in the er , which is a heterogeneous organelle .", "accordingly , notch should co - localize better with the sum of the signals of the different er markers .", "it is clear that the o - fucosylation of notch is primarily required for fringe - dependent signaling events and that ofut1 acts non - catalytically to regulate the exit of notch from the er . thus , ofut1 probably acts as a chaperone in the er to promote the proper folding of the extracellular domain of notch , as described in model 1 .", "although the catalytic and non - catalytic activities of ofut1 can be experimentally uncoupled , it is attractive to speculate that the o - fucosylation activity of ofut1 participates in the quality - control mechanism that ensures that only properly folded notch exits the er . further analysis of the trafficking of non - fucosylated notch , produced for instance by gmd mutant cells , would help address this issue ." ]

[ "<S> the precise role of the o - fucosyltransferase ofut1 in notch - receptor trafficking has remained controversial . </S>", "<S> a recent study sheds new light on the non - catalytic activity of ofut1 and provides further evidence that ofut1 acts as a chaperone in the endoplasmic reticulum . </S>" ]

[ "None", "The models and the supporting evidence", "Towards the end of a controversy", "Acknowledgements" ]

[ [ "notch proteins are evolutionarily conserved cell - surface receptors for transmembrane ligands of the dsl family ( named after the delta and serrate ligands of drosophila ) . signaling by notch regulates a broad range of cell - fate decisions during development and various human diseases , including cancers ,", "the extracellular part of notch contains 36 epidermal growth factor - like ( egf ) repeats carrying the ligand - binding region and three lin12/notch repeats ( lnrs ) that limit proteolytic cleavage of the receptor at the s2 site , and hence limit its activation .", "the egf repeats are modified by two types of o - linked glycosylation , o - glucosylation and o - fucosylation ( figure 1a ) ; both are important for notch activity [ 3 - 6 ] ( figure 1a ) .", "( a ) schematic representation of the drosophila notch receptor . the extracellular domain ( necd )", "is composed of three lin12/notch repeats ( lnrs ) ( black ) and 36 egf - like repeats ; the egf repeats are predicted to be either o - glucosylated ( cyan ) , o - fucosylated ( yellow ) or doubly modified ( green ) adapted from .", "the s2 cleavage site is indicated by an arrow . the intracellular domain ( nicd )", "contains various elements involved in transcriptional activation : a ram domain , seven ankyrin repeats ( ank ) , a transactivation domain ( tad ) and a pest domain ( p ) .", "( b , c ) two models for the roles of ofut1 in notch trafficking .", "each model is illustrated in a schematic cell that is shown as wild type on the left and ofut1 mutant on the right , gray - shaded side .", "( b ) model 1 : in the er , newly synthesized , unfolded notch ( with the necd in red and the nicd in white ) becomes properly folded ( necd in green ) and is o - fucosylated by ofut1 ( yellow diamond ) .", "it is then transported to the plasma membrane and adherens junctions ( ajs , cyan ) through the golgi . in the absence of ofut1 , notch remains unfolded and is retained in the er . ( c ) model 2 : o - fucosylated notch is first transported via the golgi to the apical membrane and then transported to ajs by a transcytosis mechanism . in ofut1 mutant cells , notch goes to the membrane , is internalized and accumulates in an uncharacterized endocytic compartment .", "o - fucosylation of notch is catalyzed by an o - fucosyltransferase ( pofut1 in mammals and ofut1 in drosophila ) that uses gdp - fucose as a substrate . the o - fucose residue added by this o - fucosyltransferase", "can be further elongated by the addition of an n - acetylglucosamine by fringe , an egf - o - fucose 1,3 n - acetylglucosamyltransferase .", "in drosophila , the activity of fringe is required for notch signaling events involved in boundary formation , in which it reduces the affinity of notch for serrate while enhancing that for delta .", "given that ofut1 is thought to be the sole enzyme that o - fucosylates notch , the activity of fringe is predicted to require the o - fucosylation activity of ofut1 .", "however , ofut1 has additional functions , and other catalytic and non - catalytic activities have been proposed [ 8,11 - 16 ] .", "these include roles in the folding of notch in the endoplasmic reticulum ( er ) , in notch - ligand binding and in endocytic trafficking of notch .", "there are several observations that support each of these models , and there has been controversy about which one is correct . a recent paper in bmc biology by kenneth irvine and colleagues ( okajima et al . )" ], [ "the first model proposes that ofut1 acts in the er , where it performs two separable functions : to o - fucosylate notch , thereby modulating notch - ligand interaction , and to promote the correct folding of the extracellular domain of notch ( a non - catalytic function ) .", "it partially co - localizes with er markers and acts , at least in part , in the er : the carboxy - terminal extremity of ofut1 contains a lys - asp - glu - leu ( kdel)-like motif that is dispensable for its catalytic activity but is required for both er retention and function .", "the notch protein accumulates in intracellular compartments marked by er markers , and knockdown of ofut1 using double - stranded rna in cultured cells inhibits the secretion of a soluble version of the notch extracellular domain ( necd ) .", "this activity of ofut1 does not require its o - fucosylation activity because expression of a mutant version of ofut1 demonstrated to be catalytically dead ( ofut1 ) mostly rescues the notch localization defects .", "in addition , ofut1 and ofut1both bind the necd , and expression of ofut1 in cultured cells can increase both the amount and the ligand - binding activity of secreted necd .", "consistent with this proposed o - fucosylation - independent activity , notch localizes at the cell cortex in gdp - mannose 4,6-dehydratase ( gmd ) mutants , which contain no gdp - fucose , indicating that non - o - fucosylated notch exits the er . together , these results led to a simple model ( model 1 , figure 1b ) in which ofut1 acts as an er chaperone to promote the proper folding of the egf repeats of notch , thereby ensuring its correct cell - surface localization and ligand - binding activity .", "the second model proposes that ofut1 acts at two post - exocytosis steps in the trafficking of notch : it regulates both the endocytosis of notch and its transcytosis from the apical plasma membrane to the adherens junctions ( ajs ) .", "first , ofut1 is required for the proper distribution of notch at ajs . in ofut1 mutant cells", ", notch does not accumulate at ajs but instead accumulates into intracellular ' dots ' that co - localize with er markers only poorly .", "in addition , surface - staining experiments indicate that a low level of notch is also present at the surface of ofut1 mutant cells .", "these data were interpreted to suggest that ofut1 is not required for the er exit and cell - surface delivery of notch .", "second , ofut1 is required to regulate the endocytic trafficking of notch , as monitored by antibody uptake .", "anti - notch antibodies are internalized in ofut1 mutant cells , albeit at a lower level than in wild - type cells , but fail to accumulate in endosomes .", "it is therefore proposed that notch accumulates in an uncharacterized endocytic compartment in ofut1 mutant cells . finally , the second model is supported by the observation that a lower level of intracellular notch is detected in gmd mutant cells than in ofut1 single - mutant or ofut1 , gmd double - mutant cells .", "this suggests that the endocytic activity of ofut1 does not depend on its catalytic activity .", "in addition , the failure of notch to localize at ajs in gmd mutant cells is interpreted in the light of a speculative model of the transcytosis of notch to suggest that ofut1 acts in a catalysis - dependent manner to regulate the transcytosis of notch .", "we feel that this transcytosis model needs further experimental validation , so we will not detail the possible role of ofut1 in this process further .", "we note , however , that this proposed role of ofut1 in the transcytosis of notch suggests that the o - fucosylation activity of ofut1 is required for fringe - independent signaling events .", "together , these observations suggest a model ( model 2 , figure 1c ) in which ofut1 regulates the endocytic trafficking of notch at a post - internalization step so that notch accumulates in an undefined endocytic compartment in the absence of ofut1 activity .", "thus , these two models offer two different interpretations of the cellular basis of the ofut1 mutant phenotype : in model 1 , the reason notch does not signal is because it is trapped in the er , whereas in model 2 , it is because notch accumulates in an undefined endocytic compartment with a low level of ( inactive ) notch at the surface .", "one implication of model 2 is that a fraction of ofut1 must escape er retention and act in the endocytic pathway .", "consistent with this possibility , experiments using cultured cells indicate that a fraction of ofut1 is secreted , interacts with the extracellular part of notch at the cell surface , and is internalized in a notch - dependent manner .", "in addition , the notch localization defects seen in cells treated with ofut1 double - stranded rna can be rescued by adding ofut1-containing conditioned medium .", "whether this happens in vivo is not clear . indeed , ofut1 acts in a cell - autonomous manner to regulate the localization of notch , arguing that secretion is not important in this regulation .", "moreover , the observed rescue of the ofut1 knockdown phenotype by extracellular ofut1 could also be consistent with model 1 , given that endocytosed ofut1 may be transported back into the er ." ], [ "so , how can we distinguish between the two models ? clearly , answering the following questions would help .", "does notch reach the cell surface in the absence of ofut1 activity ? where does notch accumulate in ofut1 mutant cells ? is the o - fucosylation activity of ofut1 required for proper notch localization and activity ? or is it only required for fringe - dependent signaling ?", "okajima et al . have addressed these issues and provide compelling evidence in favor of model 1 .", "okajima et al . have shown that the o - fucosylation activity of ofut1 is required for fringe - dependent but not for fringe - independent signaling events .", "they found that embryos lacking both maternal and zygotic contributions of gmd develop into larvae , as fringe mutant embryos do .", "the complete loss of ofut1 activity results in a strong phenotype mimicking a loss of notch activity that is rescued to larval viability by the expression of ofut1 .", "these data show that the o - fucosylation activity of ofut1 is dispensable for notch signaling in the embryo .", "this conclusion is entirely consistent with the observation that the activity of fringe is dispensable in the embryo .", "it is therefore clear that non - fucosylated notch can reach the cell surface and can signal .", "the role of the o - fucosylation activity of ofut1 during wing imaginal disc development led to identical conclusions .", "although the loss of ofut1 activity in clones leads to phenotypes mimicking a loss of notch activity , expression of ofut1rescued notch receptor activity in these ofut1 mutant cells and led to phenotypes mimicking a loss of fringe activity .", "this confirms that non - fucosylated notch can signal , and further indicates that the fucosylation activity of ofut1 is required only for fringe - dependent signaling events .", "this therefore implies that the transcytosis step of model 2 , which was proposed to be dependent on ofut1 catalytic activity , is not essential for notch signaling . because the controversy over the exact role of ofut1 is in part due to methodological differences between the various studies and also to technical limitations in subcellular localization analysis , okajima et al . ", "also re - examined the localization of notch in ofut1 mutant cells using a detergent - free cell - surface staining protocol .", "a striking difference in surface staining was observed between wild - type and ofut1 mutant cells .", "this convincingly shows that notch is not present at detectable levels at the surface of mutant cells .", "this contradicts the results obtained by sasaki et al . , who used a different protocol to assay the presence of notch at the cell surface of ofut1 mutant cells .", "whether the difference in protocols accounts for these opposite conclusions remains to be addressed experimentally .", "the second piece of evidence in support of a cell surface accumulation of notch in ofut1 mutant cells comes from antibody uptake experiments showing that anti - notch antibodies can be internalized by ofut1 mutant cells .", "however , as discussed by okijama et al . , the low level of antibody uptake can probably be accounted for by the fluid - phase uptake of anti - notch antibodies by live ofut1 mutant cells , followed by the specific retention of internalized antibodies by notch accumulating intracellularly .", "together , the published data are best interpreted as concluding that notch does not reach the cell surface in ofut1 mutant cells .", "notch was shown to partially co - localize with four different er markers that , in fact , show only partial co - localization among themselves .", "thus , the only partially overlapping distribution of er markers may explain the poor co - localization of notch with the two er markers seen by sasamura et al . .", "therefore propose that notch accumulates in ofut1 mutant cells in the er , which is a heterogeneous organelle .", "accordingly , notch should co - localize better with the sum of the signals of the different er markers .", "it is clear that the o - fucosylation of notch is primarily required for fringe - dependent signaling events and that ofut1 acts non - catalytically to regulate the exit of notch from the er . thus , ofut1 probably acts as a chaperone in the er to promote the proper folding of the extracellular domain of notch , as described in model 1 .", "although the catalytic and non - catalytic activities of ofut1 can be experimentally uncoupled , it is attractive to speculate that the o - fucosylation activity of ofut1 participates in the quality - control mechanism that ensures that only properly folded notch exits the er . further analysis of the trafficking of non - fucosylated notch , produced for instance by gmd mutant cells , would help address this issue ." ], [ "" ] ]

[ "choriocarcinoma is one of the malignant tumors arising from trophoblastic cells characterized by the secretion of human chorionic gonadotrophin ( hcg ) .", "the tumor consisted of two basic cell types : cytotrophoblast and hcg - positive syncytiotrophoblast , the typical histologic features of choriocarcinoma ( 1 - 5 ) .", "the serum hcg level is typically elevated , with resultant isosexual pseudoprecocity in children , and menstrual abnormalities , breast enlargement , androgenic changes , or combinations thereof , in adults ( 1 , 2 ) .", "it usually arises from fetal trophoblasts and rarely arises from germ cells in the testis or ovary or derives from dedifferentiation of other carcinomas ( 2 , 3 ) .", "the most common sites of systemic metastatic disease are lungs , liver and brain via hematogenous spread ( 1 ) .", "skin is a rare site for metastatic choriocarcinoma , and even more rare as the initial presenting site for the disease ( 3 - 5 ) . to the best of our knowledge ,", "we describe a case of 52-yr - old korean female patient with cutaneous metastasis of choriocarcinoma , diagnosed from a skin biopsy that provided important diagnostic clues to the primary lesion .", "a 52-yr - old korean female presented with dyspnea of 2 months duration and cutaneous nodules of 1 month duration on the left side of the neck and the right side of the upper back .", "her past medical history was significant for behet 's disease , which had been treated with colchicine for the past 4 yr . routine blood tests showed leukocytosis with a wbc of 17,780/l , anemia with a hemoglobin of 9.4 g / dl and slightly elevated hepatic enzyme levels of got / gpt at 45/51 iu / l .", "chest radiography , chest and abdomen - pelvis ct revealed pleural effusion , innumerable variable sized pulmonary metastatic nodules on both lungs and multiple metastatic lesions on the liver , left adrenal gland and left kidney .", "a skin biopsy was performed on the nodule at the right side of upper back . at low magnification", ", there were several hemorrhagic foci and multiple cellular infiltrations throughout the dermis ( fig .", "2a ) . at high magnification , the infiltrated tumor cells consisted of two cell types , and a biphasic pattern , in close proximity .", "one element had polygonal shaped , clear cytoplasm and large , irregular shaped , vesicular nuclei with prominent nucleoli , which were identified as cytotrophoblasts .", "the other cell population showed eosinophilic cytoplasm with cytoplasmic vacuoles and many dark nuclei , which were identified as syncytiotrophoblasts ( fig .", "the immunohistochemical staining for hcg antigen ( 1:600 , polyclonal rabbit , dakocytomation , denmark ) in the cytoplasm of syncytiotrophoblasts ( fig .", "magnetic resonance ( mr ) images of the abdomen - pelvis detected diffuse enlargement of the uterus body and a protruding mass in the uterine cavity , which was the primary origin of the tumor ( fig .", "her serum hcg level was 700,000 miu / ml ( upper normal limit : 10 miu / ml ) .", "she was referred to the gynecological oncology department , where combination chemotherapy was commenced . during combination chemotherapy ,", "the serum hcg level has fallen off under 2.0 miu / ml and her skin lesions have almost disappeared .", "however , 3 months after 17 cycles , total abdominal hysterectomy and bilateral salpingo - oophorectomy were performed due to elevated serum hcg level ( 4,447.7 miu / ml ) , and she is scheduled to receive post - operative adjuvant chemotherapy .", "compared to other organs , skin is an uncommon site of metastatic cancer in the body as it is usually associated with a disseminated disease and grave outcome . occasionally , however", ", as in our case , cutaneous metastasis may be the first harbinger of a visceral malignancy of unknown origin and gives an important diagnostic clue ( 3 , 4 , 6 - 8 ) .", "cutaneous metastasis of a visceral malignancy has been estimated to be in the wide range of 1.4% to 10.4% according to the type of study undertaken and the site of primary tumor studied ( 6 - 11 ) .", "involvement of the skin is likely to be near the area of the primary tumor and in proportion to the incidence of internal malignancy .", "the chest , abdomen , and scalp are common anatomic sites to which metastasis localize with the back and extremities being relatively uncommon areas .", "breast cancer in women and lung cancer in men are the most common causes of chest metastatic lesion ( 6 - 8 ) . occasionally", ", a cutaneous metastatic lesion may show typical histologic features which give a decisive diagnostic clue to the primary origin .", "such cases include mucin - containing cells in colon cancer , bile - containing glandular structure in hepatoma , clear cells in renal cell carcinoma and , like our case , cytotrophoblasts and syncytiotrophoblasts in choriocarcinoma ( 6 ) .", "choriocarcinoma , in women , is often preceded by a gravid state , which is most often hydatidiform mole ( 50% ) , less frequently abortion ( 25% ) , and normal term pregnancy ( 15% ) .", ", it commonly arises from germ cells in the testis ( 1 - 5 ) .", "the gross appearance of choriocarcinoma is often dark red or purple colored and resembles that of a friable segment of placenta ( 1 ) .", "microscopically , it is characterized by multiple foci of hemorrhage and necrosis around the neoplastic cells , by large sheets of cytotrophoblastic and syncytiotrophoblastic cells , biphasic pattern , and by the absence of hydropic villi .", "the cytotrophoblasts have cuboidal shaped , pale cytoplasm and large , irregular shaped , vesicular nuclei with prominent nucleoli .", "the syncytiotrophoblasts show eosinophilic cytoplasm with cytoplasmic vacuoles , many hyperchromatic and pleomorphic nuclei with indistinct border , looking like multinucleated giant cells , and may form syncytial knots .", "the immunohistochemical staining for hcg antigen is positive in the cytoplasm of syncytiotrophoblasts but not in cytotrophoblasts ( 1 - 5 ) .", "other germ cell tumors such as polyembryoma , embryonal carcinoma , dysgerminoma and mixed germ cell tumor can also show hcg positive syncytiotrophoblastic cells but these tumors do not demonstrate a biphasic pattern consisting of cytotrophoblasts and syncytiotrophoblasts , as in choriocarcinoma ( 1 ) .", "the most common metastatic sites are , in a descending order of frequency , lung ( 60 - 95% ) , vagina ( 40 - 50% ) , vulva ( 10 - 15% ) , brain , liver ( 10% ) , kidney and spleen ( < 5% ) .", "although choriocarcinomas respond relatively well to chemotherapy , cutaneous metastasis is an extremely uncommon presentation and a poor prognostic sign as it is associated only with disseminated disease ( 1 - 5 ) .", "reported a case of cutaneous metastatic testicular choriocarcinoma where the patient died 10 days after the initiation of chemotherapy ( 4 ) .", "shimizu et al . also reported a case of metastatic disease to the upper back of testicular choriocarcinoma , where the patient died 3 months after the appearance of a cutaneous metastatic lesion . in their literature review , of 7 patients with metastatic choriocarcinoma to the skin", ", 5 patients died within 3 months after the appearance of skin metastasis ( 3 ) .", "reported another case of choriocarcinoma with metastasis to the chest wall , which responded well to 12 cycles of combination chemotherapy ( 5 ) .", "we described an extremely rare patient with metastatic choriocarcinoma presenting initially as a cutaneous lesion .", "the diagnosis was made from the typical histological findings of a cutaneous metastatic lesion , which was confirmed by immunohistochemical staining for hcg and from the abdomen pelvis mr images .", "this case highlights the importance of a histological examination of skin metastatic lesion , which can provide conclusive evidences to the diagnosis ." ]

[ "<S> choriocarcinoma is one of the malignant tumors of trophoblastic cells characterized by the secretion of human chorionic gonadotrophin ( hcg ) ( 1 - 3 ) . </S>", "<S> cutaneous metastasis is a rare presentation of choriocarcinoma but a poor prognostic sign because it is associated only with widespread disease ( 3 - 5 ) . </S>", "<S> a 52-yr - old female complaining of dyspnea for 2 months , presented with fingertip sized erythematous nodules on the left side of the neck and the right side of the upper back of 1 month duraton . </S>", "<S> she has suffered from behet 's disease since 1999 . </S>", "<S> microscopic examination of a nodule of upper back demonstrated biphasic pattern of cytotrophoblasts and hcg - positive syncytiotrophoblasts , and the typical histologic features of choriocarcinoma . </S>", "<S> she was referred to the gynecological oncology department . </S>", "<S> after 17 cycles of combination chemotherapy , the serum hcg level has fallen from 700,000 to under 2.0 miu / ml and the skin lesions have almost disappeared . however , after 3 months , total abdominal hysterectomy and bilateral salpingo - oophorectomy were performed due to elevated serum hcg level ( 4,447.7 miu / ml ) , and she is scheduled to receive post - operative adjuvant chemotherapy . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "choriocarcinoma is one of the malignant tumors arising from trophoblastic cells characterized by the secretion of human chorionic gonadotrophin ( hcg ) .", "the tumor consisted of two basic cell types : cytotrophoblast and hcg - positive syncytiotrophoblast , the typical histologic features of choriocarcinoma ( 1 - 5 ) .", "the serum hcg level is typically elevated , with resultant isosexual pseudoprecocity in children , and menstrual abnormalities , breast enlargement , androgenic changes , or combinations thereof , in adults ( 1 , 2 ) .", "it usually arises from fetal trophoblasts and rarely arises from germ cells in the testis or ovary or derives from dedifferentiation of other carcinomas ( 2 , 3 ) .", "the most common sites of systemic metastatic disease are lungs , liver and brain via hematogenous spread ( 1 ) .", "skin is a rare site for metastatic choriocarcinoma , and even more rare as the initial presenting site for the disease ( 3 - 5 ) . to the best of our knowledge ,", "we describe a case of 52-yr - old korean female patient with cutaneous metastasis of choriocarcinoma , diagnosed from a skin biopsy that provided important diagnostic clues to the primary lesion ." ], [ "a 52-yr - old korean female presented with dyspnea of 2 months duration and cutaneous nodules of 1 month duration on the left side of the neck and the right side of the upper back .", "her past medical history was significant for behet 's disease , which had been treated with colchicine for the past 4 yr . routine blood tests showed leukocytosis with a wbc of 17,780/l , anemia with a hemoglobin of 9.4 g / dl and slightly elevated hepatic enzyme levels of got / gpt at 45/51 iu / l .", "chest radiography , chest and abdomen - pelvis ct revealed pleural effusion , innumerable variable sized pulmonary metastatic nodules on both lungs and multiple metastatic lesions on the liver , left adrenal gland and left kidney .", "a skin biopsy was performed on the nodule at the right side of upper back . at low magnification", ", there were several hemorrhagic foci and multiple cellular infiltrations throughout the dermis ( fig .", "2a ) . at high magnification , the infiltrated tumor cells consisted of two cell types , and a biphasic pattern , in close proximity .", "one element had polygonal shaped , clear cytoplasm and large , irregular shaped , vesicular nuclei with prominent nucleoli , which were identified as cytotrophoblasts .", "the other cell population showed eosinophilic cytoplasm with cytoplasmic vacuoles and many dark nuclei , which were identified as syncytiotrophoblasts ( fig .", "the immunohistochemical staining for hcg antigen ( 1:600 , polyclonal rabbit , dakocytomation , denmark ) in the cytoplasm of syncytiotrophoblasts ( fig .", "magnetic resonance ( mr ) images of the abdomen - pelvis detected diffuse enlargement of the uterus body and a protruding mass in the uterine cavity , which was the primary origin of the tumor ( fig .", "her serum hcg level was 700,000 miu / ml ( upper normal limit : 10 miu / ml ) .", "she was referred to the gynecological oncology department , where combination chemotherapy was commenced . during combination chemotherapy ,", "the serum hcg level has fallen off under 2.0 miu / ml and her skin lesions have almost disappeared .", "however , 3 months after 17 cycles , total abdominal hysterectomy and bilateral salpingo - oophorectomy were performed due to elevated serum hcg level ( 4,447.7 miu / ml ) , and she is scheduled to receive post - operative adjuvant chemotherapy ." ], [ "compared to other organs , skin is an uncommon site of metastatic cancer in the body as it is usually associated with a disseminated disease and grave outcome . occasionally , however", ", as in our case , cutaneous metastasis may be the first harbinger of a visceral malignancy of unknown origin and gives an important diagnostic clue ( 3 , 4 , 6 - 8 ) .", "cutaneous metastasis of a visceral malignancy has been estimated to be in the wide range of 1.4% to 10.4% according to the type of study undertaken and the site of primary tumor studied ( 6 - 11 ) .", "involvement of the skin is likely to be near the area of the primary tumor and in proportion to the incidence of internal malignancy .", "the chest , abdomen , and scalp are common anatomic sites to which metastasis localize with the back and extremities being relatively uncommon areas .", "breast cancer in women and lung cancer in men are the most common causes of chest metastatic lesion ( 6 - 8 ) . occasionally", ", a cutaneous metastatic lesion may show typical histologic features which give a decisive diagnostic clue to the primary origin .", "such cases include mucin - containing cells in colon cancer , bile - containing glandular structure in hepatoma , clear cells in renal cell carcinoma and , like our case , cytotrophoblasts and syncytiotrophoblasts in choriocarcinoma ( 6 ) .", "choriocarcinoma , in women , is often preceded by a gravid state , which is most often hydatidiform mole ( 50% ) , less frequently abortion ( 25% ) , and normal term pregnancy ( 15% ) .", ", it commonly arises from germ cells in the testis ( 1 - 5 ) .", "the gross appearance of choriocarcinoma is often dark red or purple colored and resembles that of a friable segment of placenta ( 1 ) .", "microscopically , it is characterized by multiple foci of hemorrhage and necrosis around the neoplastic cells , by large sheets of cytotrophoblastic and syncytiotrophoblastic cells , biphasic pattern , and by the absence of hydropic villi .", "the cytotrophoblasts have cuboidal shaped , pale cytoplasm and large , irregular shaped , vesicular nuclei with prominent nucleoli .", "the syncytiotrophoblasts show eosinophilic cytoplasm with cytoplasmic vacuoles , many hyperchromatic and pleomorphic nuclei with indistinct border , looking like multinucleated giant cells , and may form syncytial knots .", "the immunohistochemical staining for hcg antigen is positive in the cytoplasm of syncytiotrophoblasts but not in cytotrophoblasts ( 1 - 5 ) .", "other germ cell tumors such as polyembryoma , embryonal carcinoma , dysgerminoma and mixed germ cell tumor can also show hcg positive syncytiotrophoblastic cells but these tumors do not demonstrate a biphasic pattern consisting of cytotrophoblasts and syncytiotrophoblasts , as in choriocarcinoma ( 1 ) .", "the most common metastatic sites are , in a descending order of frequency , lung ( 60 - 95% ) , vagina ( 40 - 50% ) , vulva ( 10 - 15% ) , brain , liver ( 10% ) , kidney and spleen ( < 5% ) .", "although choriocarcinomas respond relatively well to chemotherapy , cutaneous metastasis is an extremely uncommon presentation and a poor prognostic sign as it is associated only with disseminated disease ( 1 - 5 ) .", "reported a case of cutaneous metastatic testicular choriocarcinoma where the patient died 10 days after the initiation of chemotherapy ( 4 ) .", "shimizu et al . also reported a case of metastatic disease to the upper back of testicular choriocarcinoma , where the patient died 3 months after the appearance of a cutaneous metastatic lesion . in their literature review , of 7 patients with metastatic choriocarcinoma to the skin", ", 5 patients died within 3 months after the appearance of skin metastasis ( 3 ) .", "reported another case of choriocarcinoma with metastasis to the chest wall , which responded well to 12 cycles of combination chemotherapy ( 5 ) .", "we described an extremely rare patient with metastatic choriocarcinoma presenting initially as a cutaneous lesion .", "the diagnosis was made from the typical histological findings of a cutaneous metastatic lesion , which was confirmed by immunohistochemical staining for hcg and from the abdomen pelvis mr images .", "this case highlights the importance of a histological examination of skin metastatic lesion , which can provide conclusive evidences to the diagnosis ." ] ]

[ "breast cancer is the second most common cancer in new zealand and the third leading cause of cancer death.1 in 2011 , the most - recent full year for which statistics are available , 2,867 new cases of breast cancer and 636 deaths were reported.2 approximately 18% of breast cancers in new zealand are human epidermal growth factor receptor-2 ( her2)-positive.3 trastuzumab is standard treatment for her2-positive early breast cancer ( ebc ) and metastatic breast cancer ( mbc ) , being administered as an intravenous ( iv ) infusion , in part with chemotherapy , as an every-3-weeks regimen for 12 months in ebc ( 17 cycles ) , or until disease progression in mbc .", "trastuzumab iv needs to be reconstituted into solution for infusion , with the dose calculated on the basis of patient bodyweight.4 iv trastuzumab treatment starts with a loading dose infused over 90 minutes , followed by subsequent maintenance doses infused over 30 minutes.4 given the requirement for up to 12 months of adjuvant or maintenance treatment , such a regimen consumes considerable health care resources , including drug preparation and administration , clinic and chair time , and physician time dedicated to patient interaction.5 furthermore , insertion of an indwelling venous catheter is common in patients requiring prolonged iv infusion therapy , adding the costs of theatre and anesthetist time ; complications such as infection or thrombosis6,7 may also be associated with additional costs in some cases .", "a subcutaneous ( sc ) formulation of trastuzumab was developed to allow drug administration over a shorter time period , with the goal of improving convenience and compliance , particularly during long - term therapy .", "the ability to do this was facilitated by the fact that trastuzumab has a wide therapeutic window8 with no known dose - limiting toxicities.9 sc administration of volumes > 1 ml has been limited by the structure and physiology of the sc layer , which contains a matrix of hyaluronan and collagen fibers.8 to overcome this issue , the trastuzumab sc formulation includes recombinant human hyaluronidase as a novel excipient.10 hyaluronidase hydrolyses hyaluronan , decreases resistance of the interstitial matrix11,12 and degrades the extracellular matrix .", "this allows monoclonal antibodies to diffuse through the channels,12 thereby facilitating sc administration of larger volumes of fluid and improving delivery of sc drugs to the systemic circulation .", "the effects of recombinant human hyaluronidase are temporary and reversible within 24 hours.12 trastuzumab sc can be administered over approximately 5 minutes , a much shorter period of time than that required for the trastuzumab iv loading and maintenance doses.4,10 in addition , trastuzumab sc is given as a fixed dose that does not require adjustment based on bodyweight.10 pharmacokinetic modeling , based on data from an open - label phase i / ib study,13 determined that a 600 mg fixed dose of trastuzumab sc provided drug exposure that was comparable to the iv trastuzumab regimen , without the need for a loading dose .", "this dose of trastuzumab sc was subsequently tested in the phase iii hannah study , which showed that trastuzumab sc was noninferior to trastuzumab iv with respect to efficacy ( pathological complete response rate ) and had an equivalent tolerability profile.14 one of the key principles guiding the new zealand ministry of health medical oncology national implementation plan15 is to effectively , equitably , and sustainably meet the future demand for medical oncology services , given the significant workforce and resource constraints that exist .", "a second aim is to maintain high quality of care and improve the quality of life for people with cancer .", "it also offers the potential to move treatment delivery to the outpatient setting and away from infusion services that are facing challenges due to increased demand for complex treatments.15 the aim of this study was to determine medical resource utilization associated with administration of trastuzumab sc injection compared with trastuzumab iv infusion in patients with her2-positive breast cancer in new zealand .", "this noninterventional , descriptive study was conducted alongside the ongoing safeher clinical trial ( nct01566721 ) , a study investigating the safety and tolerability of trastuzumab sc via handheld syringe or single - injection device . the time and motion study", "was conducted at the outpatient oncology centers at auckland city hospital ( trastuzumab iv ) and tauranga hospital ( trastuzumab sc and iv ) .", "patients receiving trastuzumab sc via handheld syringe were recruited by the health care professionals ( hcps ) who were administering trastuzumab to participants in the safeher clinical trial at tauranga hospital .", "patients with ebc were approached during a routine visit from march 2013 to may 2013 as part of their enrolment in safeher and given information about the time and motion study by a member of the clinical team .", "prior to trastuzumab administration , patient consent was sought for an observer to be present during that visit .", "patients receiving trastuzumab iv were being treated in the metastatic and adjuvant settings and were undergoing routine clinical care in the outpatient clinic of either tauranga or auckland hospital in april 2013 and may 2013 . at the start of the clinic visit", "they were given information about the time and motion study by the hcp who would be administering trastuzumab .", "under the same protocol as the sc group , patient consent to allow an observer to be present during their clinic visit was sought prior to trastuzumab administration .", "this study was an audit that does not involve the use , collection , or storage of human tissue , and therefore the health and disability ethics committees advised that the study did not require their review .", "active hcp time captured during the time and motion study related to tasks associated with administration of trastuzumab sc or iv , as detailed below .", "new zealand - specific standardized treatment observation forms were created , based on those used in a time and motion study conducted in the united kingdom.16 times for entering the bed / chair , starting trastuzumab treatment , stopping trastuzumab treatment , and exiting the bed / chair were recorded in the treatment observation form in hours and minutes ( hh : mm ) by a single trained observer .", "a stopwatch was used by the same observer to record times for active involvement in specific trastuzumab treatment - related tasks as follows : install venous catheter / line flushing ; prepare and administer premedication ( if required ) ; prepare for trastuzumab administration in the care unit ; initiate trastuzumab therapy ; monitor patient during treatment ; disconnect infusion / flush infusion line / dispose of materials ( iv only ) ; monitor patient after treatment .", "other variables recorded on the same treatment observation form were type of iv access ( iv only ) , the occurrence of any unexpected events that disrupted typical patient flow , use and type of premedication , and consumables used for each task ( including the exact product and the number of units used ) .", "trastuzumab solution for iv infusion was prepared in the hospital pharmacy , where the total time was calculated as the time from taking the prescription to leaving the prepared solution for collection .", "specific timed tasks for trastuzumab iv preparation were : prepare and label ; add trastuzumab and mix with diluent ; stand vial and check for impurities ; calculate and check dose ; add to infusion and mix ; finalize for release .", "consumables used during the preparation and delivery of both formulations were noted on the treatment observation form .", "costs for indwelling venous lines ( eg , portacath , hickman , and groshong ) were not included in cost calculations because patients who required or had these access devices in place had had them inserted for previous chemotherapy .", "unit costs for hcp and personnel time were derived from pharmaceutical management agency ( pharmac ) published costs , and were $ 45 nzd ( new zealand dollars ) per hour for a registered nurse , $ 50 nzd per hour for a hospital pharmacist , and $ 65 nzd per hour outpatient bed per chair cost.17 a simple cost - minimization model was developed to estimate cost savings associated with delivery of trastuzumab sc compared with trastuzumab iv in the outpatient setting .", "all outpatient consumables and times directly related to both routes of administration were quantified from the standardized treatment observation forms .", "average estimates for delivery of trastuzumab iv and trastuzumab sc were calculated for all hospital and pharmacy data .", "these data were used to estimate resource inputs , which were then applied to the pharmac unit cost data for hourly rates and to consumable costs to allow determination of the estimated difference in costs between the two treatment groups .", "only one trastuzumab iv formulation is available in new zealand ( herceptin , roche products ltd , auckland , new zealand ) .", "it was assumed that drug acquisition costs for trastuzumab iv and trastuzumab sc would generally be equivalent , and therefore these were not included in the analysis .", "this noninterventional , descriptive study was conducted alongside the ongoing safeher clinical trial ( nct01566721 ) , a study investigating the safety and tolerability of trastuzumab sc via handheld syringe or single - injection device . the time and motion study", "was conducted at the outpatient oncology centers at auckland city hospital ( trastuzumab iv ) and tauranga hospital ( trastuzumab sc and iv ) .", "patients receiving trastuzumab sc via handheld syringe were recruited by the health care professionals ( hcps ) who were administering trastuzumab to participants in the safeher clinical trial at tauranga hospital .", "patients with ebc were approached during a routine visit from march 2013 to may 2013 as part of their enrolment in safeher and given information about the time and motion study by a member of the clinical team .", "prior to trastuzumab administration , patient consent was sought for an observer to be present during that visit .", "patients receiving trastuzumab iv were being treated in the metastatic and adjuvant settings and were undergoing routine clinical care in the outpatient clinic of either tauranga or auckland hospital in april 2013 and may 2013 . at the start of the clinic visit they were given information about the time and motion study by the hcp who would be administering trastuzumab . under the same protocol as the sc group , patient consent to allow an observer to be present during their clinic visit was sought prior to trastuzumab administration .", "this study was an audit that does not involve the use , collection , or storage of human tissue , and therefore the health and disability ethics committees advised that the study did not require their review .", "active hcp time captured during the time and motion study related to tasks associated with administration of trastuzumab sc or iv , as detailed below .", "new zealand - specific standardized treatment observation forms were created , based on those used in a time and motion study conducted in the united kingdom.16 times for entering the bed / chair , starting trastuzumab treatment , stopping trastuzumab treatment , and exiting the bed / chair were recorded in the treatment observation form in hours and minutes ( hh : mm ) by a single trained observer .", "a stopwatch was used by the same observer to record times for active involvement in specific trastuzumab treatment - related tasks as follows : install venous catheter / line flushing ; prepare and administer premedication ( if required ) ; prepare for trastuzumab administration in the care unit ; initiate trastuzumab therapy ; monitor patient during treatment ; disconnect infusion / flush infusion line / dispose of materials ( iv only ) ; monitor patient after treatment .", "other variables recorded on the same treatment observation form were type of iv access ( iv only ) , the occurrence of any unexpected events that disrupted typical patient flow , use and type of premedication , and consumables used for each task ( including the exact product and the number of units used ) .", "trastuzumab solution for iv infusion was prepared in the hospital pharmacy , where the total time was calculated as the time from taking the prescription to leaving the prepared solution for collection .", "specific timed tasks for trastuzumab iv preparation were : prepare and label ; add trastuzumab and mix with diluent ; stand vial and check for impurities ; calculate and check dose ; add to infusion and mix ; finalize for release .", "consumables used during the preparation and delivery of both formulations were noted on the treatment observation form .", "costs for indwelling venous lines ( eg , portacath , hickman , and groshong ) were not included in cost calculations because patients who required or had these access devices in place had had them inserted for previous chemotherapy .", "unit costs for hcp and personnel time were derived from pharmaceutical management agency ( pharmac ) published costs , and were $ 45 nzd ( new zealand dollars ) per hour for a registered nurse , $ 50 nzd per hour for a hospital pharmacist , and $ 65 nzd per hour outpatient bed per chair cost.17", "a simple cost - minimization model was developed to estimate cost savings associated with delivery of trastuzumab sc compared with trastuzumab iv in the outpatient setting .", "all outpatient consumables and times directly related to both routes of administration were quantified from the standardized treatment observation forms .", "average estimates for delivery of trastuzumab iv and trastuzumab sc were calculated for all hospital and pharmacy data .", "these data were used to estimate resource inputs , which were then applied to the pharmac unit cost data for hourly rates and to consumable costs to allow determination of the estimated difference in costs between the two treatment groups .", "only one trastuzumab iv formulation is available in new zealand ( herceptin , roche products ltd , auckland , new zealand ) .", "it was assumed that drug acquisition costs for trastuzumab iv and trastuzumab sc would generally be equivalent , and therefore these were not included in the analysis .", "a total of six patients receiving trastuzumab sc as part of the ongoing safeher clinical trial , and 12 patients receiving routine clinical treatment with trastuzumab iv ( six each in tauranga and auckland ) consented to allow an observer to record data for the time and motion study during their treatment visit and were included in the final analysis .", "administration of trastuzumab sc reduced nursing time by an average of 43% and chair time by an average of 75% compared with trastuzumab iv ( table 1 ) . although the two trastuzumab formulations were prepared in different settings ( iv by the pharmacy and sc by the clinic nurse ) , the hcp time dedicated to drug preparation was also substantially lower for trastuzumab sc ( table 1 ) .", "usage of consumables was low in both the trastuzumab sc and iv groups , but some savings were associated with the use of trastuzumab sc ( table 2 ) . overall time to prepare and deliver trastuzumab therapy was markedly lower for the sc vs iv formulation ( table 1 ) .", "average hcp nurse time , chair cost , and pharmacist time savings associated with the use of trastuzumab sc injection compared with trastuzumab iv infusion were estimated to be $ 61.67 nzd per administration ( table 1 ) , and consumables cost savings were estimated as $ 15.27 nzd per administration , with the largest single item being the cost of the delivery line ( note that costs for indwelling venous lines were not included ) ( table 2 ) .", "thus , estimated cost saving achieved by switching to the sc formulation for delivery of trastuzumab in the new zealand district health board outpatient oncology clinic setting was $ 76.94 nzd per patient per cycle .", "the results of this descriptive time and motion study suggest that switching to trastuzumab sc injection from trastuzumab iv infusion in a new zealand oncology outpatient treatment setting decreases trastuzumab treatment - related tasks in the care unit , drug preparation time , chair time , and the volume and cost of consumables .", "although differences between health care systems limit the generalizability of results , these new zealand data are similar to those obtained in europe18 and the united kingdom16 showing reduced chair time and hcp resource requirement in patients treated with trastuzumab sc compared with trastuzumab iv .", "the european and uk data were obtained in patients being treated with the two different trastuzumab formulations as part of the prefher trial,19 a global , randomized , crossover preference study conducted in patients with ebc . in europe , using estimates of the typical number of ebc patients being treated in the participating centers , total estimated savings in chair time associated with switching to trastuzumab sc ranged from 4490 8-hour days annually .", "in addition , there was an associated 21%52% reduction in the time hcps spent actively involved in administering treatment.18 patients treated with trastuzumab sc in the united kingdom also spent considerably less time in the care unit ( 30.3 minutes vs 94.5 minutes ) and infusion chair ( 19.8 minutes vs 75.0 minutes ) than did those receiving trastuzumab iv.16 in addition , mean hcp time was markedly lower when the sc formulation was used ( 24.6 minutes vs 92.6 minutes for iv administration ) , and consumable costs were lower ( 2.94 vs 8.81 , respectively).16 it should be noted that the latter study included total infusion duration in hcp time , as opposed to the european study , which considered only active patient monitoring during infusion . on the basis of the results of the uk study , it was concluded that switching from trastuzumab iv to the sc formulation could substantially reduce overall treatment costs.16 these findings are similar to those documented after a switch from iv to sc rituximab in non - hodgkin lymphoma patients in the united kingdom , which was also associated with reduced active hcp and chair time and lower costs.20 in a separate trastuzumab analysis , hcps from a subset of prefher centers were asked to rate a variety of factors on their ability to impact cancer center efficiency and time required for trastuzumab administration.21 the biggest perceived impact of switching to trastuzumab sc , according to care unit respondents , was a reduced waiting list for any iv treatment at the infusion unit , followed by the potential for a more - flexible treatment schedule and the ability to treat more patients in the infusion unit.19 respondents perceived that a reduction in dosing errors , reduced drug wastage , and increased staff availability for other tasks would be the main impacts of switching from trastuzumab iv to sc.15 factors such as reduced waiting times and the flexibility to deliver treatment outside the infusion clinic offer the possibility of developing new patient care pathways defining when , where , how , and by whom trastuzumab therapy is administered .", "a switch to trastuzumab sc fits well with the new medical oncology model of care described by the new zealand ministry of health , which includes identification of patient care pathway tasks and functions that can be performed by staff other than senior medical officers.15 in this study , trastuzumab sc was prepared by a registered nurse prior to administration to the patient .", "this was considered to be the most likely way that trastuzumab sc would be prepared outside of a clinical trial situation or on a ward .", "should trastuzumab sc become available for general use within an oncology outpatient setting , preparation of the treatment dose may take place in the pharmacy or in the outpatient clinic depending on the preference or protocol of the individual hospitals .", "however , regardless of which hcp prepares trastuzumab sc prior to administration , it is reasonable to assume that the time savings achieved would be similar to those observed during this study .", "although new zealand time and motion data from the current study were consistent with those obtained in other trials , it should be noted that these preliminary assessments of the impact of switching from trastuzumab iv to the sc formulation are likely to underestimate the potential time and cost savings .", "firstly , the costs of indwelling iv catheter insertion and maintenance , and of managing complications , were not included in the analysis .", "vascular access devices are one of the most common causes of health care - associated infections , including sepsis.22 the rate of sepsis after implantation of a venous access system for chemotherapy in a new zealand population was 11%.7 catheter - related sepsis indirectly affects outcomes of chemotherapy by compromising dose intensity,23 is associated with significant morbidity and increased health care costs,2427 and is fatal in approximately 15% of patients.22 as a result , cost savings associated with switching to trastuzumab sc could be greater than those reported here . using our estimate based on costs associated with hcp time and consumables only , multiplying the cost saving per cycle by the total number of treatment cycles given ( 6,752 ) to all the patients with ebc and mbc treated with trastuzumab in new zealand in 2012 (", "the most - recent set of complete pharmac special authority data ; n=460 ) , annual cost savings to the new zealand health care system associated with a switch from trastuzumab iv infusion to trastuzumab sc injection could be more than half a million dollars ( $ 519,499 nzd ) .", "the calculations made based on the results of the current study assume that the total cost of trastuzumab therapy to the new zealand healthcare system would be equivalent whether the sc or iv formulation was used .", "these calculations could be influenced by the introduction of a biosimilar iv trastuzumab , but this is not expected to occur in new zealand until at least the first half of 2019 ( on the basis of clinical trial data timelines on clinicaltrials.gov and estimated regulatory review processes ) . on a patient - by - patient basis , the cost of trastuzumab therapy may vary , given that the dosage for trastuzumab iv is calculated based on patient bodyweight and trastuzumab sc is given as a single fixed dose . nevertheless , on a population basis ,", "overall drug costs are likely to be similar , although exact figures for the new zealand setting remain to be determined , on the basis of negotiations around access and funding .", "another reason why the current data may underestimate the resource - use impact of switching from trastuzumab iv to the sc formulation is that only in - hospital direct medical costs were considered .", "this means that costs incurred by the patient either directly ( eg , transport , parking ) or indirectly ( eg , time and loss of productivity for both patients and their caregivers ) have not been quantified .", "patient preference , well - being , and quality of life were also not assessed in this study , but the prefher trial found that patients overwhelmingly preferred trastuzumab sc over the iv formulation.19 by far the most common reason cited by patients for this preference was that receiving trastuzumab as a sc injection saved time .", "other reasons for preferring trastuzumab sc were less pain or discomfort and fewer side effects , the avoidance of problems associated with iv treatment ( ie , venous access ) , and convenience.19 these findings suggest that switching to trastuzumab sc not only offers financial and logistical advantages to the health care system but is also perceived as a more - attractive treatment option by patients .", "this study had a number of limitations , including the small sample size ( particularly in the trastuzumab sc treatment group ) and the administration of trastuzumab sc in only one of the two study centers .", "however , trastuzumab sc is not yet funded in new zealand and is therefore not administered in routine clinical practice . as a result ,", "the only available data on administration of trastuzumab sc was from consenting patients who were receiving trastuzumab sc at the single new zealand center participating in the ongoing safeher trial .", "for both the sc and iv groups , it was not possible to report patient demographic data because the patient consent obtained applied only to observation and recording of tasks and times associated with trastuzumab administration and not access to patient medical files .", "for the iv group , it is likely that the patients treated were a relatively heterogeneous group because no selection was undertaken and any patient receiving trastuzumab was eligible ( eg , ebc or mbc ) . patients in the sc group had only ebc and therefore it is possible that disease severity was a confounding factor because patients who were more unwell might have needed more hcp involvement . despite these issues , the range of values obtained was relatively narrow , and expected time savings were comparable to those from international studies with broadly similar study design and objectives .", "it is possible that the results of this study can not be generalized to centers other than the two that participated in this study .", "however , there are only six regional oncology centers in new zealand , and 40% of new zealand s cancer patients are treated at auckland city hospital , which contributed patients to this study .", "it should also be noted that in new zealand there is no regional variation in drug costs because these are mandated nationally by pharmac , and unit costs for hcp and personnel time were derived from pharmac - specified national values.17 therefore , it is likely that the findings will be relevant across the new zealand health care setting .", "the new sc formulation of trastuzumab represents a clinical opportunity to improve the efficiency of her2-positive breast cancer treatment delivery by providing time and cost savings and the potential to modify pathways of patient care .", "this contributes to new zealand ministry of health goals for increasing oncology treatment capacity and allowing treatment to be administered away from regional oncology centers.15 it should be noted that the time and equipment savings associated with a switch to trastuzumab sc are not a true financial saving but instead represent the value of the best available alternative use of a resource ( opportunity cost ) .", "fewer health care staff and infusion chairs would be needed to treat the same number of patients with trastuzumab sc , and this resource is then available to be redeployed in the treatment of other patients .", "thus , at least in the short term , the main result of switching from trastuzumab iv to trastuzumab sc would be to ease pressure at capacity - constrained treatment centers .", "data from this new zealand time and motion study provide an indication that switching from iv to sc administration of trastuzumab has beneficial effects on resource use , including hcp time and consumables needed to administer treatment .", "these reductions contribute to health care cost efficiencies , could help to address current capacity issues at new zealand district health boards , and have the potential to influence how and where her2-positive breast cancer treatment is delivered in the future ." ]

[ "<S> backgroundin new zealand , trastuzumab is standard therapy for human epidermal growth factor receptor-2 ( her2)-positive early and metastatic breast cancer . given the requirement for ongoing adjuvant or maintenance treatment and intravenous ( iv ) delivery , such a regimen consumes considerable health care resources . </S>", "<S> the development of a subcutaneous ( sc ) trastuzumab formulation with a short administration time offers the potential to reduce hospital expenditure . </S>", "<S> the aim of this study was to determine medical resource utilization associated with administration of trastuzumab sc injection via handheld syringe vs trastuzumab iv infusion in patients with her2-positive breast cancer in new zealand.methodsthis noninterventional , descriptive study was conducted at the outpatient oncology centers at auckland city and tauranga hospitals . trained </S>", "<S> observers recorded times associated with health care professional ( hcp ) tasks and consumables use associated with preparation and administration of trastuzumab iv or sc in women with early or metastatic breast cancer . </S>", "<S> the cost for each formulation was calculated as the mean cost of hcp time ( based on pharmaceutical management agency hourly rates ) plus the mean cost of consumables used.resultsuse of trastuzumab sc vs iv reduced mean chair time by 36.95 minutes and total nurse time by 6.12 minutes ; there was a 20.45-minute reduction in pharmacist time when the sc formulation was used . after adding consumable costs , the overall estimated saving with trastuzumab sc vs </S>", "<S> iv was $ 76.94 ( new zealand dollars ) per patient per cycle.conclusionscompared with trastuzumab iv infusion , administration of trastuzumab via sc injection reduced time spent in the clinic and decreased hcp resources and consumables needed to administer treatment . </S>", "<S> these reductions could contribute to a decrease in health care costs and an improvement in the efficiency of her2-positive breast cancer treatment delivery . </S>" ]

[ "Background", "Methods", "Study design", "Subjects", "Data collection", "Cost model", "Results", "Discussion", "Conclusion" ]

[ [ "breast cancer is the second most common cancer in new zealand and the third leading cause of cancer death.1 in 2011 , the most - recent full year for which statistics are available , 2,867 new cases of breast cancer and 636 deaths were reported.2 approximately 18% of breast cancers in new zealand are human epidermal growth factor receptor-2 ( her2)-positive.3 trastuzumab is standard treatment for her2-positive early breast cancer ( ebc ) and metastatic breast cancer ( mbc ) , being administered as an intravenous ( iv ) infusion , in part with chemotherapy , as an every-3-weeks regimen for 12 months in ebc ( 17 cycles ) , or until disease progression in mbc .", "trastuzumab iv needs to be reconstituted into solution for infusion , with the dose calculated on the basis of patient bodyweight.4 iv trastuzumab treatment starts with a loading dose infused over 90 minutes , followed by subsequent maintenance doses infused over 30 minutes.4 given the requirement for up to 12 months of adjuvant or maintenance treatment , such a regimen consumes considerable health care resources , including drug preparation and administration , clinic and chair time , and physician time dedicated to patient interaction.5 furthermore , insertion of an indwelling venous catheter is common in patients requiring prolonged iv infusion therapy , adding the costs of theatre and anesthetist time ; complications such as infection or thrombosis6,7 may also be associated with additional costs in some cases .", "a subcutaneous ( sc ) formulation of trastuzumab was developed to allow drug administration over a shorter time period , with the goal of improving convenience and compliance , particularly during long - term therapy .", "the ability to do this was facilitated by the fact that trastuzumab has a wide therapeutic window8 with no known dose - limiting toxicities.9 sc administration of volumes > 1 ml has been limited by the structure and physiology of the sc layer , which contains a matrix of hyaluronan and collagen fibers.8 to overcome this issue , the trastuzumab sc formulation includes recombinant human hyaluronidase as a novel excipient.10 hyaluronidase hydrolyses hyaluronan , decreases resistance of the interstitial matrix11,12 and degrades the extracellular matrix .", "this allows monoclonal antibodies to diffuse through the channels,12 thereby facilitating sc administration of larger volumes of fluid and improving delivery of sc drugs to the systemic circulation .", "the effects of recombinant human hyaluronidase are temporary and reversible within 24 hours.12 trastuzumab sc can be administered over approximately 5 minutes , a much shorter period of time than that required for the trastuzumab iv loading and maintenance doses.4,10 in addition , trastuzumab sc is given as a fixed dose that does not require adjustment based on bodyweight.10 pharmacokinetic modeling , based on data from an open - label phase i / ib study,13 determined that a 600 mg fixed dose of trastuzumab sc provided drug exposure that was comparable to the iv trastuzumab regimen , without the need for a loading dose .", "this dose of trastuzumab sc was subsequently tested in the phase iii hannah study , which showed that trastuzumab sc was noninferior to trastuzumab iv with respect to efficacy ( pathological complete response rate ) and had an equivalent tolerability profile.14 one of the key principles guiding the new zealand ministry of health medical oncology national implementation plan15 is to effectively , equitably , and sustainably meet the future demand for medical oncology services , given the significant workforce and resource constraints that exist .", "a second aim is to maintain high quality of care and improve the quality of life for people with cancer .", "it also offers the potential to move treatment delivery to the outpatient setting and away from infusion services that are facing challenges due to increased demand for complex treatments.15 the aim of this study was to determine medical resource utilization associated with administration of trastuzumab sc injection compared with trastuzumab iv infusion in patients with her2-positive breast cancer in new zealand ." ], [ "this noninterventional , descriptive study was conducted alongside the ongoing safeher clinical trial ( nct01566721 ) , a study investigating the safety and tolerability of trastuzumab sc via handheld syringe or single - injection device . the time and motion study", "was conducted at the outpatient oncology centers at auckland city hospital ( trastuzumab iv ) and tauranga hospital ( trastuzumab sc and iv ) .", "patients receiving trastuzumab sc via handheld syringe were recruited by the health care professionals ( hcps ) who were administering trastuzumab to participants in the safeher clinical trial at tauranga hospital .", "patients with ebc were approached during a routine visit from march 2013 to may 2013 as part of their enrolment in safeher and given information about the time and motion study by a member of the clinical team .", "prior to trastuzumab administration , patient consent was sought for an observer to be present during that visit .", "patients receiving trastuzumab iv were being treated in the metastatic and adjuvant settings and were undergoing routine clinical care in the outpatient clinic of either tauranga or auckland hospital in april 2013 and may 2013 . at the start of the clinic visit", "they were given information about the time and motion study by the hcp who would be administering trastuzumab .", "under the same protocol as the sc group , patient consent to allow an observer to be present during their clinic visit was sought prior to trastuzumab administration .", "this study was an audit that does not involve the use , collection , or storage of human tissue , and therefore the health and disability ethics committees advised that the study did not require their review .", "active hcp time captured during the time and motion study related to tasks associated with administration of trastuzumab sc or iv , as detailed below .", "new zealand - specific standardized treatment observation forms were created , based on those used in a time and motion study conducted in the united kingdom.16 times for entering the bed / chair , starting trastuzumab treatment , stopping trastuzumab treatment , and exiting the bed / chair were recorded in the treatment observation form in hours and minutes ( hh : mm ) by a single trained observer .", "a stopwatch was used by the same observer to record times for active involvement in specific trastuzumab treatment - related tasks as follows : install venous catheter / line flushing ; prepare and administer premedication ( if required ) ; prepare for trastuzumab administration in the care unit ; initiate trastuzumab therapy ; monitor patient during treatment ; disconnect infusion / flush infusion line / dispose of materials ( iv only ) ; monitor patient after treatment .", "other variables recorded on the same treatment observation form were type of iv access ( iv only ) , the occurrence of any unexpected events that disrupted typical patient flow , use and type of premedication , and consumables used for each task ( including the exact product and the number of units used ) .", "trastuzumab solution for iv infusion was prepared in the hospital pharmacy , where the total time was calculated as the time from taking the prescription to leaving the prepared solution for collection .", "specific timed tasks for trastuzumab iv preparation were : prepare and label ; add trastuzumab and mix with diluent ; stand vial and check for impurities ; calculate and check dose ; add to infusion and mix ; finalize for release .", "consumables used during the preparation and delivery of both formulations were noted on the treatment observation form .", "costs for indwelling venous lines ( eg , portacath , hickman , and groshong ) were not included in cost calculations because patients who required or had these access devices in place had had them inserted for previous chemotherapy .", "unit costs for hcp and personnel time were derived from pharmaceutical management agency ( pharmac ) published costs , and were $ 45 nzd ( new zealand dollars ) per hour for a registered nurse , $ 50 nzd per hour for a hospital pharmacist , and $ 65 nzd per hour outpatient bed per chair cost.17 a simple cost - minimization model was developed to estimate cost savings associated with delivery of trastuzumab sc compared with trastuzumab iv in the outpatient setting .", "all outpatient consumables and times directly related to both routes of administration were quantified from the standardized treatment observation forms .", "average estimates for delivery of trastuzumab iv and trastuzumab sc were calculated for all hospital and pharmacy data .", "these data were used to estimate resource inputs , which were then applied to the pharmac unit cost data for hourly rates and to consumable costs to allow determination of the estimated difference in costs between the two treatment groups .", "only one trastuzumab iv formulation is available in new zealand ( herceptin , roche products ltd , auckland , new zealand ) .", "it was assumed that drug acquisition costs for trastuzumab iv and trastuzumab sc would generally be equivalent , and therefore these were not included in the analysis ." ], [ "this noninterventional , descriptive study was conducted alongside the ongoing safeher clinical trial ( nct01566721 ) , a study investigating the safety and tolerability of trastuzumab sc via handheld syringe or single - injection device . the time and motion study", "was conducted at the outpatient oncology centers at auckland city hospital ( trastuzumab iv ) and tauranga hospital ( trastuzumab sc and iv ) ." ], [ "patients receiving trastuzumab sc via handheld syringe were recruited by the health care professionals ( hcps ) who were administering trastuzumab to participants in the safeher clinical trial at tauranga hospital .", "patients with ebc were approached during a routine visit from march 2013 to may 2013 as part of their enrolment in safeher and given information about the time and motion study by a member of the clinical team .", "prior to trastuzumab administration , patient consent was sought for an observer to be present during that visit .", "patients receiving trastuzumab iv were being treated in the metastatic and adjuvant settings and were undergoing routine clinical care in the outpatient clinic of either tauranga or auckland hospital in april 2013 and may 2013 . at the start of the clinic visit they were given information about the time and motion study by the hcp who would be administering trastuzumab . under the same protocol as the sc group , patient consent to allow an observer to be present during their clinic visit was sought prior to trastuzumab administration .", "this study was an audit that does not involve the use , collection , or storage of human tissue , and therefore the health and disability ethics committees advised that the study did not require their review ." ], [ "active hcp time captured during the time and motion study related to tasks associated with administration of trastuzumab sc or iv , as detailed below .", "new zealand - specific standardized treatment observation forms were created , based on those used in a time and motion study conducted in the united kingdom.16 times for entering the bed / chair , starting trastuzumab treatment , stopping trastuzumab treatment , and exiting the bed / chair were recorded in the treatment observation form in hours and minutes ( hh : mm ) by a single trained observer .", "a stopwatch was used by the same observer to record times for active involvement in specific trastuzumab treatment - related tasks as follows : install venous catheter / line flushing ; prepare and administer premedication ( if required ) ; prepare for trastuzumab administration in the care unit ; initiate trastuzumab therapy ; monitor patient during treatment ; disconnect infusion / flush infusion line / dispose of materials ( iv only ) ; monitor patient after treatment .", "other variables recorded on the same treatment observation form were type of iv access ( iv only ) , the occurrence of any unexpected events that disrupted typical patient flow , use and type of premedication , and consumables used for each task ( including the exact product and the number of units used ) .", "trastuzumab solution for iv infusion was prepared in the hospital pharmacy , where the total time was calculated as the time from taking the prescription to leaving the prepared solution for collection .", "specific timed tasks for trastuzumab iv preparation were : prepare and label ; add trastuzumab and mix with diluent ; stand vial and check for impurities ; calculate and check dose ; add to infusion and mix ; finalize for release .", "consumables used during the preparation and delivery of both formulations were noted on the treatment observation form .", "costs for indwelling venous lines ( eg , portacath , hickman , and groshong ) were not included in cost calculations because patients who required or had these access devices in place had had them inserted for previous chemotherapy .", "unit costs for hcp and personnel time were derived from pharmaceutical management agency ( pharmac ) published costs , and were $ 45 nzd ( new zealand dollars ) per hour for a registered nurse , $ 50 nzd per hour for a hospital pharmacist , and $ 65 nzd per hour outpatient bed per chair cost.17" ], [ "a simple cost - minimization model was developed to estimate cost savings associated with delivery of trastuzumab sc compared with trastuzumab iv in the outpatient setting .", "all outpatient consumables and times directly related to both routes of administration were quantified from the standardized treatment observation forms .", "average estimates for delivery of trastuzumab iv and trastuzumab sc were calculated for all hospital and pharmacy data .", "these data were used to estimate resource inputs , which were then applied to the pharmac unit cost data for hourly rates and to consumable costs to allow determination of the estimated difference in costs between the two treatment groups .", "only one trastuzumab iv formulation is available in new zealand ( herceptin , roche products ltd , auckland , new zealand ) .", "it was assumed that drug acquisition costs for trastuzumab iv and trastuzumab sc would generally be equivalent , and therefore these were not included in the analysis ." ], [ "a total of six patients receiving trastuzumab sc as part of the ongoing safeher clinical trial , and 12 patients receiving routine clinical treatment with trastuzumab iv ( six each in tauranga and auckland ) consented to allow an observer to record data for the time and motion study during their treatment visit and were included in the final analysis .", "administration of trastuzumab sc reduced nursing time by an average of 43% and chair time by an average of 75% compared with trastuzumab iv ( table 1 ) . although the two trastuzumab formulations were prepared in different settings ( iv by the pharmacy and sc by the clinic nurse ) , the hcp time dedicated to drug preparation was also substantially lower for trastuzumab sc ( table 1 ) .", "usage of consumables was low in both the trastuzumab sc and iv groups , but some savings were associated with the use of trastuzumab sc ( table 2 ) . overall time to prepare and deliver trastuzumab therapy was markedly lower for the sc vs iv formulation ( table 1 ) .", "average hcp nurse time , chair cost , and pharmacist time savings associated with the use of trastuzumab sc injection compared with trastuzumab iv infusion were estimated to be $ 61.67 nzd per administration ( table 1 ) , and consumables cost savings were estimated as $ 15.27 nzd per administration , with the largest single item being the cost of the delivery line ( note that costs for indwelling venous lines were not included ) ( table 2 ) .", "thus , estimated cost saving achieved by switching to the sc formulation for delivery of trastuzumab in the new zealand district health board outpatient oncology clinic setting was $ 76.94 nzd per patient per cycle ." ], [ "the results of this descriptive time and motion study suggest that switching to trastuzumab sc injection from trastuzumab iv infusion in a new zealand oncology outpatient treatment setting decreases trastuzumab treatment - related tasks in the care unit , drug preparation time , chair time , and the volume and cost of consumables .", "although differences between health care systems limit the generalizability of results , these new zealand data are similar to those obtained in europe18 and the united kingdom16 showing reduced chair time and hcp resource requirement in patients treated with trastuzumab sc compared with trastuzumab iv .", "the european and uk data were obtained in patients being treated with the two different trastuzumab formulations as part of the prefher trial,19 a global , randomized , crossover preference study conducted in patients with ebc . in europe , using estimates of the typical number of ebc patients being treated in the participating centers , total estimated savings in chair time associated with switching to trastuzumab sc ranged from 4490 8-hour days annually .", "in addition , there was an associated 21%52% reduction in the time hcps spent actively involved in administering treatment.18 patients treated with trastuzumab sc in the united kingdom also spent considerably less time in the care unit ( 30.3 minutes vs 94.5 minutes ) and infusion chair ( 19.8 minutes vs 75.0 minutes ) than did those receiving trastuzumab iv.16 in addition , mean hcp time was markedly lower when the sc formulation was used ( 24.6 minutes vs 92.6 minutes for iv administration ) , and consumable costs were lower ( 2.94 vs 8.81 , respectively).16 it should be noted that the latter study included total infusion duration in hcp time , as opposed to the european study , which considered only active patient monitoring during infusion . on the basis of the results of the uk study , it was concluded that switching from trastuzumab iv to the sc formulation could substantially reduce overall treatment costs.16 these findings are similar to those documented after a switch from iv to sc rituximab in non - hodgkin lymphoma patients in the united kingdom , which was also associated with reduced active hcp and chair time and lower costs.20 in a separate trastuzumab analysis , hcps from a subset of prefher centers were asked to rate a variety of factors on their ability to impact cancer center efficiency and time required for trastuzumab administration.21 the biggest perceived impact of switching to trastuzumab sc , according to care unit respondents , was a reduced waiting list for any iv treatment at the infusion unit , followed by the potential for a more - flexible treatment schedule and the ability to treat more patients in the infusion unit.19 respondents perceived that a reduction in dosing errors , reduced drug wastage , and increased staff availability for other tasks would be the main impacts of switching from trastuzumab iv to sc.15 factors such as reduced waiting times and the flexibility to deliver treatment outside the infusion clinic offer the possibility of developing new patient care pathways defining when , where , how , and by whom trastuzumab therapy is administered .", "a switch to trastuzumab sc fits well with the new medical oncology model of care described by the new zealand ministry of health , which includes identification of patient care pathway tasks and functions that can be performed by staff other than senior medical officers.15 in this study , trastuzumab sc was prepared by a registered nurse prior to administration to the patient .", "this was considered to be the most likely way that trastuzumab sc would be prepared outside of a clinical trial situation or on a ward .", "should trastuzumab sc become available for general use within an oncology outpatient setting , preparation of the treatment dose may take place in the pharmacy or in the outpatient clinic depending on the preference or protocol of the individual hospitals .", "however , regardless of which hcp prepares trastuzumab sc prior to administration , it is reasonable to assume that the time savings achieved would be similar to those observed during this study .", "although new zealand time and motion data from the current study were consistent with those obtained in other trials , it should be noted that these preliminary assessments of the impact of switching from trastuzumab iv to the sc formulation are likely to underestimate the potential time and cost savings .", "firstly , the costs of indwelling iv catheter insertion and maintenance , and of managing complications , were not included in the analysis .", "vascular access devices are one of the most common causes of health care - associated infections , including sepsis.22 the rate of sepsis after implantation of a venous access system for chemotherapy in a new zealand population was 11%.7 catheter - related sepsis indirectly affects outcomes of chemotherapy by compromising dose intensity,23 is associated with significant morbidity and increased health care costs,2427 and is fatal in approximately 15% of patients.22 as a result , cost savings associated with switching to trastuzumab sc could be greater than those reported here . using our estimate based on costs associated with hcp time and consumables only , multiplying the cost saving per cycle by the total number of treatment cycles given ( 6,752 ) to all the patients with ebc and mbc treated with trastuzumab in new zealand in 2012 (", "the most - recent set of complete pharmac special authority data ; n=460 ) , annual cost savings to the new zealand health care system associated with a switch from trastuzumab iv infusion to trastuzumab sc injection could be more than half a million dollars ( $ 519,499 nzd ) .", "the calculations made based on the results of the current study assume that the total cost of trastuzumab therapy to the new zealand healthcare system would be equivalent whether the sc or iv formulation was used .", "these calculations could be influenced by the introduction of a biosimilar iv trastuzumab , but this is not expected to occur in new zealand until at least the first half of 2019 ( on the basis of clinical trial data timelines on clinicaltrials.gov and estimated regulatory review processes ) . on a patient - by - patient basis , the cost of trastuzumab therapy may vary , given that the dosage for trastuzumab iv is calculated based on patient bodyweight and trastuzumab sc is given as a single fixed dose . nevertheless , on a population basis ,", "overall drug costs are likely to be similar , although exact figures for the new zealand setting remain to be determined , on the basis of negotiations around access and funding .", "another reason why the current data may underestimate the resource - use impact of switching from trastuzumab iv to the sc formulation is that only in - hospital direct medical costs were considered .", "this means that costs incurred by the patient either directly ( eg , transport , parking ) or indirectly ( eg , time and loss of productivity for both patients and their caregivers ) have not been quantified .", "patient preference , well - being , and quality of life were also not assessed in this study , but the prefher trial found that patients overwhelmingly preferred trastuzumab sc over the iv formulation.19 by far the most common reason cited by patients for this preference was that receiving trastuzumab as a sc injection saved time .", "other reasons for preferring trastuzumab sc were less pain or discomfort and fewer side effects , the avoidance of problems associated with iv treatment ( ie , venous access ) , and convenience.19 these findings suggest that switching to trastuzumab sc not only offers financial and logistical advantages to the health care system but is also perceived as a more - attractive treatment option by patients .", "this study had a number of limitations , including the small sample size ( particularly in the trastuzumab sc treatment group ) and the administration of trastuzumab sc in only one of the two study centers .", "however , trastuzumab sc is not yet funded in new zealand and is therefore not administered in routine clinical practice . as a result ,", "the only available data on administration of trastuzumab sc was from consenting patients who were receiving trastuzumab sc at the single new zealand center participating in the ongoing safeher trial .", "for both the sc and iv groups , it was not possible to report patient demographic data because the patient consent obtained applied only to observation and recording of tasks and times associated with trastuzumab administration and not access to patient medical files .", "for the iv group , it is likely that the patients treated were a relatively heterogeneous group because no selection was undertaken and any patient receiving trastuzumab was eligible ( eg , ebc or mbc ) . patients in the sc group had only ebc and therefore it is possible that disease severity was a confounding factor because patients who were more unwell might have needed more hcp involvement . despite these issues , the range of values obtained was relatively narrow , and expected time savings were comparable to those from international studies with broadly similar study design and objectives .", "it is possible that the results of this study can not be generalized to centers other than the two that participated in this study .", "however , there are only six regional oncology centers in new zealand , and 40% of new zealand s cancer patients are treated at auckland city hospital , which contributed patients to this study .", "it should also be noted that in new zealand there is no regional variation in drug costs because these are mandated nationally by pharmac , and unit costs for hcp and personnel time were derived from pharmac - specified national values.17 therefore , it is likely that the findings will be relevant across the new zealand health care setting .", "the new sc formulation of trastuzumab represents a clinical opportunity to improve the efficiency of her2-positive breast cancer treatment delivery by providing time and cost savings and the potential to modify pathways of patient care .", "this contributes to new zealand ministry of health goals for increasing oncology treatment capacity and allowing treatment to be administered away from regional oncology centers.15 it should be noted that the time and equipment savings associated with a switch to trastuzumab sc are not a true financial saving but instead represent the value of the best available alternative use of a resource ( opportunity cost ) .", "fewer health care staff and infusion chairs would be needed to treat the same number of patients with trastuzumab sc , and this resource is then available to be redeployed in the treatment of other patients .", "thus , at least in the short term , the main result of switching from trastuzumab iv to trastuzumab sc would be to ease pressure at capacity - constrained treatment centers ." ], [ "data from this new zealand time and motion study provide an indication that switching from iv to sc administration of trastuzumab has beneficial effects on resource use , including hcp time and consumables needed to administer treatment .", "these reductions contribute to health care cost efficiencies , could help to address current capacity issues at new zealand district health boards , and have the potential to influence how and where her2-positive breast cancer treatment is delivered in the future ." ] ]

[ "painful uterine contractions cause maternal hyperventilation and increased catecholamine concentration resulting in maternal and foetal hypoxaemia .", "an effective analgesia takes away the disadvantages and results in better maternal and foetal outcome .", "hence , the control of pain should form an integral part of labour management at any level .", "an ideal labour analgesic technique should provide adequate and satisfactory analgesia without any motor blockade or adverse maternal and foetal effects . among the variety of labour analgesia techniques ranging from parenteral and inhalational agents ,", "combined spinal epidural ( cse ) analgesia is increasingly used to provide pain relief during labour .", "it combines the advantage of rapid onset of spinal analgesia and the flexibility of the epidural catheter .", "dose adjustments and frequency of administration of the drug according to parturients requirement is possible with the epidural route which can also be extended to provide anaesthesia for caesarean delivery if need arises .", "addition of opioids to local anaesthetics reduces its requirement by synergistic effect of opioid receptors in the spinal cord .", "although fentanyl and sufentanil are the most commonly used opioids for epidural labour analgesia , studies comparing cse technique employing sufentanil and fentanyl are very few and give conflicting results . at the assumed equipotent doses of sufentanil to fentanyl", "ratio of 6:1 , there is some evidence that sufentanil is clinically superior to fentanyl as an adjunct to bupivacaine in labour epidurals .", "hence , we decided to compare the efficacy , quality and duration of analgesia and maternal and foetal effects of adding the two highly lipid - soluble opioids , sufentanil and fentanyl to low - concentration bupivacaine for cse labour analgesia .", "a randomized , prospective , single - blinded study was carried out on 60 parturients over a period of 6 months after obtaining clearance from the institutional ethical committee .", "parturients belonging to asa grade i and ii with singleton , term pregnancy in spontaneous labour with cervical dilatation of less than 4 cm , with normal foetal heart tracings requesting labour analgesia , were included in the study .", "parturients with medical disorders , obstetric complications , malpresentation of foetus and those with contraindication for regional analgesia were excluded from the study . after obtaining informed written consent , parturients were randomly allocated by a computer - generated table of random numbers by a person blinded to the procedure to avoid selection bias into two groups of 30 each as group s ( n=30 ) and group f ( n=30 ) . before institution of cse , parturients were administered 1000 ml ringer lactate solution and monitors were connected and baseline readings recorded .", "patients were placed in the left lateral position and , under aseptic precautions , cse analgesia was administered through a midline approach ( l2-l3 or l3-l4 level ) .", "lumbar epidural space was identified with an 18 g tuohy needle using loss of resistance to saline technique .", "after negative aspiration for blood and cerebrospinal fluid , the epidural catheter was threaded on through the tuohy needle and the needle was carefully withdrawn .", "spinal analgesia was instituted using a 25 g whitacre needle one space below . on identification of subarachnoid space , 1 ml of drug containing 0.5 ml 0.5% bupivacaine heavy ( 2.5 mg ) and 0.5 ml sufentanil ( 5 mcg ) for group s or 0.5 ml fentanyl ( 25 mcg ) for group f", "the parturient was turned supine immediately after subarachnoid block and the uterus was displaced to the left using a wedge .", "pulse rate , blood pressure , respiratory rate and spo2 were monitored at 0 , 5 , 10 , 15 and 30 min and thereafter every 30 min until the women delivered .", "motor block was assessed by modified bromage scale and the level of sensory block was recorded .", "pain relief was assessed using visual analogue scores ( vas ) ( 0 = no pain , 10 = worst possible pain experienced ) . when the parturient experienced pain equivalent to a vas score of 5 , epidural top - up was administered . the time interval between initiation of intrathecal analgesia and patient developing pain equivalent to vas", "parturients of group s received 10 ml of 0.125% bupivacaine with 0.30 mcg / ml of sufentanil and group f received 10 ml of 0.125% bupivacaine with fentanyl 2.5 mcg / ml through an epidural catheter .", "time of onset of epidural analgesia , total number of top - ups and duration between successive top - ups were recorded .", "the mode of delivery in the form of normal vaginal delivery , instrumental delivery or caesarean section was observed .", "side - effects such as pruritis ( rated as none , minimal , moderate and severe ) , hypotension ( fall of > 20% from baseline systolic reading ) , motor blockade of limbs , shivering , sedation ( categorized as none , mild , moderate and severe for awake , drowsy , sleepy and unarousable ) and bradycardia ( heart rate<60 ) were noted . neonatal outcome in the form of apgar scores at 1 min and 5 min and need for intensive care were noted .", "all the data were recorded by a resident blinded to the drug administered during conduct of labour analgesia throughout the study period .", "the parturient was monitored for 2 h following delivery and the epidural catheter was removed .", "they were questioned after 24 h about their views on the procedure and the satisfaction .", "enquiry about the symptoms related to post - dural puncture headache ( pdph ) was made .", "sample size of 60 with 30 parturients in each group was determined with power of study of 80% .", "sample size of 60 with 30 parturients in each group was determined with power of study of 80% .", "there were no significant differences between the groups with respect to maternal demographic characteristics , parity , cervical dilation at initiation of labour analgesia , duration of labour and delivery characteristics [ table 1 ] .", "maternal demographic characteristics the mode of delivery data suggests no significant difference between the groups regarding incidence of caesarean , instrumental or vaginal deliveries [ table 2 ] .", "the mean duration of intrathecal analgesia was 109.7 ( 6.3 ) min for group s and 73.63 ( 15.76 ) min for group f. the mean time of onset of epidural analgesia was 2.48 ( 0.28 ) min and 2.55 ( 0.77 ) min for group s and group f , respectively .", "the mean duration between epidural top - ups was 90.51 ( 15.8 ) min for group s and 83.54 ( 24.64 ) min for group f [ table 3 ] .", "the number of additional bolus supplementary analgesic top - ups were comparatively less in the sufentanil group .", "eight women among group s and nine among group f experienced transient initial motor blockade ( bromage score 1 ) .", "none of the women had any motor blockade at the end of 1 h. the highest level of sensory block attained was t6 in 63.33% , t8 in 36.66% among group s and 66.66% and 33.33% among group f , respectively .", "onset and duration of analgesia the pain scores during the first and second stages of labour were comparable without any significant difference between the groups using vas scores ( vas : 0 = no pain , 10 = worst pain experienced ) ; 93.3% and 90.0% women had vas scores less than 1 , indicating excellent pain relief by the cse technique in both groups [ table 4 ] .", "mean visual analogue scale scores no significant alterations in intrapartum vital parameters like spo2 , mean arterial pressure , pulse rate and fhr were noted between the groups .", "the incidence of side - effects did not differ between the groups [ table 5 ] .", "pruritis , shivering , nausea and vomiting , sedation , bradycardia and hypotension were reported and treated accordingly .", "the most common side - effect was pruritis , which was reported among 20% and 30% of the women belonging to groups s and f , respectively .", "nine parturients ( four in group s and five in group f ; p = ns ) experienced mild sedation during the study period .", "neonatal outcome was assessed based on apgar scores at the 1 and the 5 minutes , which did not differ between the groups .", "maternal satisfaction was assessed based on subjective assessment as excellent , good , satisfactory and poor .", "93.33% of the women of group s and 90% of the women of group f opined labour analgesia as excellent .", "highly lipid - soluble synthetic opioids such as sufentanil and fentanyl are being increasingly used along with very low concentrations of local anaesthetic agents such as bupivacaine ( 0.06250.125% ) and ropivacaine ( 0.20.25% ) to provide excellent relief of pain during labour . unlike hydrophilic opiates ( morphine ) or intermediate lipid - soluble opioids ( meperidine ) , lipophillic opioids do not spread rostrally in cerebrospinal fluid to any great extent , and they tend to have fairly segmental analgesic profiles .", "quality of analgesia and overall satisfaction scores were found to be comparable in both groups in our study . in a dose response study ,", "the analgesic potency of epidural sufentanil was reported to be approximately five - times that of fentanyl when it was administered as the sole analgesic after lidocaine anaesthesia for caesarean delivery .", "addition of bupivacaine to intrathecal opioid prolongs duration of labour analgesia compared with either drug used alone .", "evidence suggests that bupivacaine potentiates the binding of morphine to opioid receptors , especially the highly dense kappa receptors , as the result of an associated conformational change in opioid receptors .", "we observed that duration of analgesia provided by intrathecal sufentanil and bupivacaine was 109.706.37 min as compared with 73.6315.76 min by group f. this difference was statistically significant .", "these are similar to previously reported results using sufentanil : 11426 min and 99 min .", "this prolonged duration of analgesia with sufentanil could be attributed to the known superiority of sufentanil over fentanyl in terms of potency .", "similarly , the number of supplementary bupivacaine top - ups was comparatively less in the sufentanil group ( group s 1.220.75 and group f 2.060.081 ) .", "addition of bupivacaine 2.5 mg and sufentanil 10 g for labour analgesia showed that the combination , although effective , was associated with a higher incidence of hypotension and impairment of muscle power of the lower limbs , although mild in some parturients .", "another investigation by halving the total amount of intrathecal sufentanil and bupivacaine reduced the incidence of side - effects .", "however , this reduced dose regimen was associated with slower onset and shorter duration of labour analgesia . as 10 mcg sufentanil is associated with severe hypotension and 2.5 mcg sufentanil produces analgesia of shorter duration and slower onset , in our study , we added 5 mcg sufentanil to bupivacaine 2.5 mg intrathecal .", "cse has an added advantage of initiating labour analgesia even at the late stages of labour .", "we found that analgesia through intrathecal route resulted in immediate and predictable onset of analgesia enhancing the parturient 's cooperation for the insertion of epidural catheter .", "the duration of first and second stage of labour and the mode of delivery between the groups did not differ significantly .", "the incidence of caesarean delivery was 20% and 16.66% between group s and f , respectively .", "higher concentration of bupivacaine ( 0.25% ) was used in the past , which resulted in a fairly higher incidence of motor block causing pelvic muscle relaxation , foetal malposition and maternal inability to push and a higher incidence of instrumental delivery .", "incidence of motor blockade in our study groups was very minimal and was of bromage scale 1 , which did not persist beyond the first hour .", "the pain relief during labour was assessed by vas scoring and the pain scores were comparable between the groups .", "it was graded as excellent in 93.33% parturients of group s and 90.0% parturients of group f , as in previous studies .", "the dose of opioids administered did not adversely affect the apgar scores or cause respiratory depression among newborns in our study .", "pruritus was the most common side - effect between the two groups , which was not of a serious nature .", "the incidence of sedation and nausea was similar in the two groups , with no parturient in either group requiring any intervention .", "continuous infusion of a dilute mixture of local anaesthetics and opioids offers the advantage of stable level of analgesia and increased maternal haemodynamic stability .", "however , automated regular bolus delivery of epidural analgesia when compared with continuous infusion is found to decrease the incidence of breakthrough pain and increased maternal satisfaction . because of logistic constraints , we used intermittent epidural dosing on demand by the parturients , which , we found , could be comparable to continuous and automated delivery system . the limitation in our study was to combine primiparous and multiparous parturients , which could have altered the results of mode of delivery .", "we conclude that cse using sufentanil and fentanyl achieved high patient satisfaction and excellent labour analgesia without serious maternal or neonatal side - effects ." ]

[ "<S> objectives : the highly lipid - soluble opioids , sufentanil and fentanyl , are used in combination with low - concentration bupivacaine to provide combined spinal epidural ( cse ) analgesia during labour . </S>", "<S> we designed a prospective , randomized , single - blind study to compare the efficacy of these two opioids with bupivacaine in terms of the quality of analgesia , side - effects and maternal and foetal outcome.methods:sixty parturients requesting labour analgesia were divided into two groups randomly . </S>", "<S> group s ( n=30 ) received bupivacaine heavy ( 2.5 mg ) and sufentanil ( 5 mcg ) intrathecally and 10 ml intermittent bolus of sufentanil 0.30 mcg / ml in bupivacaine 0.125% as epidural top - ups . </S>", "<S> group f ( n=30 ) received bupivacaine heavy ( 2.5 mg ) and fentanyl ( 25 mcg ) intrathecally and 10 ml intermittent bolus of fentanyl 2.5 mcg / ml in bupivacaine 0.125% as epidural top - ups . </S>", "<S> duration of intrathecal and epidural analgesia , mean duration between epidural top - ups and total analgesic requirements were noted . </S>", "<S> pain and overall satisfaction scores were assessed with a 10-point visual scale . </S>", "<S> mode of delivery and neonatal apgar scores were recorded.results:maternal demographic characteristics were comparable between the groups . </S>", "<S> although cse provided satisfactory analgesia in both the groups , parturients of group s had a significant prolongation of analgesia through the intrathecal route compared with parturients of group f. incidence of caesarean , instrumental delivery did not differ between the groups . </S>", "<S> no difference in the incidence of motor blockade or cephalad extent of sensory analgesia was observed . neonatal outcome and incidence of side - effects </S>", "<S> were similar in both the groups.conclusion:we conclude that combined spinal epidural using sufentanil and fentanyl achieved high patient satisfaction and excellent labour analgesia without serious maternal or neonatal side - effects . </S>", "<S> sufentanil provided a significantly longer duration of labour analgesia compared with fentanyl . </S>" ]

[ "INTRODUCTION", "METHODS", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSION" ]

[ [ "painful uterine contractions cause maternal hyperventilation and increased catecholamine concentration resulting in maternal and foetal hypoxaemia .", "an effective analgesia takes away the disadvantages and results in better maternal and foetal outcome .", "hence , the control of pain should form an integral part of labour management at any level .", "an ideal labour analgesic technique should provide adequate and satisfactory analgesia without any motor blockade or adverse maternal and foetal effects . among the variety of labour analgesia techniques ranging from parenteral and inhalational agents ,", "combined spinal epidural ( cse ) analgesia is increasingly used to provide pain relief during labour .", "it combines the advantage of rapid onset of spinal analgesia and the flexibility of the epidural catheter .", "dose adjustments and frequency of administration of the drug according to parturients requirement is possible with the epidural route which can also be extended to provide anaesthesia for caesarean delivery if need arises .", "addition of opioids to local anaesthetics reduces its requirement by synergistic effect of opioid receptors in the spinal cord .", "although fentanyl and sufentanil are the most commonly used opioids for epidural labour analgesia , studies comparing cse technique employing sufentanil and fentanyl are very few and give conflicting results . at the assumed equipotent doses of sufentanil to fentanyl", "ratio of 6:1 , there is some evidence that sufentanil is clinically superior to fentanyl as an adjunct to bupivacaine in labour epidurals .", "hence , we decided to compare the efficacy , quality and duration of analgesia and maternal and foetal effects of adding the two highly lipid - soluble opioids , sufentanil and fentanyl to low - concentration bupivacaine for cse labour analgesia ." ], [ "a randomized , prospective , single - blinded study was carried out on 60 parturients over a period of 6 months after obtaining clearance from the institutional ethical committee .", "parturients belonging to asa grade i and ii with singleton , term pregnancy in spontaneous labour with cervical dilatation of less than 4 cm , with normal foetal heart tracings requesting labour analgesia , were included in the study .", "parturients with medical disorders , obstetric complications , malpresentation of foetus and those with contraindication for regional analgesia were excluded from the study . after obtaining informed written consent , parturients were randomly allocated by a computer - generated table of random numbers by a person blinded to the procedure to avoid selection bias into two groups of 30 each as group s ( n=30 ) and group f ( n=30 ) . before institution of cse , parturients were administered 1000 ml ringer lactate solution and monitors were connected and baseline readings recorded .", "patients were placed in the left lateral position and , under aseptic precautions , cse analgesia was administered through a midline approach ( l2-l3 or l3-l4 level ) .", "lumbar epidural space was identified with an 18 g tuohy needle using loss of resistance to saline technique .", "after negative aspiration for blood and cerebrospinal fluid , the epidural catheter was threaded on through the tuohy needle and the needle was carefully withdrawn .", "spinal analgesia was instituted using a 25 g whitacre needle one space below . on identification of subarachnoid space , 1 ml of drug containing 0.5 ml 0.5% bupivacaine heavy ( 2.5 mg ) and 0.5 ml sufentanil ( 5 mcg ) for group s or 0.5 ml fentanyl ( 25 mcg ) for group f", "the parturient was turned supine immediately after subarachnoid block and the uterus was displaced to the left using a wedge .", "pulse rate , blood pressure , respiratory rate and spo2 were monitored at 0 , 5 , 10 , 15 and 30 min and thereafter every 30 min until the women delivered .", "motor block was assessed by modified bromage scale and the level of sensory block was recorded .", "pain relief was assessed using visual analogue scores ( vas ) ( 0 = no pain , 10 = worst possible pain experienced ) . when the parturient experienced pain equivalent to a vas score of 5 , epidural top - up was administered . the time interval between initiation of intrathecal analgesia and patient developing pain equivalent to vas", "parturients of group s received 10 ml of 0.125% bupivacaine with 0.30 mcg / ml of sufentanil and group f received 10 ml of 0.125% bupivacaine with fentanyl 2.5 mcg / ml through an epidural catheter .", "time of onset of epidural analgesia , total number of top - ups and duration between successive top - ups were recorded .", "the mode of delivery in the form of normal vaginal delivery , instrumental delivery or caesarean section was observed .", "side - effects such as pruritis ( rated as none , minimal , moderate and severe ) , hypotension ( fall of > 20% from baseline systolic reading ) , motor blockade of limbs , shivering , sedation ( categorized as none , mild , moderate and severe for awake , drowsy , sleepy and unarousable ) and bradycardia ( heart rate<60 ) were noted . neonatal outcome in the form of apgar scores at 1 min and 5 min and need for intensive care were noted .", "all the data were recorded by a resident blinded to the drug administered during conduct of labour analgesia throughout the study period .", "the parturient was monitored for 2 h following delivery and the epidural catheter was removed .", "they were questioned after 24 h about their views on the procedure and the satisfaction .", "enquiry about the symptoms related to post - dural puncture headache ( pdph ) was made .", "sample size of 60 with 30 parturients in each group was determined with power of study of 80% ." ], [ "sample size of 60 with 30 parturients in each group was determined with power of study of 80% ." ], [ "there were no significant differences between the groups with respect to maternal demographic characteristics , parity , cervical dilation at initiation of labour analgesia , duration of labour and delivery characteristics [ table 1 ] .", "maternal demographic characteristics the mode of delivery data suggests no significant difference between the groups regarding incidence of caesarean , instrumental or vaginal deliveries [ table 2 ] .", "the mean duration of intrathecal analgesia was 109.7 ( 6.3 ) min for group s and 73.63 ( 15.76 ) min for group f. the mean time of onset of epidural analgesia was 2.48 ( 0.28 ) min and 2.55 ( 0.77 ) min for group s and group f , respectively .", "the mean duration between epidural top - ups was 90.51 ( 15.8 ) min for group s and 83.54 ( 24.64 ) min for group f [ table 3 ] .", "the number of additional bolus supplementary analgesic top - ups were comparatively less in the sufentanil group .", "eight women among group s and nine among group f experienced transient initial motor blockade ( bromage score 1 ) .", "none of the women had any motor blockade at the end of 1 h. the highest level of sensory block attained was t6 in 63.33% , t8 in 36.66% among group s and 66.66% and 33.33% among group f , respectively .", "onset and duration of analgesia the pain scores during the first and second stages of labour were comparable without any significant difference between the groups using vas scores ( vas : 0 = no pain , 10 = worst pain experienced ) ; 93.3% and 90.0% women had vas scores less than 1 , indicating excellent pain relief by the cse technique in both groups [ table 4 ] .", "mean visual analogue scale scores no significant alterations in intrapartum vital parameters like spo2 , mean arterial pressure , pulse rate and fhr were noted between the groups .", "the incidence of side - effects did not differ between the groups [ table 5 ] .", "pruritis , shivering , nausea and vomiting , sedation , bradycardia and hypotension were reported and treated accordingly .", "the most common side - effect was pruritis , which was reported among 20% and 30% of the women belonging to groups s and f , respectively .", "nine parturients ( four in group s and five in group f ; p = ns ) experienced mild sedation during the study period .", "neonatal outcome was assessed based on apgar scores at the 1 and the 5 minutes , which did not differ between the groups .", "maternal satisfaction was assessed based on subjective assessment as excellent , good , satisfactory and poor .", "93.33% of the women of group s and 90% of the women of group f opined labour analgesia as excellent ." ], [ "highly lipid - soluble synthetic opioids such as sufentanil and fentanyl are being increasingly used along with very low concentrations of local anaesthetic agents such as bupivacaine ( 0.06250.125% ) and ropivacaine ( 0.20.25% ) to provide excellent relief of pain during labour . unlike hydrophilic opiates ( morphine ) or intermediate lipid - soluble opioids ( meperidine ) , lipophillic opioids do not spread rostrally in cerebrospinal fluid to any great extent , and they tend to have fairly segmental analgesic profiles .", "quality of analgesia and overall satisfaction scores were found to be comparable in both groups in our study . in a dose response study ,", "the analgesic potency of epidural sufentanil was reported to be approximately five - times that of fentanyl when it was administered as the sole analgesic after lidocaine anaesthesia for caesarean delivery .", "addition of bupivacaine to intrathecal opioid prolongs duration of labour analgesia compared with either drug used alone .", "evidence suggests that bupivacaine potentiates the binding of morphine to opioid receptors , especially the highly dense kappa receptors , as the result of an associated conformational change in opioid receptors .", "we observed that duration of analgesia provided by intrathecal sufentanil and bupivacaine was 109.706.37 min as compared with 73.6315.76 min by group f. this difference was statistically significant .", "these are similar to previously reported results using sufentanil : 11426 min and 99 min .", "this prolonged duration of analgesia with sufentanil could be attributed to the known superiority of sufentanil over fentanyl in terms of potency .", "similarly , the number of supplementary bupivacaine top - ups was comparatively less in the sufentanil group ( group s 1.220.75 and group f 2.060.081 ) .", "addition of bupivacaine 2.5 mg and sufentanil 10 g for labour analgesia showed that the combination , although effective , was associated with a higher incidence of hypotension and impairment of muscle power of the lower limbs , although mild in some parturients .", "another investigation by halving the total amount of intrathecal sufentanil and bupivacaine reduced the incidence of side - effects .", "however , this reduced dose regimen was associated with slower onset and shorter duration of labour analgesia . as 10 mcg sufentanil is associated with severe hypotension and 2.5 mcg sufentanil produces analgesia of shorter duration and slower onset , in our study , we added 5 mcg sufentanil to bupivacaine 2.5 mg intrathecal .", "cse has an added advantage of initiating labour analgesia even at the late stages of labour .", "we found that analgesia through intrathecal route resulted in immediate and predictable onset of analgesia enhancing the parturient 's cooperation for the insertion of epidural catheter .", "the duration of first and second stage of labour and the mode of delivery between the groups did not differ significantly .", "the incidence of caesarean delivery was 20% and 16.66% between group s and f , respectively .", "higher concentration of bupivacaine ( 0.25% ) was used in the past , which resulted in a fairly higher incidence of motor block causing pelvic muscle relaxation , foetal malposition and maternal inability to push and a higher incidence of instrumental delivery .", "incidence of motor blockade in our study groups was very minimal and was of bromage scale 1 , which did not persist beyond the first hour .", "the pain relief during labour was assessed by vas scoring and the pain scores were comparable between the groups .", "it was graded as excellent in 93.33% parturients of group s and 90.0% parturients of group f , as in previous studies .", "the dose of opioids administered did not adversely affect the apgar scores or cause respiratory depression among newborns in our study .", "pruritus was the most common side - effect between the two groups , which was not of a serious nature .", "the incidence of sedation and nausea was similar in the two groups , with no parturient in either group requiring any intervention .", "continuous infusion of a dilute mixture of local anaesthetics and opioids offers the advantage of stable level of analgesia and increased maternal haemodynamic stability .", "however , automated regular bolus delivery of epidural analgesia when compared with continuous infusion is found to decrease the incidence of breakthrough pain and increased maternal satisfaction . because of logistic constraints , we used intermittent epidural dosing on demand by the parturients , which , we found , could be comparable to continuous and automated delivery system . the limitation in our study was to combine primiparous and multiparous parturients , which could have altered the results of mode of delivery ." ], [ "we conclude that cse using sufentanil and fentanyl achieved high patient satisfaction and excellent labour analgesia without serious maternal or neonatal side - effects ." ] ]

[ "it stimulates the dopamine ( d2 ) receptors and also inhibits the gamma amino butyric acid release in central nervous system .", "common therapeutic doses of tramadol are 50 mg orally and 100 mg with parenteral and rectal route of administration up to 400 mg / day .", "the main adverse drug reactions of tramadol are nausea , dizziness , somnolence , drowsiness , increased sweating , vomiting , and dry mouth .", "seizure and apnea are the most important life - threatening clinical presentations of tramadol in therapeutic and toxic doses .", "from this view , evaluation of laboratory findings including plasma electrolytes , kidney and liver function tests , and blood gas analysis have a critical role for patient monitoring .", "as there are few studies about laboratory findings in tramadol acute poisoning , we evaluated the clinical and laboratory findings in acute tramadol - intoxicated cases and their role in the prediction of seizure .", "this was a retrospective study on patients with acute tramadol poisoning who referred to loghman hakim hospital poison center , tehran , iran from january to april 2012 .", "the exclusion criteria were co - ingestion , intoxication with unknown dose of tramadol , uncertainty about time of tramadol ingestion , onset of a seizure before admission on hospital , the past medical history of epilepsy and history of drug / substance abuse .", "data such as patients age , sex , time of ingestion , ingested dose , cause of intoxication , respiratory rate , pulse rate , systolic and diastolic blood pressure , temperature , coma grade scale on admission time , and therapeutic interventions and patients outcome were extracted from the medical records .", "laboratory findings including blood sugar , blood urea nitrogen ( bun ) , creatinin ( cr ) , sodium ( na ) , potassium ( k ) , liver function tests , cell blood count , and blood gas on admission time were retrieved from patients medical records .", "the data were expressed as mean standard deviation / standard error ( se ) for continuous or discrete variables and as frequency and percentage for categorical variables .", "we used the student 's t - test and mann whitney u - test for statistical analyses of continuous variables with and without normal distribution , respectively .", "linear correlations between variables were assessed by spearman and expressed as the spearman correlation coefficient .", "the data were expressed as mean standard deviation / standard error ( se ) for continuous or discrete variables and as frequency and percentage for categorical variables .", "we used the student 's t - test and mann whitney u - test for statistical analyses of continuous variables with and without normal distribution , respectively .", "linear correlations between variables were assessed by spearman and expressed as the spearman correlation coefficient .", "we used spss software ( version 13 , spss inc . , chicago , il , usa ) .", "a total of 144 patients including 111 men ( 77% ) and 33 ( 23% ) women with the mean age of 23.7 6.9 ( range = 15 - 57 ) years old included in this study .", "the average time between ingestion and admission on hospital ( mean se ) was 292.2 30 min ( range = 30 - 3600 min ) .", "mean duration of hospitalization was 17.9 10.6 h ( range = 3.6 - 80 h ) . in all of the cases , the route of exposure was oral , and the most drug dosage form was tablet ( n = 142 ) and then capsule ( n = 2 ) .", "the mean ingested dose ( mean se ) was 1971.2 233.4 mg ( range = 100 - 20000 mg ) . in most of the cases ( n = 99 , 68.8% ) the cause of intoxication was suicide and then abuse ( n = 45 , 31.2% ) [ table 1 ] .", "demographic findings in tramadol - intoxicated patients major cases had stable vital signs on admission and the related data are summarized in table 2 .", "128 ( 88.9% ) of patients were conscious , and 16 of them had a decreased level of consciousness .", "seizure ( 47.9% ) , nausea ( 29.9% ) , vomiting ( 22.2% ) , drowsiness ( 20.1% ) , dizziness ( 18.1% ) , lethargy ( 6.3% ) , apnea ( 5.6% ) , agitation ( 4.2% ) , headache ( 1.4% ) , blurred vision ( 1.4% ) , ataxia ( 0.7% ) , anxiety ( 0.7% ) , sweating ( 0.7% ) , and nystagmus ( 0.7% ) were the most clinical findings in tramadol - intoxicated patients [ table 2 ] .", "laboratory findings on admission time in the intoxicated patients have been summarized in table 3 .", "clinical findings in tramadol - intoxicated patients on admission time laboratory findings in tramadol - intoxicated patients on admission time we divided the cases with regard to occurrence of seizure during hospitalization .", "the results showed that there were significant differences between cases with seizure and cases without seizure according to time interval between tramadol ingestion and hospital admission ( tibtiha ) ( mean se ) ( 330.3 53.2 vs. 257.3 30.1 min , p = 0.01 ) , ingested dose ( mean se ) ( 1395.7 218.3 vs. 2500.7 390.7 mg , p = 0.006 ) , with odds ratio 2.7 ( 1.03 - 7.09 , 95% confidence interval [ ci ] ) , dizziness ( 3 cases vs. 23 cases , p = <", "0.0001 ) , with odds ratio 0.1 ( 0.29 - 0.36 , 95% ci ) , pco2 ( 51.2 8.5 vs. 48.4 8.6 mmhg , p = 0.03 ) , with odds ratio 0.58 ( 0.27 - 1.24 , 95% ci ) , and total bilirubin ( 0.5 0.2 vs. 0.7 0.4 mg / dl , p = 0.002 ) . there was a correlation between ingested dose ( r = 0.2 , p = 0.006 ) , paco2 ( r = 0.2 , p = 0.03 ) , tibtiha ( r = 0.2 , p = 0.01 ) , total bilirubin ( r = 0.3 , p = 0.002 ) , dizziness ( r = 0.3 , p = 0.000 ) , and seizure .", "tramadol abuse and overdose is one of the most frequent health problems in iran and worldwide . in this study , we report clinical and paraclinical findings in 144 cases with pure tramadol poisoning who referred to a referral - poisoning center in tehran , iran .", "furthermore , the results indicated that oral route is the most common route of exposure , which is similar with the findings of our previous studies .", "the frequency of other clinical manifestations including lethargy , coma , nausea , vomiting , agitation , and respiratory depression in our study were different with previous studies .", "tramadol is metabolized by cytochrome p450 ( cyp450 ) enzymes ( mainly 2d6 isoenzyme ) to its active metabolites m1 ( o - desmethyl tramadol ) , m2 ( n - desmethyl tramadol ) , m3 ( n , n - didesmethyl tramadol ) , m4 ( o , n , n - tridesmethyl tramadol ) , and m5 ( o , n - didesmethyl tramadol ) .", "m1 metabolite has more affinity ( 200 times ) for the -opioid receptors and also it has more inhibitory effect on biogenic amine reuptake than that of parent drug molecule and may be responsible for tramadol induce analgesia or seizure in intoxicated patients . in this", "regard , genetic polymorphism in humans may affect the tramadol metabolism and its peak blood concentration resulting to a different frequency of tramadol adverse effects or clinical presentations during therapeutic doses or intoxication . although the increasing of liver function tests , serum bun and cr due to liver and kidney damage have been demonstrated in chronic administration of tramadol in experimental model , but in our study due to acute onset of toxicity we did not observe any increase in liver function tests , bun and cr and this is in concordance of the previous study .", "rhabdomyolysis and rise of creatine phosphokinase ( cpk ) have been reported as a rare and serious complication in tramadol intoxication in the previous studies , which was observed in our study too .", "although in the previous studies , prolong immobilization and multiple seizures have been described as one of the reasons for the rise of cpk and rhabdomyolysis , but in our study there was no significant difference in level of cpk in the tramadol - intoxicated patients with seizure in comparison to cases without seizure .", "le berre et al . , reported tramadol induced hyponatremia which described as a result of inappropriate antidiuretic hormone secretion . in this study , the level of na , and k was within the normal range .", "the pco2 level was above normal range which could be attributed to tramadol - induced respiratory depression , which has been reported previously .", "in this study , the mean of ingested dose in the seizure group was less than those cases without seizure which is in contrasts with the result of the previous study .", "one of the explanations is the difference between two groups with regard to tibtiha , which was significantly longer in seizure group .", "furthermore , as mentioned previously , the other reason could be the genetic polymorphism in patients .", "the main limitation of this study is its retrospective design , which should be considered in the interpretation of the results . with this regard", "there were significant differences between seizure and nonseizure cases with regard to tibtiha , ingested dose , and pco2 .", "furthermore , we showed poor correlation between tramadol ingested dose , tibtiha , pco2 , and seizure in tramadol - intoxicated cases .", "hrr involving in the data collection and data analysis . ss and ksn involving in the study design , conducting of the study and data analysis ." ]

[ "<S> background : tramadol is a centrally acting analgesic with opioid and nonopioid properties , which extensively used in the relief of mild to moderate pain . </S>", "<S> tramadol poisoning is a common cause of acute pharmaceutical poisoning in iran . </S>", "<S> there are a few studies about clinical and laboratory findings related to acute tramadol poisoning . therefore , the aim of this study was to demonstrate the clinical and laboratory findings in tramadol acute poisoning cases.materials and methods : this was a retrospective descriptive study of patients with acute tramadol poisoning who referred to loghman hakim hospital poison center during january to april 2012 . </S>", "<S> data such as patient 's age , sex , time of ingestion , ingested dose , cause of poisoning , mean duration of hospitalization , patient 's clinical presentations , laboratory findings , therapeutic measures , and patient 's outcome have collected in a predesigned checklist.results:a total of 144 patients including 111 men ( 77% ) and 33 women ( 23% ) with acute tramadol poisoning was included in this study . </S>", "<S> the mean ingested dose was 1971.2 mg ( 100 - 20000 mg ) . </S>", "<S> seizure ( 47.91% ) was the most frequent clinical symptom . </S>", "<S> blood gas on admission showed ph ( 7.3 0.1 ) , pco2 ( 49.7 8.6 mmhg ) and hco3 ( 24.1 3.8 </S>", "<S> meq / l ) , indicating pure acute respiratory acidosis may be occurred in tramadol - intoxicated patients . there were significant differences between tramadol - intoxicated cases with and without a seizure with regard to the time interval between ingestion and admission on hospital , ingested dose and pco2.conclusion:seizure and rise of pco2 were the most findings in this study . </S>" ]

[ "INTRODUCTION", "METHODS", "Statistical analyses", "RESULTS", "DISCUSSION", "CONCLUSION", "AUTHOR'S CONTRIBUTION" ]

[ [ "tramadol has low affinity for - and -opioid receptors and inhibits the reuptake of both nor - epinephrine and serotonin ( 5-hydroxytryptamine ) neurotransmitters .", "it stimulates the dopamine ( d2 ) receptors and also inhibits the gamma amino butyric acid release in central nervous system .", "common therapeutic doses of tramadol are 50 mg orally and 100 mg with parenteral and rectal route of administration up to 400 mg / day .", "the main adverse drug reactions of tramadol are nausea , dizziness , somnolence , drowsiness , increased sweating , vomiting , and dry mouth .", "seizure and apnea are the most important life - threatening clinical presentations of tramadol in therapeutic and toxic doses .", "from this view , evaluation of laboratory findings including plasma electrolytes , kidney and liver function tests , and blood gas analysis have a critical role for patient monitoring .", "as there are few studies about laboratory findings in tramadol acute poisoning , we evaluated the clinical and laboratory findings in acute tramadol - intoxicated cases and their role in the prediction of seizure ." ], [ "this was a retrospective study on patients with acute tramadol poisoning who referred to loghman hakim hospital poison center , tehran , iran from january to april 2012 .", "the exclusion criteria were co - ingestion , intoxication with unknown dose of tramadol , uncertainty about time of tramadol ingestion , onset of a seizure before admission on hospital , the past medical history of epilepsy and history of drug / substance abuse .", "data such as patients age , sex , time of ingestion , ingested dose , cause of intoxication , respiratory rate , pulse rate , systolic and diastolic blood pressure , temperature , coma grade scale on admission time , and therapeutic interventions and patients outcome were extracted from the medical records .", "laboratory findings including blood sugar , blood urea nitrogen ( bun ) , creatinin ( cr ) , sodium ( na ) , potassium ( k ) , liver function tests , cell blood count , and blood gas on admission time were retrieved from patients medical records .", "the data were expressed as mean standard deviation / standard error ( se ) for continuous or discrete variables and as frequency and percentage for categorical variables .", "we used the student 's t - test and mann whitney u - test for statistical analyses of continuous variables with and without normal distribution , respectively .", "linear correlations between variables were assessed by spearman and expressed as the spearman correlation coefficient ." ], [ "the data were expressed as mean standard deviation / standard error ( se ) for continuous or discrete variables and as frequency and percentage for categorical variables .", "we used the student 's t - test and mann whitney u - test for statistical analyses of continuous variables with and without normal distribution , respectively .", "linear correlations between variables were assessed by spearman and expressed as the spearman correlation coefficient .", "we used spss software ( version 13 , spss inc . , chicago , il , usa ) ." ], [ "a total of 144 patients including 111 men ( 77% ) and 33 ( 23% ) women with the mean age of 23.7 6.9 ( range = 15 - 57 ) years old included in this study .", "the average time between ingestion and admission on hospital ( mean se ) was 292.2 30 min ( range = 30 - 3600 min ) .", "mean duration of hospitalization was 17.9 10.6 h ( range = 3.6 - 80 h ) . in all of the cases , the route of exposure was oral , and the most drug dosage form was tablet ( n = 142 ) and then capsule ( n = 2 ) .", "the mean ingested dose ( mean se ) was 1971.2 233.4 mg ( range = 100 - 20000 mg ) . in most of the cases ( n = 99 , 68.8% ) the cause of intoxication was suicide and then abuse ( n = 45 , 31.2% ) [ table 1 ] .", "demographic findings in tramadol - intoxicated patients major cases had stable vital signs on admission and the related data are summarized in table 2 .", "128 ( 88.9% ) of patients were conscious , and 16 of them had a decreased level of consciousness .", "seizure ( 47.9% ) , nausea ( 29.9% ) , vomiting ( 22.2% ) , drowsiness ( 20.1% ) , dizziness ( 18.1% ) , lethargy ( 6.3% ) , apnea ( 5.6% ) , agitation ( 4.2% ) , headache ( 1.4% ) , blurred vision ( 1.4% ) , ataxia ( 0.7% ) , anxiety ( 0.7% ) , sweating ( 0.7% ) , and nystagmus ( 0.7% ) were the most clinical findings in tramadol - intoxicated patients [ table 2 ] .", "laboratory findings on admission time in the intoxicated patients have been summarized in table 3 .", "clinical findings in tramadol - intoxicated patients on admission time laboratory findings in tramadol - intoxicated patients on admission time we divided the cases with regard to occurrence of seizure during hospitalization .", "the results showed that there were significant differences between cases with seizure and cases without seizure according to time interval between tramadol ingestion and hospital admission ( tibtiha ) ( mean se ) ( 330.3 53.2 vs. 257.3 30.1 min , p = 0.01 ) , ingested dose ( mean se ) ( 1395.7 218.3 vs. 2500.7 390.7 mg , p = 0.006 ) , with odds ratio 2.7 ( 1.03 - 7.09 , 95% confidence interval [ ci ] ) , dizziness ( 3 cases vs. 23 cases , p = <", "0.0001 ) , with odds ratio 0.1 ( 0.29 - 0.36 , 95% ci ) , pco2 ( 51.2 8.5 vs. 48.4 8.6 mmhg , p = 0.03 ) , with odds ratio 0.58 ( 0.27 - 1.24 , 95% ci ) , and total bilirubin ( 0.5 0.2 vs. 0.7 0.4 mg / dl , p = 0.002 ) . there was a correlation between ingested dose ( r = 0.2 , p = 0.006 ) , paco2 ( r = 0.2 , p = 0.03 ) , tibtiha ( r = 0.2 , p = 0.01 ) , total bilirubin ( r = 0.3 , p = 0.002 ) , dizziness ( r = 0.3 , p = 0.000 ) , and seizure ." ], [ "tramadol abuse and overdose is one of the most frequent health problems in iran and worldwide . in this study , we report clinical and paraclinical findings in 144 cases with pure tramadol poisoning who referred to a referral - poisoning center in tehran , iran .", "furthermore , the results indicated that oral route is the most common route of exposure , which is similar with the findings of our previous studies .", "the frequency of other clinical manifestations including lethargy , coma , nausea , vomiting , agitation , and respiratory depression in our study were different with previous studies .", "tramadol is metabolized by cytochrome p450 ( cyp450 ) enzymes ( mainly 2d6 isoenzyme ) to its active metabolites m1 ( o - desmethyl tramadol ) , m2 ( n - desmethyl tramadol ) , m3 ( n , n - didesmethyl tramadol ) , m4 ( o , n , n - tridesmethyl tramadol ) , and m5 ( o , n - didesmethyl tramadol ) .", "m1 metabolite has more affinity ( 200 times ) for the -opioid receptors and also it has more inhibitory effect on biogenic amine reuptake than that of parent drug molecule and may be responsible for tramadol induce analgesia or seizure in intoxicated patients . in this", "regard , genetic polymorphism in humans may affect the tramadol metabolism and its peak blood concentration resulting to a different frequency of tramadol adverse effects or clinical presentations during therapeutic doses or intoxication . although the increasing of liver function tests , serum bun and cr due to liver and kidney damage have been demonstrated in chronic administration of tramadol in experimental model , but in our study due to acute onset of toxicity we did not observe any increase in liver function tests , bun and cr and this is in concordance of the previous study .", "rhabdomyolysis and rise of creatine phosphokinase ( cpk ) have been reported as a rare and serious complication in tramadol intoxication in the previous studies , which was observed in our study too .", "although in the previous studies , prolong immobilization and multiple seizures have been described as one of the reasons for the rise of cpk and rhabdomyolysis , but in our study there was no significant difference in level of cpk in the tramadol - intoxicated patients with seizure in comparison to cases without seizure .", "le berre et al . , reported tramadol induced hyponatremia which described as a result of inappropriate antidiuretic hormone secretion . in this study , the level of na , and k was within the normal range .", "the pco2 level was above normal range which could be attributed to tramadol - induced respiratory depression , which has been reported previously .", "in this study , the mean of ingested dose in the seizure group was less than those cases without seizure which is in contrasts with the result of the previous study .", "one of the explanations is the difference between two groups with regard to tibtiha , which was significantly longer in seizure group .", "furthermore , as mentioned previously , the other reason could be the genetic polymorphism in patients .", "the main limitation of this study is its retrospective design , which should be considered in the interpretation of the results . with this regard" ], [ "there were significant differences between seizure and nonseizure cases with regard to tibtiha , ingested dose , and pco2 .", "furthermore , we showed poor correlation between tramadol ingested dose , tibtiha , pco2 , and seizure in tramadol - intoxicated cases ." ], [ "hrr involving in the data collection and data analysis . ss and ksn involving in the study design , conducting of the study and data analysis ." ] ]

[ "familial glucocorticoid deficiency ( fgd ) is a rare autosomal recessive disorder resulting from adrenal unresponsiveness to adrenocorticotropic hormone ( acth ) .", "this disease is characterized by low serum cortisol concentrations in the presence of grossly elevated plasma acth levels .", "affected individuals typically present with hyperpigmentation , hypoglycemic seizures , failure to thrive , failure to thrive and recurrent infections .", "causal mutations of fgd have been identified in mc2r , mrap , mcm4 , txnrd2 , star and nnt .", "mc2r accessory protein ( mrap ) , a 19-kda single - transmembrane domain protein , is essential for trafficking of mc2r from the endoplasmic reticulum ( er ) to the cell surface and subsequent signaling in response to acth .", "mutations in mrap are responsible for 1520% of fgd cases , named fgd type 2 . until now", ", this mutation has not been reported in chinese han cases of fgd . in the current paper", ", we describe a chinese han child who presented with isolated hyperpigmentation at birth and a homozygous splice mutation ( c.106 + 1delg ) in mrap .", "a female infant was referred to our neonatal unit at the age of nine days for evaluation of hyperpigmentation of the skin . she was the product of an uncomplicated pregnancy and was born by caesarian section for concerning cardiotocographic changes during labor .", "her sibling had died on the third day of life with hyperpigmentation of the skin .", "birth size was normal ( weight 2800 g , 0.99 sds ; length 46 cm , 1.69 sds ; head circumference 33 cm , 0.74 sds ) and apgar scores was 8 and 9 at 1st and 5th minute , respectively . on physical examination ,", "weight was 2740 g ( 1.31 sds ) , length 47 cm ( 2.47 sds ) , and head circumference was 34.5 cm ( 1.5 sds ) .", "endocrine investigations revealed low baseline 8 am serum cortisol ( < 1.0 g / dl ) with extremely elevated acth ( 1250 pg / ml ) .", "testosterone , 17-oh progesterone , estradiol , progesterone , luteinizing hormone , folliculin , and prolactin , free t4 , and tsh levels were normal .", "based on these findings , a familial glucocorticoid deficiency was suspected and oral hydrocortisone treatment at dose of 20 mg / m per day was started .", "hydrocortisone dose ( varying from 10 to 15 mg / m per day ) adjustment was based on acth and cortisol concentrations .", "after 4 weeks , acth level was 278.0 pg / ml and cortisol level was 42.4 g / dl , and hydrocortisone dose was reduced to 15 mg / m per day ( given in divided doses twice daily ) .", "the acth level was suppressed to slightly above the normal limit and the cortisol level is normal during the follow - up period . on her most recent visit , at the age of 12 months ,", "hydrocortisone dose was 11 mg / m per day ( 5 mg was give once daily in the morning ) , the acth level was 25.10 pg / ml and cortisol level was 33.4 g / dl , and her skin was slightly lightened ( fig .", "her weight was 10 kg ( 0.84 sds ) , length 70 cm ( 1.67 sds ) , and head circumference was 46.5 cm ( 1.12 sds ) .", "genomic dna was extracted from peripheral blood leucocytes from the infant and her family after informed consent was obtained . for the molecular diagnosis", ", a custom panel - based next - generation sequencing approach has been used to sequence all known adrenal gland diseases - associated genes in this child .", "we found a homozygous deletion of one nucleotide at the canonical 5 donor splice site ( c.106 + 1delg ) in intron 3 of mrap gene .", "this would result in the skipping of exon 3 and a prematurely terminated translation product ( ( 1 ) ) .", "her parents and maternal grandmother were found to be heterozygous carrier for the same mutation ( fig .", "we report a case of fgd with homozygous mutation of mrap in a chinese han neonate .", "this girl presented with isolated hyperpigmentation , have low cortisol and high acth with normal electrolytes . a homozygous splice mutation ( c.106 + 1delg ) of mrap confirmed the diagnosis of fgd type 2 . her parents and maternal grandmother had the same mutation .", "until now , this mutation has not been reported in chinese han cases of fgd .", "fgd is a rare autosomal recessive disorder characterized in laboratory testing by glucocorticoid deficiency and markedly elevated acth levels .", "patients with fgd usually present during neonatal period to late childhood with symptoms related to cortisol deficiency and acth excess . in the neonatal period", ", these symptoms may include hyperpigmentation , hypoglycemia , irritability , jitteriness , lethargy , respiratory distress , cyanosis , apnea , hypotonia , seizures , shock , or sudden death .", "newborns can present with a positive family history of early - unexplained infant deaths or other affected family members supports a diagnosis .", "the severe pigmentation of the skin is due to the over - stimulation of mc1r ( cutaneous msh receptors ) by high circulating msh which is a byproduct of acth synthesis from proopiomelanocortin .", "this hyperpigmentation fades once proper treatment is initiated with glucocorticoids , which reduce acth concentrations .", "this would suggest that the fetal corticotrophs could produce excessive plasma acth in response to low fetal cortisol , which in turn acts on melanocytes to promote eumelanin synthesis before birth . in most of the fgd patients suppression of acth levels", "is difficult , and therefore is not used as a goal for therapy . in this case , replacement treatment with hydrocortisone suppressed acth level to slightly above the normal limit and partially resolved the hyperpigmentation . acquired causes of adrenal insufficiency such as adrenal hemorrhage , trauma and infections were excluded by history and laboratory findings .", "congenital adrenal hyperplasia was excluded by hormone analysis ; congenital adrenal hypoplasia was also unlikely , because she had normal serum 17-oh progesterone and no mutation of the dax1 gene .", "mrap , located at 21q22.1 , is a small single - pass transmembrane domain protein , which is essential for the processing of the acth receptor ( melanocortin 2 receptor , mc2r ) and its trafficking from the er to the cell surface .", "this mutation will lead to skipping of exon 3 and early truncation of the protein and the absence of the mc2r interacting transmembrane domain . as a result", ", mc2r is retained within the er and fails to reach the cell surface which lead to acth resistance and adrenal insufficiency .", "mutations in mrap causing fgd2 were first described in 2005 . so far over 9 different mutations of mrap in fgd type 2 patients", "have been documented all of which are splice site or nonsense mutations and are predicted to produce proteins lacking the transmembrane domain essential for interaction with mc2r , . in our patient , we found a newborn with fgd type 2 , presenting with severe hyperpigmentation .", "she was found to have a splice - site mutation in the mrap gene , hence explaining the early presentation seen in the case . in their study , metherell et al . identified the 1-bp deletion , c. 106 + 1delg , in 6 individuals from 5 families with glucocorticoid deficiency , making this the second frequent mutation causing fgd unrelated to defects in the mc2r gene . to our current knowledge , 1-bp deletion ( c. 106 + 1delg ) in intron 3 of mrap gene , identified in the dna of the patient ,", "cases of the condition have been reported in white , , black , indian , and middle eastern populations . to our knowledge", ", our patient is the first reported chinese han patient with fgd type 2 , with a known mrap mutation .", "the patient was the offspring of non - consanguineous parents and her sibling had died in the neonatal period , possibly due to glucocorticoid insufficiency .", "prolonged acth excess or glucocorticoid deficiency increases linear growth , while early diagnosis and appropriate therapy in this case enabled the patient to achieve normal developmental milestones .", "further studies describing new cases and mutations causing fgd will contribute to understanding the mechanism of this rare and potentially life - threatening disease ." ]

[ "<S> familial glucocorticoid deficiency ( fgd ) is a rare autosomal recessive disorder in which the adrenal cortex fails to respond appropriately to stimulation by adrenocorticotropic hormone ( acth ) to produce cortisol . </S>", "<S> the disease is characterized in laboratory testing by glucocorticoid deficiency and markedly elevated acth levels . </S>", "<S> fgd may present in infancy or early childhood with symptoms related to low cortisol and high acth , such as hyperpigmentation , severe hypoglycemia , failure to thrive and recurrent infections . </S>", "<S> mutations in the mc2r accessory protein ( mrap ) cause fgd types 2 , which accounts for approximately 1520% of fgd cases . here , we report a female neonate of chinese han origin , who presented with noted hyperpigmentation at birth . </S>", "<S> laboratory investigations revealed hypocortisolaemia ( cortisol < 1.0 g / dl ) and elevated plasma acth ( 1051 pg / ml ) . </S>", "<S> she responded to hydrocortisone treatment . </S>", "<S> genetic studies confirmed the diagnosis showing homozygous deletion ( c. 106 + 1delg ) in intron 3 of mrap gene , a mutation already reported as responsible for fdg type 2 . </S>", "<S> this mutation can cause complete lack of acth response thus explaining the early presentation in this case . </S>", "<S> her parents and maternal grandmother were heterozygous for the same mutation . </S>", "<S> to our knowledge , this is the first chinese han patient reported with fgd type 2 due to a known mrap mutation . </S>" ]

[ "Introduction", "Case presentation", "Discussion" ]

[ [ "familial glucocorticoid deficiency ( fgd ) is a rare autosomal recessive disorder resulting from adrenal unresponsiveness to adrenocorticotropic hormone ( acth ) .", "this disease is characterized by low serum cortisol concentrations in the presence of grossly elevated plasma acth levels .", "affected individuals typically present with hyperpigmentation , hypoglycemic seizures , failure to thrive , failure to thrive and recurrent infections .", "causal mutations of fgd have been identified in mc2r , mrap , mcm4 , txnrd2 , star and nnt .", "mc2r accessory protein ( mrap ) , a 19-kda single - transmembrane domain protein , is essential for trafficking of mc2r from the endoplasmic reticulum ( er ) to the cell surface and subsequent signaling in response to acth .", "mutations in mrap are responsible for 1520% of fgd cases , named fgd type 2 . until now", ", this mutation has not been reported in chinese han cases of fgd . in the current paper", ", we describe a chinese han child who presented with isolated hyperpigmentation at birth and a homozygous splice mutation ( c.106 + 1delg ) in mrap ." ], [ "a female infant was referred to our neonatal unit at the age of nine days for evaluation of hyperpigmentation of the skin . she was the product of an uncomplicated pregnancy and was born by caesarian section for concerning cardiotocographic changes during labor .", "her sibling had died on the third day of life with hyperpigmentation of the skin .", "birth size was normal ( weight 2800 g , 0.99 sds ; length 46 cm , 1.69 sds ; head circumference 33 cm , 0.74 sds ) and apgar scores was 8 and 9 at 1st and 5th minute , respectively . on physical examination ,", "weight was 2740 g ( 1.31 sds ) , length 47 cm ( 2.47 sds ) , and head circumference was 34.5 cm ( 1.5 sds ) .", "endocrine investigations revealed low baseline 8 am serum cortisol ( < 1.0 g / dl ) with extremely elevated acth ( 1250 pg / ml ) .", "testosterone , 17-oh progesterone , estradiol , progesterone , luteinizing hormone , folliculin , and prolactin , free t4 , and tsh levels were normal .", "based on these findings , a familial glucocorticoid deficiency was suspected and oral hydrocortisone treatment at dose of 20 mg / m per day was started .", "hydrocortisone dose ( varying from 10 to 15 mg / m per day ) adjustment was based on acth and cortisol concentrations .", "after 4 weeks , acth level was 278.0 pg / ml and cortisol level was 42.4 g / dl , and hydrocortisone dose was reduced to 15 mg / m per day ( given in divided doses twice daily ) .", "the acth level was suppressed to slightly above the normal limit and the cortisol level is normal during the follow - up period . on her most recent visit , at the age of 12 months ,", "hydrocortisone dose was 11 mg / m per day ( 5 mg was give once daily in the morning ) , the acth level was 25.10 pg / ml and cortisol level was 33.4 g / dl , and her skin was slightly lightened ( fig .", "her weight was 10 kg ( 0.84 sds ) , length 70 cm ( 1.67 sds ) , and head circumference was 46.5 cm ( 1.12 sds ) .", "genomic dna was extracted from peripheral blood leucocytes from the infant and her family after informed consent was obtained . for the molecular diagnosis", ", a custom panel - based next - generation sequencing approach has been used to sequence all known adrenal gland diseases - associated genes in this child .", "we found a homozygous deletion of one nucleotide at the canonical 5 donor splice site ( c.106 + 1delg ) in intron 3 of mrap gene .", "this would result in the skipping of exon 3 and a prematurely terminated translation product ( ( 1 ) ) .", "her parents and maternal grandmother were found to be heterozygous carrier for the same mutation ( fig ." ], [ "we report a case of fgd with homozygous mutation of mrap in a chinese han neonate .", "this girl presented with isolated hyperpigmentation , have low cortisol and high acth with normal electrolytes . a homozygous splice mutation ( c.106 + 1delg ) of mrap confirmed the diagnosis of fgd type 2 . her parents and maternal grandmother had the same mutation .", "until now , this mutation has not been reported in chinese han cases of fgd .", "fgd is a rare autosomal recessive disorder characterized in laboratory testing by glucocorticoid deficiency and markedly elevated acth levels .", "patients with fgd usually present during neonatal period to late childhood with symptoms related to cortisol deficiency and acth excess . in the neonatal period", ", these symptoms may include hyperpigmentation , hypoglycemia , irritability , jitteriness , lethargy , respiratory distress , cyanosis , apnea , hypotonia , seizures , shock , or sudden death .", "newborns can present with a positive family history of early - unexplained infant deaths or other affected family members supports a diagnosis .", "the severe pigmentation of the skin is due to the over - stimulation of mc1r ( cutaneous msh receptors ) by high circulating msh which is a byproduct of acth synthesis from proopiomelanocortin .", "this hyperpigmentation fades once proper treatment is initiated with glucocorticoids , which reduce acth concentrations .", "this would suggest that the fetal corticotrophs could produce excessive plasma acth in response to low fetal cortisol , which in turn acts on melanocytes to promote eumelanin synthesis before birth . in most of the fgd patients suppression of acth levels", "is difficult , and therefore is not used as a goal for therapy . in this case , replacement treatment with hydrocortisone suppressed acth level to slightly above the normal limit and partially resolved the hyperpigmentation . acquired causes of adrenal insufficiency such as adrenal hemorrhage , trauma and infections were excluded by history and laboratory findings .", "congenital adrenal hyperplasia was excluded by hormone analysis ; congenital adrenal hypoplasia was also unlikely , because she had normal serum 17-oh progesterone and no mutation of the dax1 gene .", "mrap , located at 21q22.1 , is a small single - pass transmembrane domain protein , which is essential for the processing of the acth receptor ( melanocortin 2 receptor , mc2r ) and its trafficking from the er to the cell surface .", "this mutation will lead to skipping of exon 3 and early truncation of the protein and the absence of the mc2r interacting transmembrane domain . as a result", ", mc2r is retained within the er and fails to reach the cell surface which lead to acth resistance and adrenal insufficiency .", "mutations in mrap causing fgd2 were first described in 2005 . so far over 9 different mutations of mrap in fgd type 2 patients", "have been documented all of which are splice site or nonsense mutations and are predicted to produce proteins lacking the transmembrane domain essential for interaction with mc2r , . in our patient , we found a newborn with fgd type 2 , presenting with severe hyperpigmentation .", "she was found to have a splice - site mutation in the mrap gene , hence explaining the early presentation seen in the case . in their study , metherell et al . identified the 1-bp deletion , c. 106 + 1delg , in 6 individuals from 5 families with glucocorticoid deficiency , making this the second frequent mutation causing fgd unrelated to defects in the mc2r gene . to our current knowledge , 1-bp deletion ( c. 106 + 1delg ) in intron 3 of mrap gene , identified in the dna of the patient ,", "cases of the condition have been reported in white , , black , indian , and middle eastern populations . to our knowledge", ", our patient is the first reported chinese han patient with fgd type 2 , with a known mrap mutation .", "the patient was the offspring of non - consanguineous parents and her sibling had died in the neonatal period , possibly due to glucocorticoid insufficiency .", "prolonged acth excess or glucocorticoid deficiency increases linear growth , while early diagnosis and appropriate therapy in this case enabled the patient to achieve normal developmental milestones .", "further studies describing new cases and mutations causing fgd will contribute to understanding the mechanism of this rare and potentially life - threatening disease ." ] ]

[ "pulmonary arteriovenous malformation ( pavm ) , first described in 1897 by churton , is characterized by abnormal communications between pulmonary veins and arteries , and is known to disturb the filtering action of pulmonary capillaries , causing thromboembolic event in systemic circulation .", "most of the pavms have no symptoms , and are detected as abnormal shadow in the chest .", "conclusive diagnosis is generally made by means of pulmonary arteriography or three - dimensional computed tomography ( 3d - ct ) angiography .", "while hypervascular lesions can mimic pavm , we observed a rare case of a lung granuloma mimicking pavm , and performed video - assisted thoracic surgery ( vats ) .", "a 76-year - old woman , who was otherwise symptom - free , was admitted to our hospital because an abnormal shadow was detected in the left lung field on her chest x - ray .", "chest computed tomography ( ct ) revealed a 20 mm 14 mm nodule with well - defined margins and smooth contours in the left upper segment ( fig .", "an enhanced solitary lung nodule , connected with linear structures suggestive of feeding artery and drainage vein , was revealed by contrast - enhanced 3d - ct ( fig .", "arterial blood gas analysis showed hypoxemia , with arterial oxygen pressure ( pao2 ) 56 mmhg in room air .", "first , the patient was placed in the right lateral decubitus position , and the left lung was deflated .", "intraoperatively , elastic hard nodule was palpable in the left upper segment and bruit was not convincing .", "the nodule was nontortuous in shape , covered with the visceral pleura , and neither the feeding artery nor the drainage vein was detected .", "histopathological findings revealed multiple foci of coagulative necrosis surrounded by epithelioid cell granuloma containing langhans - type multinucleated giant cells , involving the medium - sized blood vessels in the pulmonary parenchyma .", "the lung granuloma was difficult to be preoperatively distinguished from pavm , because hypervascular lesion such , as inflammatory changes can present as strongly enhanced nodules after injection of contrast material .", "the patient had an uneventful postoperative course and was discharged 14 days after the operation .", "causes of acquired or secondary pavm include chest trauma , thoracic surgery , hepatic cirrhosis , infections ( actinomycosis , schistomiasis ) , metastatic carcinoma and systemic disease . twenty eight percent of cases are considered to have no symptoms .", "it was reported that when a single isolated pulmonary arteriovenous malformation is 2 cm or smaller , no symptoms appear . generally ,", "when pavm is 2 cm or more , symptoms such as breathing difficulty , cyanosis , hypoxemia , finger clubbing and polycythemia occur , and the rate of occurrence of severe complications , such as rupture of the malformation , hemoptysis , cerebral infarction and cerebral abscess , is approximately 30% .", "the patient in the present case was asymptomatic and had no history of chest trauma , surgery , infection or systemic disease .", "abnormal vascular structures , such as pavm are not convincing in histopathological findings . coincidentally , the inflammatory granuloma of the lung looked almost identical to pavm because of the nontortuous shape with linear structures suggestive of feeding artery and drainage vein .", "the resection of lung granuloma is highly successful for an isolated malformation , and mortality rate is low in this surgery .", "embolization is an appropriate treatment modality in multiple pavm for which surgery is not suitable .", "complications of embolization include pleuritic chest pain , pulmonary infection , air embolism , migration of coils and paradoxical embolism .", "although pulmonary angiography or contrast - enhanced 3d - ct has been the standard diagnostic tool for pavm , it has limitations in detecting pavms presenting as small nodules and enhanced nodules . in our case , since the enhanced lung nodule size was 22 mm in diameter , it mimicked pavm . in this patient , it was difficult to distinguish the lung granuloma from pavm , preoperatively . though there is a report which shows lung cancer mimicking pavm , to our knowledge , this is the first report of a lung granuloma mimicking pavm .", "in conclusion , clinicians should pay particular attention to enhanced nodules to rule out a variety of disorders , including neoplasm , infection , inflammatory and vascular abnormality when pavm was suspected .", "for the purpose of not only diagnosis but also for safety in treatment of pavm , the surgical approach seems appropriate .", "written informed consent was obtained from the patient for publication of this case report and accompanying images .", "a copy of the written consent is available for review by the editor - in - chief of this journal on request .", "yoshinobu ichiki : study design , data collections , data analysis , writing ; junji kawasaki : data collections ; takayuki hamatsu : data collections ; taketoshi suehiro : data collections ; makiko koike : data collections ; fumihiro tanaka : study design , data collections , data analysis ; keizo sugimachi : data collections ." ]

[ "<S> introductionwhile hypervascular lesions in the lung are known to mimic pulmonary arteriovenous malformation ( pavm ) , here we report a rare case of lung granuloma mimicking pavm , on which video - assisted thoracic surgery ( vats ) was performed.presentation of casea 76-year - old woman without any symptom was admitted to our hospital because of abnormal shadow in the left lung field on chest x - ray . </S>", "<S> a 20 mm 14 mm nodule with well - defined margins and smooth contours in the left upper segment was detected in her chest computed tomography ( ct ) . </S>", "<S> contrast - enhanced three - dimensional ct ( 3d - ct ) revealed an enhanced solitary lung nodule , which was connected with linear structures suggestive of feeding artery and drainage vein . </S>", "<S> thus , we made a preoperative diagnosis of pavm by performing partial pulmonary resection by vats . </S>", "<S> intraoperatively , elastic hard nodule was palpable in the left upper segment and bruit was not convincing . </S>", "<S> histopathological findings revealed multiple foci of coagulative necrosis surrounded by epithelioid cell granuloma containing langhans - type multinucleated giant cells , involving the medium - sized blood vessels in the pulmonary parenchyma . </S>", "<S> abnormal vascular structures , such as pavm were not convincing . </S>", "<S> based on these findings , a diagnosis of left lung granuloma was made.discussionpreoperatively , it was difficult to distinguish the left lung granuloma from pavm , because hypervascular lesion , such as inflammatory changes can present as strongly enhanced nodules after injection of contrast material.conclusionsurgical approach seems appropriate , not only for the purpose of diagnosis , but also for the safety in treatment of a pavm . </S>" ]

[ "Introduction", "Case report", "Discussion", "Conclusion", "Conflict of interest", "Funding", "Ethical approval", "Author contributions" ]

[ [ "pulmonary arteriovenous malformation ( pavm ) , first described in 1897 by churton , is characterized by abnormal communications between pulmonary veins and arteries , and is known to disturb the filtering action of pulmonary capillaries , causing thromboembolic event in systemic circulation .", "most of the pavms have no symptoms , and are detected as abnormal shadow in the chest .", "conclusive diagnosis is generally made by means of pulmonary arteriography or three - dimensional computed tomography ( 3d - ct ) angiography .", "while hypervascular lesions can mimic pavm , we observed a rare case of a lung granuloma mimicking pavm , and performed video - assisted thoracic surgery ( vats ) ." ], [ "a 76-year - old woman , who was otherwise symptom - free , was admitted to our hospital because an abnormal shadow was detected in the left lung field on her chest x - ray .", "chest computed tomography ( ct ) revealed a 20 mm 14 mm nodule with well - defined margins and smooth contours in the left upper segment ( fig .", "an enhanced solitary lung nodule , connected with linear structures suggestive of feeding artery and drainage vein , was revealed by contrast - enhanced 3d - ct ( fig .", "arterial blood gas analysis showed hypoxemia , with arterial oxygen pressure ( pao2 ) 56 mmhg in room air .", "first , the patient was placed in the right lateral decubitus position , and the left lung was deflated .", "intraoperatively , elastic hard nodule was palpable in the left upper segment and bruit was not convincing .", "the nodule was nontortuous in shape , covered with the visceral pleura , and neither the feeding artery nor the drainage vein was detected .", "histopathological findings revealed multiple foci of coagulative necrosis surrounded by epithelioid cell granuloma containing langhans - type multinucleated giant cells , involving the medium - sized blood vessels in the pulmonary parenchyma .", "the lung granuloma was difficult to be preoperatively distinguished from pavm , because hypervascular lesion such , as inflammatory changes can present as strongly enhanced nodules after injection of contrast material .", "the patient had an uneventful postoperative course and was discharged 14 days after the operation ." ], [ "causes of acquired or secondary pavm include chest trauma , thoracic surgery , hepatic cirrhosis , infections ( actinomycosis , schistomiasis ) , metastatic carcinoma and systemic disease . twenty eight percent of cases are considered to have no symptoms .", "it was reported that when a single isolated pulmonary arteriovenous malformation is 2 cm or smaller , no symptoms appear . generally ,", "when pavm is 2 cm or more , symptoms such as breathing difficulty , cyanosis , hypoxemia , finger clubbing and polycythemia occur , and the rate of occurrence of severe complications , such as rupture of the malformation , hemoptysis , cerebral infarction and cerebral abscess , is approximately 30% .", "the patient in the present case was asymptomatic and had no history of chest trauma , surgery , infection or systemic disease .", "abnormal vascular structures , such as pavm are not convincing in histopathological findings . coincidentally , the inflammatory granuloma of the lung looked almost identical to pavm because of the nontortuous shape with linear structures suggestive of feeding artery and drainage vein .", "the resection of lung granuloma is highly successful for an isolated malformation , and mortality rate is low in this surgery .", "embolization is an appropriate treatment modality in multiple pavm for which surgery is not suitable .", "complications of embolization include pleuritic chest pain , pulmonary infection , air embolism , migration of coils and paradoxical embolism .", "although pulmonary angiography or contrast - enhanced 3d - ct has been the standard diagnostic tool for pavm , it has limitations in detecting pavms presenting as small nodules and enhanced nodules . in our case , since the enhanced lung nodule size was 22 mm in diameter , it mimicked pavm . in this patient , it was difficult to distinguish the lung granuloma from pavm , preoperatively . though there is a report which shows lung cancer mimicking pavm , to our knowledge , this is the first report of a lung granuloma mimicking pavm ." ], [ "in conclusion , clinicians should pay particular attention to enhanced nodules to rule out a variety of disorders , including neoplasm , infection , inflammatory and vascular abnormality when pavm was suspected .", "for the purpose of not only diagnosis but also for safety in treatment of pavm , the surgical approach seems appropriate ." ], [ "" ], [ "" ], [ "written informed consent was obtained from the patient for publication of this case report and accompanying images .", "a copy of the written consent is available for review by the editor - in - chief of this journal on request ." ], [ "yoshinobu ichiki : study design , data collections , data analysis , writing ; junji kawasaki : data collections ; takayuki hamatsu : data collections ; taketoshi suehiro : data collections ; makiko koike : data collections ; fumihiro tanaka : study design , data collections , data analysis ; keizo sugimachi : data collections ." ] ]

[ "although chronic hepatitis c ( chc ) in children is usually asymptomatic , it is often persistent and is a possible cause of morbidity in later life . moreover ,", "progressive liver disease , including cirrhosis , has been reported during childhood . although new direct - acting antivirals ( daas ) are now the cornerstones in the treatment of hcv infection in adults , combination therapy with pegylated interferon ( peg - ifn ) alpha and ribavirin ( rbv ) ( peg / rbv therapy ) is still the current standard therapy in children [ 2 , 3 ] .", "although this regimen was found to be highly effective in children with genotypes 2 and 3 , it has limited efficacy for those with genotype 4 [ 5 , 6 ] , the prevalent genotype in egypt .", "therefore , the ability to predict response to this costly with many side effects antiviral regimen remains an important research goal . in spite of", "the extensive researches in this field , knowledge is still defective . on defining predictors of treatment response , many studies concentrated on virus genotype , gender , age , race , and fibrosis stage , among others . nevertheless ,", "all of these are nonmodifiable factors but only give a prediction about the likelihood of sustained virologic response ( svr ) . moreover , some of them are expensive and invasive . on investigating the role of interleukin-28b ( il28b ) polymorphism in pediatric patients with chc ,", "one study suggested that il28b genotype was not a strong predictor of svr , while other studies have shown usefulness of il28b genotype as a predictor of treatment response [ 1012 ] .", "virological responses during therapy , such as rapid virologic response ( rvr ) and early virologic response ( evr ) , are widely used for predicting end of treatment response ( etr ) and svr . nonetheless , it is obvious that predictions made before administration of therapy are more desirable than those done during treatment course .", "we aimed to investigate some inexpensive , easy to perform , and noninvasive modifiable ( as adiponectin and vitamin d ) and nonmodifiable ( as alpha - fetoprotein afp ) factors which may have a relation to treatment response in children with chc .", "if we found a significant relation to successful treatment , we can use them not just to predict the likelihood of treatment response but also to improve the svr rates by modulating the modifiable ones .", "this knowledge can also constitute a base for later on researches when daa became approved in pediatric age .", "this prospective cohort study included fifty children with chc recruited from pediatric hepatology department , national liver institute ( a tertiary level institute ) , menofiya university , egypt , between june 2012 and june 2014 .", "their mean age was 11.46 3.48 years and thirty three ( 66% ) of them were males .", "another 21 healthy children with comparable age ( mean ; 9.14 3.31 years ) and sex ( males ; 13 61.9% ) to the disease group were enrolled as controls .", "chc was diagnosed on the basis of positive anti - hcv antibodies and positive hcv - rna for more than 6 months duration , together with the pathological picture of chc . any case with associated liver disease ( either identified by laboratory or histological examination ) or out of the inclusion criteria for peg / rbv therapy , as defined by el naghi et al .", "a signed informed consent was obtained from parents of all recruited children before enrollment in the study .", "the study was approved by the research ethics committee of the national liver institute , menofiya university , and conforms to the 1975 declaration of helsinki and its later amendments .", "alanine transaminase ( alt ) , aspartate transaminase ( ast ) , prothrombin time ( pt ) , complete blood count ( cbc ) , thyroid stimulating hormone ( tsh ) , and serum autoantibodies ( anti - nuclear antibodies , anti - smooth muscle antibodies , and liver - kidney microsomal antibodies ) were performed for every patient .", "viral markers ( anti - hcv , hepatitis b virus ( hbv ) surface antigen , hbv core immunoglobulin ( ig)m , and igg antibodies ) were performed for all patients using chemiluminescence immunoassay ( roche diagnostic inc . ,", "real - time pcr for hcv - rna was performed using abbott m2000 real - time system ( abbott molecular inc .", ", des plaines , illinois , usa ) for every patient before starting therapy and at week 12 of therapy . for those who continued therapy ,", "abdominal ultrasound was performed using 25 mhz curved linear and 48 mhz linear transducers ( xario xg ; toshiba , tokyo , japan ) .", "an ultrasonographic - guided liver biopsy was performed for all patients using a true - cut needle , size 16 g. biopsy specimens were fixed in formalin , embedded in paraffin , and finally the slides obtained were stained by hematoxylin and eosin ( h&e ) , perls , and orcein for routine histopathological evaluation .", "grades of necroinflammatory activity 13 were ascribed as minimal , grades 47 as mild , and grades 812 as moderate , whereas grades > 13 were ascribed as severe chronic hepatitis .", "stages of fibrosis of 0 - 1 indicated absent / minimal fibrosis , stages 2 - 3 indicated significant fibrosis , and stages 46 indicated advanced fibrosis .", "all children with chc received peg - ifn-2b ( pegintron ; schering - plough , new jersey , usa ) at a dosage of 60 g / m / week subcutaneously and rbv orally at a dosage of 15 mg / kg / day on two divided doses .", "thirty - six ( 72% ) children attained etr and svr with no relapses , while 14 ( 28% ) children were nonresponders .", "serum samples were collected from every patient before starting peg / rbv therapy and from healthy controls then stored at 80c until used .", "serum adiponectin level was determined by the human adiponectin ( acrp30 ) enzyme - linked immunosorbent assay ( elisa ) kit ( orgenium laboratories , vantaa finland ) .", "serum vitamin d was measured using 25-oh vitamin d enzyme - immunoassay ( eia ) kit ( immundiagnostik , bensheim and biomedica , wien austria ) intended for the quantitative determination of the 25-oh vitamin d in plasma or serum .", "lastly , serum afp was measured by quantikine , a human afp eia kit ( r&d systems inc . , minneapolis , usa ) .", "serum levels of the three test parameters were expressed as nanograms per milliliter ( ng / ml ) .", "values were expressed as mean standard deviation ( range ) or number ( percentage ) of individuals with a condition . for quantitative data ,", "statistical significance was tested by either independent samples t - test or nonparametric mann - whitney u test according to the nature of the data . for qualitative data ,", "a multivariate analysis was performed using a binary logistic regression analysis for factors that significantly associated with treatment response on univariate analysis .", "the cutoff values for optimal clinical performance of adiponectin , vitamin d , afp , and level of hcv - rna for differentiation between responders and nonresponders were determined from the receiver - operating characteristic ( roc ) curves .", "the diagnostic performance was presented as sensitivity , specificity , negative predictive value ( npv ) , positive predictive value ( ppv ) , and accuracy .", "body mass index of children with chc was within average with a mean of 18.56 2.79 kg / m .", "level of hepatitis c viremia ranged from 8.04 10 to 6.23 10 with a mean of 6.28 10 1.23 10 iu / ml .", "forty - five ( 90% ) patients with chc showed mild stage of fibrosis and 5 ( 10% ) cases showed moderate fibrosis .", "most of ( 86% ) chc children had mild activity , while 7 ( 14% ) cases had minimal activity .", "serum adiponectin was significantly higher in the chc group than healthy controls ( 8.92 2.85 and 6.049 1.04 ", "ng / ml , resp . ; p < 0.0001 ) . also , in spite of being insignificant , serum vitamin d and afp were higher in the chc group ( 71.6 49.1 ng / ml and 3.6 2.96 ng / ml , resp . ) than healthy controls ( 46.4 21.8 ng / ml and 3.0 0.39 ng / ml , resp . )", "pretreatment factors that could be associated with response to peg / rbv therapy were compared between treatment responders ( svr ) and treatment nonresponders ( figure 2 ) .", "it was found that adiponectin was significantly higher in those with svr ( 9.79 2.7 versus 6.69 1.77 ng / ml ; p < 0.0001 ) . on the other hand", "it was found that both afp and viremia were significantly lower in the treatment responders ( 2.84 0.51 ng / ml and 4.18 10 1.03 10 iu / ml , resp . ) than in nonresponders ( 5.69 5.1 ng / ml and 1.16 10 1.57 10 iu / ml , resp . ) with p < 0.0001 and p = 0.0003 , respectively .", "lastly , vitamin d was found to be higher in the treatment responders ( 77.2 46.6 ng / ml ) than nonresponders ( 57.2 53.9 ng / ml ) , with borderline significance , p = 0.076 .", "other studied pretreatment parameters showed no difference between responders and nonresponders ( table 1 ) , while in multivariate analysis adiponectin was shown to be the only significant independent predictor of treatment response ( p = 0.044 ) ( table 3 ) .", "cutoff points for variables showing significant associations with treatment response were analyzed by the roc curves ( figure 3 ) .", "for adiponectin it was found that at a cutoff value of > 8.04 ng / ml , it can predict treatment response by 77.8% sensitivity , 92.9% specificity , 96.6% ppv , 61.9% npv , and 82.3% accuracy , while afp and hcv - rna at cutoff values < 3.265 ng / ml and < 235,384 iu / ml , respectively , can predict treatment response with a sensitivity of 83.3% and 83.3% , specificity of 85.7% and 78.6% , ppv of 93.75% and 90.9% , npv of 66.7% and 64.7% , and accuracy of 82.36% and 79.38% , respectively .", "adiponectin was found to be significantly negatively correlated with both afp ( r = 0.29 and p = 0.043 ) and level of viremia ( r = 0.39 and p = 0.005 ) , with no significant correlation with other studied parameters .", "also , there was no significant correlation between afp , vitamin d , and level of viremia and all other studied parameters ( table 2 ) .", "this study is , to our knowledge , the first to show the predictive value of baseline serum levels of adiponectin , vitamin d , and afp for the treatment response of chc in children .", "it is a protein hormone that modulates a number of metabolic processes including glucose and fatty acid catabolism .", "the anti - inflammatory effects of adiponectin could protect the liver from the development of inflammation and cell injury . in the present work , the significantly higher adiponectin in the chc group than the control group may be due to an anti - inflammatory role of adiponectin in those with chc . in previous studies ,", "adiponectin was found to directly affect the inflammatory response by regulating both production and activity of cytokines .", "in addition , hypoadiponectinemia has been reported to enhance hepatic steatosis , inflammation , fibrosis , and hepatocarcinogenesis in animal models of liver diseases [ 1719 ] . moreover ,", "nonalcoholic steatohepatitis patients show lower levels of adiponectin with higher grades of inflammation . in this work ,", "the pretreatment serum level of adiponectin was significantly higher in the treatment responders ( svr ) than nonresponders , and at a cutoff value of > 8.04 ng / ml it can predict the treatment response by a sensitivity of 77.8% and a specificity of 92.9% .", ". found that lower adiponectin was an independent predictor of no virological response at the end of treatment ( p < 0.001 ) .", "this may indicate the benefit of the anti - inflammatory role of adiponectin in those with chc .", "adiponectin administration , in both alcoholic and nonalcoholic fatty liver in mice , was found to suppress hepatic production and the circulating levels of tumor necrosis factor- and ameliorates hepatic steatosis .", "moreover , the significant negative correlation found in this work between serum adiponectin and viremia may indicate an antiviral role of adiponectin . on the other hand", "this later concept was suggested by the study of zografos et al . who found a significant increase of adiponectin at the end of hcv treatment for those with etr .", "abdel latif et al . found that serum adiponectin levels were lower in hcv - infected patients with steatosis than in those without steatosis and these levels tend to decrease with the increase in the grade of steatosis , the advance in the grade of histological activity , and the stage of fibrosis . in our work", ", there was no significant correlation between adiponectin and stage of fibrosis or grade of necroinflammatory activity .", "this difference may relate to the difference in age range and the relatively mild histological affection of our chc group . according to the previous results , not only", "can adiponectin be used as a reliable pretreatment predictor of treatment response in combination with other defined parameters but also it can be tried as an adjuvant therapy with peg / rbv especially for those with pretreatment lower serum levels . to prove this", ", it needs a well controlled clinical trial . beside its action in calcium homeostasis , vitamin d has a significant immunomodulatory action and is an important mediator of innate and adaptive immune systems . in spite of many researches ,", "generally , in relation to vitamin d synthesis in the liver , mild to moderate liver dysfunction causes malabsorption of vitamin d. moreover , liver dysfunction of 90% or more results in inability to make sufficient 25-oh vitamin d .", "some researchers showed that adults with chc have higher incidence of severe 25-oh vitamin d deficiency compared to the normal control . on the contrary , in the present study we found that vitamin d was higher in children with chc than normal controls with borderline significance ( p = 0.071 ) .", "study may be due to younger age and milder liver affection in our children compared to their adult population .", "hypothetically , this reported vitamin d increase in children with chc reflects a possible antiviral role of vitamin d. this is supported by matsumura et al . who demonstrated in in vitro study that 25-oh vitamin d is an anti - hcv agent that targets viral particle assembly step .", ". found that low vitamin d serum level is related to severe fibrosis in adults with chc .", "however , we did not find significant correlation between vitamin d and any of the stage of fibrosis or grade of activity or any other studied pretreatment parameter .", "however , no previous data are available for the pediatric age group . in the present work", ", a higher vitamin d level was found in hcv treatment responders than nonresponders but with borderline significance .", ", bitetto et al . , and nimer and mouch detected an association between lower vitamin d serum levels and failure to achieve svr in adults with chc .", "on the other hand , lange et al . found that pretreatment serum level of vitamin d is not an optimal predictor of treatment response in hcv genotype 1 .", "hypothetically , in the presence of vitamin d deficiency , it might be preferable to correct the deficiency before starting antiviral therapy .", "nevertheless , to date there are few published reports on the role of vitamin d supplementation in patients with chc .", ". found that vitamin d supplementation , in adults with recurrent hepatitis c postliver transplant , improves the probability of achieving a svr . also , nimer and mouch found that adding vitamin d to conventional peg / rbv therapy for patients with hcv genotypes 2 - 3 significantly improved viral response", "the reported higher vitamin d among treatment responders in the present study together with the known immunomodulatory action of vitamin d and previous clinical trials of vitamin d supplementation in adults [ 29 , 30 ] suggest that adding vitamin d to peg / rbv therapy in children may increase svr rates without serious adverse events .", "however , to prove these findings , well designed and large prospective studies are needed .", "following birth , afp levels decrease rapidly to less than 20 ng / ml and increase significantly in certain pathologic conditions .", ", significant elevations of afp are commonly seen in nonhepatic malignancies and benign conditions , such as acute and chronic viral hepatitis .", "previous studies reported that the prevalence of increased serum afp varies from 10% to 43% in adult patients with chc and suggested an association between an increased serum afp and advanced fibrosis or cirrhosis [ 3336 ] . in agreement with the above reports", ", we found that afp was higher in children with chc than healthy controls with statistically insignificant difference .", "the absence of a significant correlation between afp and stage of fibrosis in the present work may be due to the milder liver affection with low stages of fibrosis in reported cases when compared to the adult series .", "some studies found significant correlation of pretreatment low afp serum level and treatment response in adults with chc [ 3739 ] . in accordance with the previous studies", ", we found that lower serum afp was significantly related to svr in children with chc but with borderline significance in multivariate analysis .", "the svr was 93.75% among children with afp below 3.265 ng / ml and 33.3% for children with afp 3.265 ", "increased production of afp in hepatitis and cirrhosis was first thought to reflect the process of surviving hepatocytes , but this hypothesis has been refuted by other reports [ 33 , 40 ] .", "more recent hypothesis ascribes the increased serum afp to the hepatic damage per se with selective transcriptional activation of the afp gene . in the present study ,", "the reported negative correlation between adiponectin with its known hepatoprotective role and afp ( p = 0.043 ) is supporting this hypothesis and may give an explanation to the link between the baseline serum afp and the treatment response .", "in the present study viral load in univariate analysis was found to be a significant pretreatment predictor of treatment response as it was shown in previous reports .", "however , in multivariate analysis , it was not an independent predictor of treatment response .", "the emergence of the new daas for the treatment of chc [ 41 , 42 ] may be considered a limitation in the current study .", "however , peg / rbv is the still approved standard therapy for children with chc [ 5 , 43 ] .", "moreover , peg - ifn and rbv are still included with daas in the treatment regimens of those with genotype 4 , the prevalent genotype in egypt .", "a major limitation of this study is the low number of cases and controls enrolled .", "also , absence of testing for hcv genotype and il28b genotype could be considered another limitation of this work .", "in conclusion , serum adiponectin can be added to the list of the pretreatment determinants of svr in children with chc , with the advantage of being easy to perform , noninvasive , and modifiable . in spite of its significant prediction for the likelihood of response", ", we can not state to screen and select the patients to be treated or not based on their pretreatment levels of this predictor .", "instead , it can be used to prioritize patients to treatment when resources are limited , thus avoiding toxicities and cost for those unlikely to respond to treatment . also as it is a modifiable factor , supplementation for deficient cases" ]

[ "<S> \n background & aims . </S>", "<S> the currently available treatment for chronic hepatitis c ( chc ) in children is costly and with much toxicity . </S>", "<S> so , predicting the likelihood of response before starting therapy is important </S>", "<S> . methods . </S>", "<S> serum adiponectin , vitamin d , and alpha - fetoprotein ( afp ) were measured before starting pegylated - interferon / ribavirin therapy for 50 children with chc . </S>", "<S> another 21 healthy children were recruited as controls . </S>", "<S> results . </S>", "<S> serum adiponectin , vitamin d , and afp were higher in the chc group than healthy controls ( p < 0.0001 , p = 0.071 , and p = 0.87 , resp . ) . </S>", "<S> in univariate analysis , serum adiponectin was significantly higher in responders than nonresponders ( p < 0.0001 ) and at a cutoff value 8.04 ng / ml it can predict treatment response by 77.8% sensitivity and 92.9% specificity , while both afp and viremia were significantly lower in responders than nonresponders , p < 0.0001 and p = 0.0003 , respectively , and at cutoff values 3.265 ng / ml and 235,384 iu / ml , respectively , they can predict treatment response with a sensitivity of 83.3% for both and specificity of 85.7% and 78.6% , respectively . in multivariate analysis , </S>", "<S> adiponectin was found to be the only independent predictor of treatment response ( p = 0.044 ) . </S>", "<S> conclusions . </S>", "<S> the pretreatment serum level of adiponectin can predict the likelihood of treatment response , thus avoiding toxicities for those unlikely to respond to therapy . </S>" ]

[ "1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusions" ]

[ [ "although chronic hepatitis c ( chc ) in children is usually asymptomatic , it is often persistent and is a possible cause of morbidity in later life . moreover ,", "progressive liver disease , including cirrhosis , has been reported during childhood . although new direct - acting antivirals ( daas ) are now the cornerstones in the treatment of hcv infection in adults , combination therapy with pegylated interferon ( peg - ifn ) alpha and ribavirin ( rbv ) ( peg / rbv therapy ) is still the current standard therapy in children [ 2 , 3 ] .", "although this regimen was found to be highly effective in children with genotypes 2 and 3 , it has limited efficacy for those with genotype 4 [ 5 , 6 ] , the prevalent genotype in egypt .", "therefore , the ability to predict response to this costly with many side effects antiviral regimen remains an important research goal . in spite of", "the extensive researches in this field , knowledge is still defective . on defining predictors of treatment response , many studies concentrated on virus genotype , gender , age , race , and fibrosis stage , among others . nevertheless ,", "all of these are nonmodifiable factors but only give a prediction about the likelihood of sustained virologic response ( svr ) . moreover , some of them are expensive and invasive . on investigating the role of interleukin-28b ( il28b ) polymorphism in pediatric patients with chc ,", "one study suggested that il28b genotype was not a strong predictor of svr , while other studies have shown usefulness of il28b genotype as a predictor of treatment response [ 1012 ] .", "virological responses during therapy , such as rapid virologic response ( rvr ) and early virologic response ( evr ) , are widely used for predicting end of treatment response ( etr ) and svr . nonetheless , it is obvious that predictions made before administration of therapy are more desirable than those done during treatment course .", "we aimed to investigate some inexpensive , easy to perform , and noninvasive modifiable ( as adiponectin and vitamin d ) and nonmodifiable ( as alpha - fetoprotein afp ) factors which may have a relation to treatment response in children with chc .", "if we found a significant relation to successful treatment , we can use them not just to predict the likelihood of treatment response but also to improve the svr rates by modulating the modifiable ones .", "this knowledge can also constitute a base for later on researches when daa became approved in pediatric age ." ], [ "this prospective cohort study included fifty children with chc recruited from pediatric hepatology department , national liver institute ( a tertiary level institute ) , menofiya university , egypt , between june 2012 and june 2014 .", "their mean age was 11.46 3.48 years and thirty three ( 66% ) of them were males .", "another 21 healthy children with comparable age ( mean ; 9.14 3.31 years ) and sex ( males ; 13 61.9% ) to the disease group were enrolled as controls .", "chc was diagnosed on the basis of positive anti - hcv antibodies and positive hcv - rna for more than 6 months duration , together with the pathological picture of chc . any case with associated liver disease ( either identified by laboratory or histological examination ) or out of the inclusion criteria for peg / rbv therapy , as defined by el naghi et al .", "a signed informed consent was obtained from parents of all recruited children before enrollment in the study .", "the study was approved by the research ethics committee of the national liver institute , menofiya university , and conforms to the 1975 declaration of helsinki and its later amendments .", "alanine transaminase ( alt ) , aspartate transaminase ( ast ) , prothrombin time ( pt ) , complete blood count ( cbc ) , thyroid stimulating hormone ( tsh ) , and serum autoantibodies ( anti - nuclear antibodies , anti - smooth muscle antibodies , and liver - kidney microsomal antibodies ) were performed for every patient .", "viral markers ( anti - hcv , hepatitis b virus ( hbv ) surface antigen , hbv core immunoglobulin ( ig)m , and igg antibodies ) were performed for all patients using chemiluminescence immunoassay ( roche diagnostic inc . ,", "real - time pcr for hcv - rna was performed using abbott m2000 real - time system ( abbott molecular inc .", ", des plaines , illinois , usa ) for every patient before starting therapy and at week 12 of therapy . for those who continued therapy ,", "abdominal ultrasound was performed using 25 mhz curved linear and 48 mhz linear transducers ( xario xg ; toshiba , tokyo , japan ) .", "an ultrasonographic - guided liver biopsy was performed for all patients using a true - cut needle , size 16 g. biopsy specimens were fixed in formalin , embedded in paraffin , and finally the slides obtained were stained by hematoxylin and eosin ( h&e ) , perls , and orcein for routine histopathological evaluation .", "grades of necroinflammatory activity 13 were ascribed as minimal , grades 47 as mild , and grades 812 as moderate , whereas grades > 13 were ascribed as severe chronic hepatitis .", "stages of fibrosis of 0 - 1 indicated absent / minimal fibrosis , stages 2 - 3 indicated significant fibrosis , and stages 46 indicated advanced fibrosis .", "all children with chc received peg - ifn-2b ( pegintron ; schering - plough , new jersey , usa ) at a dosage of 60 g / m / week subcutaneously and rbv orally at a dosage of 15 mg / kg / day on two divided doses .", "thirty - six ( 72% ) children attained etr and svr with no relapses , while 14 ( 28% ) children were nonresponders .", "serum samples were collected from every patient before starting peg / rbv therapy and from healthy controls then stored at 80c until used .", "serum adiponectin level was determined by the human adiponectin ( acrp30 ) enzyme - linked immunosorbent assay ( elisa ) kit ( orgenium laboratories , vantaa finland ) .", "serum vitamin d was measured using 25-oh vitamin d enzyme - immunoassay ( eia ) kit ( immundiagnostik , bensheim and biomedica , wien austria ) intended for the quantitative determination of the 25-oh vitamin d in plasma or serum .", "lastly , serum afp was measured by quantikine , a human afp eia kit ( r&d systems inc . , minneapolis , usa ) .", "serum levels of the three test parameters were expressed as nanograms per milliliter ( ng / ml ) .", "values were expressed as mean standard deviation ( range ) or number ( percentage ) of individuals with a condition . for quantitative data ,", "statistical significance was tested by either independent samples t - test or nonparametric mann - whitney u test according to the nature of the data . for qualitative data ,", "a multivariate analysis was performed using a binary logistic regression analysis for factors that significantly associated with treatment response on univariate analysis .", "the cutoff values for optimal clinical performance of adiponectin , vitamin d , afp , and level of hcv - rna for differentiation between responders and nonresponders were determined from the receiver - operating characteristic ( roc ) curves .", "the diagnostic performance was presented as sensitivity , specificity , negative predictive value ( npv ) , positive predictive value ( ppv ) , and accuracy ." ], [ "body mass index of children with chc was within average with a mean of 18.56 2.79 kg / m .", "level of hepatitis c viremia ranged from 8.04 10 to 6.23 10 with a mean of 6.28 10 1.23 10 iu / ml .", "forty - five ( 90% ) patients with chc showed mild stage of fibrosis and 5 ( 10% ) cases showed moderate fibrosis .", "most of ( 86% ) chc children had mild activity , while 7 ( 14% ) cases had minimal activity .", "serum adiponectin was significantly higher in the chc group than healthy controls ( 8.92 2.85 and 6.049 1.04 ", "ng / ml , resp . ; p < 0.0001 ) . also , in spite of being insignificant , serum vitamin d and afp were higher in the chc group ( 71.6 49.1 ng / ml and 3.6 2.96 ng / ml , resp . ) than healthy controls ( 46.4 21.8 ng / ml and 3.0 0.39 ng / ml , resp . )", "pretreatment factors that could be associated with response to peg / rbv therapy were compared between treatment responders ( svr ) and treatment nonresponders ( figure 2 ) .", "it was found that adiponectin was significantly higher in those with svr ( 9.79 2.7 versus 6.69 1.77 ng / ml ; p < 0.0001 ) . on the other hand", "it was found that both afp and viremia were significantly lower in the treatment responders ( 2.84 0.51 ng / ml and 4.18 10 1.03 10 iu / ml , resp . ) than in nonresponders ( 5.69 5.1 ng / ml and 1.16 10 1.57 10 iu / ml , resp . ) with p < 0.0001 and p = 0.0003 , respectively .", "lastly , vitamin d was found to be higher in the treatment responders ( 77.2 46.6 ng / ml ) than nonresponders ( 57.2 53.9 ng / ml ) , with borderline significance , p = 0.076 .", "other studied pretreatment parameters showed no difference between responders and nonresponders ( table 1 ) , while in multivariate analysis adiponectin was shown to be the only significant independent predictor of treatment response ( p = 0.044 ) ( table 3 ) .", "cutoff points for variables showing significant associations with treatment response were analyzed by the roc curves ( figure 3 ) .", "for adiponectin it was found that at a cutoff value of > 8.04 ng / ml , it can predict treatment response by 77.8% sensitivity , 92.9% specificity , 96.6% ppv , 61.9% npv , and 82.3% accuracy , while afp and hcv - rna at cutoff values < 3.265 ng / ml and < 235,384 iu / ml , respectively , can predict treatment response with a sensitivity of 83.3% and 83.3% , specificity of 85.7% and 78.6% , ppv of 93.75% and 90.9% , npv of 66.7% and 64.7% , and accuracy of 82.36% and 79.38% , respectively .", "adiponectin was found to be significantly negatively correlated with both afp ( r = 0.29 and p = 0.043 ) and level of viremia ( r = 0.39 and p = 0.005 ) , with no significant correlation with other studied parameters .", "also , there was no significant correlation between afp , vitamin d , and level of viremia and all other studied parameters ( table 2 ) ." ], [ "this study is , to our knowledge , the first to show the predictive value of baseline serum levels of adiponectin , vitamin d , and afp for the treatment response of chc in children .", "it is a protein hormone that modulates a number of metabolic processes including glucose and fatty acid catabolism .", "the anti - inflammatory effects of adiponectin could protect the liver from the development of inflammation and cell injury . in the present work , the significantly higher adiponectin in the chc group than the control group may be due to an anti - inflammatory role of adiponectin in those with chc . in previous studies ,", "adiponectin was found to directly affect the inflammatory response by regulating both production and activity of cytokines .", "in addition , hypoadiponectinemia has been reported to enhance hepatic steatosis , inflammation , fibrosis , and hepatocarcinogenesis in animal models of liver diseases [ 1719 ] . moreover ,", "nonalcoholic steatohepatitis patients show lower levels of adiponectin with higher grades of inflammation . in this work ,", "the pretreatment serum level of adiponectin was significantly higher in the treatment responders ( svr ) than nonresponders , and at a cutoff value of > 8.04 ng / ml it can predict the treatment response by a sensitivity of 77.8% and a specificity of 92.9% .", ". found that lower adiponectin was an independent predictor of no virological response at the end of treatment ( p < 0.001 ) .", "this may indicate the benefit of the anti - inflammatory role of adiponectin in those with chc .", "adiponectin administration , in both alcoholic and nonalcoholic fatty liver in mice , was found to suppress hepatic production and the circulating levels of tumor necrosis factor- and ameliorates hepatic steatosis .", "moreover , the significant negative correlation found in this work between serum adiponectin and viremia may indicate an antiviral role of adiponectin . on the other hand", "this later concept was suggested by the study of zografos et al . who found a significant increase of adiponectin at the end of hcv treatment for those with etr .", "abdel latif et al . found that serum adiponectin levels were lower in hcv - infected patients with steatosis than in those without steatosis and these levels tend to decrease with the increase in the grade of steatosis , the advance in the grade of histological activity , and the stage of fibrosis . in our work", ", there was no significant correlation between adiponectin and stage of fibrosis or grade of necroinflammatory activity .", "this difference may relate to the difference in age range and the relatively mild histological affection of our chc group . according to the previous results , not only", "can adiponectin be used as a reliable pretreatment predictor of treatment response in combination with other defined parameters but also it can be tried as an adjuvant therapy with peg / rbv especially for those with pretreatment lower serum levels . to prove this", ", it needs a well controlled clinical trial . beside its action in calcium homeostasis , vitamin d has a significant immunomodulatory action and is an important mediator of innate and adaptive immune systems . in spite of many researches ,", "generally , in relation to vitamin d synthesis in the liver , mild to moderate liver dysfunction causes malabsorption of vitamin d. moreover , liver dysfunction of 90% or more results in inability to make sufficient 25-oh vitamin d .", "some researchers showed that adults with chc have higher incidence of severe 25-oh vitamin d deficiency compared to the normal control . on the contrary , in the present study we found that vitamin d was higher in children with chc than normal controls with borderline significance ( p = 0.071 ) .", "study may be due to younger age and milder liver affection in our children compared to their adult population .", "hypothetically , this reported vitamin d increase in children with chc reflects a possible antiviral role of vitamin d. this is supported by matsumura et al . who demonstrated in in vitro study that 25-oh vitamin d is an anti - hcv agent that targets viral particle assembly step .", ". found that low vitamin d serum level is related to severe fibrosis in adults with chc .", "however , we did not find significant correlation between vitamin d and any of the stage of fibrosis or grade of activity or any other studied pretreatment parameter .", "however , no previous data are available for the pediatric age group . in the present work", ", a higher vitamin d level was found in hcv treatment responders than nonresponders but with borderline significance .", ", bitetto et al . , and nimer and mouch detected an association between lower vitamin d serum levels and failure to achieve svr in adults with chc .", "on the other hand , lange et al . found that pretreatment serum level of vitamin d is not an optimal predictor of treatment response in hcv genotype 1 .", "hypothetically , in the presence of vitamin d deficiency , it might be preferable to correct the deficiency before starting antiviral therapy .", "nevertheless , to date there are few published reports on the role of vitamin d supplementation in patients with chc .", ". found that vitamin d supplementation , in adults with recurrent hepatitis c postliver transplant , improves the probability of achieving a svr . also , nimer and mouch found that adding vitamin d to conventional peg / rbv therapy for patients with hcv genotypes 2 - 3 significantly improved viral response", "the reported higher vitamin d among treatment responders in the present study together with the known immunomodulatory action of vitamin d and previous clinical trials of vitamin d supplementation in adults [ 29 , 30 ] suggest that adding vitamin d to peg / rbv therapy in children may increase svr rates without serious adverse events .", "however , to prove these findings , well designed and large prospective studies are needed .", "following birth , afp levels decrease rapidly to less than 20 ng / ml and increase significantly in certain pathologic conditions .", ", significant elevations of afp are commonly seen in nonhepatic malignancies and benign conditions , such as acute and chronic viral hepatitis .", "previous studies reported that the prevalence of increased serum afp varies from 10% to 43% in adult patients with chc and suggested an association between an increased serum afp and advanced fibrosis or cirrhosis [ 3336 ] . in agreement with the above reports", ", we found that afp was higher in children with chc than healthy controls with statistically insignificant difference .", "the absence of a significant correlation between afp and stage of fibrosis in the present work may be due to the milder liver affection with low stages of fibrosis in reported cases when compared to the adult series .", "some studies found significant correlation of pretreatment low afp serum level and treatment response in adults with chc [ 3739 ] . in accordance with the previous studies", ", we found that lower serum afp was significantly related to svr in children with chc but with borderline significance in multivariate analysis .", "the svr was 93.75% among children with afp below 3.265 ng / ml and 33.3% for children with afp 3.265 ", "increased production of afp in hepatitis and cirrhosis was first thought to reflect the process of surviving hepatocytes , but this hypothesis has been refuted by other reports [ 33 , 40 ] .", "more recent hypothesis ascribes the increased serum afp to the hepatic damage per se with selective transcriptional activation of the afp gene . in the present study ,", "the reported negative correlation between adiponectin with its known hepatoprotective role and afp ( p = 0.043 ) is supporting this hypothesis and may give an explanation to the link between the baseline serum afp and the treatment response .", "in the present study viral load in univariate analysis was found to be a significant pretreatment predictor of treatment response as it was shown in previous reports .", "however , in multivariate analysis , it was not an independent predictor of treatment response .", "the emergence of the new daas for the treatment of chc [ 41 , 42 ] may be considered a limitation in the current study .", "however , peg / rbv is the still approved standard therapy for children with chc [ 5 , 43 ] .", "moreover , peg - ifn and rbv are still included with daas in the treatment regimens of those with genotype 4 , the prevalent genotype in egypt .", "a major limitation of this study is the low number of cases and controls enrolled .", "also , absence of testing for hcv genotype and il28b genotype could be considered another limitation of this work ." ], [ "in conclusion , serum adiponectin can be added to the list of the pretreatment determinants of svr in children with chc , with the advantage of being easy to perform , noninvasive , and modifiable . in spite of its significant prediction for the likelihood of response", ", we can not state to screen and select the patients to be treated or not based on their pretreatment levels of this predictor .", "instead , it can be used to prioritize patients to treatment when resources are limited , thus avoiding toxicities and cost for those unlikely to respond to treatment . also as it is a modifiable factor , supplementation for deficient cases" ] ]

[ "radical prostatectomy ( rp ) is considered to be a standard curative procedure for patients with organ - confined prostate cancer .", "recently , the advent of pioneering nerve - sparing robot - assisted laparoscopic radical prostatectomy ( ns ralp ) has significantly increased the potency rate after rp [ 1 - 3 ] . however , even if ns ralp is performed , a large number of patients still experience erectile dysfunction ( ed ) after surgery . therefore , the need for better penile rehabilitation is anticipated . this increase in need for penile rehabilitation stems from two pillars .", "first , the introduction of prostate - specific antigen ( psa ) testing in the 1990s made early detection of prostate cancer possible .", "the early screening using psa testing led to a significant drop in patient age , and younger prostate cancer patients have great interest in and need for erectile function ( ef ) after surgery .", "second , recent advances in surgical techniques have motivated more prostate cancer patients to consider penile rehabilitation .", "ns ralp by an experienced surgeon can minimize nerve injury , and early penile rehabilitation with oral administration of phosphodiesterase type 5 inhibitors ( pde5-is ) can maximize penile recovery in ed patients after rp . after the advent of the orally administered pde5-is in 1998 , there was a huge shift in the ed treatment algorithm .", "recent literature shows that pde5-is are safe and effective in treating ed patients after rp .", "once daily administration of pde5-is has been experimentally proven to have a protective role in ed , and early administration of pde5-is helps to prevent cavernosal hypoxia , which leads to smooth muscle apoptosis and penile fibrosis [ 8 - 11 ] .", "although pde5-is are one of the standard treatments for ed after rp , consensus has not been reached on their use , such as when to start , which drug is best , which dosage to use , how long to use , and when to stop . in the case of tadalafil ,", "doses of 10 and 20 mg have been administered on demand to ed patients , and once - a - day dosing of 5 mg has been used since the korea food and drug administration approval in 2008 . moreover , in a study that compared the efficacy , safety , and tolerability of on - demand tadalafil and daily dosed tadalafil for the treatment of ed , 72% of the patients preferred daily dosed tadalafil .", "however , clinical studies on the daily administration of low - dose pde5-is and the effect of such treatment on ed patients after rp are sorely lacking .", "the present study was therefore conducted to evaluate the efficacy and safety of once daily use of tadalafil 5 mg in the treatment of ed patients after ralp .", "we evaluated the records of 116 patients who , between september 2007 and december 2011 , had undergone ralp by an experienced surgeon for localized prostate cancer at our institution .", "ralp was first performed in 2004 as a joint effort between our institution and singapore general hospital , which was the site of the initial korean experience .", "standard ralp techniques including bladder neck preservation and posterior urethral reconstruction were used because these techniques during ralp can have favorable impacts on the early postoperative recovery of continence .", "the 116 patients who developed ed after ns ralp , who had normal preoperative ef , and whose local clinical stage was t2 or lower with a gleason score lower than 8 and a serum psa of less than 20 ng before surgery were included in the study .", "the additional exclusion of 24 patients who underwent hormonal or radiation therapy preoperatively or postoperatively , who received any kind of preoperative ed treatment , or to whom pde5-is could not be prescribed resulted in 92 patients .", "the 92 patients were retrospectively evaluated in accordance with the ethical standards of the institutional review board at dong - a university hospital . in a study of the 92 patients ,", "47 patients were prescribed tadalafil at a dose of 5 mg per day . in the tadalafil group , all patients took 5 mg tadalafil per day 1 hour before bedtime .", "for all 47 patients , oral intake of tadalafil 5 mg was initiated early after foley catheter removal , between 2 and 3 weeks after the surgery .", "the tadalafil group was then stratified depending on the ns procedure they had undergone : bilateral ns ( n=30 ) and unilateral ns ( n=17 ) .", "postoperative ef was assessed through the patients ' responses to the korean version of the five - item international index of erectile function ( iief-5 ) questionnaire , which is also referred to as the sexual health inventory of men .", "the iief-5 questionnaire is the abridged version of the iief-15 and consists of 5 questions related to ed diagnosis and ef improvement .", "the iief-5 questionnaire is a self - administered questionnaire that can sensitively detect subtle changes in ef recovery .", "each of the 5 questions is scored from 0 to 5 or from 1 to 5 , where 0 , the absence of sexual activity ; 1 , very low / almost never or never / extremely difficult ; 2 , low / a few times / very difficult ; 3 , moderate / sometimes / difficult ; 4 , high / most times / slightly difficult ; and 5 , very high / almost always or always / not difficult . the total iief-5 score is calculated by summing each score from the five questions and varies from 1 to 25 , where a score of 1 to 7 , severe ed ; 8 to 11 , moderate ed ; 12 to 16 , mild - moderate ed ; 17 to 21 , mild ed ; and 22 to 25 , no ed . in the present study , the iief-5 questionnaire was conducted 3 times : before surgery , at 6 months after surgery , and at 1 year after surgery .", "all patients were also counseled on ed , tadalafil intake , and its side effects at each visit . in the present study ,", "positive responders were defined as those patients whose combined score for q2 , \" when you had erections with sexual stimulation , how often were your erections hard enough for penetration ? \" and q3 , \" during sexual intercourse , how often were you able to maintain your erection after you had penetrated your partner ? \" on the iief-5 questionnaire was 8 or more .", "outcomes from the tadalafil group and the non - tadalafil group were compared and analyzed according to ns status on the basis of the patients ' iief-5 score .", "data were summarized by using descriptive statistics : frequency and percentage for categorical variables and meanstandard deviation and median ( range ) for continuous variables .", "differences in the patients ' demographic and clinical characteristics were compared across subgroups with fisher exact test for categorical variables or mann - whitney u - test for continuous variables .", "wilcoxon 's rank sum test was performed to test differences in iief-5 domains between time points .", "the cumulative incidence of recovery of erection firm enough for sexual intercourse was estimated by the kaplan - meier product limit method .", "the log - rank test was also employed to investigate the difference in recovery of erection between groups .", "a p - value lower than 0.05 was considered statistically significant . by its nature , this study was explorative and , therefore , no adjustment for multiple testing was applied .", "all statistical analyses were carried out by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) and medcalc 11.6.1.0 ( medcalc , ostend , belgium ) .", "demographic and clinical variables at baseline were well balanced between the tadalafil group and the non - tadalafil group ( table 1 ) .", "the 92 patients who were screened for eligibility according to the inclusion and exclusion criteria were retrospectively assessed in the present study .", "the patients in the tadalafil group ( n=47 ) and those in the non - tadalafil group ( n=45 ) were also stratified according to ns status . in the tadalafil group , 63.8% of the patients ( n=30 ) underwent a bilateral ns procedure and 36.2% of the patients ( n=17 ) underwent a unilateral ns procedure . in the non - tadalafil group , bilateral ns", "was performed for 60% of the patients ( n=27 ) and unilateral ns was performed for 40% of the patients ( n=18 ) .", "compared with the non - tadalafil group , the tadalafil group regardless of ns procedure reported a statistically significant improvement in ef ( p=0.0049 , log - rank test ) . at 6 months after the surgery", ", 11% of the patients in the tadalafil group ( n=5 ) responded positively to tadalafil , whereas there were no positive responders in the non - tadalafil group . at 12 months after the surgery ,", "32% of the patients in the tadalafil group ( n=15 ) were able to perform intercourse , and 9% of the patients in the non - tadalafil group ( n=4 ) had erection sufficient for sexual intercourse .", "once - daily use of tadalafil 5 mg resulted in ef improvement during the course of 1 year after ns ralp ( fig .", "the mean iief-5 score at 1 year also differed between the tadalafil group and the non - tadalafil group as shown in table 2 ( p<0.05 , wilcoxon 's rank sum test ) and in fig .", "most of the patients who had undergone ns ralp experienced ed after the surgery . at 6 months , the total iief-5 scores of the tadalafil group and the non - tadalafil group were 10.03.4 and 7.04.0 , respectively ( p<0.0001 , mann - whitney u test ) . at 1 year after the surgery , the total iief-5 score in the tadalafil group was significantly greater than that in the non - tadalafil group ( 13.25.6 vs 7.74.8 , p<0.0001 ; mann - whitney u test ) .", "moreover , there was a significant increase in all 5 domains of the iief-5 in the tadalafil group , whereas in the non - tadalafil group no significant increases were observed in any of the domains at 1 year ( q4 , p<0.0001 ; q1 , p<0.0001 ; q3 , p<0.0001 ; q2 , p=0.006 ; q5 , p<0.0001 ) . in the tadalafil group ,", "the iief-5 score of the bilateral ns group was greater on all 5 domains of the iief-5 questionnaire than the score in the unilateral ns group at 1 year after surgery ( table 3 ) .", "1 , the patients ' ns status largely influenced the response rate to tadalafil ( p=0.0098 , log - rank test ) . among the patients who were prescribed", "tadalafil 5 mg per day , four patients ( 13% ) in the bilateral ns group and one patient ( 6% ) in the unilateral ns group positively responded to tadalafil at 6 months after the surgery . at 1 year , 12 patients ( 40% ) in the bilateral ns group and 3 patients ( 18% ) in the unilateral ns group responded positively to tadalafil and were able to perform intercourse . in the non - tadalafil group ,", "only three patients ( 11% ) for whom bilateral ns was performed and one patient ( 6% ) for whom unilateral ns was performed had erections sufficient for vaginal intercourse . in the tadalafil group , two patients did not attempt sexual intercourse even though they had sufficient erections after the surgery . according to the results of the study , the once - daily dosage of tadalafil 5 mg was most effective when bilateral ns was performed .", "daily use of tadalafil was well tolerated in the present study . at 6 months , flushing and dizziness", "were the most commonly reported adverse events . in the tadalafil group , four patients ( 8.5% ) experienced flushing and one patient ( 2.1% ) experienced dizziness at 6 months . at 1 year , two patients ( 4.3% ) had headache , one patient ( 2.1% ) experienced dizziness , and three patients ( 6.4% ) were reported to experience flushing after taking the drug .", "in our retrospective review , we learned that the dropout rate was 8% ( 4 of 51 ) over a period of 1 year ; two of the four patients had discontinued treatment owing to the high cost of the medication and the other two patients owing to a lack of efficacy .", "the results of the current study of daily administration of tadalafil 5 mg for 1 year demonstrate the efficacy and safety of once - a - day dosing of tadalafil 5 mg in ed patients after ralp .", "after the surgery , patients in the tadalafil group showed greater improvement in ef than did those in the non - tadalafil group .", "the potency rate in the tadalafil groups who underwent either unilateral or bilateral ns continued to increase after the surgery , whereas the magnitude of improvement in the bilateral ns group was far greater than in the unilateral ns group . according to the iief-5 scores at 6 months and 1 year in the tadalafil group , the scores of the bilateral ns group ( 10.73.3 and 15.05.3 , respectively ) were significantly higher than the scores in the unilateral ns group ( 8.73.2 and 10.14.7 , respectively ) .", "erection sufficient enough for sexual intercourse with the administration of tadalafil was observed in 40% of the patients in the bilateral ns group and 18% of the patients in the unilateral ns group .", "the efficacy of daily administration of tadalafil was highly dependent on the degree of neurovascular bundle preservation .", "reported that the efficacy of sildenafil citrate for ed patients who underwent bilateral , unilateral , and non - ns retropubic rp was 71.7% , 50% , and 15.4% , respectively . in another study that evaluated 91 ed patients after rp", ", 52% of the patients ( n=48 ) responded positively to sildenafil citrate and were able to have successful vaginal intercourse 1 year after the surgery . in a randomized , double - blind , placebo - controlled , parallel study , montorsi et al . reported that tadalafil once - a - day significantly improved ef compared with the placebo group .", "after 12 weeks of drug administration , iief - ef domain scores 26 were obtained for 67 patients ( 45.9% ) in the tadalafil group and 21 patients ( 31.3% ) in the placebo group . however , in a randomized , double - blind , placebo - controlled study on the effects of nightly sildenafil citrate for the prevention of ed after bilateral ns rp , erection good enough for satisfactory sexual activity occurred only in 27% of the sildenafil group ( n=14 of 51 ) and in 4% of the placebo group ( n=1 of 25 ) . moreover , montorsi et al . conducted a prospective , randomized , double - blind , double - dummy , multinational , multicenter , parallel group study and evaluated 628 patients after bilateral ns rp .", "they compared the efficacy of on - demand versus nightly dosages of vardenafil and reported that iief scores 22 were obtained in 24.8% , 32.0% , and 48.2% of the placebo , vardenafil nightly , and vardenafil on - demand groups , respectively .", "response rates to sildenafil after ns rp varied from 10% to 76% , whereas the response rates for the non - ns group varied from 0% to 15% .", "the potency rate of 32% in the tadalafil group in the current study is slightly lower than the rates in previously published studies . the slightly lower response rate ( 32% ) in the tadalafil group may stem from the rather stringent criteria applied in this study . for instance , several other studies did not provide a clear definition or criteria for a positive response , and in those studies a positive response did not necessarily mean the ability to achieve sexual intercourse . in the current study , a summed score of 8 or more on q2 ( \" when you had erections with sexual stimulation , how often were your erections hard enough for penetration ? \" ) and q3 ( \" during sexual intercourse , how often were you able to maintain your erection after you had penetrated your partner ? \" ) of the iief-5 questionnaire was required for patients to be classified as positive responders .", "the relatively older age of the patients in the present study ( 67.96.8 years ) might have had a negative impact on the potency rate .", "furthermore , 2 patients in the tadalafil group did not attempt sexual intercourse even though they had sufficient erections after the surgery .", "lastly , the biopsy gleason score , which is known to be related to psa failure , was slightly higher in the tadalafil group than in the non - tadalafil group even though a high gleason score of 8 or more was observed in only three of the patients ( one in the non - tadalafil group and two in the tadalafil group ) .", "diverse factors including preoperative ef , frequency of sexual intercourse before surgery , type of ns procedure , degree of postoperative potency , and the interval between surgery and the administration of pde5-is might have affected the potency results .", "the discrepancy among published results suggests that further confirmatory studies are needed on the effects of pde5-is and the optimal regimen .", "the mechanism of action of pde5-is is related to the etiology of ed after rp .", "even though the ns technique with the advent of ralp significantly improved the potency rate , neurapraxia and nerve injury still persist even 1 year after surgery .", "the patients ' iief-5 score at 1 year was significantly lower than the patients ' iief-5 score at preoperative baseline , and most patients ( n=116 ) experienced ed after the surgery even though ns ralp was performed .", "when neurologic injury occurs , penile hypoxia and fibrosis lead to the absence of spontaneous nocturnal erections , which decreases the release of nitric oxide .", "this decrease in nitric oxide production leads to a drop in the amount of available cyclic guanosine monophosphate ( cgmp ) .", "pde5-is inhibit pde5 , which metabolizes cgmp , and this results in an increase in cgmp levels .", "this increase in the amount of cgmp coupled with nitric oxide induces corporal smooth muscle relaxation , and this leads to subsequent erection by allowing blood flow to the penis [ 21 - 24 ] .", "the whole process requires nerve preservation , because if there is no cgmp , there is no substrate for pde5 to metabolize .", "this mechanism of action explains why the effect of tadalafil was much greater in the bilateral ns group than in the unilateral ns group in the current study .", "persistent use of pde5-is inhibits hypoxia - associated fibrosis by amplifying the depressed nitric oxide signaling pathway , and this leads to erectile recovery after rp . in a recent study that compared the effects of different pde5-is ,", "tadalafil was the most effective agent , followed by vardenafil , although there was no significant difference in the safety profile of the drugs . in long - term studies", ", once daily administration of tadalafil 5 mg was effective and safe and thus became a viable alternative to the widely used on - demand administration of tadalafil for ed patients .", "moreover , the mean half - life of tadalafil ( 17.5 hours ) is longer than that of sildenafil and vardenafil ( about 4 hours ) .", "the longer duration of the drug may allow patients with ed to enjoy sexual activity free from the burden of planning drug intake before intercourse , which can improve the patients ' sexual quality of life [ 27 - 29 ] .", "previously published studies maintain that pde5-is can be administered as early as at the time of removal of the foley catheter or within 1 month after rp .", "early administration of pde5-is helps to prevent cavernosal hypoxia , which leads to smooth muscle apoptosis and penile fibrosis . in our study , all patients in the tadalafil group initiated oral intake of tadalafil 5 mg right after foley catheter removal , between 2 to 3 weeks after the surgery , and tadalafil was administered for a period of 1 year .", "the potency rate of the bilateral ns group who took tadalafil ( 40% ) was significantly higher than that of the non - tadalafil group ( 11% ) at 1 year after the surgery .", "the results of our study suggest that ed after ralp can be well treated with once daily dosage of tadalafil 5 mg if ns ralp is performed .", "therefore , tadalafil should be considered as a first - line treatment for a well - selected subgroup of ed patients after rp .", "the current study was neither randomized nor placebo - controlled because it was carried out in a retrospective manner .", "although the results of the study should be applied with caution , the findings suggest that once daily dosage of tadalafil 5 mg was effective and well - tolerated by ed patients after ralp at 1 year after the surgery .", "a second limitation of the current study is related to the relatively small number of patients .", "however , the current study can be a pilot for further studies that have larger randomized samples of patients . despite these limitations , this study is the first long - term study that evaluated the efficacy and safety of tadalafil 5 mg once daily use in ed patients after ns ralp .", "more confirmatory studies that are prospective , crossover , randomized , placebo - controlled , and double - blind are clearly required to determine the effects and the optimal regimen of tadalafil in ed patients after ralp .", "this study is the first long - term study that evaluated the efficacy and safety of tadalafil 5 mg once daily use in ed patients after ns ralp .", "the results of the study indicate that once - a - day dosing of tadalafil 5 mg is effective and safe in properly selected ed patients after ns ralp . at 1 year after the surgery", ", a recovery rate of 40% was attained in the bilateral ns group who took tadalafil 5 mg once per day before bedtime .", "once daily administration of tadalafil 5 mg was effective and safe for ed patients who had undergone ns ralp , and the patients showed gradual improvement in their ef over a period of 1 year ." ]

[ "<S> purposeto evaluate the efficacy and safety of tadalafil 5 mg once daily use in the treatment of erectile dysfunction ( ed ) after robot - assisted laparoscopic radical prostatectomy ( ralp).materials and methodsthe study retrospectively evaluated 92 patients who underwent ralp at dong - a university hospital . </S>", "<S> the patients were surveyed by use of the abridged five - item version of the international index of erectile function ( iief-5 ) questionnaire , which was self - administered before surgery and at 6 months and 1 year after surgery . </S>", "<S> the 92 patients were classified into the tadalafil group ( n=47 ) and the non - tadalafil group ( n=45 ) . </S>", "<S> each group was then classified depending on the nerve - sparing ( ns ) procedure used : bilateral ns or unilateral ns.resultsat 6 months , the total iief-5 scores of the tadalafil group and the non - tadalafil group were 10.03.4 and 7.04.0 , respectively . </S>", "<S> at 1 year , the total iief-5 score in the tadalafil group was significantly greater than that in the non - tadalafil group ( 13.25.6 vs. 7.74.8 , p<0.0001 ) . </S>", "<S> statistically significant improvements ( p<0.05 ) were observed in the tadalafil group for all 5 domains of the iief-5 score , whereas in the non - tadalafil group there was no significant improvement in any of the domains at 1 year . </S>", "<S> the reported side effects were flushing ( 8.5% , n=4 ) , headache ( 4.3% , n=2 ) , and dizziness ( 2.1% , n=1).conclusionsin ed patients after ns ralp , a once - daily dose of tadalafil 5 mg was well tolerated and significantly improved ef compared with that in the non - tadalafil group . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS" ]

[ [ "radical prostatectomy ( rp ) is considered to be a standard curative procedure for patients with organ - confined prostate cancer .", "recently , the advent of pioneering nerve - sparing robot - assisted laparoscopic radical prostatectomy ( ns ralp ) has significantly increased the potency rate after rp [ 1 - 3 ] . however , even if ns ralp is performed , a large number of patients still experience erectile dysfunction ( ed ) after surgery . therefore , the need for better penile rehabilitation is anticipated . this increase in need for penile rehabilitation stems from two pillars .", "first , the introduction of prostate - specific antigen ( psa ) testing in the 1990s made early detection of prostate cancer possible .", "the early screening using psa testing led to a significant drop in patient age , and younger prostate cancer patients have great interest in and need for erectile function ( ef ) after surgery .", "second , recent advances in surgical techniques have motivated more prostate cancer patients to consider penile rehabilitation .", "ns ralp by an experienced surgeon can minimize nerve injury , and early penile rehabilitation with oral administration of phosphodiesterase type 5 inhibitors ( pde5-is ) can maximize penile recovery in ed patients after rp . after the advent of the orally administered pde5-is in 1998 , there was a huge shift in the ed treatment algorithm .", "recent literature shows that pde5-is are safe and effective in treating ed patients after rp .", "once daily administration of pde5-is has been experimentally proven to have a protective role in ed , and early administration of pde5-is helps to prevent cavernosal hypoxia , which leads to smooth muscle apoptosis and penile fibrosis [ 8 - 11 ] .", "although pde5-is are one of the standard treatments for ed after rp , consensus has not been reached on their use , such as when to start , which drug is best , which dosage to use , how long to use , and when to stop . in the case of tadalafil ,", "doses of 10 and 20 mg have been administered on demand to ed patients , and once - a - day dosing of 5 mg has been used since the korea food and drug administration approval in 2008 . moreover , in a study that compared the efficacy , safety , and tolerability of on - demand tadalafil and daily dosed tadalafil for the treatment of ed , 72% of the patients preferred daily dosed tadalafil .", "however , clinical studies on the daily administration of low - dose pde5-is and the effect of such treatment on ed patients after rp are sorely lacking .", "the present study was therefore conducted to evaluate the efficacy and safety of once daily use of tadalafil 5 mg in the treatment of ed patients after ralp ." ], [ "we evaluated the records of 116 patients who , between september 2007 and december 2011 , had undergone ralp by an experienced surgeon for localized prostate cancer at our institution .", "ralp was first performed in 2004 as a joint effort between our institution and singapore general hospital , which was the site of the initial korean experience .", "standard ralp techniques including bladder neck preservation and posterior urethral reconstruction were used because these techniques during ralp can have favorable impacts on the early postoperative recovery of continence .", "the 116 patients who developed ed after ns ralp , who had normal preoperative ef , and whose local clinical stage was t2 or lower with a gleason score lower than 8 and a serum psa of less than 20 ng before surgery were included in the study .", "the additional exclusion of 24 patients who underwent hormonal or radiation therapy preoperatively or postoperatively , who received any kind of preoperative ed treatment , or to whom pde5-is could not be prescribed resulted in 92 patients .", "the 92 patients were retrospectively evaluated in accordance with the ethical standards of the institutional review board at dong - a university hospital . in a study of the 92 patients ,", "47 patients were prescribed tadalafil at a dose of 5 mg per day . in the tadalafil group , all patients took 5 mg tadalafil per day 1 hour before bedtime .", "for all 47 patients , oral intake of tadalafil 5 mg was initiated early after foley catheter removal , between 2 and 3 weeks after the surgery .", "the tadalafil group was then stratified depending on the ns procedure they had undergone : bilateral ns ( n=30 ) and unilateral ns ( n=17 ) .", "postoperative ef was assessed through the patients ' responses to the korean version of the five - item international index of erectile function ( iief-5 ) questionnaire , which is also referred to as the sexual health inventory of men .", "the iief-5 questionnaire is the abridged version of the iief-15 and consists of 5 questions related to ed diagnosis and ef improvement .", "the iief-5 questionnaire is a self - administered questionnaire that can sensitively detect subtle changes in ef recovery .", "each of the 5 questions is scored from 0 to 5 or from 1 to 5 , where 0 , the absence of sexual activity ; 1 , very low / almost never or never / extremely difficult ; 2 , low / a few times / very difficult ; 3 , moderate / sometimes / difficult ; 4 , high / most times / slightly difficult ; and 5 , very high / almost always or always / not difficult . the total iief-5 score is calculated by summing each score from the five questions and varies from 1 to 25 , where a score of 1 to 7 , severe ed ; 8 to 11 , moderate ed ; 12 to 16 , mild - moderate ed ; 17 to 21 , mild ed ; and 22 to 25 , no ed . in the present study , the iief-5 questionnaire was conducted 3 times : before surgery , at 6 months after surgery , and at 1 year after surgery .", "all patients were also counseled on ed , tadalafil intake , and its side effects at each visit . in the present study ,", "positive responders were defined as those patients whose combined score for q2 , \" when you had erections with sexual stimulation , how often were your erections hard enough for penetration ? \" and q3 , \" during sexual intercourse , how often were you able to maintain your erection after you had penetrated your partner ? \" on the iief-5 questionnaire was 8 or more .", "outcomes from the tadalafil group and the non - tadalafil group were compared and analyzed according to ns status on the basis of the patients ' iief-5 score .", "data were summarized by using descriptive statistics : frequency and percentage for categorical variables and meanstandard deviation and median ( range ) for continuous variables .", "differences in the patients ' demographic and clinical characteristics were compared across subgroups with fisher exact test for categorical variables or mann - whitney u - test for continuous variables .", "wilcoxon 's rank sum test was performed to test differences in iief-5 domains between time points .", "the cumulative incidence of recovery of erection firm enough for sexual intercourse was estimated by the kaplan - meier product limit method .", "the log - rank test was also employed to investigate the difference in recovery of erection between groups .", "a p - value lower than 0.05 was considered statistically significant . by its nature , this study was explorative and , therefore , no adjustment for multiple testing was applied .", "all statistical analyses were carried out by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) and medcalc 11.6.1.0 ( medcalc , ostend , belgium ) ." ], [ "demographic and clinical variables at baseline were well balanced between the tadalafil group and the non - tadalafil group ( table 1 ) .", "the 92 patients who were screened for eligibility according to the inclusion and exclusion criteria were retrospectively assessed in the present study .", "the patients in the tadalafil group ( n=47 ) and those in the non - tadalafil group ( n=45 ) were also stratified according to ns status . in the tadalafil group , 63.8% of the patients ( n=30 ) underwent a bilateral ns procedure and 36.2% of the patients ( n=17 ) underwent a unilateral ns procedure . in the non - tadalafil group , bilateral ns", "was performed for 60% of the patients ( n=27 ) and unilateral ns was performed for 40% of the patients ( n=18 ) .", "compared with the non - tadalafil group , the tadalafil group regardless of ns procedure reported a statistically significant improvement in ef ( p=0.0049 , log - rank test ) . at 6 months after the surgery", ", 11% of the patients in the tadalafil group ( n=5 ) responded positively to tadalafil , whereas there were no positive responders in the non - tadalafil group . at 12 months after the surgery ,", "32% of the patients in the tadalafil group ( n=15 ) were able to perform intercourse , and 9% of the patients in the non - tadalafil group ( n=4 ) had erection sufficient for sexual intercourse .", "once - daily use of tadalafil 5 mg resulted in ef improvement during the course of 1 year after ns ralp ( fig .", "the mean iief-5 score at 1 year also differed between the tadalafil group and the non - tadalafil group as shown in table 2 ( p<0.05 , wilcoxon 's rank sum test ) and in fig .", "most of the patients who had undergone ns ralp experienced ed after the surgery . at 6 months , the total iief-5 scores of the tadalafil group and the non - tadalafil group were 10.03.4 and 7.04.0 , respectively ( p<0.0001 , mann - whitney u test ) . at 1 year after the surgery , the total iief-5 score in the tadalafil group was significantly greater than that in the non - tadalafil group ( 13.25.6 vs 7.74.8 , p<0.0001 ; mann - whitney u test ) .", "moreover , there was a significant increase in all 5 domains of the iief-5 in the tadalafil group , whereas in the non - tadalafil group no significant increases were observed in any of the domains at 1 year ( q4 , p<0.0001 ; q1 , p<0.0001 ; q3 , p<0.0001 ; q2 , p=0.006 ; q5 , p<0.0001 ) . in the tadalafil group ,", "the iief-5 score of the bilateral ns group was greater on all 5 domains of the iief-5 questionnaire than the score in the unilateral ns group at 1 year after surgery ( table 3 ) .", "1 , the patients ' ns status largely influenced the response rate to tadalafil ( p=0.0098 , log - rank test ) . among the patients who were prescribed", "tadalafil 5 mg per day , four patients ( 13% ) in the bilateral ns group and one patient ( 6% ) in the unilateral ns group positively responded to tadalafil at 6 months after the surgery . at 1 year , 12 patients ( 40% ) in the bilateral ns group and 3 patients ( 18% ) in the unilateral ns group responded positively to tadalafil and were able to perform intercourse . in the non - tadalafil group ,", "only three patients ( 11% ) for whom bilateral ns was performed and one patient ( 6% ) for whom unilateral ns was performed had erections sufficient for vaginal intercourse . in the tadalafil group , two patients did not attempt sexual intercourse even though they had sufficient erections after the surgery . according to the results of the study , the once - daily dosage of tadalafil 5 mg was most effective when bilateral ns was performed .", "daily use of tadalafil was well tolerated in the present study . at 6 months , flushing and dizziness", "were the most commonly reported adverse events . in the tadalafil group , four patients ( 8.5% ) experienced flushing and one patient ( 2.1% ) experienced dizziness at 6 months . at 1 year , two patients ( 4.3% ) had headache , one patient ( 2.1% ) experienced dizziness , and three patients ( 6.4% ) were reported to experience flushing after taking the drug .", "in our retrospective review , we learned that the dropout rate was 8% ( 4 of 51 ) over a period of 1 year ; two of the four patients had discontinued treatment owing to the high cost of the medication and the other two patients owing to a lack of efficacy ." ], [ "the results of the current study of daily administration of tadalafil 5 mg for 1 year demonstrate the efficacy and safety of once - a - day dosing of tadalafil 5 mg in ed patients after ralp .", "after the surgery , patients in the tadalafil group showed greater improvement in ef than did those in the non - tadalafil group .", "the potency rate in the tadalafil groups who underwent either unilateral or bilateral ns continued to increase after the surgery , whereas the magnitude of improvement in the bilateral ns group was far greater than in the unilateral ns group . according to the iief-5 scores at 6 months and 1 year in the tadalafil group , the scores of the bilateral ns group ( 10.73.3 and 15.05.3 , respectively ) were significantly higher than the scores in the unilateral ns group ( 8.73.2 and 10.14.7 , respectively ) .", "erection sufficient enough for sexual intercourse with the administration of tadalafil was observed in 40% of the patients in the bilateral ns group and 18% of the patients in the unilateral ns group .", "the efficacy of daily administration of tadalafil was highly dependent on the degree of neurovascular bundle preservation .", "reported that the efficacy of sildenafil citrate for ed patients who underwent bilateral , unilateral , and non - ns retropubic rp was 71.7% , 50% , and 15.4% , respectively . in another study that evaluated 91 ed patients after rp", ", 52% of the patients ( n=48 ) responded positively to sildenafil citrate and were able to have successful vaginal intercourse 1 year after the surgery . in a randomized , double - blind , placebo - controlled , parallel study , montorsi et al . reported that tadalafil once - a - day significantly improved ef compared with the placebo group .", "after 12 weeks of drug administration , iief - ef domain scores 26 were obtained for 67 patients ( 45.9% ) in the tadalafil group and 21 patients ( 31.3% ) in the placebo group . however , in a randomized , double - blind , placebo - controlled study on the effects of nightly sildenafil citrate for the prevention of ed after bilateral ns rp , erection good enough for satisfactory sexual activity occurred only in 27% of the sildenafil group ( n=14 of 51 ) and in 4% of the placebo group ( n=1 of 25 ) . moreover , montorsi et al . conducted a prospective , randomized , double - blind , double - dummy , multinational , multicenter , parallel group study and evaluated 628 patients after bilateral ns rp .", "they compared the efficacy of on - demand versus nightly dosages of vardenafil and reported that iief scores 22 were obtained in 24.8% , 32.0% , and 48.2% of the placebo , vardenafil nightly , and vardenafil on - demand groups , respectively .", "response rates to sildenafil after ns rp varied from 10% to 76% , whereas the response rates for the non - ns group varied from 0% to 15% .", "the potency rate of 32% in the tadalafil group in the current study is slightly lower than the rates in previously published studies . the slightly lower response rate ( 32% ) in the tadalafil group may stem from the rather stringent criteria applied in this study . for instance , several other studies did not provide a clear definition or criteria for a positive response , and in those studies a positive response did not necessarily mean the ability to achieve sexual intercourse . in the current study , a summed score of 8 or more on q2 ( \" when you had erections with sexual stimulation , how often were your erections hard enough for penetration ? \" ) and q3 ( \" during sexual intercourse , how often were you able to maintain your erection after you had penetrated your partner ? \" ) of the iief-5 questionnaire was required for patients to be classified as positive responders .", "the relatively older age of the patients in the present study ( 67.96.8 years ) might have had a negative impact on the potency rate .", "furthermore , 2 patients in the tadalafil group did not attempt sexual intercourse even though they had sufficient erections after the surgery .", "lastly , the biopsy gleason score , which is known to be related to psa failure , was slightly higher in the tadalafil group than in the non - tadalafil group even though a high gleason score of 8 or more was observed in only three of the patients ( one in the non - tadalafil group and two in the tadalafil group ) .", "diverse factors including preoperative ef , frequency of sexual intercourse before surgery , type of ns procedure , degree of postoperative potency , and the interval between surgery and the administration of pde5-is might have affected the potency results .", "the discrepancy among published results suggests that further confirmatory studies are needed on the effects of pde5-is and the optimal regimen .", "the mechanism of action of pde5-is is related to the etiology of ed after rp .", "even though the ns technique with the advent of ralp significantly improved the potency rate , neurapraxia and nerve injury still persist even 1 year after surgery .", "the patients ' iief-5 score at 1 year was significantly lower than the patients ' iief-5 score at preoperative baseline , and most patients ( n=116 ) experienced ed after the surgery even though ns ralp was performed .", "when neurologic injury occurs , penile hypoxia and fibrosis lead to the absence of spontaneous nocturnal erections , which decreases the release of nitric oxide .", "this decrease in nitric oxide production leads to a drop in the amount of available cyclic guanosine monophosphate ( cgmp ) .", "pde5-is inhibit pde5 , which metabolizes cgmp , and this results in an increase in cgmp levels .", "this increase in the amount of cgmp coupled with nitric oxide induces corporal smooth muscle relaxation , and this leads to subsequent erection by allowing blood flow to the penis [ 21 - 24 ] .", "the whole process requires nerve preservation , because if there is no cgmp , there is no substrate for pde5 to metabolize .", "this mechanism of action explains why the effect of tadalafil was much greater in the bilateral ns group than in the unilateral ns group in the current study .", "persistent use of pde5-is inhibits hypoxia - associated fibrosis by amplifying the depressed nitric oxide signaling pathway , and this leads to erectile recovery after rp . in a recent study that compared the effects of different pde5-is ,", "tadalafil was the most effective agent , followed by vardenafil , although there was no significant difference in the safety profile of the drugs . in long - term studies", ", once daily administration of tadalafil 5 mg was effective and safe and thus became a viable alternative to the widely used on - demand administration of tadalafil for ed patients .", "moreover , the mean half - life of tadalafil ( 17.5 hours ) is longer than that of sildenafil and vardenafil ( about 4 hours ) .", "the longer duration of the drug may allow patients with ed to enjoy sexual activity free from the burden of planning drug intake before intercourse , which can improve the patients ' sexual quality of life [ 27 - 29 ] .", "previously published studies maintain that pde5-is can be administered as early as at the time of removal of the foley catheter or within 1 month after rp .", "early administration of pde5-is helps to prevent cavernosal hypoxia , which leads to smooth muscle apoptosis and penile fibrosis . in our study , all patients in the tadalafil group initiated oral intake of tadalafil 5 mg right after foley catheter removal , between 2 to 3 weeks after the surgery , and tadalafil was administered for a period of 1 year .", "the potency rate of the bilateral ns group who took tadalafil ( 40% ) was significantly higher than that of the non - tadalafil group ( 11% ) at 1 year after the surgery .", "the results of our study suggest that ed after ralp can be well treated with once daily dosage of tadalafil 5 mg if ns ralp is performed .", "therefore , tadalafil should be considered as a first - line treatment for a well - selected subgroup of ed patients after rp .", "the current study was neither randomized nor placebo - controlled because it was carried out in a retrospective manner .", "although the results of the study should be applied with caution , the findings suggest that once daily dosage of tadalafil 5 mg was effective and well - tolerated by ed patients after ralp at 1 year after the surgery .", "a second limitation of the current study is related to the relatively small number of patients .", "however , the current study can be a pilot for further studies that have larger randomized samples of patients . despite these limitations , this study is the first long - term study that evaluated the efficacy and safety of tadalafil 5 mg once daily use in ed patients after ns ralp .", "more confirmatory studies that are prospective , crossover , randomized , placebo - controlled , and double - blind are clearly required to determine the effects and the optimal regimen of tadalafil in ed patients after ralp ." ], [ "this study is the first long - term study that evaluated the efficacy and safety of tadalafil 5 mg once daily use in ed patients after ns ralp .", "the results of the study indicate that once - a - day dosing of tadalafil 5 mg is effective and safe in properly selected ed patients after ns ralp . at 1 year after the surgery", ", a recovery rate of 40% was attained in the bilateral ns group who took tadalafil 5 mg once per day before bedtime .", "once daily administration of tadalafil 5 mg was effective and safe for ed patients who had undergone ns ralp , and the patients showed gradual improvement in their ef over a period of 1 year ." ] ]

[ "many older adults have substantial vision problems such as cataracts , glaucoma , and macular \n degeneration , which are the leading causes of reduced visual acuity1 .", "poor vision is one of the most important risk factors \n associated with falls among older people2 . \n", "vision plays a key role in maintaining balance by providing information about the \n environment and constant information to the central nervous system about body orientation in \n space and relative to other body parts3 .", "achieving effectively maintained balance during functional dynamic balance tasks requires \n energy efficiency to minimize fatigue , which is associated with postural control deficits , \n and stability to prevent falling or injury4 . among the clinical balance tests ,", "the single - limb stand ( sls ) is a \n commonly used clinical tool for assessing postural stability in the elderly and individuals \n with various balance disorders .", "the y - balance test ( ybt ) , which was developed by clinical \n applications of the star excursion balance test , involves maintaining a sls while reaching \n as far as possible with the other leg . as a dynamic sls balance test , the ybt has been used \n to prospectively identify individuals who have chronic ankle instability and greater risk of \n lower extremity injury in sports and as a post - rehabilitation test5 .", "recently , the test has been used as a screening tool for \n elderly participation6 .", "however , no \n studies have investigated energy expenditure during the dynamic single - limb balance test in \n older adults with different visual acuities .", "therefore , this study compared energy \n expenditure in the ybt , the functional dynamic single - limb balance test , in elderly people \n with good and poor binocular visual acuity ( bva ) .", "a total of 21 elderly females from community housing were enrolled in the present study . \n", "visual acuity was measured by use of a , jin s vision test chart ( jv institute , seoul , south \n korea ) .", "each participant s bva was evaluated with and without their own spectacles for \n classification into two groups : poor bva ( corrected bva 0.4 logarithm of the minimum angle \n of resolution [ logmar ] ) and good bva ( corrected bva 0.3 logmar)7 . in the poor bva group , the participants average age \n ( meansd ) , height , and weight were 78.555.24 years , 148.862.77 cm , and 49.466.05 kg , \n respectively ; they had a left side va and right side va of 0.530.15 logmar and 0.540.16 \n logmar , respectively . in the good bva group , the participants average age , height , and \n weight were 76.406.13 years , 147.004.00 cm , and 48.994.91 kg , respectively", "; they had a \n left side va and right side va of 0.230.07 logmar and 0.230.09 logmar , respectively .", "all \n participants could walk independently without an assistive device and scored greater than 24 \n points on the korean version of the mini - mental state examination .", "they had no past or \n present neurological disorder , no musculoskeletal disease that could interfere with daily \n activities , and no significant auditory impairments , and they were not taking drugs that \n could have influenced the results of this study .", "ethical approval was obtained from the inje \n university faculty of health science human ethics committee , and all subjects signed an \n informed consent form prior to their participation . a triaxial accelerometer ( fit . life , suwon , south korea ) was used to measure energy \n expenditure during the ybt .", "we measured the raw data using the x , y , and z variables of \n acceleration reformed as acceleration due to activity by removing gravitational \n acceleration .", "moreover , we calibrated the single vector magnitude ( svm ) by summing the \n acceleration of the three axes8:svm = in the present study , a range of 2 g was selected .", "data were collected at a sampling rate \n of 32 hz . before the task ,", "the participant s dominant leg was determined by kicking a soccer ball . \n for normalization , the lower limb lengths of all participants were measured in supine \n position from the anterior superior iliac spine to ipsilateral medial malleolus using a \n cloth tape measure .", "reach distance was normalized to limb length , and the maximum reach \n distance was expressed as a percentage of limb length ( % maxd ) .", "the normalized value was \n calculated as reach distance divided by limb length and then multiplied by 100 .", "composite \n reach distance was the sum of the three reach directions divided by 3 times the limb length , \n which was then multiplied by 100 .", "the accelerometer was fixed with double - sided adhesive tape \n over the l3 spinous process9 . for the test", ", the participants stood with one foot on the center footplate from the ybt \n kit ( move2perform , evansville , in , usa ) at the starting line and their hands \n on their pelvis .", "they were asked to push the reach - indicator block with the free limb in the \n anterior ( a ) , posterior medial ( pm ) , and posterior lateral ( pl ) directions in relation to \n the stance foot on the central footplate .", "the testing order was trial standing on the right \n foot reaching in the a direction ( right a reach ) followed by trial standing on the left foot \n reaching in the a direction .", "after the test trials were \n completed , each participant was given a 2-minute rest period and then conducted three test \n trials in each direction11 .", "a trial was classified as invalid if the \n participant did not return to the starting position , placed the reach foot on the ground on \n either side of the line or tube , raised or moved the stance foot during the test , or kicked \n the plate with the reach foot to gain more distance .", "if an invalid trial occurred , the data \n were discarded , and the participant repeated the trial .", "statistical analysis was performed using the spss software ( version 18.0 for windows , spss \n inc . ,", "a paired t - test was used to examine differences between right and \n left limb reach distances .", "as no differences were found , for the data analysis , we used the \n dominant limb distance in each direction ( right side ) .", "the differences in reach distance and \n energy expenditure were analyzed using the independent t - test for comparisons between \n groups .", "effect sizes ( cohen s d ) for test differences were calculated by \n determining the difference between the poor bva and good bva group mean values and dividing \n by the pooled standard deviation12 . \n", "the normalized reach distance ( % maxd ) in the good bva group ( a , 60.84.3 ; pm , 94.36.1 ; pl , \n 88.97.8 ) during the ybt in the three directions and composite reach ( 81.34.8 ) were \n significantly longer ( p < 0.05 ) compared with the values in the poor bva group ( a , \n 55.65.9 ; pm , 84.29.9 ; pl , 81.75.4 ; composite , 73.86.2 ) . the energy expenditure \n ( cm / s ) in the good bva group during the ybt in the three directions ( a , \n 413.8145.2 ; pm , 490.0189.4 ; pl , 585.8209.7 ) was significantly reduced ( p < 0.05 ) \n compared with the values in the poor bva group ( a , 697.4231.6 ; pm , 698.8227.3 ; pl , \n 791.6182.7 ) .", "the effect size value of the reach distance and energy expenditure showed that \n the results were large for the a ( 1.064 and 1.525 ) , pm ( 1.268 and 1.044 ) , pl directions \n ( 1.187 and 1.104 ) and composite reach ( 1.412 ) in terms of the differences between the good \n and poor bva groups .", "this study was designed to compare energy expenditure and reach distance during the ybt in \n elderly women with good bva and poor bva .", "it showed that normalized reach distances in the \n a , pm , and pl directions and the composite reach of the good bva group were significantly \n greater compared with the values in the poor bva group .", "the reach distance during the ybt \n reflected the degree of dynamic balance control . a longer reach", "thus , our findings suggest that visual acuity affects the \n functional dynamic sls in the a , pm , and pl directions .", "moreover , older adults with poor bva \n are more balance challenged compared with those with good bva . in the a reach direction , participants received visual feedback during the ybt .", "wang et \n al.13 showed that reduction of the \n stability boundary of the multi - joint coordination patterns in maintaining the sls in the \n absence of vision could lead to increased rotation of segments around the center of mass . \n", "hallemans et al.14 reported that no \n vision conditions limited movements of the hip and ankle in the sagittal plane compared with \n a full vision condition .", "although , visual awareness is reduced in the pm and pl directions \n compared with the a direction , participants with good bva have a longer reach compared with \n those with poor bva . when one sense diminishes , it is common to find a greater dependence on \n other sensory cues to preserve balance15 . \n however , hazime et al.16 reported that \n vision had a key role in the sls , requiring greater balance ability , whereas perturbation in \n proprioception showed effects only in the double- limb stance .", "the stance platform of the \n ybt kit is elevated 1 inch , and its width is 5.2 inches ; thus , maintaining \n balance in the dynamic sls on the balance platform of the ybt kit is greater \n challenge than doing so on the ground in elderly subjects .", "the results show that elderly \n people with good and poor bva rely more on visual input than other sensory inputs for \n postural control .", "in addition , in this study , the energy expenditure during the ybt in three \n directions in the poor bva group was significantly increased compared with the values in the \n good bva group .", "an accelerometer was used as an objective tool for assessing physical \n activity level related to energy expenditure .", "accelerometry has been shown to have \n intra - rater reliabilities ( 0.9250.994 ) and correlation with scores on the commonly used \n physical activity questionnaire ( r = 0.830)17 .", "our results imply that the velocity of trunk segment motion \n frequently changes and fluctuates , and that ineffective biomechanical cost strategies in \n terms of energy efficiency are performed .", "individuals with poor bva may habituated to \n discomforts visual acuity and rely more on ankle proprioception to control their posture \n during quiet standing . when balance challenged with conditions such as the sls , vision play \n a major role compared with in a normal standing position .", "thus we surmise that these \n individuals compensate with trunk rotation during ybt in order to maintain postural \n stability16 , 18 .", "hence , participants with good bva have a longer reach in the ybt \n compared with those with poor bva .", "first , it only measured reaching distances and energy \n expenditure . measurements of muscle activation and range of motion in the trunk and lower \n extremities were not taken .", "thus , we could not explain the connection between energy \n expenditure and fatigue or trunk lean during the ybt .", "second , the valid of the triaxial \n accelerometry in measurement of energy expenditure during a short period of activity has not \n been verified .", "in addition , the sample size was small , so caution should be used when \n generalizing the results .", "further studies should investigate kinematic and kinetic data , as \n well as the correlation of the ybt with gait parameters in elderly subjects with poor \n bva ." ]

[ "<S> [ purpose ] this study compared the energy expenditure during the y - balance test ( ybt ) \n between elderly women with good binocular visual acuity ( bva ) and those with poor bva . \n </S>", "<S> [ subjects ] twenty - one elderly women who could walk independently were recruited from a \n community dwelling . </S>", "<S> eleven participants had a bva equal to or less than 0.4 logarithm of \n the minimum angle of resolution ( logmar ) , and the other 10 participants had a bva equal to \n or better than 0.3 logmar . </S>", "<S> [ methods ] the participants had an accelerometer attached over \n the l3 spinous process for measurement of energy expenditure and performed the ybt in the \n anterior , posteromedial , and posterolateral directions . [ results ] the normalized reach \n distance in the good bva group during the ybt in three directions and composite reach \n distance were significantly longer compared with the values in the poor bva group . </S>", "<S> the \n energy expenditure in the good bva group during the ybt in the three directions was \n significantly reduced compared with the values in the poor bva group . </S>", "<S> [ conclusion ] we \n suggest that visual acuity in the elderly influences dynamic balance and energy \n expenditure . elderly subjects with poor bva showed poor dynamic balance control and an \n inefficient biomechanical cost strategy compared to subjects with good bva . </S>" ]

[ "INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "many older adults have substantial vision problems such as cataracts , glaucoma , and macular \n degeneration , which are the leading causes of reduced visual acuity1 .", "poor vision is one of the most important risk factors \n associated with falls among older people2 . \n", "vision plays a key role in maintaining balance by providing information about the \n environment and constant information to the central nervous system about body orientation in \n space and relative to other body parts3 .", "achieving effectively maintained balance during functional dynamic balance tasks requires \n energy efficiency to minimize fatigue , which is associated with postural control deficits , \n and stability to prevent falling or injury4 . among the clinical balance tests ,", "the single - limb stand ( sls ) is a \n commonly used clinical tool for assessing postural stability in the elderly and individuals \n with various balance disorders .", "the y - balance test ( ybt ) , which was developed by clinical \n applications of the star excursion balance test , involves maintaining a sls while reaching \n as far as possible with the other leg . as a dynamic sls balance test , the ybt has been used \n to prospectively identify individuals who have chronic ankle instability and greater risk of \n lower extremity injury in sports and as a post - rehabilitation test5 .", "recently , the test has been used as a screening tool for \n elderly participation6 .", "however , no \n studies have investigated energy expenditure during the dynamic single - limb balance test in \n older adults with different visual acuities .", "therefore , this study compared energy \n expenditure in the ybt , the functional dynamic single - limb balance test , in elderly people \n with good and poor binocular visual acuity ( bva ) ." ], [ "a total of 21 elderly females from community housing were enrolled in the present study . \n", "visual acuity was measured by use of a , jin s vision test chart ( jv institute , seoul , south \n korea ) .", "each participant s bva was evaluated with and without their own spectacles for \n classification into two groups : poor bva ( corrected bva 0.4 logarithm of the minimum angle \n of resolution [ logmar ] ) and good bva ( corrected bva 0.3 logmar)7 . in the poor bva group , the participants average age \n ( meansd ) , height , and weight were 78.555.24 years , 148.862.77 cm , and 49.466.05 kg , \n respectively ; they had a left side va and right side va of 0.530.15 logmar and 0.540.16 \n logmar , respectively . in the good bva group , the participants average age , height , and \n weight were 76.406.13 years , 147.004.00 cm , and 48.994.91 kg , respectively", "; they had a \n left side va and right side va of 0.230.07 logmar and 0.230.09 logmar , respectively .", "all \n participants could walk independently without an assistive device and scored greater than 24 \n points on the korean version of the mini - mental state examination .", "they had no past or \n present neurological disorder , no musculoskeletal disease that could interfere with daily \n activities , and no significant auditory impairments , and they were not taking drugs that \n could have influenced the results of this study .", "ethical approval was obtained from the inje \n university faculty of health science human ethics committee , and all subjects signed an \n informed consent form prior to their participation . a triaxial accelerometer ( fit . life , suwon , south korea ) was used to measure energy \n expenditure during the ybt .", "we measured the raw data using the x , y , and z variables of \n acceleration reformed as acceleration due to activity by removing gravitational \n acceleration .", "moreover , we calibrated the single vector magnitude ( svm ) by summing the \n acceleration of the three axes8:svm = in the present study , a range of 2 g was selected .", "data were collected at a sampling rate \n of 32 hz . before the task ,", "the participant s dominant leg was determined by kicking a soccer ball . \n for normalization , the lower limb lengths of all participants were measured in supine \n position from the anterior superior iliac spine to ipsilateral medial malleolus using a \n cloth tape measure .", "reach distance was normalized to limb length , and the maximum reach \n distance was expressed as a percentage of limb length ( % maxd ) .", "the normalized value was \n calculated as reach distance divided by limb length and then multiplied by 100 .", "composite \n reach distance was the sum of the three reach directions divided by 3 times the limb length , \n which was then multiplied by 100 .", "the accelerometer was fixed with double - sided adhesive tape \n over the l3 spinous process9 . for the test", ", the participants stood with one foot on the center footplate from the ybt \n kit ( move2perform , evansville , in , usa ) at the starting line and their hands \n on their pelvis .", "they were asked to push the reach - indicator block with the free limb in the \n anterior ( a ) , posterior medial ( pm ) , and posterior lateral ( pl ) directions in relation to \n the stance foot on the central footplate .", "the testing order was trial standing on the right \n foot reaching in the a direction ( right a reach ) followed by trial standing on the left foot \n reaching in the a direction .", "after the test trials were \n completed , each participant was given a 2-minute rest period and then conducted three test \n trials in each direction11 .", "a trial was classified as invalid if the \n participant did not return to the starting position , placed the reach foot on the ground on \n either side of the line or tube , raised or moved the stance foot during the test , or kicked \n the plate with the reach foot to gain more distance .", "if an invalid trial occurred , the data \n were discarded , and the participant repeated the trial .", "statistical analysis was performed using the spss software ( version 18.0 for windows , spss \n inc . ,", "a paired t - test was used to examine differences between right and \n left limb reach distances .", "as no differences were found , for the data analysis , we used the \n dominant limb distance in each direction ( right side ) .", "the differences in reach distance and \n energy expenditure were analyzed using the independent t - test for comparisons between \n groups .", "effect sizes ( cohen s d ) for test differences were calculated by \n determining the difference between the poor bva and good bva group mean values and dividing \n by the pooled standard deviation12 . \n" ], [ "the normalized reach distance ( % maxd ) in the good bva group ( a , 60.84.3 ; pm , 94.36.1 ; pl , \n 88.97.8 ) during the ybt in the three directions and composite reach ( 81.34.8 ) were \n significantly longer ( p < 0.05 ) compared with the values in the poor bva group ( a , \n 55.65.9 ; pm , 84.29.9 ; pl , 81.75.4 ; composite , 73.86.2 ) . the energy expenditure \n ( cm / s ) in the good bva group during the ybt in the three directions ( a , \n 413.8145.2 ; pm , 490.0189.4 ; pl , 585.8209.7 ) was significantly reduced ( p < 0.05 ) \n compared with the values in the poor bva group ( a , 697.4231.6 ; pm , 698.8227.3 ; pl , \n 791.6182.7 ) .", "the effect size value of the reach distance and energy expenditure showed that \n the results were large for the a ( 1.064 and 1.525 ) , pm ( 1.268 and 1.044 ) , pl directions \n ( 1.187 and 1.104 ) and composite reach ( 1.412 ) in terms of the differences between the good \n and poor bva groups ." ], [ "this study was designed to compare energy expenditure and reach distance during the ybt in \n elderly women with good bva and poor bva .", "it showed that normalized reach distances in the \n a , pm , and pl directions and the composite reach of the good bva group were significantly \n greater compared with the values in the poor bva group .", "the reach distance during the ybt \n reflected the degree of dynamic balance control . a longer reach", "thus , our findings suggest that visual acuity affects the \n functional dynamic sls in the a , pm , and pl directions .", "moreover , older adults with poor bva \n are more balance challenged compared with those with good bva . in the a reach direction , participants received visual feedback during the ybt .", "wang et \n al.13 showed that reduction of the \n stability boundary of the multi - joint coordination patterns in maintaining the sls in the \n absence of vision could lead to increased rotation of segments around the center of mass . \n", "hallemans et al.14 reported that no \n vision conditions limited movements of the hip and ankle in the sagittal plane compared with \n a full vision condition .", "although , visual awareness is reduced in the pm and pl directions \n compared with the a direction , participants with good bva have a longer reach compared with \n those with poor bva . when one sense diminishes , it is common to find a greater dependence on \n other sensory cues to preserve balance15 . \n however , hazime et al.16 reported that \n vision had a key role in the sls , requiring greater balance ability , whereas perturbation in \n proprioception showed effects only in the double- limb stance .", "the stance platform of the \n ybt kit is elevated 1 inch , and its width is 5.2 inches ; thus , maintaining \n balance in the dynamic sls on the balance platform of the ybt kit is greater \n challenge than doing so on the ground in elderly subjects .", "the results show that elderly \n people with good and poor bva rely more on visual input than other sensory inputs for \n postural control .", "in addition , in this study , the energy expenditure during the ybt in three \n directions in the poor bva group was significantly increased compared with the values in the \n good bva group .", "an accelerometer was used as an objective tool for assessing physical \n activity level related to energy expenditure .", "accelerometry has been shown to have \n intra - rater reliabilities ( 0.9250.994 ) and correlation with scores on the commonly used \n physical activity questionnaire ( r = 0.830)17 .", "our results imply that the velocity of trunk segment motion \n frequently changes and fluctuates , and that ineffective biomechanical cost strategies in \n terms of energy efficiency are performed .", "individuals with poor bva may habituated to \n discomforts visual acuity and rely more on ankle proprioception to control their posture \n during quiet standing . when balance challenged with conditions such as the sls , vision play \n a major role compared with in a normal standing position .", "thus we surmise that these \n individuals compensate with trunk rotation during ybt in order to maintain postural \n stability16 , 18 .", "hence , participants with good bva have a longer reach in the ybt \n compared with those with poor bva .", "first , it only measured reaching distances and energy \n expenditure . measurements of muscle activation and range of motion in the trunk and lower \n extremities were not taken .", "thus , we could not explain the connection between energy \n expenditure and fatigue or trunk lean during the ybt .", "second , the valid of the triaxial \n accelerometry in measurement of energy expenditure during a short period of activity has not \n been verified .", "in addition , the sample size was small , so caution should be used when \n generalizing the results .", "further studies should investigate kinematic and kinetic data , as \n well as the correlation of the ybt with gait parameters in elderly subjects with poor \n bva ." ] ]

[ "c57bl/6j male mice ( 25 months of age ) were housed individually on a 12 hr/12 hr light / dark schedule with lights on at 7 a.m. ( zt0 ) and handled for 6 days .", "mice were sleep - deprived ( sd ) in their home cages for 5 hours by gentle handling beginning at zt5 or left undisturbed ( non - sleep - deprived mice , nsd ) . for contextual fear conditioning experiments , animals were placed in a novel chamber for 3 minutes , and received a 2-second , 1.5 ma footshock after 2.5 minutes .", "mice received intra - peritoneal injections of rolipram ( rol ; 1 mg / kg ) or vehicle ( 2% dmso in 0.9% saline ) immediately and 2.5 hours post - training .", "testing of contextual memory was performed 24 hours after training in the trained context and 48 hours after training in a novel chamber .", "1-train ltp was induced by a single 100 hz , 1-second duration train of stimuli .", "4-train ltp consisted of 4 trains applied with a 5-minute inter - train interval ; for massed 4-train ltp a 5-second inter - train interval was used .", "theta - burst stimulation ( tbs ) consisted of 40-ms duration , 100 hz bursts delivered at 5 hz for 3 seconds ( 15 bursts of 4 pulses per burst , for a total of 60 pulses ) .", "chemical ltp was induced by treatment of slices for 15 minutes with 5 m forskolin ( fsk ) in 0.1% ethanol , or a combination of 50 m forskolin and 30 m 3-isobutyl-1-methylxanthine ( ibmx , in water ) .", "rolipram ( 0.1 m in 0.1% dmso ) was applied for 60 minutes , beginning 30 minutes before tetanization .", "camp assays on ca1 regions of hippocampal slices 10 minutes after treatment for 15 minutes with forskolin ( 50 m ) , forskolin + ibmx ( 30 m ) , or vehicle ( 0.1% etoh ) were performed by radioimmunoassay according to kit instructions .", "camp - specific pde activity assays29 and western blots for pde4a530 were performed as previously described .", "full methods and any associated references are available in the online version of the paper at www.nature.com/nature ." ]

[ "<S> millions of people regularly obtain insufficient sleep1 . given the impact of sleep deprivation on our lives , understanding the cellular and molecular pathways affected by sleep deprivation is clearly of social and clinical importance . </S>", "<S> one of the major effects of sleep deprivation on the brain is to produce memory deficits in learning paradigms that are dependent on the hippocampus25 . in this study , we have identified a molecular mechanism by which brief sleep deprivation alters hippocampal function . </S>", "<S> sleep deprivation selectively impaired camp / pka - dependent forms of synaptic plasticity6 in the hippocampus , reduced camp signaling , and increased activity and protein levels of phosphodiesterase-4 ( pde4 ) , an enzyme that degrades camp . treatment with pde inhibitors rescued the sleep deprivation - induced deficits in camp signaling , synaptic plasticity , and hippocampus - dependent memory . </S>", "<S> these findings demonstrate that brief sleep deprivation disrupts hippocampal function by interfering with camp signaling through increased pde4 activity . </S>", "<S> thus drugs that enhance camp signaling may provide a novel therapeutic approach to counteract the cognitive effects of sleep deprivation . </S>" ]

[ "Methods Summary", "Supplementary Material" ]

[ [ "c57bl/6j male mice ( 25 months of age ) were housed individually on a 12 hr/12 hr light / dark schedule with lights on at 7 a.m. ( zt0 ) and handled for 6 days .", "mice were sleep - deprived ( sd ) in their home cages for 5 hours by gentle handling beginning at zt5 or left undisturbed ( non - sleep - deprived mice , nsd ) . for contextual fear conditioning experiments , animals were placed in a novel chamber for 3 minutes , and received a 2-second , 1.5 ma footshock after 2.5 minutes .", "mice received intra - peritoneal injections of rolipram ( rol ; 1 mg / kg ) or vehicle ( 2% dmso in 0.9% saline ) immediately and 2.5 hours post - training .", "testing of contextual memory was performed 24 hours after training in the trained context and 48 hours after training in a novel chamber .", "1-train ltp was induced by a single 100 hz , 1-second duration train of stimuli .", "4-train ltp consisted of 4 trains applied with a 5-minute inter - train interval ; for massed 4-train ltp a 5-second inter - train interval was used .", "theta - burst stimulation ( tbs ) consisted of 40-ms duration , 100 hz bursts delivered at 5 hz for 3 seconds ( 15 bursts of 4 pulses per burst , for a total of 60 pulses ) .", "chemical ltp was induced by treatment of slices for 15 minutes with 5 m forskolin ( fsk ) in 0.1% ethanol , or a combination of 50 m forskolin and 30 m 3-isobutyl-1-methylxanthine ( ibmx , in water ) .", "rolipram ( 0.1 m in 0.1% dmso ) was applied for 60 minutes , beginning 30 minutes before tetanization .", "camp assays on ca1 regions of hippocampal slices 10 minutes after treatment for 15 minutes with forskolin ( 50 m ) , forskolin + ibmx ( 30 m ) , or vehicle ( 0.1% etoh ) were performed by radioimmunoassay according to kit instructions .", "camp - specific pde activity assays29 and western blots for pde4a530 were performed as previously described .", "full methods and any associated references are available in the online version of the paper at www.nature.com/nature ." ], [ "" ] ]

[ "cell metabolism governing the growth and functioning of each cell and a whole organism refers to chemical transformations and enzyme - catalyzed energy producing and energy utilizing reactions of carbohydrates , proteins , and lipids . amongst the most metabolically active organs are liver , brain , gut , kidneys , and heart [ 13 ] .", "although the rate of metabolic reactions is lower in skeletal muscles , they account for around 20% of the total energy expenditure due to a 5060% contribution to a total body mass .", "several micrornas were reported to control processes related to metabolism such as insulin secretion ( mir-9 , mir-375 ) , adipocyte differentiation ( mir-143 ) , fatty acid metabolism ( mir-122 ) , and myogenesis ( mir-1 , mir-133a , mir-133b , and mir-206 ) ( reviewed in ) .", "of potential meaning is also mir-378a , located in the gene encoding master metabolic regulator , peroxisome proliferator - activated receptor gamma , coactivator 1 beta ( pgc-1 ) .", "mir-378a was found to affect lipid and xenobiotic metabolism , lipid storage , mitochondrial function , and shift towards a glycolytic pathway ( warburg effect ) [ 5 , 6 ] .", "because nutrients supply for metabolic processes is a matter of circulation , metabolically active tissues require high vascular density .", "recently , mir-378a was reported to regulate tumor angiogenesis mainly via inhibition of tumor suppressors sufu and fus-1 [ 8 , 9 ] .", "thus , a growing body of evidence suggests a role of mir-378a as a mediator controlling reciprocally dependent processes such as metabolism , muscle differentiation / regeneration , and angiogenesis .", "micrornas ( mirnas ; mirs ) are small noncoding rna molecules with an average length of 21 - 22 nucleotides which can regulate gene expression posttranscriptionally by targeting mostly the 3untranslated region ( 3utr ) of mrnas .", "however , mirna target sites were also found on the 5utr regions of human mrna . since their discovery in c. elegans in 1993 ,", "mirnas currently can be recognized as potent players in wide spectrum of biological processes like development , differentiation , cellular defense mechanisms , and others .", "conservative estimates state that over 30% of mrna expression is regulated by mirnas [ 12 , 13 ] .", "however , others suggest that even up to 60% of the mrna expression is targeted by mirnas .", "mirnas are often located in the introns of coding genes or noncoding sequences but can also be located in exons .", "intronic mirnas can be expressed together with their host gene mrna being derived from a common rna transcript [ 15 , 16 ] .", "other mirnas can also have their own promoters , which enable independent expression , or can be organized in clusters sharing a common transcriptional regulation [ 17 , 18 ] .", "mirnas transcription is rna polymerase ii - dependent . in the case of mirnas that are encoded in their own genes , the primary mirna transcript ( pri - mirna )", "is several kilobases long , while mirnas encoded in intronic regions of other genes ( mirtrons ) have shorter transcripts .", "the mirna stem loop is excised from pri - mirna by endoribonuclease drosha / dgcr8 ( microprocessor complex ) and a hairpin called pre - mirna is exported from the nucleus by exportin-5 in a ran - gtp dependent manner . an endoribonuclease dicer removes the hairpin loop sequence from pre - mirna , creating a double stranded mirna duplex . depending on the relative stability of the mirna duplex , one or , more rarely , both strands can be incorporated in a multiprotein rna - induced silencing complex ( risc ) .", "when there is perfect pairing between the mirna sequence and its target site , mrna is cleaved by a protein part of the risc called argonaute ( ago ) .", "if the pairing is partial , deadenylation of the mrna via recruitment of the ccr4-not complex by the gw182 proteins inside the risc takes place and the poly - a tail is lost , leaving the mrna vulnerable to rnase activity , ubiquitination , and mrna degradation .", "alternatively , mirna - induced risc can also cause repression of translation by mechanisms such as , for example , the promotion of ribosome drop - off from the mrna transcript or destabilization of the mrna binding cap protein ( figure 1 ) ( reviewed in [ 20 , 21 ] ) .", "the pre - mir gives rise to a leading strand ( mir-378a-3p , previous ids for murine sequence : mmu - mir-422b , mmu - mir-378 , and mmu - mir-378 - 3p ; for human : hsa - mir-422b and hsa - mir-378 ) and a passenger strand ( mir-378a-5p , previous ids for murine sequence : mmu - mir-378 , mmu - mir-378 , and mmu - mir-378 - 5p ; for human : hsa - mir-378 and hsa - mir-378 ) .", "mirna-378a-3p mature strand was first identified in 2004 in humans ( originally named mir-422b ) .", "recently , other mirs with similar sequences but other localizations in the genome have been discovered and named : mmu - mir-378b , c , d in mouse and hsa - mir-378-b , c , d1,d2,e , f , g , h , i , j in human [ 2327 ] ( table 1 ) . in humans , mir-378a is by far the most expressed of the mir-378 sequences , with 7030 reads per million , in 78 experiments during deep sequencing , compared with 1013220 reads per million , in 4272 experiments for the other forms , respectively . in mice , mir-378a and mir-378b", "have similar expression levels , at 11700 and 11000 reads per million ( mirbase , version 21 , september 2015 ) .", "the sequence of mir-378a mature strands is highly conserved between species , with the mir-378a-5p strand being identical in both human and mice and the mir-378a-3p strand only differing in one nucleotide ( table 2 ) [ 6 , 27 ] .", "pgc-1 may regulate several facets of energy metabolism such as mitochondrial biogenesis , thermogenesis , and glucose and fatty acid metabolism .", "both strands of mir-378a are coexpressed with pgc-1 as shown , for example , in the liver and during adipocyte differentiation [ 6 , 29 ] .", "the coexpression of mir-378a with its host gene implies they may share the same transcriptional activators , and mir-378a might be involved in similar processes as pgc-1. accordingly , high levels of ( porcine ) mir-378 - 1 ( table 2 ) expression are found in developing muscle , postnatal muscle , and myocardium and in brown adipose tissue [ 29 , 30 ] . to date", ", only a limited number of mir-378a targets , which can be predicted based on in silico analysis , have been experimentally validated .", "the latter , however , imply a role of mir-378a in mitochondrial energy homeostasis , glycolysis , and skeletal muscle development and in tumor angiogenesis and other processes ( table 3 ) .", "a major source of energy production comprises oxidation of glucose in glycolysis followed by oxidation of pyruvate in well - oxygenated cells ( or followed by lactic acid fermentation in cancer , the warburg effect ) and from -oxidation of lipids , which yields even more atp per gram then carbohydrates metabolism .", "a location of mir-378a in the gene encoding pgc-1 implies an involvement of mir-378a in metabolic pathways . unlike its homologue , pgc-1 , the expression of pgc-1", "is not elevated in response to cold exposure but occurs in response to hypoxia , exercise , caloric restriction , or aging ( reviewed in ) .", "pgc-1 is preferentially expressed in tissues with relatively high mitochondrial content , such as heart , skeletal muscle , and brown adipose tissue . in 2002", ", pgc-1 was first cloned and shown to be upregulated in the liver during fasting .", "pgc-1 strongly activates hepatic nuclear factor 4 ( hnf4 ) and ppar , both of these nuclear receptors being important for the adaptation of hepatocytes to the effects of fasting .", "these findings could hint to a possible role of pgc-1 in the regulation of gluconeogenesis and fatty acid oxidation in the liver .", "pgc-1 is also involved in the regulation of energy expenditure or in the pathway of estrogen receptor - related receptors ( errs ) [ 3337 ] . since mirnas originating in the introns of host genes", "may modulate the protein encoded by their parental genes and may be involved in the same mechanisms [ 3840 ] , mir-378a is proposed to be involved in the metabolic pathways affected by pgc-1 .", "it was reported that mice lacking the first intron of the ppargc1b gene ( and thus mir-378a ) have a significantly higher oxygen capacity and mitochondrial function .", "they identified a mediator complex subunit 13 ( med13 ) , involved in nuclear receptor signaling , and carnitine acetyltransferase ( crat ) , a mitochondrial enzyme involved in fatty acid metabolism , as targets of mir-378a-5p and mir-378a-3p , respectively .", "regulated also cytochrome p450 2e1 ( cyp2e1 ) being involved in the metabolism of , for example , drugs and toxins . in addition , it has been discovered that transcription factor nuclear respiratory factor-1 ( nrf-1 ) , a critical regulator of the expression of some important metabolic genes in mitochondria regulating cellular growth , is inhibited by mir-378a-3p .", "thus , mir-378a can be considered as a regulator of mitochondrial function in cells overexpressing mir-378a .", "moreover , mir-378a-5p inhibits the mrnas of err and ga - binding protein- in breast cancer , which both interact with pgc-1 and together control oxidative metabolism .", "this leads to a reduction of tricarboxylic acid gene expression and oxygen consumption and an increase in lactate production , which shifts cells from an oxidative towards a glycolytic pathway . in this way", ", mir-378a-5p is believed to be a switch regulating the warburg effect in breast cancer .", "moreover , in situ hybridization experiments in this study showed that mir-378a-5p expression correlates with progression of breast cancer .", "the proposed regulating role of mir-378a-5p on the warburg effect is in parallel with the effects of pgc-1 , which mediates gluconeogenesis and fatty acid metabolism after periods of fasting or intense exercise .", "coactivation by pgc-1 of err and ppar makes muscle fibers in pgc-1 transgenic mice more rich in mitochondria and highly oxidative . accordingly", "increased glycolytic rates and increased cell proliferation can be related to lactate production by lactate dehydrogenase ( ldh ) .", "ldha was found to be a direct target of mir-378a in the study of mallat et al . .", "hsa - mir-378a-3p represses cell growth and increases cell death by targeting ldha . of note , hsa - mir-378a-3p and hsa - mir-378a-5p had opposite effects on ldha expression .", "in addition , mir-378a is also considered as an important factor in adipogenesis and lipid storage .", "there is a complex family of factors regulating those processes such as insulin , insulin - like growth factors ( igfs ) , glucagon , and thyroid hormones t3 and t4 ( reviewed in [ 4649 ] ) .", "as mentioned before , it was demonstrated that mir-378a - knockout mice do not get fat after 8 weeks of high fat diet .", "such animals show an enhanced mitochondrial fatty acid metabolism and have elevated oxidative capacity of tissues targeted by insulin ( e.g. , liver , muscles , and adipose tissues ) . in accordance with that , it was shown that mature strands of bta - mir-378 - 1 ( table 1 ) are expressed at higher level in cows with high ( versus low ) amount of back fat .", "similarly , an inhibition of both mmu - mir-378a-3p and its host gene , pgc-1 , by the flavonoid fisetin lowered the accumulation of fat in the liver .", "interestingly , mmu - mir-378a-5p was downregulated in mice that were fed a high fat diet for five months .", "overexpression of mir-378a-3p/-5p during adipogenesis increased the transcriptional activity of ccaat / enhancer - binding proteins ( cebp ) alpha and beta , which can stimulate the expression of leptin , a hormone produced mainly by adipocytes which controls the homeostasis of body weight ( reviewed in [ 52 , 53 ] ) . on the other hand , tnf- , il-6 , and", "leptin are reported to increase the expression of mir-378a-3p in mature human adipocytes in vitro .", "these cytokines are mainly secreted in the adipose tissue and are suggested to be involved in development of insulin resistance [ 55 , 56 ] . in addition , mir-378a-3p was shown to target insulin growth factor 1 receptor ( igf1r ) and reduce the akt signaling cascade in cardiomyocytes during cardiac development . moreover , in tissues where igf1 levels were high ( e.g. , fibroblasts and fetal hearts ) , mir-378 - 3p levels were very low , showing an inverse relation and suggesting a negative feedback loop between mir-378a-3p , igf1r , and igf1 .", "the latter functions as a master regulator of adipogenesis and is involved in the formation of peroxisomes and the catabolism of very long chain fatty acids [ 58 , 59 ] .", "accordingly , the amount of adipose tissue does not increase in mice lacking ppar when they are fed a high fat diet .", "it was also reported that in cultured adipocytes mmu - mir-378a and pgc-1 expression is ppar , or rosiglitazone ( a ppar ligand ) , dependent , finding two peroxisome proliferator response elements in the mir-378a loci . on the other hand , overexpression of mir-378a elevated the expression of ppar isoform 2 , suggesting positive feedback loop and confirming the involvement of mir-378a in the storage of fat .", "there are several activators known to induce expression of ppar such as the members of the e2f transcription factor family and prostaglandin j-2 ( pgj-2 ) [ 6365 ] .", "the latter may act through rar - related orphan receptor alpha ( rora ) , which is frequently found in myocardium .", "in addition to ppar , rora regulates also myod , a major transcription factor involved in skeletal muscle differentiation [ 67 , 68 ] .", "interestingly , rora is a possible ( but not yet validated ) target for mir-378a-3p .", "a proteomics - based study revealed several other proteins that are potentially targeted by rat mir-378a-3p or mir-378a-5p .", "mir-378a-3p was shown to regulate mannose-1-phosphate guanylyltransferase ( gdp ) , dimethylarginine dimethylaminohydrolase 1 ( ddah1 ) , and lactate dehydrogenase a ( ldha ) ; all those proteins are participating in metabolic processes . on the other hand , tropomyosin beta chain , which is involved in the regulation of atpase activity ,", "high levels of murine and rat mir-378a-3p , mir-378a-5p , and porcine mir-378 - 1 are reported in both developing and adult skeletal muscles [ 7 , 30 , 44 ] .", "myod and myog play a role in the processes of myogenesis and muscle regeneration , in which dormant satellite cells are activated upon muscle damage and start proliferating and differentiating into muscle fibers ( reviewed in [ 70 , 71 ] ) .", "it has been shown that mir-378a-3p targets the myogenic repressor myor during myoblast differentiation , which directly inhibits myod .", "on the other hand , myod is upregulated in response to mir-378a-3p overexpression and , conversely , the level of mir-378a-3p may be enhanced by myod .", "thus , there is evidence for a feedback loop in which mir-378a-3p regulates muscle differentiation via inhibiting myor , leading to an increase of myod , which in turn enhances mir-378a-3p .", "that mir-378a may also control the development of skeletal muscle mass after training . in this study , mir-378a ( strand not specified ) was significantly downregulated in men who obtained low training - induced muscle mass gain compared to men who obtained high training - induced muscle mass gain .", "mir-378a-3p is expressed mostly by cardiomyocytes , but not by nonmuscle cells , whereas the level of mir-378a-5p was reported to be very low in the heart .", "fang et al . showed that mir-378 - 3p is significantly downregulated both in vitro in cardiomyocytes cell cultures exposed to hypoxia and in vivo during myocardial injury in rats .", "overexpression of mir-378a-3p enhanced cell viability and inhibited apoptosis via caspase-3 inhibition . in contrast to this finding , another study found that mir-378a-3p downregulation enhanced the survival of cardiac stem cells via focal adhesion kinase activation and releasing connective tissue growth factor ( ctgf ) , the latter being a target of mir-378a-3p .", "overexpression of mir-378a-3p in the study of knezevic et al . increased apoptosis of cardiomyocytes via the direct targeting of igf1r leading to a decrease of akt signaling .", "this is in opposition to the previously mentioned study of fang et al . which showed apoptosis", "the converse findings of the studies could be explained by different models used by knezevic et al . and", "because of those discrepancies , the role of mir-378a in apoptosis of cardiomyocytes requires further investigation .", "the finding that mir-378a-3p affects both igf1r and the akt pathway was confirmed in a study which found that overexpression of mir-378a-3p in rhabdomyosarcoma suppressed igf1r expression and affected phosphorylation of the akt protein .", "mir-378a-5p was shown to target heat shock protein 70.3 ( hsp70.3 ) in mouse hearts in normoxic conditions , but in hypoxic conditions a transcript variant of hsp70.3 without mir-378a-5p target site in its 3-utr is not repressed and can exert its cytoprotective properties .", "mir-378a-3p prevented cardiac hypertrophy by targeting either ras signaling or the mitogen - activated protein kinase ( mapk ) pathway [ 77 , 78 ] .", "more studies on the effect of mir-378a expression in muscle disorders would also be desirable . in both golden retriever muscular dystrophy dogs and duchenne muscular dystrophy patients ,", "all in all , these findings suggest mir-378a-3p can be considered as an important player in cardiac development , remodeling , and hypertrophy .", "angiogenesis comprises development of new blood vessels from existing ones , regulated by cytokines and growth factors such as , for example , vascular endothelial growth factor ( vegf ) , platelet - derived growth factor ( pdgf ) , and angiopoietin-1 ( ang-1 ) .", "their expression can be posttranscriptionally controlled by micrornas such as mir-126 , mir-296 , mir-210 , mir-21 , and the mir-17~92 cluster ( reviewed in ) .", "skeletal muscles and heart muscle are tissues which , due to their oxygen and energy consumption , need to be sufficiently vascularized .", "one of the major regulators of angiogenesis is the hypoxia - inducible factor-1 ( hif-1 ) , which controls over 100 genes involved mainly in the glycolytic pathway and blood vessel formation , including vegf - a or interleukin-8 [ 8385 ] .", "vegf is generally induced by hypoxia , while il-8 in at least some cancers and endothelial cells can be diminished by hif-1 via inhibition of c - myc and sp-1 transcription factors [ 86 , 87 ] .", "c - myc , known as a regulator of cell cycle progression , apoptosis , and cellular transformation , is also a potent activator of pgc-1 and , in turn , mir-378a-3p , upregulating their expression .", "in addition , mir-378a has been shown to affect vegf - a in two ways .", "human hsa - mir-378a-5p ( by the study of hua et al . named as mir-378 ) can directly affect vegf - a by competing with hsa - mir-125a for the same seed - region in the vegf - a 3utr causing upregulation of vegf - a .", "mir-378a-5p can also indirectly regulate vegf - a affecting sonic hedgehog ( shh ) signaling via sufu inhibition , which is an inhibitory component of this signaling pathway .", "the shh pathway in turn can upregulate vegf - a and also other regulators of blood vessels formation , ang-1 and ang-2 expression [ 9092 ] .", "increased expression of vegf - a , as well as pdgf and tgf1 , was also seen in mesenchymal stromal cells ( mscs ) transfected with rno - mir-378a-5p . in skeletal muscles , vegf - induced angiogenesis appears not to be regulated by the well - known hif pathway but by pgc-1 , which coactivates estrogen - related receptor alpha ( err- ) on binding sites in the promoter and the first intron of the vegf gene , inducing its expression .", "this angiogenic pathway shows new roles for pgc-1 and err- , which are important regulators of mitochondrial activity in response to stimuli like exercise .", "if there might be a role for pgc-1 in this pathway , it is yet to be examined .", "it is noteworthy , however , that mir-378a-5p is known to affect the estrogen receptors by inhibiting err , another estrogen - related receptor .", "a role for mir-378a in cell cycle regulation and stimulation of cell growth is also proposed . in human mammary epithelial and breast cancer cell lines", ", mir-378a-3p can target the antiproliferative protein tob2 , which is a suppressor of cyclin d1 , which in turn is required for cell cycle g1-phase to s - phase progression .", "whether mir-378 affects endothelial cell proliferation by regulation of cell cycle remains to be established .", "the role of mir-378a in the formation of blood vessels nourishing tumor and enabling tumor growth was revealed .", "mir-378a was found to be differentially regulated in different types of cancers being downregulated in gastric cancer [ 96 , 97 ] , oral , and colon carcinoma , while being upregulated in renal and lung cancer [ 9 , 101 ] . since it is also changed in serum or plasma of patients with prostate cancer , renal cancer [ 100 , 103 ] , and gastric cancer and frequently found to be overexpressed in cryopreserved bone marrow mononuclear cells from acute myeloid leukemia patients , mir-378a might be considered as a biomarker .", "the role of mir-378a in tumorigenesis , tumor growth , and tumor vascularization was revealed for the first time by lee and coworkers in glioblastoma .", "they showed that mir-378a-5p enhances cell survival , reduces caspase-3 activity , and promotes tumor growth and angiogenesis , through repression of two tumor suppressors , sufu and fus-1 .", "strikingly , nude mice injected with mir-378a-5p transfected cancer cells formed tumors of bigger volume and with larger blood vessels compared to gfp - transfected cells . on the other hand , high expression of mir-378a-5p in nsclc correlated with brain metastases due to higher cell migration , invasion , and tumor angiogenesis .", "another study confirmed the downregulation of fus-1 by mir-378a-5p and showed that in the hepg2 liver cancer cells mir-378a-5p overexpression enhanced proliferation , migration , and , when injected in mice , invasion .", "also in rhabdomyosarcoma , enhanced expression of mir-378a , vegf , and mmp9 correlated with increased vascularization and metastasis . taken together", ", these studies suggest that mir-378a may serve as a prognostic marker in cancer due to its effects on angiogenesis .", "our recent data confirmed the proangiogenic effect of mir-378a ( both strands ) in non - small cell lung carcinoma ( nsclc ) and pointed at its correlation with heme - degrading enzyme , heme oxygenase-1 ( ho-1 ) .", "an involvement of ho-1 in angiogenesis and vegf - a as well as il-8 signaling was shown by us previously ; however , its action in tumors seems to be complex . in nci - h292 cell line overexpressing ho-1 , mir-378a ( both strands )", "also overexpression of the mir-378a precursor sequence diminished ho-1 expression . conditioned medium from nci - h292 cells overexpressing mir-378a enhanced angiogenic potential of hmec-1 endothelial cell line .", "tumors formed by such cells in subcutaneous xenografts showed enhanced growth , vascularization , oxygenation , and distal metastasis in vivo .", "these interactions between mir-378a and ho-1 were confirmed in our studies on the role of the nrf-2 transcription factor / ho-1 axis in nsclc cell lines [ 110 , 111 ] . on the other hand ,", "enhanced expression of mmu - mir-378a-5p in 4t1 murine breast cancer cells decreased the proliferation , migration , and invasiveness of these cancer cells in vitro and in vivo by targeting fibronectin , resulting in inhibition of tumor growth .", "recent study showed that mir-378a may act as a biomarker for response to antiangiogenic treatment in ovarian cancer .", "low expression of mir-378a was associated with longer progressive - free survival in patients with recurrent ovarian cancer treated with the antiangiogenic drug bevacizumab .", "overexpression of the mir-378a precursor in ovarian cancer cells altered expression of genes associated with angiogenesis ( alcam , ehd1 , elk3 , and tln1 ) , apoptosis ( rpn2 , hipk3 ) , and cell cycle regulation ( swap-70 , lsm14a , and rdx ) .", "high mir-378a ( strand not specified ) expression in renal carcinoma correlated with higher levels of endothelial surface marker cd34 in these tumors .", "notably , a recent study suggested clinical relevance for mir-378a in metastatic colorectal cancer , in which enhanced mir-378a expression significantly improved the sensitivity to cetuximab treatment in these patients .", "mir-378a-5p transfection of mscs has been shown to enhance their survival and angiogenic potential under hypoxic conditions in vitro .", "in coculture with human umbilical vein endothelial cells ( huvecs ) , mir-378a-5p - transfected mscs formed a larger number of vascular branches on matrigel . in the mscs transfected with mir-378a-5p , the expression of bcl-2-associated x protein ( bax ) , which is an important proapoptotic regulator , was decreased , leading to a better survival .", "it still has to be determined if the proangiogenic effect of mir-378a in vivo is confined to tumor angiogenesis , or if this effect is also present in physiological angiogenesis and regenerative neovascularization .", "recently , it was reported that anti - mir-378a-5p enhances wound healing process by upregulating integrin beta-3 and vimentin .", "it was reported that pgc-1 induces angiogenesis in skeletal muscle , enhancing the expression of vegf both in vitro and in vivo after ( transgenic ) overexpression .", "accordingly , it was also found that vegfa is upregulated in c2c12 myoblast cell line with pgc-1 overexpression . however , after a pcr - based gene array of 84 known angiogenic factors and further rt - pcr of individual genes , they concluded that pgc-1 triggered an antiangiogenic program .", "after inducing hind limb ischemia in pgc-1 overexpressing mice , an impaired reperfusion was noticed when compared to wild type littermates .", "the role of inflammation in angiogenesis is studied the most in the context of cancer ( e.g. , reviewed in [ 119 , 120 ] ) but is certainly not limited to this pathology .", "both lymphoid ( reviewed in [ 121 , 122 ] ) and myeloid ( reviewed in ) derived inflammatory cells affect angiogenesis in a stimulating or inhibitory manner .", "the role of mir-378a in inflammatory cells was reported and its anti - inflammatory effect could be suggested .", "nk cells exert potent cytotoxic effects when activated by type i ifn from the host once infected .", "mir-378a was found to be downregulated in activated nk cells and further proved to target granzyme b. thus , ifn- activation decreases mir-378a expression and in turn augments nk cell cytotoxicity .", "accordingly , suppression of mir-378 targeting granzyme b in nk cells resulted in inhibition of dengue virus replication in vivo .", "macrophages are known to play either inhibitory or stimulatory roles in angiogenesis ( reviewed by ) .", "mirnas have been proposed to regulate activation and polarization of macrophages ( reviewed by [ 127 , 128 ] ) . in a study of rckerl", "mir-378a-3p was identified as a part of the il-4-driven activation program of anti - inflammatory macrophages ( m2 ) .", "mir-378a-3p was highly upregulated after stimulation with il-4 of peritoneal exudate cells of mice injected with the parasite brugia malayi compared to controls and infected il-4-knockout mice .", "the study identified several targets for mir-378a-3p within the pi3 k / akt signaling pathway , which are important for proliferation but only partially responsible for m2 phenotype .", "another study found mir-378a ( strand not specified ) expression upregulated after stimulation with cytokines like , for example , tnf- and il-6 . in line with its potential role in macrophages ,", "mmu - mir-378a ( strand not specified ) has been found to be upregulated during osteoclastogenesis in vitro .", "furthermore , serum levels of mir-378a-3p have been shown to correlate with bone metastasis burden in mice injected with mouse mammary tumor cell lines 4t1 and 4t1.2 .", "a growing body of evidence suggests a role for mir-378a as a mediator controlling reciprocally dependent processes in metabolism , muscle differentiation / regeneration , and angiogenesis .", "as mir-378a was found to be differentially regulated in different types of cancers and its level is changed in serum of prostate , renal , and gastric cancer patients , it can be considered as a biomarker for those diseases . the correlation between mir-378a expression and disease progression in lung cancer , liver cancer , and rhabdomyosarcoma suggests a further role of this microrna as a prognostic marker .", "if more research will be done to the mechanisms of action , possibilities for therapeutic use of mir-378a could be sought in the field of metabolic disorders , obesity , or tumors .", "more studies on the effect of mir-378a expression in muscle disorders would also be desirable .", "the proangiogenic effect of mir-378a was observed in tumors ; however , no studies have been performed on the angiogenic effects of mir-378a in physiological settings or diseases where angiogenesis plays important roles , such as diabetes and cardiovascular diseases .", "more study has to be done to assess the mechanisms of mir-378a function in blood vessel formation . of note , in contrast with proangiogenic role of mir-378a , inhibition of mir-378a-5p enhanced wound healing process .", "this might suggest a role for mir-378a-5p in diseases such as diabetes or in decubitus ulcers , in which wound healing is impaired . of note is", "the confusion that has arises because of a disarray in nomenclature with studies describing the same molecule , mir-378a , as mir-422b , mir-378 , or mir-378 .", "in addition , it is not always clear which of the two mature strands of mir-378a is studied", ". this could lead to misunderstandings and errors in interpreting the data published so far ." ]

[ "<S> microrna-378a ( mir-378a , previously known as mir-378 ) is one of the small noncoding rna molecules able to regulate gene expression at posttranscriptional level . </S>", "<S> its two mature strands , mir-378a-3p and mir-378a-5p , originate from the first intron of the peroxisome proliferator - activated receptor gamma , coactivator 1 beta ( ppargc1b ) gene encoding pgc-1. embedding in the sequence of this transcriptional regulator of oxidative energy metabolism implies involvement of mir-378a in metabolic pathways , mitochondrial energy homeostasis , and related biological processes such as muscle development , differentiation , and regeneration . on the other hand , </S>", "<S> modulating the expression of proangiogenic factors such as vascular endothelial growth factor , angiopoietin-1 , or interleukin-8 , influencing inflammatory reaction , and affecting tumor suppressors , such as sufu and fus-1 , mir-378a is considered as a part of an angiogenic network in tumors . in the latter </S>", "<S> , mir-378a can evoke broader actions by enhancing cell survival , reducing apoptosis , and promoting cell migration and invasion . </S>", "<S> this review describes the current knowledge on mir-378a linking oxidative / lipid metabolism , muscle biology , and blood vessel formation . </S>" ]

[ "1. Introduction", "2. MicroRNAs", "3. miR-378a: Basics", "4. miR-378a in Metabolism", "5. miR-378a in Muscle Development, Differentiation, and Regeneration", "6. miR-378a in Angiogenesis", "7. miR-378a in Inflammation ", "8. Conclusions " ]

[ [ "cell metabolism governing the growth and functioning of each cell and a whole organism refers to chemical transformations and enzyme - catalyzed energy producing and energy utilizing reactions of carbohydrates , proteins , and lipids . amongst the most metabolically active organs are liver , brain , gut , kidneys , and heart [ 13 ] .", "although the rate of metabolic reactions is lower in skeletal muscles , they account for around 20% of the total energy expenditure due to a 5060% contribution to a total body mass .", "several micrornas were reported to control processes related to metabolism such as insulin secretion ( mir-9 , mir-375 ) , adipocyte differentiation ( mir-143 ) , fatty acid metabolism ( mir-122 ) , and myogenesis ( mir-1 , mir-133a , mir-133b , and mir-206 ) ( reviewed in ) .", "of potential meaning is also mir-378a , located in the gene encoding master metabolic regulator , peroxisome proliferator - activated receptor gamma , coactivator 1 beta ( pgc-1 ) .", "mir-378a was found to affect lipid and xenobiotic metabolism , lipid storage , mitochondrial function , and shift towards a glycolytic pathway ( warburg effect ) [ 5 , 6 ] .", "because nutrients supply for metabolic processes is a matter of circulation , metabolically active tissues require high vascular density .", "recently , mir-378a was reported to regulate tumor angiogenesis mainly via inhibition of tumor suppressors sufu and fus-1 [ 8 , 9 ] .", "thus , a growing body of evidence suggests a role of mir-378a as a mediator controlling reciprocally dependent processes such as metabolism , muscle differentiation / regeneration , and angiogenesis ." ], [ "micrornas ( mirnas ; mirs ) are small noncoding rna molecules with an average length of 21 - 22 nucleotides which can regulate gene expression posttranscriptionally by targeting mostly the 3untranslated region ( 3utr ) of mrnas .", "however , mirna target sites were also found on the 5utr regions of human mrna . since their discovery in c. elegans in 1993 ,", "mirnas currently can be recognized as potent players in wide spectrum of biological processes like development , differentiation , cellular defense mechanisms , and others .", "conservative estimates state that over 30% of mrna expression is regulated by mirnas [ 12 , 13 ] .", "however , others suggest that even up to 60% of the mrna expression is targeted by mirnas .", "mirnas are often located in the introns of coding genes or noncoding sequences but can also be located in exons .", "intronic mirnas can be expressed together with their host gene mrna being derived from a common rna transcript [ 15 , 16 ] .", "other mirnas can also have their own promoters , which enable independent expression , or can be organized in clusters sharing a common transcriptional regulation [ 17 , 18 ] .", "mirnas transcription is rna polymerase ii - dependent . in the case of mirnas that are encoded in their own genes , the primary mirna transcript ( pri - mirna )", "is several kilobases long , while mirnas encoded in intronic regions of other genes ( mirtrons ) have shorter transcripts .", "the mirna stem loop is excised from pri - mirna by endoribonuclease drosha / dgcr8 ( microprocessor complex ) and a hairpin called pre - mirna is exported from the nucleus by exportin-5 in a ran - gtp dependent manner . an endoribonuclease dicer removes the hairpin loop sequence from pre - mirna , creating a double stranded mirna duplex . depending on the relative stability of the mirna duplex , one or , more rarely , both strands can be incorporated in a multiprotein rna - induced silencing complex ( risc ) .", "when there is perfect pairing between the mirna sequence and its target site , mrna is cleaved by a protein part of the risc called argonaute ( ago ) .", "if the pairing is partial , deadenylation of the mrna via recruitment of the ccr4-not complex by the gw182 proteins inside the risc takes place and the poly - a tail is lost , leaving the mrna vulnerable to rnase activity , ubiquitination , and mrna degradation .", "alternatively , mirna - induced risc can also cause repression of translation by mechanisms such as , for example , the promotion of ribosome drop - off from the mrna transcript or destabilization of the mrna binding cap protein ( figure 1 ) ( reviewed in [ 20 , 21 ] ) ." ], [ "the pre - mir gives rise to a leading strand ( mir-378a-3p , previous ids for murine sequence : mmu - mir-422b , mmu - mir-378 , and mmu - mir-378 - 3p ; for human : hsa - mir-422b and hsa - mir-378 ) and a passenger strand ( mir-378a-5p , previous ids for murine sequence : mmu - mir-378 , mmu - mir-378 , and mmu - mir-378 - 5p ; for human : hsa - mir-378 and hsa - mir-378 ) .", "mirna-378a-3p mature strand was first identified in 2004 in humans ( originally named mir-422b ) .", "recently , other mirs with similar sequences but other localizations in the genome have been discovered and named : mmu - mir-378b , c , d in mouse and hsa - mir-378-b , c , d1,d2,e , f , g , h , i , j in human [ 2327 ] ( table 1 ) . in humans , mir-378a is by far the most expressed of the mir-378 sequences , with 7030 reads per million , in 78 experiments during deep sequencing , compared with 1013220 reads per million , in 4272 experiments for the other forms , respectively . in mice , mir-378a and mir-378b", "have similar expression levels , at 11700 and 11000 reads per million ( mirbase , version 21 , september 2015 ) .", "the sequence of mir-378a mature strands is highly conserved between species , with the mir-378a-5p strand being identical in both human and mice and the mir-378a-3p strand only differing in one nucleotide ( table 2 ) [ 6 , 27 ] .", "pgc-1 may regulate several facets of energy metabolism such as mitochondrial biogenesis , thermogenesis , and glucose and fatty acid metabolism .", "both strands of mir-378a are coexpressed with pgc-1 as shown , for example , in the liver and during adipocyte differentiation [ 6 , 29 ] .", "the coexpression of mir-378a with its host gene implies they may share the same transcriptional activators , and mir-378a might be involved in similar processes as pgc-1. accordingly , high levels of ( porcine ) mir-378 - 1 ( table 2 ) expression are found in developing muscle , postnatal muscle , and myocardium and in brown adipose tissue [ 29 , 30 ] . to date", ", only a limited number of mir-378a targets , which can be predicted based on in silico analysis , have been experimentally validated .", "the latter , however , imply a role of mir-378a in mitochondrial energy homeostasis , glycolysis , and skeletal muscle development and in tumor angiogenesis and other processes ( table 3 ) ." ], [ "a major source of energy production comprises oxidation of glucose in glycolysis followed by oxidation of pyruvate in well - oxygenated cells ( or followed by lactic acid fermentation in cancer , the warburg effect ) and from -oxidation of lipids , which yields even more atp per gram then carbohydrates metabolism .", "a location of mir-378a in the gene encoding pgc-1 implies an involvement of mir-378a in metabolic pathways . unlike its homologue , pgc-1 , the expression of pgc-1", "is not elevated in response to cold exposure but occurs in response to hypoxia , exercise , caloric restriction , or aging ( reviewed in ) .", "pgc-1 is preferentially expressed in tissues with relatively high mitochondrial content , such as heart , skeletal muscle , and brown adipose tissue . in 2002", ", pgc-1 was first cloned and shown to be upregulated in the liver during fasting .", "pgc-1 strongly activates hepatic nuclear factor 4 ( hnf4 ) and ppar , both of these nuclear receptors being important for the adaptation of hepatocytes to the effects of fasting .", "these findings could hint to a possible role of pgc-1 in the regulation of gluconeogenesis and fatty acid oxidation in the liver .", "pgc-1 is also involved in the regulation of energy expenditure or in the pathway of estrogen receptor - related receptors ( errs ) [ 3337 ] . since mirnas originating in the introns of host genes", "may modulate the protein encoded by their parental genes and may be involved in the same mechanisms [ 3840 ] , mir-378a is proposed to be involved in the metabolic pathways affected by pgc-1 .", "it was reported that mice lacking the first intron of the ppargc1b gene ( and thus mir-378a ) have a significantly higher oxygen capacity and mitochondrial function .", "they identified a mediator complex subunit 13 ( med13 ) , involved in nuclear receptor signaling , and carnitine acetyltransferase ( crat ) , a mitochondrial enzyme involved in fatty acid metabolism , as targets of mir-378a-5p and mir-378a-3p , respectively .", "regulated also cytochrome p450 2e1 ( cyp2e1 ) being involved in the metabolism of , for example , drugs and toxins . in addition , it has been discovered that transcription factor nuclear respiratory factor-1 ( nrf-1 ) , a critical regulator of the expression of some important metabolic genes in mitochondria regulating cellular growth , is inhibited by mir-378a-3p .", "thus , mir-378a can be considered as a regulator of mitochondrial function in cells overexpressing mir-378a .", "moreover , mir-378a-5p inhibits the mrnas of err and ga - binding protein- in breast cancer , which both interact with pgc-1 and together control oxidative metabolism .", "this leads to a reduction of tricarboxylic acid gene expression and oxygen consumption and an increase in lactate production , which shifts cells from an oxidative towards a glycolytic pathway . in this way", ", mir-378a-5p is believed to be a switch regulating the warburg effect in breast cancer .", "moreover , in situ hybridization experiments in this study showed that mir-378a-5p expression correlates with progression of breast cancer .", "the proposed regulating role of mir-378a-5p on the warburg effect is in parallel with the effects of pgc-1 , which mediates gluconeogenesis and fatty acid metabolism after periods of fasting or intense exercise .", "coactivation by pgc-1 of err and ppar makes muscle fibers in pgc-1 transgenic mice more rich in mitochondria and highly oxidative . accordingly", "increased glycolytic rates and increased cell proliferation can be related to lactate production by lactate dehydrogenase ( ldh ) .", "ldha was found to be a direct target of mir-378a in the study of mallat et al . .", "hsa - mir-378a-3p represses cell growth and increases cell death by targeting ldha . of note , hsa - mir-378a-3p and hsa - mir-378a-5p had opposite effects on ldha expression .", "in addition , mir-378a is also considered as an important factor in adipogenesis and lipid storage .", "there is a complex family of factors regulating those processes such as insulin , insulin - like growth factors ( igfs ) , glucagon , and thyroid hormones t3 and t4 ( reviewed in [ 4649 ] ) .", "as mentioned before , it was demonstrated that mir-378a - knockout mice do not get fat after 8 weeks of high fat diet .", "such animals show an enhanced mitochondrial fatty acid metabolism and have elevated oxidative capacity of tissues targeted by insulin ( e.g. , liver , muscles , and adipose tissues ) . in accordance with that , it was shown that mature strands of bta - mir-378 - 1 ( table 1 ) are expressed at higher level in cows with high ( versus low ) amount of back fat .", "similarly , an inhibition of both mmu - mir-378a-3p and its host gene , pgc-1 , by the flavonoid fisetin lowered the accumulation of fat in the liver .", "interestingly , mmu - mir-378a-5p was downregulated in mice that were fed a high fat diet for five months .", "overexpression of mir-378a-3p/-5p during adipogenesis increased the transcriptional activity of ccaat / enhancer - binding proteins ( cebp ) alpha and beta , which can stimulate the expression of leptin , a hormone produced mainly by adipocytes which controls the homeostasis of body weight ( reviewed in [ 52 , 53 ] ) . on the other hand , tnf- , il-6 , and", "leptin are reported to increase the expression of mir-378a-3p in mature human adipocytes in vitro .", "these cytokines are mainly secreted in the adipose tissue and are suggested to be involved in development of insulin resistance [ 55 , 56 ] . in addition , mir-378a-3p was shown to target insulin growth factor 1 receptor ( igf1r ) and reduce the akt signaling cascade in cardiomyocytes during cardiac development . moreover , in tissues where igf1 levels were high ( e.g. , fibroblasts and fetal hearts ) , mir-378 - 3p levels were very low , showing an inverse relation and suggesting a negative feedback loop between mir-378a-3p , igf1r , and igf1 .", "the latter functions as a master regulator of adipogenesis and is involved in the formation of peroxisomes and the catabolism of very long chain fatty acids [ 58 , 59 ] .", "accordingly , the amount of adipose tissue does not increase in mice lacking ppar when they are fed a high fat diet .", "it was also reported that in cultured adipocytes mmu - mir-378a and pgc-1 expression is ppar , or rosiglitazone ( a ppar ligand ) , dependent , finding two peroxisome proliferator response elements in the mir-378a loci . on the other hand , overexpression of mir-378a elevated the expression of ppar isoform 2 , suggesting positive feedback loop and confirming the involvement of mir-378a in the storage of fat .", "there are several activators known to induce expression of ppar such as the members of the e2f transcription factor family and prostaglandin j-2 ( pgj-2 ) [ 6365 ] .", "the latter may act through rar - related orphan receptor alpha ( rora ) , which is frequently found in myocardium .", "in addition to ppar , rora regulates also myod , a major transcription factor involved in skeletal muscle differentiation [ 67 , 68 ] .", "interestingly , rora is a possible ( but not yet validated ) target for mir-378a-3p .", "a proteomics - based study revealed several other proteins that are potentially targeted by rat mir-378a-3p or mir-378a-5p .", "mir-378a-3p was shown to regulate mannose-1-phosphate guanylyltransferase ( gdp ) , dimethylarginine dimethylaminohydrolase 1 ( ddah1 ) , and lactate dehydrogenase a ( ldha ) ; all those proteins are participating in metabolic processes . on the other hand , tropomyosin beta chain , which is involved in the regulation of atpase activity ," ], [ "high levels of murine and rat mir-378a-3p , mir-378a-5p , and porcine mir-378 - 1 are reported in both developing and adult skeletal muscles [ 7 , 30 , 44 ] .", "myod and myog play a role in the processes of myogenesis and muscle regeneration , in which dormant satellite cells are activated upon muscle damage and start proliferating and differentiating into muscle fibers ( reviewed in [ 70 , 71 ] ) .", "it has been shown that mir-378a-3p targets the myogenic repressor myor during myoblast differentiation , which directly inhibits myod .", "on the other hand , myod is upregulated in response to mir-378a-3p overexpression and , conversely , the level of mir-378a-3p may be enhanced by myod .", "thus , there is evidence for a feedback loop in which mir-378a-3p regulates muscle differentiation via inhibiting myor , leading to an increase of myod , which in turn enhances mir-378a-3p .", "that mir-378a may also control the development of skeletal muscle mass after training . in this study , mir-378a ( strand not specified ) was significantly downregulated in men who obtained low training - induced muscle mass gain compared to men who obtained high training - induced muscle mass gain .", "mir-378a-3p is expressed mostly by cardiomyocytes , but not by nonmuscle cells , whereas the level of mir-378a-5p was reported to be very low in the heart .", "fang et al . showed that mir-378 - 3p is significantly downregulated both in vitro in cardiomyocytes cell cultures exposed to hypoxia and in vivo during myocardial injury in rats .", "overexpression of mir-378a-3p enhanced cell viability and inhibited apoptosis via caspase-3 inhibition . in contrast to this finding , another study found that mir-378a-3p downregulation enhanced the survival of cardiac stem cells via focal adhesion kinase activation and releasing connective tissue growth factor ( ctgf ) , the latter being a target of mir-378a-3p .", "overexpression of mir-378a-3p in the study of knezevic et al . increased apoptosis of cardiomyocytes via the direct targeting of igf1r leading to a decrease of akt signaling .", "this is in opposition to the previously mentioned study of fang et al . which showed apoptosis", "the converse findings of the studies could be explained by different models used by knezevic et al . and", "because of those discrepancies , the role of mir-378a in apoptosis of cardiomyocytes requires further investigation .", "the finding that mir-378a-3p affects both igf1r and the akt pathway was confirmed in a study which found that overexpression of mir-378a-3p in rhabdomyosarcoma suppressed igf1r expression and affected phosphorylation of the akt protein .", "mir-378a-5p was shown to target heat shock protein 70.3 ( hsp70.3 ) in mouse hearts in normoxic conditions , but in hypoxic conditions a transcript variant of hsp70.3 without mir-378a-5p target site in its 3-utr is not repressed and can exert its cytoprotective properties .", "mir-378a-3p prevented cardiac hypertrophy by targeting either ras signaling or the mitogen - activated protein kinase ( mapk ) pathway [ 77 , 78 ] .", "more studies on the effect of mir-378a expression in muscle disorders would also be desirable . in both golden retriever muscular dystrophy dogs and duchenne muscular dystrophy patients ,", "all in all , these findings suggest mir-378a-3p can be considered as an important player in cardiac development , remodeling , and hypertrophy ." ], [ "angiogenesis comprises development of new blood vessels from existing ones , regulated by cytokines and growth factors such as , for example , vascular endothelial growth factor ( vegf ) , platelet - derived growth factor ( pdgf ) , and angiopoietin-1 ( ang-1 ) .", "their expression can be posttranscriptionally controlled by micrornas such as mir-126 , mir-296 , mir-210 , mir-21 , and the mir-17~92 cluster ( reviewed in ) .", "skeletal muscles and heart muscle are tissues which , due to their oxygen and energy consumption , need to be sufficiently vascularized .", "one of the major regulators of angiogenesis is the hypoxia - inducible factor-1 ( hif-1 ) , which controls over 100 genes involved mainly in the glycolytic pathway and blood vessel formation , including vegf - a or interleukin-8 [ 8385 ] .", "vegf is generally induced by hypoxia , while il-8 in at least some cancers and endothelial cells can be diminished by hif-1 via inhibition of c - myc and sp-1 transcription factors [ 86 , 87 ] .", "c - myc , known as a regulator of cell cycle progression , apoptosis , and cellular transformation , is also a potent activator of pgc-1 and , in turn , mir-378a-3p , upregulating their expression .", "in addition , mir-378a has been shown to affect vegf - a in two ways .", "human hsa - mir-378a-5p ( by the study of hua et al . named as mir-378 ) can directly affect vegf - a by competing with hsa - mir-125a for the same seed - region in the vegf - a 3utr causing upregulation of vegf - a .", "mir-378a-5p can also indirectly regulate vegf - a affecting sonic hedgehog ( shh ) signaling via sufu inhibition , which is an inhibitory component of this signaling pathway .", "the shh pathway in turn can upregulate vegf - a and also other regulators of blood vessels formation , ang-1 and ang-2 expression [ 9092 ] .", "increased expression of vegf - a , as well as pdgf and tgf1 , was also seen in mesenchymal stromal cells ( mscs ) transfected with rno - mir-378a-5p . in skeletal muscles , vegf - induced angiogenesis appears not to be regulated by the well - known hif pathway but by pgc-1 , which coactivates estrogen - related receptor alpha ( err- ) on binding sites in the promoter and the first intron of the vegf gene , inducing its expression .", "this angiogenic pathway shows new roles for pgc-1 and err- , which are important regulators of mitochondrial activity in response to stimuli like exercise .", "if there might be a role for pgc-1 in this pathway , it is yet to be examined .", "it is noteworthy , however , that mir-378a-5p is known to affect the estrogen receptors by inhibiting err , another estrogen - related receptor .", "a role for mir-378a in cell cycle regulation and stimulation of cell growth is also proposed . in human mammary epithelial and breast cancer cell lines", ", mir-378a-3p can target the antiproliferative protein tob2 , which is a suppressor of cyclin d1 , which in turn is required for cell cycle g1-phase to s - phase progression .", "whether mir-378 affects endothelial cell proliferation by regulation of cell cycle remains to be established .", "the role of mir-378a in the formation of blood vessels nourishing tumor and enabling tumor growth was revealed .", "mir-378a was found to be differentially regulated in different types of cancers being downregulated in gastric cancer [ 96 , 97 ] , oral , and colon carcinoma , while being upregulated in renal and lung cancer [ 9 , 101 ] . since it is also changed in serum or plasma of patients with prostate cancer , renal cancer [ 100 , 103 ] , and gastric cancer and frequently found to be overexpressed in cryopreserved bone marrow mononuclear cells from acute myeloid leukemia patients , mir-378a might be considered as a biomarker .", "the role of mir-378a in tumorigenesis , tumor growth , and tumor vascularization was revealed for the first time by lee and coworkers in glioblastoma .", "they showed that mir-378a-5p enhances cell survival , reduces caspase-3 activity , and promotes tumor growth and angiogenesis , through repression of two tumor suppressors , sufu and fus-1 .", "strikingly , nude mice injected with mir-378a-5p transfected cancer cells formed tumors of bigger volume and with larger blood vessels compared to gfp - transfected cells . on the other hand , high expression of mir-378a-5p in nsclc correlated with brain metastases due to higher cell migration , invasion , and tumor angiogenesis .", "another study confirmed the downregulation of fus-1 by mir-378a-5p and showed that in the hepg2 liver cancer cells mir-378a-5p overexpression enhanced proliferation , migration , and , when injected in mice , invasion .", "also in rhabdomyosarcoma , enhanced expression of mir-378a , vegf , and mmp9 correlated with increased vascularization and metastasis . taken together", ", these studies suggest that mir-378a may serve as a prognostic marker in cancer due to its effects on angiogenesis .", "our recent data confirmed the proangiogenic effect of mir-378a ( both strands ) in non - small cell lung carcinoma ( nsclc ) and pointed at its correlation with heme - degrading enzyme , heme oxygenase-1 ( ho-1 ) .", "an involvement of ho-1 in angiogenesis and vegf - a as well as il-8 signaling was shown by us previously ; however , its action in tumors seems to be complex . in nci - h292 cell line overexpressing ho-1 , mir-378a ( both strands )", "also overexpression of the mir-378a precursor sequence diminished ho-1 expression . conditioned medium from nci - h292 cells overexpressing mir-378a enhanced angiogenic potential of hmec-1 endothelial cell line .", "tumors formed by such cells in subcutaneous xenografts showed enhanced growth , vascularization , oxygenation , and distal metastasis in vivo .", "these interactions between mir-378a and ho-1 were confirmed in our studies on the role of the nrf-2 transcription factor / ho-1 axis in nsclc cell lines [ 110 , 111 ] . on the other hand ,", "enhanced expression of mmu - mir-378a-5p in 4t1 murine breast cancer cells decreased the proliferation , migration , and invasiveness of these cancer cells in vitro and in vivo by targeting fibronectin , resulting in inhibition of tumor growth .", "recent study showed that mir-378a may act as a biomarker for response to antiangiogenic treatment in ovarian cancer .", "low expression of mir-378a was associated with longer progressive - free survival in patients with recurrent ovarian cancer treated with the antiangiogenic drug bevacizumab .", "overexpression of the mir-378a precursor in ovarian cancer cells altered expression of genes associated with angiogenesis ( alcam , ehd1 , elk3 , and tln1 ) , apoptosis ( rpn2 , hipk3 ) , and cell cycle regulation ( swap-70 , lsm14a , and rdx ) .", "high mir-378a ( strand not specified ) expression in renal carcinoma correlated with higher levels of endothelial surface marker cd34 in these tumors .", "notably , a recent study suggested clinical relevance for mir-378a in metastatic colorectal cancer , in which enhanced mir-378a expression significantly improved the sensitivity to cetuximab treatment in these patients .", "mir-378a-5p transfection of mscs has been shown to enhance their survival and angiogenic potential under hypoxic conditions in vitro .", "in coculture with human umbilical vein endothelial cells ( huvecs ) , mir-378a-5p - transfected mscs formed a larger number of vascular branches on matrigel . in the mscs transfected with mir-378a-5p , the expression of bcl-2-associated x protein ( bax ) , which is an important proapoptotic regulator , was decreased , leading to a better survival .", "it still has to be determined if the proangiogenic effect of mir-378a in vivo is confined to tumor angiogenesis , or if this effect is also present in physiological angiogenesis and regenerative neovascularization .", "recently , it was reported that anti - mir-378a-5p enhances wound healing process by upregulating integrin beta-3 and vimentin .", "it was reported that pgc-1 induces angiogenesis in skeletal muscle , enhancing the expression of vegf both in vitro and in vivo after ( transgenic ) overexpression .", "accordingly , it was also found that vegfa is upregulated in c2c12 myoblast cell line with pgc-1 overexpression . however , after a pcr - based gene array of 84 known angiogenic factors and further rt - pcr of individual genes , they concluded that pgc-1 triggered an antiangiogenic program .", "after inducing hind limb ischemia in pgc-1 overexpressing mice , an impaired reperfusion was noticed when compared to wild type littermates ." ], [ "the role of inflammation in angiogenesis is studied the most in the context of cancer ( e.g. , reviewed in [ 119 , 120 ] ) but is certainly not limited to this pathology .", "both lymphoid ( reviewed in [ 121 , 122 ] ) and myeloid ( reviewed in ) derived inflammatory cells affect angiogenesis in a stimulating or inhibitory manner .", "the role of mir-378a in inflammatory cells was reported and its anti - inflammatory effect could be suggested .", "nk cells exert potent cytotoxic effects when activated by type i ifn from the host once infected .", "mir-378a was found to be downregulated in activated nk cells and further proved to target granzyme b. thus , ifn- activation decreases mir-378a expression and in turn augments nk cell cytotoxicity .", "accordingly , suppression of mir-378 targeting granzyme b in nk cells resulted in inhibition of dengue virus replication in vivo .", "macrophages are known to play either inhibitory or stimulatory roles in angiogenesis ( reviewed by ) .", "mirnas have been proposed to regulate activation and polarization of macrophages ( reviewed by [ 127 , 128 ] ) . in a study of rckerl", "mir-378a-3p was identified as a part of the il-4-driven activation program of anti - inflammatory macrophages ( m2 ) .", "mir-378a-3p was highly upregulated after stimulation with il-4 of peritoneal exudate cells of mice injected with the parasite brugia malayi compared to controls and infected il-4-knockout mice .", "the study identified several targets for mir-378a-3p within the pi3 k / akt signaling pathway , which are important for proliferation but only partially responsible for m2 phenotype .", "another study found mir-378a ( strand not specified ) expression upregulated after stimulation with cytokines like , for example , tnf- and il-6 . in line with its potential role in macrophages ,", "mmu - mir-378a ( strand not specified ) has been found to be upregulated during osteoclastogenesis in vitro .", "furthermore , serum levels of mir-378a-3p have been shown to correlate with bone metastasis burden in mice injected with mouse mammary tumor cell lines 4t1 and 4t1.2 ." ], [ "a growing body of evidence suggests a role for mir-378a as a mediator controlling reciprocally dependent processes in metabolism , muscle differentiation / regeneration , and angiogenesis .", "as mir-378a was found to be differentially regulated in different types of cancers and its level is changed in serum of prostate , renal , and gastric cancer patients , it can be considered as a biomarker for those diseases . the correlation between mir-378a expression and disease progression in lung cancer , liver cancer , and rhabdomyosarcoma suggests a further role of this microrna as a prognostic marker .", "if more research will be done to the mechanisms of action , possibilities for therapeutic use of mir-378a could be sought in the field of metabolic disorders , obesity , or tumors .", "more studies on the effect of mir-378a expression in muscle disorders would also be desirable .", "the proangiogenic effect of mir-378a was observed in tumors ; however , no studies have been performed on the angiogenic effects of mir-378a in physiological settings or diseases where angiogenesis plays important roles , such as diabetes and cardiovascular diseases .", "more study has to be done to assess the mechanisms of mir-378a function in blood vessel formation . of note , in contrast with proangiogenic role of mir-378a , inhibition of mir-378a-5p enhanced wound healing process .", "this might suggest a role for mir-378a-5p in diseases such as diabetes or in decubitus ulcers , in which wound healing is impaired . of note is", "the confusion that has arises because of a disarray in nomenclature with studies describing the same molecule , mir-378a , as mir-422b , mir-378 , or mir-378 .", "in addition , it is not always clear which of the two mature strands of mir-378a is studied", ". this could lead to misunderstandings and errors in interpreting the data published so far ." ] ]

[ "a large number of patients ( 70 - 80% ) with head and neck cancers are diagnosed having locally advanced primary disease along with high lymph nodal metastases . in recent years", ", intensity modulated radiotherapy ( imrt ) has shown benefits in lowering the toxicities as well as improvements in loco regional control in these patients.[35 ] a crucial step in radiotherapy treatment planning process is to determine the tumor location and its extent .", "modern imaging modalities , such as computed tomography ( ct ) , magnetic resonance imaging ( mri ) , ultrasound , single - photon emission computer tomography , and positron emission tomography ( pet ) assist the radiation oncologists in localization of the target volume .", "ct scan is the principle source of imaging data used for defining gross tumor volume ( gtv ) for planning conformal therapy for most sites .", "recurrence pattern following imrt shows that most of the recurrences occur only in high - dose region .", "so apart from anatomical delineation , functional imaging may improve outcome with imrt . with the advent of pet scan , it is possible to demonstrate abnormal glucose metabolism in tumor cells using [ 18f]-2fluoro , 2deoxy d - glucose([18f]-fdg ) .", "recently , [ 18f]-fdg - pet - ct scan has been applied to tumor volume delineation for many cancer sites in addition to diagnostic purpose . in this study , we compared quantitatively gtv delineated on [ 18f]-fdg - pet - ct scan to those delineated on contrast - enhanced ct scan ( cect scan ) in primary and nodal areas of head and neck cancers .", "twenty - six patients with head and neck cancers underwent [ 18f]-fdg - pet and cect scans in a dedicated pet - ct scanner in single session from august 2008 to march 2010 .", "this study was approved by the institutional review board and the patients were enrolled for the study after an informed consent had been obtained .", "characteristics of 26 patients underwent [ 18f]-2fluoro , 2deoxy d - glucose - positron emission tomography / computed tomography pet / ct was performed using the siemens biograph 40 true point pet / ct scanner ( luetium oxyorthosilicate based 40 slice scanner ) .", "following administration of 375 mbq of 18f - fdg , the patients were asked to wait in a quiet room for 1 h. we performed diagnostic pet / ct and radiotherapy planning pet / ct in subsequent sessions on the same day .", "the field of view for diagnostic pet / ct was from the skull to the upper thigh .", "after the completion of attenuation correction ct scan ( 120 kv , 80 ma ) , pet acquisition was performed .", "after completion of diagnostic pet / ct , the table top was changed to flat one for imrt planning pet / ct .", "the patients were immobilized with face mask and were properly aligned in radiotherapy treatment position with the help of lasers .", "the pet acquisition was performed with the field of view from frontal sinus to d4 vertebral level and acquisition time was 120 - 180 s / bed position . immediately following the acquisition of the pet scan", "cect scan was performed using the same slice position with a slice thickness of 5 mm .", "all data were sent through digital imaging and communications in medicine to a workstation treatment planning system . then cect scans were fused with pet images acquired in the treatment planning position using inbuilt software in the siemens coherence - oncologist treatment planning system [ figure 1 ] . while evaluating cect images , pet images were blinded .", "target volumes and organs at risk were contoured and transferred to oncentra treatment planning system for volume analysis .", "positron emission tomography / computed tomography of 64 years female with nasal cavity cancer showing clear demarcation of tumor with thickened mucosa the primary tumor and abnormal lymph nodes in the neck were delineated by an experienced radiation oncologist . gtv - ct scan ( gtv ct ) was delineated on the imrt planning cect scans according to standard protocols using all available clinical information from physical palpation , available imaging including ct and mri and direct laryngoscope findings [ figure 2 ] .", "lymph nodes with > 10 mm in shortest dimension , perinodal extension and necrosis in center were included in gtv ct .", "gtv cts were contoured by a radiation oncologist after consulting with an experienced radiologist . while contouring gtv ct , fdg - pet images were completely blinded .", "but ct does not show clear demarcation of tumor with thickened mucosa gtv-[18f]-fdg - pet / ct ( gtv pet ) was delineated on fused images using the visual interpretation method along with the opinion of a nuclear medicine physician .", "abnormal fdg uptake areas in imrt planning pet / ct scans above normal background uptake were included in gtv pet .", "the gtvs obtained from the pet / ct scans were compared with the ct - based gtv using student 's t - test with the help of spss 16.0 software .", "the comparative analysis of two volumes is displayed as a schematic diagram [ figure 3 ] .", "the mismatch between two volumes was analyzed by the following method : mismatch a to b = ( a - intersection volume)/b 100 .", "the coverage between two volumes was analyzed by the following method : coverage a to b = intersection volume / b 100 .", "gross tumor volume ( gtv ) positron emission tomography ( pet ) ( black ) , gtv ct ( red ) , pet out of ct ( purple ) , ct out of pet ( green ) and intersection volume ( turquoise ) were analyzed", "pet / ct was performed using the siemens biograph 40 true point pet / ct scanner ( luetium oxyorthosilicate based 40 slice scanner ) .", "following administration of 375 mbq of 18f - fdg , the patients were asked to wait in a quiet room for 1 h. we performed diagnostic pet / ct and radiotherapy planning pet / ct in subsequent sessions on the same day .", "the field of view for diagnostic pet / ct was from the skull to the upper thigh .", "after the completion of attenuation correction ct scan ( 120 kv , 80 ma ) , pet acquisition was performed .", "after completion of diagnostic pet / ct , the table top was changed to flat one for imrt planning pet / ct .", "the patients were immobilized with face mask and were properly aligned in radiotherapy treatment position with the help of lasers .", "the pet acquisition was performed with the field of view from frontal sinus to d4 vertebral level and acquisition time was 120 - 180 s / bed position . immediately following the acquisition of the pet scan", "cect scan was performed using the same slice position with a slice thickness of 5 mm .", "all data were sent through digital imaging and communications in medicine to a workstation treatment planning system . then cect scans were fused with pet images acquired in the treatment planning position using inbuilt software in the siemens coherence - oncologist treatment planning system [ figure 1 ] . while evaluating cect images , pet images were blinded .", "target volumes and organs at risk were contoured and transferred to oncentra treatment planning system for volume analysis .", "positron emission tomography / computed tomography of 64 years female with nasal cavity cancer showing clear demarcation of tumor with thickened mucosa", "the primary tumor and abnormal lymph nodes in the neck were delineated by an experienced radiation oncologist . gtv - ct scan ( gtv ct ) was delineated on the imrt planning cect scans according to standard protocols using all available clinical information from physical palpation , available imaging including ct and mri and direct laryngoscope findings [ figure 2 ] .", "lymph nodes with > 10 mm in shortest dimension , perinodal extension and necrosis in center were included in gtv ct .", "gtv cts were contoured by a radiation oncologist after consulting with an experienced radiologist . while contouring gtv ct , fdg - pet images were completely blinded .", "but ct does not show clear demarcation of tumor with thickened mucosa gtv-[18f]-fdg - pet / ct ( gtv pet ) was delineated on fused images using the visual interpretation method along with the opinion of a nuclear medicine physician .", "abnormal fdg uptake areas in imrt planning pet / ct scans above normal background uptake were included in gtv pet .", "the gtvs obtained from the pet / ct scans were compared with the ct - based gtv using student 's t - test with the help of spss 16.0 software .", "the comparative analysis of two volumes is displayed as a schematic diagram [ figure 3 ] .", "the mismatch between two volumes was analyzed by the following method : mismatch a to b = ( a - intersection volume)/b 100 .", "the coverage between two volumes was analyzed by the following method : coverage a to b = intersection volume / b 100 .", "gross tumor volume ( gtv ) positron emission tomography ( pet ) ( black ) , gtv ct ( red ) , pet out of ct ( purple ) , ct out of pet ( green ) and intersection volume ( turquoise ) were analyzed", "the distribution of all staging patterns for t , n , and m categories is shown in table 2 .", "pet scan changed the ct scan - based staging in 8 cases ( 30.76% ) .", "seven cases ( 26.92% ) were up - staged by pet / ct scan , and only one ( 0.38% ) was down - staged by pet / ct scan . in one patient ( patient", "12 ) with the unknown primary with secondaries in neck , pet / ct identified primary in soft palate .", "pet / ct identified one second primary tumor in left pyriform sinus in the case of right pyriform sinus ( patient no .", "3 ) . impact of positron emission tomography / computed tomography on staging and comparison of gross tumor volume the impact of pet / ct on gtvs is shown in table 3 .", "the median value of gtv ct was 32.00 cc ( range : 0.00 - 309.10 cc ) and the mean was 54.78 cc 64.47 cc .", "the median value of gtv pet was 35.00 cc ( range : 3.20 - 269.50 cc ) and the mean was 48.43 cc 53.21 cc .", "the gtv ct was larger than the gtv pet in nine cases ( 37.5% ) and was smaller in 15 cases ( 62.5% ) .", "impact of positron emission tomography / computed tomography in primary and nodal gross tumor volume the median value of percentage of primary gtv pet covered by ct was 70.07% ( range : 32.42 - 95.52% ) and the mean was 65.18% 18.84% .", "the median value of percentage of primary gtv ct covered by gtv pet was 68.39% ( range : 34.58 - 88.94% ) and the mean was 69.12% 14.64% .", "the median value percentage of mismatch of primary gtv pet to ct was 25.01% ( range : 5.00 - 141.41% ) and the mean was 33.63% 28.96% .", "the median value of percentage of mismatch of primary gtv ct to pet was 31.97% ( range : 2.60 - 142.67% ) and the mean percentage was 46.05% 36.92% .", "pet scan changed the gtv ct with a median value of -12.52% ( range : -131.11 - 53.74% ) and with a mean value of 15.18 41.49% . in most of the cases , pet scan identified gtvs outside the volume delineated by ct scan .", "the median value of primary gtv pet outside ct scan volume was 12.10 cc ( range : 1.50 - 29.80 cc ) and the mean value was 12.17 cc 7.81 cc .", "the median value of gtv pet was 7.00 cc ( range : 0.40 - 71.70 cc ) and the mean was 12.72 cc 15.46 cc .", "the median value of gtv ct was 6.50 cc ( range : 0.20 - 67.00 cc ) and the mean was 11.04 cc 14.87 cc .", "the gtv ct was larger than the gtv pet in nine cases ( 37.5% ) and was smaller in 15 cases ( 62.5% ) .", "the median value percentage of nodal gtv pet covered by ct was 83.73% ( range : 31.18 - 100.00% ) and the mean was 81.46 19.48% .", "the median value of percentage of nodal gtv ct covered by gtv pet was 61.30% ( range : 25.81 - 100.00% ) and the mean was 61.02 18.00% .", "the median value of percentage of mismatch of nodal gtv pet to ct was 61.88% ( range : 0.00 - 168.42% ) and the mean was 63.90 47.53% .", "the median value percentage of mismatch of nodal gtv ct to pet was 10.86% ( range : 0.00 - 81.82% ) and the mean percentage was 18.66 24.65% .", "mismatch pet to ct was significantly smaller than ct to pet ( p = 0.04 ) .", "the median value of primary gtv pet outside ct scan volume was 3.20 cc ( range : 0.00 - 18.20 cc ) and the mean value was 4.44 4.52 cc .", "the median value of gtv ct was 32.00 cc ( range : 0.00 - 309.10 cc ) and the mean was 54.78 cc 64.47 cc .", "the median value of gtv pet was 35.00 cc ( range : 3.20 - 269.50 cc ) and the mean was 48.43 cc 53.21 cc .", "the gtv ct was larger than the gtv pet in nine cases ( 37.5% ) and was smaller in 15 cases ( 62.5% ) .", "impact of positron emission tomography / computed tomography in primary and nodal gross tumor volume the median value of percentage of primary gtv pet covered by ct was 70.07% ( range : 32.42 - 95.52% ) and the mean was 65.18% 18.84% .", "the median value of percentage of primary gtv ct covered by gtv pet was 68.39% ( range : 34.58 - 88.94% ) and the mean was 69.12% 14.64% .", "the median value percentage of mismatch of primary gtv pet to ct was 25.01% ( range : 5.00 - 141.41% ) and the mean was 33.63% 28.96% .", "the median value of percentage of mismatch of primary gtv ct to pet was 31.97% ( range : 2.60 - 142.67% ) and the mean percentage was 46.05% 36.92% .", "pet scan changed the gtv ct with a median value of -12.52% ( range : -131.11 - 53.74% ) and with a mean value of 15.18 41.49% . in most of the cases , pet scan identified gtvs outside the volume delineated by ct scan .", "the median value of primary gtv pet outside ct scan volume was 12.10 cc ( range : 1.50 - 29.80 cc ) and the mean value was 12.17 cc 7.81 cc .", "the median value of gtv pet was 7.00 cc ( range : 0.40 - 71.70 cc ) and the mean was 12.72 cc 15.46 cc .", "the median value of gtv ct was 6.50 cc ( range : 0.20 - 67.00 cc ) and the mean was 11.04 cc 14.87 cc .", "the gtv ct was larger than the gtv pet in nine cases ( 37.5% ) and was smaller in 15 cases ( 62.5% ) .", "the median value percentage of nodal gtv pet covered by ct was 83.73% ( range : 31.18 - 100.00% ) and the mean was 81.46 19.48% .", "the median value of percentage of nodal gtv ct covered by gtv pet was 61.30% ( range : 25.81 - 100.00% ) and the mean was 61.02 18.00% .", "the median value of percentage of mismatch of nodal gtv pet to ct was 61.88% ( range : 0.00 - 168.42% ) and the mean was 63.90 47.53% .", "the median value percentage of mismatch of nodal gtv ct to pet was 10.86% ( range : 0.00 - 81.82% ) and the mean percentage was 18.66 24.65% .", "mismatch pet to ct was significantly smaller than ct to pet ( p = 0.04 ) .", "the median value of primary gtv pet outside ct scan volume was 3.20 cc ( range : 0.00 - 18.20 cc ) and the mean value was 4.44 4.52 cc .", "in our study , all patients underwent imrt planning cect scans along with pet / ct .", "the use of non - ionic contrast material did not interfere in interpretation of data .", "fused cect scan studies with pet scans for better anatomical delineation for various types of solid cancers .", "incorporation of pet / ct in the management of head and neck cancers has resulted in significant changes in clinical staging.[1014 ] wang , et al .", ", evaluated the impact of fdg - pet fused with planning ct scans on tumor localization .", "pet / ct changed ct - based staging in 16 of the 28 ( 57% ) patients . in 12 cases ,", "the ct - based t - stage was upgraded by the pet / ct . in six cases ,", "the ct - based nodal information was up - staged by the pet / ct .", ", analyzed the use of [ 18f]-pet / ct images for staging and target volume delineation of patients with head and neck carcinoma .", "pet / ct imaging changed the tnm categories in 5 of the 22 ( 22% ) cases when compared with ct alone .", "t - stage changed in 3 of 22 ( 14% ) and n - stage in 2 of 22 cases ( 10% ) .", "similarly , in this study , pet / ct resulted significant discrepancy with ct - based staging in 7 of the 26 patients ( up - staged in 6 and down - staged in 1 ) .", "various methods were proposed for accurate contouring of the gtv in pet / ct fusion - guided radiotherapy .", "visual interpretation method,[1517 ] a fixed 50% threshold of the background subtracted tumor maximum uptake ( thr ) , an appropriate thr level for an individual patient , software - based automated segmentation , were most commonly used for pet / ct - based contouring in clinical studies . the most appropriate method for pet / ct - based gtv delineation", "various studies compared the gtvs contoured in pet / ct scans with ct scans and demonstrated the role of pet / ct fusion for radiotherapy planning.[111516182226 ] scarfone , et al .", ", prospectively studied the impact of hybrid pet - ct simulation in tumor and normal tissue delineation for rt planning in patients with head and neck cancer . they contoured abnormal pet uptake areas using the visual interpretation method .", "we also adopted visual interpretation method to define gtvs in primary and nodal areas for our study .", "heron , et al . , prospectively studied the impact of hybrid pet - ct simulation in tumor and normal tissue delineation for rt planning in patients with head and neck cancers . for primary disease , pet - based target volumes ( mean = 42.7 cm ) were significantly ( p = 0.002 ) smaller compared with ct - based target volumes ( mean = 65 cm ) for all patients .", ", compared ct , mri , and [ 18f]-fdg - pet for delineation of gtv in pharyngolaryngeal squamous cell carcinoma . for oropharyngeal tumors and for laryngeal or hypopharyngeal tumors ,", "average gtvs delineated at ct were 32.0 and 21.4 cm , respectively , whereas average gtvs at pet were smaller , 20.3 [ p = 0.10 ] and 16.4 cm [ p = 0.01 ] , respectively . in various studies ,", "pet - based gtvs were smaller than ct - based gtvs . in this study ,", "gtv pet was significantly smaller than gtv ct ( p < 0.001 ) and consistent with above studies .", "in contrast , nishioka , et al . showed that gtv volumes were not changed by image fusion between 18f - fdg - pet and mri / ct in majority of cases and ashish , et al .", "variations in image acquisition procedures , registration methods , and target volume delineation methods are potential factors for this variation .", "mismatch analysis is a potential tool to study the disagreement between ct and pet / ct in contouring of gtv .", "many studies analyzed this issue and found that there is a significant mismatch existing between gtv pet and gtv ct . in a study by daisne , et al .", ", the average mismatch ct to fdg - pet was 73% and fdg - pet to ct was 14% for oropharyngeal tumors .", "for laryngeal tumors , average mismatch ct to fdg - pet was 48% and fdg - pet to ct was 17% .", ", found the mean value for mismatch of gtv pet / gtv ct as 28.9% s32.9% and the mismatch of gtv ct / gtv pet as 70.9% 50.9% . in this study ,", "the mismatch gtv pet / gtv ct was significantly smaller than mismatch gtv ct / gtv pet ( p = 0.03 ) . in this study ,", "the coverage of gtv pet to gtv ct and gtv ct to gtv pet was analyzed to evaluate the difference between two gtv volumes .", "this was comparable with results of studies done by ei - bassiouni , et al . , and schinagl et al . , in a study by ei - bassiouni , et al", ". , the median percentage of the gtv pet covered by gtv ct was of 99.5% ( range : 41.6 - 100% ) and a mean of 92.4% 16.4% , whereas the median percentage of gtvct covered by ptvpet was 95.1% ( range : 44.3 - 100% ) and a mean was 88.2 - 16.2% ( p = 0.2 ) .", "the major clinical implication of our study is delineation of the metabolically active volumes outside the ct scan .", "these volumes of diseases could have been missed during radiotherapy treatment planning and could result in in - field recurrences and marginal recurrences .", "the median value of primary gtv pet outside ct scan volume was 12.10 cc ( range : 1.50 - 29.80 cc ) and the mean value was 12.17 7.81 cc .", "pet / ct has the advantage to identify metabolically active volumes that are used in simultaneous integrated boost sib - imrt and these volumes are also utilized for dose escalation of gtv .", "performed studies in phantoms filled with radioactive material and proposed threshold - based contouring for pet / ct scans in head and neck cancers .", "however , we adopted the visual interpretation method for target volume delineation mainly because of limitations in our planning systems .", "twenty - six patients were included in this study . in spite of these limitations ,", "though this study demonstrated that pet / ct could improve target volume delineation for radiation treatment planning , we feel that more studies are needed to substantiate the importance of pet / ct .", "pet / ct significantly alters the staging of tumor ( t staging ) and lymph node metastases ( n staging ) in head and neck cancers .", "pet / ct also has the potential to identify gtv outside ct - based gtv that increases the accuracy of radiation planning .", "we believe that in the era of imrt / image - guided radiotherapy ( igrt ) , pet / ct will increasingly be used for the radiotherapy planning ." ]

[ "<S> purpose : to compare quantitatively gross tumor volume ( gtv ) , both primary and nodal areas of head and neck cancers , delineated on [ 18f]-2fluoro , 2deoxy d - glucose - positron emission tomography / computed tomography ( [ 18f]-fdg - pet - ct ) scan to those delineated on contrast - enhanced ct scan ( cect scan).methods : a total of 26 consecutive patients with squamous cell cancers of head and neck were included in this study . </S>", "<S> the primary sites were oropharynx ( n = 7 ) , hypopharynx ( n = 6 ) , paranasal sinus ( n = 6 ) , nasopharynx ( n = 4 ) , oral cavity ( n = 2 ) , and one with unknown primary and secondary neck node . </S>", "<S> all patients underwent routine staging work - up . </S>", "<S> fdg - pet and cect scans were performed with dedicated pet - ct scanner in single session as a part of the radiotherapy treatment planning for intensity modulated radiotherapy / image - guided radiotherapy.results:all patients had abnormal increased uptake in pet - ct scans . </S>", "<S> pet - ct resulted in changes of ct - based staging in 8 of 26 patients ( up - staged in 7 and down - staged in 1 ) . </S>", "<S> the mean primary and nodal gtv volumes on pet - ct and ct were significantly different ( primary : pet - gtv : 48.43 53.21 cc vs. ct 54.78 64.47 cc , p < 0.001 ; nodes : pet - gtv : 12.72 15.46 cc vs. 11.04 14.87 cc , p < 0.001 ) . </S>", "<S> the mismatch between two target volumes was statistically significant ( p = 0.03 for gtv primary , p = 0.04 for gtv node).conclusion : accuracy of delineation of gtv can be improved along with functional imaging using [ 18f]-fdg . </S>", "<S> these metabolically active volumes are significantly smaller than ct - based volumes and could be missed during conventional ct - based target delineations of gtvs . </S>" ]

[ "INTRODUCTION", "METHODS", "Study protocol", "Delineation of GTV", "Statistical analysis", "RESULTS", "Primary GTV changes", "Nodal GTV changes", "DISCUSSION", "CONCLUSION" ]

[ [ "a large number of patients ( 70 - 80% ) with head and neck cancers are diagnosed having locally advanced primary disease along with high lymph nodal metastases . in recent years", ", intensity modulated radiotherapy ( imrt ) has shown benefits in lowering the toxicities as well as improvements in loco regional control in these patients.[35 ] a crucial step in radiotherapy treatment planning process is to determine the tumor location and its extent .", "modern imaging modalities , such as computed tomography ( ct ) , magnetic resonance imaging ( mri ) , ultrasound , single - photon emission computer tomography , and positron emission tomography ( pet ) assist the radiation oncologists in localization of the target volume .", "ct scan is the principle source of imaging data used for defining gross tumor volume ( gtv ) for planning conformal therapy for most sites .", "recurrence pattern following imrt shows that most of the recurrences occur only in high - dose region .", "so apart from anatomical delineation , functional imaging may improve outcome with imrt . with the advent of pet scan , it is possible to demonstrate abnormal glucose metabolism in tumor cells using [ 18f]-2fluoro , 2deoxy d - glucose([18f]-fdg ) .", "recently , [ 18f]-fdg - pet - ct scan has been applied to tumor volume delineation for many cancer sites in addition to diagnostic purpose . in this study , we compared quantitatively gtv delineated on [ 18f]-fdg - pet - ct scan to those delineated on contrast - enhanced ct scan ( cect scan ) in primary and nodal areas of head and neck cancers ." ], [ "twenty - six patients with head and neck cancers underwent [ 18f]-fdg - pet and cect scans in a dedicated pet - ct scanner in single session from august 2008 to march 2010 .", "this study was approved by the institutional review board and the patients were enrolled for the study after an informed consent had been obtained .", "characteristics of 26 patients underwent [ 18f]-2fluoro , 2deoxy d - glucose - positron emission tomography / computed tomography pet / ct was performed using the siemens biograph 40 true point pet / ct scanner ( luetium oxyorthosilicate based 40 slice scanner ) .", "following administration of 375 mbq of 18f - fdg , the patients were asked to wait in a quiet room for 1 h. we performed diagnostic pet / ct and radiotherapy planning pet / ct in subsequent sessions on the same day .", "the field of view for diagnostic pet / ct was from the skull to the upper thigh .", "after the completion of attenuation correction ct scan ( 120 kv , 80 ma ) , pet acquisition was performed .", "after completion of diagnostic pet / ct , the table top was changed to flat one for imrt planning pet / ct .", "the patients were immobilized with face mask and were properly aligned in radiotherapy treatment position with the help of lasers .", "the pet acquisition was performed with the field of view from frontal sinus to d4 vertebral level and acquisition time was 120 - 180 s / bed position . immediately following the acquisition of the pet scan", "cect scan was performed using the same slice position with a slice thickness of 5 mm .", "all data were sent through digital imaging and communications in medicine to a workstation treatment planning system . then cect scans were fused with pet images acquired in the treatment planning position using inbuilt software in the siemens coherence - oncologist treatment planning system [ figure 1 ] . while evaluating cect images , pet images were blinded .", "target volumes and organs at risk were contoured and transferred to oncentra treatment planning system for volume analysis .", "positron emission tomography / computed tomography of 64 years female with nasal cavity cancer showing clear demarcation of tumor with thickened mucosa the primary tumor and abnormal lymph nodes in the neck were delineated by an experienced radiation oncologist . gtv - ct scan ( gtv ct ) was delineated on the imrt planning cect scans according to standard protocols using all available clinical information from physical palpation , available imaging including ct and mri and direct laryngoscope findings [ figure 2 ] .", "lymph nodes with > 10 mm in shortest dimension , perinodal extension and necrosis in center were included in gtv ct .", "gtv cts were contoured by a radiation oncologist after consulting with an experienced radiologist . while contouring gtv ct , fdg - pet images were completely blinded .", "but ct does not show clear demarcation of tumor with thickened mucosa gtv-[18f]-fdg - pet / ct ( gtv pet ) was delineated on fused images using the visual interpretation method along with the opinion of a nuclear medicine physician .", "abnormal fdg uptake areas in imrt planning pet / ct scans above normal background uptake were included in gtv pet .", "the gtvs obtained from the pet / ct scans were compared with the ct - based gtv using student 's t - test with the help of spss 16.0 software .", "the comparative analysis of two volumes is displayed as a schematic diagram [ figure 3 ] .", "the mismatch between two volumes was analyzed by the following method : mismatch a to b = ( a - intersection volume)/b 100 .", "the coverage between two volumes was analyzed by the following method : coverage a to b = intersection volume / b 100 .", "gross tumor volume ( gtv ) positron emission tomography ( pet ) ( black ) , gtv ct ( red ) , pet out of ct ( purple ) , ct out of pet ( green ) and intersection volume ( turquoise ) were analyzed" ], [ "pet / ct was performed using the siemens biograph 40 true point pet / ct scanner ( luetium oxyorthosilicate based 40 slice scanner ) .", "following administration of 375 mbq of 18f - fdg , the patients were asked to wait in a quiet room for 1 h. we performed diagnostic pet / ct and radiotherapy planning pet / ct in subsequent sessions on the same day .", "the field of view for diagnostic pet / ct was from the skull to the upper thigh .", "after the completion of attenuation correction ct scan ( 120 kv , 80 ma ) , pet acquisition was performed .", "after completion of diagnostic pet / ct , the table top was changed to flat one for imrt planning pet / ct .", "the patients were immobilized with face mask and were properly aligned in radiotherapy treatment position with the help of lasers .", "the pet acquisition was performed with the field of view from frontal sinus to d4 vertebral level and acquisition time was 120 - 180 s / bed position . immediately following the acquisition of the pet scan", "cect scan was performed using the same slice position with a slice thickness of 5 mm .", "all data were sent through digital imaging and communications in medicine to a workstation treatment planning system . then cect scans were fused with pet images acquired in the treatment planning position using inbuilt software in the siemens coherence - oncologist treatment planning system [ figure 1 ] . while evaluating cect images , pet images were blinded .", "target volumes and organs at risk were contoured and transferred to oncentra treatment planning system for volume analysis .", "positron emission tomography / computed tomography of 64 years female with nasal cavity cancer showing clear demarcation of tumor with thickened mucosa" ], [ "the primary tumor and abnormal lymph nodes in the neck were delineated by an experienced radiation oncologist . gtv - ct scan ( gtv ct ) was delineated on the imrt planning cect scans according to standard protocols using all available clinical information from physical palpation , available imaging including ct and mri and direct laryngoscope findings [ figure 2 ] .", "lymph nodes with > 10 mm in shortest dimension , perinodal extension and necrosis in center were included in gtv ct .", "gtv cts were contoured by a radiation oncologist after consulting with an experienced radiologist . while contouring gtv ct , fdg - pet images were completely blinded .", "but ct does not show clear demarcation of tumor with thickened mucosa gtv-[18f]-fdg - pet / ct ( gtv pet ) was delineated on fused images using the visual interpretation method along with the opinion of a nuclear medicine physician .", "abnormal fdg uptake areas in imrt planning pet / ct scans above normal background uptake were included in gtv pet ." ], [ "the gtvs obtained from the pet / ct scans were compared with the ct - based gtv using student 's t - test with the help of spss 16.0 software .", "the comparative analysis of two volumes is displayed as a schematic diagram [ figure 3 ] .", "the mismatch between two volumes was analyzed by the following method : mismatch a to b = ( a - intersection volume)/b 100 .", "the coverage between two volumes was analyzed by the following method : coverage a to b = intersection volume / b 100 .", "gross tumor volume ( gtv ) positron emission tomography ( pet ) ( black ) , gtv ct ( red ) , pet out of ct ( purple ) , ct out of pet ( green ) and intersection volume ( turquoise ) were analyzed" ], [ "the distribution of all staging patterns for t , n , and m categories is shown in table 2 .", "pet scan changed the ct scan - based staging in 8 cases ( 30.76% ) .", "seven cases ( 26.92% ) were up - staged by pet / ct scan , and only one ( 0.38% ) was down - staged by pet / ct scan . in one patient ( patient", "12 ) with the unknown primary with secondaries in neck , pet / ct identified primary in soft palate .", "pet / ct identified one second primary tumor in left pyriform sinus in the case of right pyriform sinus ( patient no .", "3 ) . impact of positron emission tomography / computed tomography on staging and comparison of gross tumor volume the impact of pet / ct on gtvs is shown in table 3 .", "the median value of gtv ct was 32.00 cc ( range : 0.00 - 309.10 cc ) and the mean was 54.78 cc 64.47 cc .", "the median value of gtv pet was 35.00 cc ( range : 3.20 - 269.50 cc ) and the mean was 48.43 cc 53.21 cc .", "the gtv ct was larger than the gtv pet in nine cases ( 37.5% ) and was smaller in 15 cases ( 62.5% ) .", "impact of positron emission tomography / computed tomography in primary and nodal gross tumor volume the median value of percentage of primary gtv pet covered by ct was 70.07% ( range : 32.42 - 95.52% ) and the mean was 65.18% 18.84% .", "the median value of percentage of primary gtv ct covered by gtv pet was 68.39% ( range : 34.58 - 88.94% ) and the mean was 69.12% 14.64% .", "the median value percentage of mismatch of primary gtv pet to ct was 25.01% ( range : 5.00 - 141.41% ) and the mean was 33.63% 28.96% .", "the median value of percentage of mismatch of primary gtv ct to pet was 31.97% ( range : 2.60 - 142.67% ) and the mean percentage was 46.05% 36.92% .", "pet scan changed the gtv ct with a median value of -12.52% ( range : -131.11 - 53.74% ) and with a mean value of 15.18 41.49% . in most of the cases , pet scan identified gtvs outside the volume delineated by ct scan .", "the median value of primary gtv pet outside ct scan volume was 12.10 cc ( range : 1.50 - 29.80 cc ) and the mean value was 12.17 cc 7.81 cc .", "the median value of gtv pet was 7.00 cc ( range : 0.40 - 71.70 cc ) and the mean was 12.72 cc 15.46 cc .", "the median value of gtv ct was 6.50 cc ( range : 0.20 - 67.00 cc ) and the mean was 11.04 cc 14.87 cc .", "the gtv ct was larger than the gtv pet in nine cases ( 37.5% ) and was smaller in 15 cases ( 62.5% ) .", "the median value percentage of nodal gtv pet covered by ct was 83.73% ( range : 31.18 - 100.00% ) and the mean was 81.46 19.48% .", "the median value of percentage of nodal gtv ct covered by gtv pet was 61.30% ( range : 25.81 - 100.00% ) and the mean was 61.02 18.00% .", "the median value of percentage of mismatch of nodal gtv pet to ct was 61.88% ( range : 0.00 - 168.42% ) and the mean was 63.90 47.53% .", "the median value percentage of mismatch of nodal gtv ct to pet was 10.86% ( range : 0.00 - 81.82% ) and the mean percentage was 18.66 24.65% .", "mismatch pet to ct was significantly smaller than ct to pet ( p = 0.04 ) .", "the median value of primary gtv pet outside ct scan volume was 3.20 cc ( range : 0.00 - 18.20 cc ) and the mean value was 4.44 4.52 cc ." ], [ "the median value of gtv ct was 32.00 cc ( range : 0.00 - 309.10 cc ) and the mean was 54.78 cc 64.47 cc .", "the median value of gtv pet was 35.00 cc ( range : 3.20 - 269.50 cc ) and the mean was 48.43 cc 53.21 cc .", "the gtv ct was larger than the gtv pet in nine cases ( 37.5% ) and was smaller in 15 cases ( 62.5% ) .", "impact of positron emission tomography / computed tomography in primary and nodal gross tumor volume the median value of percentage of primary gtv pet covered by ct was 70.07% ( range : 32.42 - 95.52% ) and the mean was 65.18% 18.84% .", "the median value of percentage of primary gtv ct covered by gtv pet was 68.39% ( range : 34.58 - 88.94% ) and the mean was 69.12% 14.64% .", "the median value percentage of mismatch of primary gtv pet to ct was 25.01% ( range : 5.00 - 141.41% ) and the mean was 33.63% 28.96% .", "the median value of percentage of mismatch of primary gtv ct to pet was 31.97% ( range : 2.60 - 142.67% ) and the mean percentage was 46.05% 36.92% .", "pet scan changed the gtv ct with a median value of -12.52% ( range : -131.11 - 53.74% ) and with a mean value of 15.18 41.49% . in most of the cases , pet scan identified gtvs outside the volume delineated by ct scan .", "the median value of primary gtv pet outside ct scan volume was 12.10 cc ( range : 1.50 - 29.80 cc ) and the mean value was 12.17 cc 7.81 cc ." ], [ "the median value of gtv pet was 7.00 cc ( range : 0.40 - 71.70 cc ) and the mean was 12.72 cc 15.46 cc .", "the median value of gtv ct was 6.50 cc ( range : 0.20 - 67.00 cc ) and the mean was 11.04 cc 14.87 cc .", "the gtv ct was larger than the gtv pet in nine cases ( 37.5% ) and was smaller in 15 cases ( 62.5% ) .", "the median value percentage of nodal gtv pet covered by ct was 83.73% ( range : 31.18 - 100.00% ) and the mean was 81.46 19.48% .", "the median value of percentage of nodal gtv ct covered by gtv pet was 61.30% ( range : 25.81 - 100.00% ) and the mean was 61.02 18.00% .", "the median value of percentage of mismatch of nodal gtv pet to ct was 61.88% ( range : 0.00 - 168.42% ) and the mean was 63.90 47.53% .", "the median value percentage of mismatch of nodal gtv ct to pet was 10.86% ( range : 0.00 - 81.82% ) and the mean percentage was 18.66 24.65% .", "mismatch pet to ct was significantly smaller than ct to pet ( p = 0.04 ) .", "the median value of primary gtv pet outside ct scan volume was 3.20 cc ( range : 0.00 - 18.20 cc ) and the mean value was 4.44 4.52 cc ." ], [ "in our study , all patients underwent imrt planning cect scans along with pet / ct .", "the use of non - ionic contrast material did not interfere in interpretation of data .", "fused cect scan studies with pet scans for better anatomical delineation for various types of solid cancers .", "incorporation of pet / ct in the management of head and neck cancers has resulted in significant changes in clinical staging.[1014 ] wang , et al .", ", evaluated the impact of fdg - pet fused with planning ct scans on tumor localization .", "pet / ct changed ct - based staging in 16 of the 28 ( 57% ) patients . in 12 cases ,", "the ct - based t - stage was upgraded by the pet / ct . in six cases ,", "the ct - based nodal information was up - staged by the pet / ct .", ", analyzed the use of [ 18f]-pet / ct images for staging and target volume delineation of patients with head and neck carcinoma .", "pet / ct imaging changed the tnm categories in 5 of the 22 ( 22% ) cases when compared with ct alone .", "t - stage changed in 3 of 22 ( 14% ) and n - stage in 2 of 22 cases ( 10% ) .", "similarly , in this study , pet / ct resulted significant discrepancy with ct - based staging in 7 of the 26 patients ( up - staged in 6 and down - staged in 1 ) .", "various methods were proposed for accurate contouring of the gtv in pet / ct fusion - guided radiotherapy .", "visual interpretation method,[1517 ] a fixed 50% threshold of the background subtracted tumor maximum uptake ( thr ) , an appropriate thr level for an individual patient , software - based automated segmentation , were most commonly used for pet / ct - based contouring in clinical studies . the most appropriate method for pet / ct - based gtv delineation", "various studies compared the gtvs contoured in pet / ct scans with ct scans and demonstrated the role of pet / ct fusion for radiotherapy planning.[111516182226 ] scarfone , et al .", ", prospectively studied the impact of hybrid pet - ct simulation in tumor and normal tissue delineation for rt planning in patients with head and neck cancer . they contoured abnormal pet uptake areas using the visual interpretation method .", "we also adopted visual interpretation method to define gtvs in primary and nodal areas for our study .", "heron , et al . , prospectively studied the impact of hybrid pet - ct simulation in tumor and normal tissue delineation for rt planning in patients with head and neck cancers . for primary disease , pet - based target volumes ( mean = 42.7 cm ) were significantly ( p = 0.002 ) smaller compared with ct - based target volumes ( mean = 65 cm ) for all patients .", ", compared ct , mri , and [ 18f]-fdg - pet for delineation of gtv in pharyngolaryngeal squamous cell carcinoma . for oropharyngeal tumors and for laryngeal or hypopharyngeal tumors ,", "average gtvs delineated at ct were 32.0 and 21.4 cm , respectively , whereas average gtvs at pet were smaller , 20.3 [ p = 0.10 ] and 16.4 cm [ p = 0.01 ] , respectively . in various studies ,", "pet - based gtvs were smaller than ct - based gtvs . in this study ,", "gtv pet was significantly smaller than gtv ct ( p < 0.001 ) and consistent with above studies .", "in contrast , nishioka , et al . showed that gtv volumes were not changed by image fusion between 18f - fdg - pet and mri / ct in majority of cases and ashish , et al .", "variations in image acquisition procedures , registration methods , and target volume delineation methods are potential factors for this variation .", "mismatch analysis is a potential tool to study the disagreement between ct and pet / ct in contouring of gtv .", "many studies analyzed this issue and found that there is a significant mismatch existing between gtv pet and gtv ct . in a study by daisne , et al .", ", the average mismatch ct to fdg - pet was 73% and fdg - pet to ct was 14% for oropharyngeal tumors .", "for laryngeal tumors , average mismatch ct to fdg - pet was 48% and fdg - pet to ct was 17% .", ", found the mean value for mismatch of gtv pet / gtv ct as 28.9% s32.9% and the mismatch of gtv ct / gtv pet as 70.9% 50.9% . in this study ,", "the mismatch gtv pet / gtv ct was significantly smaller than mismatch gtv ct / gtv pet ( p = 0.03 ) . in this study ,", "the coverage of gtv pet to gtv ct and gtv ct to gtv pet was analyzed to evaluate the difference between two gtv volumes .", "this was comparable with results of studies done by ei - bassiouni , et al . , and schinagl et al . , in a study by ei - bassiouni , et al", ". , the median percentage of the gtv pet covered by gtv ct was of 99.5% ( range : 41.6 - 100% ) and a mean of 92.4% 16.4% , whereas the median percentage of gtvct covered by ptvpet was 95.1% ( range : 44.3 - 100% ) and a mean was 88.2 - 16.2% ( p = 0.2 ) .", "the major clinical implication of our study is delineation of the metabolically active volumes outside the ct scan .", "these volumes of diseases could have been missed during radiotherapy treatment planning and could result in in - field recurrences and marginal recurrences .", "the median value of primary gtv pet outside ct scan volume was 12.10 cc ( range : 1.50 - 29.80 cc ) and the mean value was 12.17 7.81 cc .", "pet / ct has the advantage to identify metabolically active volumes that are used in simultaneous integrated boost sib - imrt and these volumes are also utilized for dose escalation of gtv .", "performed studies in phantoms filled with radioactive material and proposed threshold - based contouring for pet / ct scans in head and neck cancers .", "however , we adopted the visual interpretation method for target volume delineation mainly because of limitations in our planning systems .", "twenty - six patients were included in this study . in spite of these limitations ,", "though this study demonstrated that pet / ct could improve target volume delineation for radiation treatment planning , we feel that more studies are needed to substantiate the importance of pet / ct ." ], [ "pet / ct significantly alters the staging of tumor ( t staging ) and lymph node metastases ( n staging ) in head and neck cancers .", "pet / ct also has the potential to identify gtv outside ct - based gtv that increases the accuracy of radiation planning .", "we believe that in the era of imrt / image - guided radiotherapy ( igrt ) , pet / ct will increasingly be used for the radiotherapy planning ." ] ]

[ "there has been considerable interest in the possibility that the eradication of persistent viral reservoirs in hiv-1-infected patients could be achieved through specific upregulation of viral expression from quiescently infected reservoir cells [ 16 ] .", "these silent viral reservoirs , largely comprised of hiv-1-infected , resting cd4 + t cells , are long - lived despite continuous and lengthy administration of haart or antiretroviral therapy [ 3 , 4 , 7 ] .", "eradication of these persistent reservoirs may be possible if a sufficient level of viral expression could be induced from the latent proviruses in order to trigger immune clearance or apoptosis of infected reservoir cells [ 36 ] . a number of diverse agents upregulate viral transcription from latent hiv-1 proviral templates in vitro and in vivo .", "these compounds include small chemical cellular activators such as prostratin , a member within the phorbol ester family , the macrolide lactone bryostatin-1 , chromatin - remodeling agents , and select cytokines ( primarily interleukins and interferons ) [ 1 , 5 , 812 ] .", "upregulation of latent hiv-1 expression via the phorbol ester family of compounds or bryostatin-1 occurs as a consequence of activating or modulating the protein kinase c pathway [ 2 , 8 , 9 , 11 , 12 ] .", "the ability of such compounds to upregulate hiv-1 transcription has been well documented in several cellular systems including the latently infected u1 or ach 2 cell - lines , primary t lymphocytes from haart patients as well as the scid - hu mouse model for hiv-1 infection [ 2 , 8 ] .", "the concentration ranges of the pkc activating agents investigated in this study effectively upregulate latent hiv-1 expression in primary cells of infected patients .", "however , developmental and toxicological effects associated with administration of these agents in an intact whole animal model have not been thoroughly evaluated . ", "one purpose of this study was to rapidly assess the gross effects of pkc activation or modulation on zebrafish embryos and larvae , particularly morbidity or lethality , as major indicators of whether nontumor promoting phorbols , including prostratin , or the lactone bryostatin-1 , could be regarded as serious candidates for use in humans at reasonable and effective doses .", "the tumor - promoting property of some phorbol esters essentially excludes these agents from consideration for clinical administration .", "the observation that zebrafish are able to uptake pkc activating agents from the media also provides impetus to observe the effects of modulating protein kinase c pathway activity on zebrafish tissue formation and function .", "modulation of the pkc signal cascade can have widely diverse effects upon cells and whole tissue systems .", "notable effects , apart from the potential of hiv-1 reservoir eradication in humans , include the apparent extension of memory which may bear relevance toward the treatment of alzheimer 's disease as well as the amelioration of certain malignancies [ 1416 ] .", "wild - type ab strain and fli-1 transgenic strain zebrafish were maintained in separate tank systems for embryo and larvae harvesting .", "fli-1 transgenic zebrafish bear an enhanced green fluorescent ( egfp ) open reading frame linked to the fli-1 promoter which drives zebrafish expression of the egfp transgene in blood vessels .", "embryos a few hours after fertilization were distributed into 60 15 mm sterile culture dishes containing equal volumes of embryo media comprised 5 mm nacl , 0.17 mm kcl , 0.33 mm cacl22h2o , and 0.33 mm mgso47h2o . typically , each dish contained 4050 embryos per dish then treated once daily by replenishing the media with the concentrations of agents as stated for three consecutive days or as stated otherwise .", "samples included a negative vehicle control , 1% dimethyl sulfoxide ( dmso ) and varying concentrations of prostratin or a phorbol-12 myristate-13 acetate ( pma ) positive control at 10 um , each suspended in dmso .", "the treated embryos were then observed for developmental abnormalities or mortality using fluorescent or light microscopy .", "fluorescent images under blue light were captured using an olympus ck40 fluorescent microscope equipped with a digital camera .", "visible light photos were obtained using a fujifilm finepix a610 digital camera with the lens placed onto one eyepiece of dissecting microscope bearing an illuminated specimen base .", "hatched larvae were evenly distributed into 60 15 mm sterile culture dishes at a minimum of 20 larvae / dish containing embryo media .", "the larvae were treated once with the concentrations of agents as indicated and harvested 6075 minutes after treatment then stored at 70 degrees c for subsequent page / western analysis .", "typically , fourteen larvae from each treated population were harvested by transfer into individual 1.5 ml microfuge tubes on ice using 1 ml plastic transfer pipets .", "the remaining specimens remained in the media and were photographed either immediately or 24 hours after treatment using 0.4% tricaine as an anesthetic for surviving specimens .", "zebrafish specimens harvested and frozen at 70c were thawed and suspended in 50 ul of laemmli protein loading buffer ( biorad , hercules , ca , usa ) .", "equivalent quantities of protein were loaded to each lane , quantified by using the lowry protein assay ( biorad , hercules , ca , usa ) .", "prior to gel loading , the samples were heated to 90c for 5 minutes and centrifuged at 12,000 g for 3 minutes .", "samples were electrophoresed on 4%15% gradient gels ( biorad , hercules , ca , usa ) and proteins transferred to pvdf membranes using trans - blot semi dry transfer cell ( biorad , hercules , ca , usa ) at 15 v for 1 - 2 hours .", "protein blots were incubated in the presence of anti - gfp antisera ( bio - rad , hercules , ca , usa ) or anti - mapk-8 zebrafish - specific antisera obtained from anaspec , freemont , ca , usa .", "pvdf membranes were developed using the westernbreeze chromogenic western blot immunodetection kit as per instructions using anti - rabbit igg conjugated with alkaline phosphatase .", "images of blots were captured by scanning with an hp officejet pro combination printer / scanner .", "as shown in figure 1 , no abnormalities or increased mortality were observed one day after treatment of day 1 postfertilization embryos with varying concentrations of prostratin and 10 um pma compared to embryos treated with an equivalent concentration of the control vehicle , dmso .", "the development of the embryonic vascular systems appeared similar in all treated samples as assessed by egfp fluorescence of vascular endothelial cells which are visibly highlighted via expression of the fli-1 promoter / egfp transgene .", "the egfp fluorescence notably highlights the vascular endothelium in the anterior head portion and extending down the developing vertebral column of the embryos in these images . ", "as shown in figure 2 , observations by light microscopy of treated , unhatched embryos on day 3 postfertilization looked much the same with regard to development as those noted for the previous day treatments .", "these later - stage embryos , exposed to low ( 0.1 m ) and medium ( 1 m ) concentrations of prostratin , were not observably different from those treated with dmso . as well , exposure to 10 m prostratin elicited no observable degree of developmental abnormality .", "this was very much in contrast to specimens treated with 10 m pma treatment .", "as shown in the bottom right panel of figure 2 , this representative pma - treated embryo was deformed and failed to survive . ", "as shown in figure 3 , overall development of the 4-day postfertilization embryos and newly hatched larvae continued to proceed without observable gross defects with increasing concentrations of prostratin .", "differences began to emerge shortly after day 3 after fertilization and generally into day 4 when hatching is generally initiated .", "the most common effects observed were death and/or trapping in the egg sac with exposure to 10 m pma .", "the concentration - dependent effects of phorbol treatment were next compared on hatched , nontransgenic seven - day - old ab larvae as shown in figure 4 .", "pma treatment at 10 m resulted in rapid and complete lethality as is clearly apparent for the decaying embryo specimen shown to the right side of figure 4 which was photographed 24 hours after treatment .", "however , very limited gross morphological deformities were apparent in larvae treated with 10 m prostratin at least from the time of treatment to visual inspection 24 hours later .", "indeed , the 10 m prostratin - treated larvae appeared morphologically similar to those treated with the dmso vehicle control ( middle and left panels of figure 4 ) with high survival rates 24 hours after treatment .", "protein gel electrophoresis ( page ) of total proteins from treated larvae was employed to assess any major differences in gene expression as a consequence of phorbol ester exposure .", "page comparing treated and vehicle control showed no remarkable difference in overall expression among treated or control embryos ( data not shown ) or larvae as shown in figure 5(a ) .", "abundant structural proteins in larvae were similar in presence and abundance among all the phorbol - treated larvae samples versus the dmso control .", "pma , known to be a potent apoptotic mitogen for most cells and tissue systems [ 15 , 16 ] , exhibited the highest degree of inducing developmental defects and lethality relative to prostratin - treated specimens at the same concentration .", ", western analysis was performed on hatched ab larvae treated with dmso , prostratin and pma using mapk-8 antisera in the expectation that phorbol exposure may alter the expression level of this specific signaling protein . as shown in figure 5(b ) , mapk-8 exhibited modest levels of upregulation following treatment with prostratin or pma relative to dmso - control - treated larvae in this representative blot .", "western analysis was also employed to assess any changes in the expression of the egfp protein which remained at similar levels among fli-1 larvae treated with prostratin or pma versus dmso control ( data not shown ) .", "the lethal effects of an expanded panel of pkc modulators were next compared in a concentration - dependent fashion using larvae eight days after fertilization .", "as shown in figure 6 , treatment with different pkc modulators exhibited defined threshold doses for lethality .", "the phorbol ester , 12-deoxyphorbol-13-phenylacetate ( dpp ) , had a concentration - dependent lethal effect at 10 m , similar to pma .", "prostratin only elicited rapid death at 100 m which occurred in the same time frame as dosage with 10 m pma and 10 m dpp , usually within 6075 minutes after treatment .", "of interest is the ability of larvae to tolerate concentrations of bryostatin-1 up to 10 m .", "although 10 m bryostatin-1 treatment did not induce lethality in the short term , the fish appeared very lethargic 1 hour after treatment and no survivors were observed 24 hours after treatment .", "the treated fish were unaffected by concentrations of bryostatin-1 at or below 1 m . ", "the survival profiles for each individual compound shown in figure 6 represent an average of four data sets for treated larvae eight days after fertilization with less than 2% standard deviation from the mean for each set .", "the lethal effects at the concentrations noted for this larval set also occurred reproducibly for either ab or fli-1 larvae treated even at earlier postfertilization times .", "in addition , these survival frequencies were similar for unhatched 4-day postfertilization ab or fli-1 embryos .", "survival frequency for embryos and larvae , regardless of strain was always reproducibly ~94%99% at nonlethal agent doses , and lethality was consistently very near or at 100% with higher doses of the compounds at all times of treatment beyond three days after fertilization .", "the nontumor - promoting phorbol ester prostratin and the lactone bryostatin-1 modulate pkc cascade activity .", "these agents have been forwarded as potential candidates for the eradication of haart - persistent cellular reservoirs bearing silent hiv-1 proviral dna [ 24 , 8 , 9 , 11 ] .", "these pkc modulators , including prostratin , upregulate latent viral expression in a number of cell - based systems [ 2 , 8 , 9 , 11 ] but have not had widespread clinical use in patients except for bryostatin-1 . ", "the primary objective of this study was to determine whether treatment with pkc modulators or activators , particularly the phorbol ester prostratin , might elicit a severe cytotoxic profile or induce development effects in tissue systems within a tractable vertebrate model .", "specificity was further noted by modest upregulation in the expression of the signaling factor , mapk-8 , within phorbol treated larvae . ", "the map kinases ( mapks ) including mapk-8 ( p38 ) are known to respond to mitogen stimulation , proinflammatory cytokines and environmental stress although this response is primarily mediated through a series of phosphorylation events of preexisting proteins [ 17 , 18 ] .", "our data suggest that some upregulation of mapk-8 via de novo synthesis can occur by exogeneous phorbol treatment of whole zebrafish larvae .", "this contrasts the expression of the egfp whose levels remained unchanged in fli-1 larvae treated similarly with the equivalent concentrations of prostratin and pma . ", "we have some preliminary evidence that pkc modulating compounds induce apoptosis at high doses likely contributing to their obvious lethal effect .", "this is consistent with the action of phorbol esters and mapk-8 which can participate in a mitogen - activated cascade to initiate an apoptotic effect [ 16 , 20 ] . assessing", "the effects of pkc modulators using the zebrafish model are of interest given the ongoing concerns regarding the use of pkc activators or modulators as clinical candidates for administration to humans .", "this caution may be warranted , since this diverse class of compounds can broadly activate multiple cell - types and can rapidly advance cell - type specific differentiation , maturation , or apoptosis [ 2 , 8 , 19 ] .", "for instance , prostratin rapidly advances monocyte differentiation and bryostatin-1 induces accelerated maturation of human cord - blood derived dendritic cells .", "interestingly , the broad effects of such properties induced by the phorbol ester family are unknown in a whole developing animal model .", "in contrast , bryostatin-1 has been evaluated clinically at low doses for the treatment of certain human cancers [ 2123 ] .", "bryostatin-1 is also regarded as a potential candidate for the treatment of alzheimer 's disease , as it appears that exposure to the compound can extend memory and rescue retrograde or anterograde long - term memory following cerebral ischemia / hypoxia [ 24 , 25 ] . ", "the data in this paper might be regarded as encouraging in that low concentrations of pkc modulators , including the phorbol esters , which upregulate latent hiv-1 expression in human cells within a range of 1 to 10 m [ 2 , 9 ] , showed no obvious effect upon zebrafish embryo or larvae development .", "in contrast , higher concentrations of pkc activators , elicited almost complete lethality in both zebrafish embryos and early - term larvae . ", "zebrafish embryos and larvae were particularly useful to study the effects of pkc activators or modulators in a whole animal model , since the compounds can be absorbed directly from the media and have clear effects on this organism 's development and survival . ", "interestingly , effects on embryos were not observable until after day 3 into day 4 of agent treatment and were most notable with pma exposure .", "it is not clear why this very potent tumor - promoting phorbol did not elicit lethal effects on early - stage embryos as quickly as it did for hatched larvae .", "zebrafish are recognized as a useful model to rapidly establish paradigms for the investigation and treatment of disabling human diseases .", "the absorption of exogenous phorbol esters by zebrafish at varying developmental stages appears to be a feasible way to begin to modulate pkc signaling in vivo to assess effects on specific tissue systems .", "importantly , such processes affected by pkc modulators include , but are not limited to , memory extension and tumorigenesis as noted in other vertebrate systems .", "[ 22 , 24 , 25 ] . these studies also demonstrate that the nontumor promoting phorbol ester prostratin had no obvious deleterious effects on zebrafish development at concentrations below 10 m , which is sufficient to upregulate latent hiv-1 expression in human cellular systems [ 2 , 8 , 9 ] .", "this compound or related agents may deserve further consideration in clinical protocols toward the eradication of hiv-1 latent reservoirs ." ]

[ "<S> clinical interventions using protein kinase c ( pkc ) modulators have been proposed for eradication of hiv-1-infected cellular reservoirs which persist in patients despite prolonged antiretroviral therapy . </S>", "<S> the effects of some of these agents have not been assessed in a developing vertebrate model . </S>", "<S> this study examines the developmental and toxicological effects of these compounds on zebrafish embryos and larvae . </S>", "<S> treatment of zebrafish through the first week of development with various pkc pathway modulators did not elicit gross physical defects or elevated incidences of death at lower doses . </S>", "<S> higher concentrations resulted in rapid death for both later - stage embryos and larvae . </S>", "<S> each compound had a threshold dose for lethality . </S>", "<S> the defined nonlethal doses may be useful toward assessing the effects of modulating pkc activity on zebrafish development . </S>", "<S> they may further provide some guidance for the potential dosing of pkc modulators in clinical trials toward the goal of hiv-1 reservoir eradication . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion" ]

[ [ "there has been considerable interest in the possibility that the eradication of persistent viral reservoirs in hiv-1-infected patients could be achieved through specific upregulation of viral expression from quiescently infected reservoir cells [ 16 ] .", "these silent viral reservoirs , largely comprised of hiv-1-infected , resting cd4 + t cells , are long - lived despite continuous and lengthy administration of haart or antiretroviral therapy [ 3 , 4 , 7 ] .", "eradication of these persistent reservoirs may be possible if a sufficient level of viral expression could be induced from the latent proviruses in order to trigger immune clearance or apoptosis of infected reservoir cells [ 36 ] . a number of diverse agents upregulate viral transcription from latent hiv-1 proviral templates in vitro and in vivo .", "these compounds include small chemical cellular activators such as prostratin , a member within the phorbol ester family , the macrolide lactone bryostatin-1 , chromatin - remodeling agents , and select cytokines ( primarily interleukins and interferons ) [ 1 , 5 , 812 ] .", "upregulation of latent hiv-1 expression via the phorbol ester family of compounds or bryostatin-1 occurs as a consequence of activating or modulating the protein kinase c pathway [ 2 , 8 , 9 , 11 , 12 ] .", "the ability of such compounds to upregulate hiv-1 transcription has been well documented in several cellular systems including the latently infected u1 or ach 2 cell - lines , primary t lymphocytes from haart patients as well as the scid - hu mouse model for hiv-1 infection [ 2 , 8 ] .", "the concentration ranges of the pkc activating agents investigated in this study effectively upregulate latent hiv-1 expression in primary cells of infected patients .", "however , developmental and toxicological effects associated with administration of these agents in an intact whole animal model have not been thoroughly evaluated . ", "one purpose of this study was to rapidly assess the gross effects of pkc activation or modulation on zebrafish embryos and larvae , particularly morbidity or lethality , as major indicators of whether nontumor promoting phorbols , including prostratin , or the lactone bryostatin-1 , could be regarded as serious candidates for use in humans at reasonable and effective doses .", "the tumor - promoting property of some phorbol esters essentially excludes these agents from consideration for clinical administration .", "the observation that zebrafish are able to uptake pkc activating agents from the media also provides impetus to observe the effects of modulating protein kinase c pathway activity on zebrafish tissue formation and function .", "modulation of the pkc signal cascade can have widely diverse effects upon cells and whole tissue systems .", "notable effects , apart from the potential of hiv-1 reservoir eradication in humans , include the apparent extension of memory which may bear relevance toward the treatment of alzheimer 's disease as well as the amelioration of certain malignancies [ 1416 ] ." ], [ "wild - type ab strain and fli-1 transgenic strain zebrafish were maintained in separate tank systems for embryo and larvae harvesting .", "fli-1 transgenic zebrafish bear an enhanced green fluorescent ( egfp ) open reading frame linked to the fli-1 promoter which drives zebrafish expression of the egfp transgene in blood vessels .", "embryos a few hours after fertilization were distributed into 60 15 mm sterile culture dishes containing equal volumes of embryo media comprised 5 mm nacl , 0.17 mm kcl , 0.33 mm cacl22h2o , and 0.33 mm mgso47h2o . typically , each dish contained 4050 embryos per dish then treated once daily by replenishing the media with the concentrations of agents as stated for three consecutive days or as stated otherwise .", "samples included a negative vehicle control , 1% dimethyl sulfoxide ( dmso ) and varying concentrations of prostratin or a phorbol-12 myristate-13 acetate ( pma ) positive control at 10 um , each suspended in dmso .", "the treated embryos were then observed for developmental abnormalities or mortality using fluorescent or light microscopy .", "fluorescent images under blue light were captured using an olympus ck40 fluorescent microscope equipped with a digital camera .", "visible light photos were obtained using a fujifilm finepix a610 digital camera with the lens placed onto one eyepiece of dissecting microscope bearing an illuminated specimen base .", "hatched larvae were evenly distributed into 60 15 mm sterile culture dishes at a minimum of 20 larvae / dish containing embryo media .", "the larvae were treated once with the concentrations of agents as indicated and harvested 6075 minutes after treatment then stored at 70 degrees c for subsequent page / western analysis .", "typically , fourteen larvae from each treated population were harvested by transfer into individual 1.5 ml microfuge tubes on ice using 1 ml plastic transfer pipets .", "the remaining specimens remained in the media and were photographed either immediately or 24 hours after treatment using 0.4% tricaine as an anesthetic for surviving specimens .", "zebrafish specimens harvested and frozen at 70c were thawed and suspended in 50 ul of laemmli protein loading buffer ( biorad , hercules , ca , usa ) .", "equivalent quantities of protein were loaded to each lane , quantified by using the lowry protein assay ( biorad , hercules , ca , usa ) .", "prior to gel loading , the samples were heated to 90c for 5 minutes and centrifuged at 12,000 g for 3 minutes .", "samples were electrophoresed on 4%15% gradient gels ( biorad , hercules , ca , usa ) and proteins transferred to pvdf membranes using trans - blot semi dry transfer cell ( biorad , hercules , ca , usa ) at 15 v for 1 - 2 hours .", "protein blots were incubated in the presence of anti - gfp antisera ( bio - rad , hercules , ca , usa ) or anti - mapk-8 zebrafish - specific antisera obtained from anaspec , freemont , ca , usa .", "pvdf membranes were developed using the westernbreeze chromogenic western blot immunodetection kit as per instructions using anti - rabbit igg conjugated with alkaline phosphatase .", "images of blots were captured by scanning with an hp officejet pro combination printer / scanner ." ], [ "as shown in figure 1 , no abnormalities or increased mortality were observed one day after treatment of day 1 postfertilization embryos with varying concentrations of prostratin and 10 um pma compared to embryos treated with an equivalent concentration of the control vehicle , dmso .", "the development of the embryonic vascular systems appeared similar in all treated samples as assessed by egfp fluorescence of vascular endothelial cells which are visibly highlighted via expression of the fli-1 promoter / egfp transgene .", "the egfp fluorescence notably highlights the vascular endothelium in the anterior head portion and extending down the developing vertebral column of the embryos in these images . ", "as shown in figure 2 , observations by light microscopy of treated , unhatched embryos on day 3 postfertilization looked much the same with regard to development as those noted for the previous day treatments .", "these later - stage embryos , exposed to low ( 0.1 m ) and medium ( 1 m ) concentrations of prostratin , were not observably different from those treated with dmso . as well , exposure to 10 m prostratin elicited no observable degree of developmental abnormality .", "this was very much in contrast to specimens treated with 10 m pma treatment .", "as shown in the bottom right panel of figure 2 , this representative pma - treated embryo was deformed and failed to survive . ", "as shown in figure 3 , overall development of the 4-day postfertilization embryos and newly hatched larvae continued to proceed without observable gross defects with increasing concentrations of prostratin .", "differences began to emerge shortly after day 3 after fertilization and generally into day 4 when hatching is generally initiated .", "the most common effects observed were death and/or trapping in the egg sac with exposure to 10 m pma .", "the concentration - dependent effects of phorbol treatment were next compared on hatched , nontransgenic seven - day - old ab larvae as shown in figure 4 .", "pma treatment at 10 m resulted in rapid and complete lethality as is clearly apparent for the decaying embryo specimen shown to the right side of figure 4 which was photographed 24 hours after treatment .", "however , very limited gross morphological deformities were apparent in larvae treated with 10 m prostratin at least from the time of treatment to visual inspection 24 hours later .", "indeed , the 10 m prostratin - treated larvae appeared morphologically similar to those treated with the dmso vehicle control ( middle and left panels of figure 4 ) with high survival rates 24 hours after treatment .", "protein gel electrophoresis ( page ) of total proteins from treated larvae was employed to assess any major differences in gene expression as a consequence of phorbol ester exposure .", "page comparing treated and vehicle control showed no remarkable difference in overall expression among treated or control embryos ( data not shown ) or larvae as shown in figure 5(a ) .", "abundant structural proteins in larvae were similar in presence and abundance among all the phorbol - treated larvae samples versus the dmso control .", "pma , known to be a potent apoptotic mitogen for most cells and tissue systems [ 15 , 16 ] , exhibited the highest degree of inducing developmental defects and lethality relative to prostratin - treated specimens at the same concentration .", ", western analysis was performed on hatched ab larvae treated with dmso , prostratin and pma using mapk-8 antisera in the expectation that phorbol exposure may alter the expression level of this specific signaling protein . as shown in figure 5(b ) , mapk-8 exhibited modest levels of upregulation following treatment with prostratin or pma relative to dmso - control - treated larvae in this representative blot .", "western analysis was also employed to assess any changes in the expression of the egfp protein which remained at similar levels among fli-1 larvae treated with prostratin or pma versus dmso control ( data not shown ) .", "the lethal effects of an expanded panel of pkc modulators were next compared in a concentration - dependent fashion using larvae eight days after fertilization .", "as shown in figure 6 , treatment with different pkc modulators exhibited defined threshold doses for lethality .", "the phorbol ester , 12-deoxyphorbol-13-phenylacetate ( dpp ) , had a concentration - dependent lethal effect at 10 m , similar to pma .", "prostratin only elicited rapid death at 100 m which occurred in the same time frame as dosage with 10 m pma and 10 m dpp , usually within 6075 minutes after treatment .", "of interest is the ability of larvae to tolerate concentrations of bryostatin-1 up to 10 m .", "although 10 m bryostatin-1 treatment did not induce lethality in the short term , the fish appeared very lethargic 1 hour after treatment and no survivors were observed 24 hours after treatment .", "the treated fish were unaffected by concentrations of bryostatin-1 at or below 1 m . ", "the survival profiles for each individual compound shown in figure 6 represent an average of four data sets for treated larvae eight days after fertilization with less than 2% standard deviation from the mean for each set .", "the lethal effects at the concentrations noted for this larval set also occurred reproducibly for either ab or fli-1 larvae treated even at earlier postfertilization times .", "in addition , these survival frequencies were similar for unhatched 4-day postfertilization ab or fli-1 embryos .", "survival frequency for embryos and larvae , regardless of strain was always reproducibly ~94%99% at nonlethal agent doses , and lethality was consistently very near or at 100% with higher doses of the compounds at all times of treatment beyond three days after fertilization ." ], [ "the nontumor - promoting phorbol ester prostratin and the lactone bryostatin-1 modulate pkc cascade activity .", "these agents have been forwarded as potential candidates for the eradication of haart - persistent cellular reservoirs bearing silent hiv-1 proviral dna [ 24 , 8 , 9 , 11 ] .", "these pkc modulators , including prostratin , upregulate latent viral expression in a number of cell - based systems [ 2 , 8 , 9 , 11 ] but have not had widespread clinical use in patients except for bryostatin-1 . ", "the primary objective of this study was to determine whether treatment with pkc modulators or activators , particularly the phorbol ester prostratin , might elicit a severe cytotoxic profile or induce development effects in tissue systems within a tractable vertebrate model .", "specificity was further noted by modest upregulation in the expression of the signaling factor , mapk-8 , within phorbol treated larvae . ", "the map kinases ( mapks ) including mapk-8 ( p38 ) are known to respond to mitogen stimulation , proinflammatory cytokines and environmental stress although this response is primarily mediated through a series of phosphorylation events of preexisting proteins [ 17 , 18 ] .", "our data suggest that some upregulation of mapk-8 via de novo synthesis can occur by exogeneous phorbol treatment of whole zebrafish larvae .", "this contrasts the expression of the egfp whose levels remained unchanged in fli-1 larvae treated similarly with the equivalent concentrations of prostratin and pma . ", "we have some preliminary evidence that pkc modulating compounds induce apoptosis at high doses likely contributing to their obvious lethal effect .", "this is consistent with the action of phorbol esters and mapk-8 which can participate in a mitogen - activated cascade to initiate an apoptotic effect [ 16 , 20 ] . assessing", "the effects of pkc modulators using the zebrafish model are of interest given the ongoing concerns regarding the use of pkc activators or modulators as clinical candidates for administration to humans .", "this caution may be warranted , since this diverse class of compounds can broadly activate multiple cell - types and can rapidly advance cell - type specific differentiation , maturation , or apoptosis [ 2 , 8 , 19 ] .", "for instance , prostratin rapidly advances monocyte differentiation and bryostatin-1 induces accelerated maturation of human cord - blood derived dendritic cells .", "interestingly , the broad effects of such properties induced by the phorbol ester family are unknown in a whole developing animal model .", "in contrast , bryostatin-1 has been evaluated clinically at low doses for the treatment of certain human cancers [ 2123 ] .", "bryostatin-1 is also regarded as a potential candidate for the treatment of alzheimer 's disease , as it appears that exposure to the compound can extend memory and rescue retrograde or anterograde long - term memory following cerebral ischemia / hypoxia [ 24 , 25 ] . ", "the data in this paper might be regarded as encouraging in that low concentrations of pkc modulators , including the phorbol esters , which upregulate latent hiv-1 expression in human cells within a range of 1 to 10 m [ 2 , 9 ] , showed no obvious effect upon zebrafish embryo or larvae development .", "in contrast , higher concentrations of pkc activators , elicited almost complete lethality in both zebrafish embryos and early - term larvae . ", "zebrafish embryos and larvae were particularly useful to study the effects of pkc activators or modulators in a whole animal model , since the compounds can be absorbed directly from the media and have clear effects on this organism 's development and survival . ", "interestingly , effects on embryos were not observable until after day 3 into day 4 of agent treatment and were most notable with pma exposure .", "it is not clear why this very potent tumor - promoting phorbol did not elicit lethal effects on early - stage embryos as quickly as it did for hatched larvae .", "zebrafish are recognized as a useful model to rapidly establish paradigms for the investigation and treatment of disabling human diseases .", "the absorption of exogenous phorbol esters by zebrafish at varying developmental stages appears to be a feasible way to begin to modulate pkc signaling in vivo to assess effects on specific tissue systems .", "importantly , such processes affected by pkc modulators include , but are not limited to , memory extension and tumorigenesis as noted in other vertebrate systems .", "[ 22 , 24 , 25 ] . these studies also demonstrate that the nontumor promoting phorbol ester prostratin had no obvious deleterious effects on zebrafish development at concentrations below 10 m , which is sufficient to upregulate latent hiv-1 expression in human cellular systems [ 2 , 8 , 9 ] .", "this compound or related agents may deserve further consideration in clinical protocols toward the eradication of hiv-1 latent reservoirs ." ] ]

[ "why have we not been able to use neuroimaging in the clinical management of psychoses ?", "after 30 years of using this approach , we have established that structural brain alterations are present in individuals with psychoses . while this information has advanced academic knowledge on the pathophysiology of these disorders , it has had limited utility in clinical practice .", "the main reason can be found in the nature of brain alterations in psychoses : they are subtle and spatially distributed .", "still , the time may now have come when quick and noninvasive structural neuroimaging abandons the academic solitude in which it has been operating , and brings its novel methods and findings into the clinical arena .", "this paper aims to appraise this evidence , and discuss it in the context of potential future applications of magnetic resonance imaging ( mri ) in the clinical management of psychoses .", "the first part of this review will present evidence on the presence of neuroanatomical alterations in psychoses .", "it will then discuss studies that have examined the relationship between brain alterations and various medium- to long - term clinical and functional outcomes . in the third section", ", it will discuss more recent evidence on the relationship between brain alterations and early outcomes , with a particular focus on the relationship between symptomatic responses and initial treatment with antipsychotic drugs .", "the paper will then present a discussion of more novel approaches for the analysis of mri data , such as support vector machine .", "findings from these approaches have started to suggest that mri has the potential to provide clinically meaningful information .", "finally , it will discuss the implications of having strong and valid neuroimaging markers of psychosis outcomes in developing targeted , as well as novel , treatment interventions for these disorders .", "moving from the first evidence that individuals with established schizophrenia have subtle structural brain alterations , most notably an enlargement of the lateral ventricles , we have been studying individuals earlier and earlier along the disease course .", "more than 10 years ago , the seminal work by pantelis and colleagues showed that individuals at high risk of developing psychosis , but not ill yet , already show smaller volumes of the same brain areas that have been reported as smaller in individuals with an established psychosis , namely frontal and temporal cortices .", "even more novel , however , was their report that this is particularly the case for individuals who then actually develop the illness , and that some of these alterations could be specific to the type of psychosis that these subjects developed .", "for example , they found that subjects who develop an affective type of psychosis showed reductions in the subgenual anterior cingulate cortex , an area involved in the generation of affective symptoms .", "complementary to these gray - matter changes , ultra - high - risk individuals also have white matter alterations similar to , albeit less extensive than , first - episode patients .", "these include poorer integrity of the major associative fibers that connect fronto - parieto - temporal ( superior longitudinal fasciculus ) and fronto - parieto - occipital ( inferior fronto - occipital fasciculus ) regions , commissural fibers ( corpus callosum ) , and cortico - subcortical pathways ( corona radiata , corticospinal tract , and corticopontine tract ) .", "these findings , integrated with evidence from many reviews and meta - analyses , suggest that brain alterations become more extensive at the time of a first full psychotic episode , and then possibly even more marked over the years , when the illness becomes established .", "these are indeed two main factors that have limited our ability to use this information about brain alterations in the clinical management of the individual patient .", "therefore , many studies have tried to establish whether these distributed brain changes are more likely to occur in individuals with a more severe illness course or a worse clinical and functional outcome .", "for example , a poorer clinical outcome has been variously defined as response to early treatment , number of subsequent episodes , severity of symptoms , hospitalizations , or duration of remissions ; while functional outcome has been defined as the ability to live independently , maintain employment , or be in a relationship .", "nevertheless , many studies have tried to establish if structural brain alterations are related to illness characteristics by comparing brain structure in patients and healthy controls and then evaluating the relationship with outcomes at a single time point ; or conducting repeated evaluations of brain structure , and then investigating the relationship between longitudinal brain changes and outcomes .", "most of these studies have evaluated the relationship with medium to long - term outcomes , and only more recently has the attention shifted to the evaluation of the relationship between brain alterations and early outcomes such as response to the first treatment .", "studies that have evaluated brain structure at the time of illness onset and again following treatment , have often reported smaller volumes of gray matter in association with indicators of subsequent poorer clinical outcomes ( eg , higher number of admissions or worse symptom severity ) , and with worse social or occupational outcomes .", "for example , smaller volumes of prefrontal areas have been found to predict poorer functioning ( defined as a combination of social , occupational , and psychological functioning ) 1 year later in a small sample of first - episode psychosis patients . similarly , in a sample of patients with schizophrenia , drawn from an unselected general population sample , jskelinen and colleagues found that individuals showing higher density ( an indirect measure of volume ) of frontal and limbic areas had an overall better clinical ( defined as a lower number of hospitalizations ) and functional ( defined as not being on a disability pension ) outcome in the subsequent 16 years . wassink and colleagues reported an association between smaller volumes of another brain area , the cerebellum , and longer negative and psychotic symptom duration , as well as psychosocial impairment ( eg , quality of relationships , sexual activity , enjoyment of recreation , and work performance ) in individuals with schizophrenia spectrum disorders after a 7-year follow - up .", "still , others have not found an association between brain volumes at onset and subsequent outcomes .", "for example , the prospective study by van ilaren and colleagues found no relationship between brain volume measurements and symptom severity , level of functioning ( defined as the ability to maintain a variety of social roles ) , need for care , and illness course after 2 years .", "mitelman and colleagues examined white matter integrity using diffusion tensor imaging ( dti ) , in a group of 104 patients with an average 4-year schizophrenia duration .", "they classified them as having a poor or good outcome , based on both symptom severity and level of functioning at this time point .", "they found that although both patient groups showed reduced overall white matter integrity of the prefrontal and temporal areas ( as a measure of fractional anisotropy [ fa ] ) , these alterations were more extensive in patients with a poor outcome than in those with a good outcome .", "longitudinal studies that have evaluated brain structure at multiple time points after illness onset can help clarify if brain alterations , evident at illness onset , progress differently in individuals who then developed a poorer outcome . for example , a larger decrease in the volume of the lingual gyrus , insula , and cerebellum has been reported in patients with a worse functional outcome ( a combination of social , occupational , and psychological functioning ) 4 years after the first psychotic episode . with a similar duration of follow - up , van ilaren and colleagues also reported a greater gray matter volume reduction , and a greater thinning of temporal and frontal cortices in individuals who had poorer outcomes ( including a higher number of hospitalizations and a lower level of social and personal functioning ) 5 years after illness onset .", "similarly , in a sample of children and adolescents with early onset first - episode schizophrenia , arango and colleagues showed that more weeks of hospitalization and higher severity of negative symptoms were correlated with greater left frontal gray matter volume loss and greater cerebral spinal fluid increase , respectively .", "dynamic changes in relation to outcomes were also observed by our group in a 6-year follow - up study of hippocampal volume in first - episode psychosis patients . in this sample , we specifically found that individuals who had an increase in hippocampal volume over the first 6 years of illness were the ones who showed a less severe illness course and lower symptom severity , as well as a better functional outcome at follow - up , thus suggesting an important role for brain neuroplasticity .", "taken together , these findings suggest that brain alterations are present at illness onset and may be associated with worse medium- to long - term illness outcomes and ability to function .", "still , two important considerations need to be made in the interpretation of these findings .", "first , it is not possible to establish what the casual link is between brain structure and illness course . while brain alterations could be the substrate of a more severe illness , the more severe illness itself could affect brain plasticity by increasing exposure to substance abuse , worse living conditions , or a less stimulating environment .", "it is , therefore , possible that patients with a more severe illness are exposed to longer periods of treatment or higher doses of antipsychotics , which may themselves affect brain volumes . from a clinical perspective", ", it may be more informative to study the relationship between brain alterations at illness onset and outcome measures that occur earlier in the course of illness , which is the topic of the next section .", "if brain alterations could be used to predict , as early as possible , which patients are destined to have a poorer response to the first few weeks or months of treatment with antipsychotic medications ( the main stake for the treatment of psychosis ) , we could implement interventional strategies targeted at this specific subgroup .", "currently , we treat the first psychotic episode on a trialand - error basis , and we simply have no way of saying who will respond to the first antipsychotic drug and who might benefit from longer or different interventions . in fact , we know that only -55% of patients respond to antipsychotics in the first 12 months of treatment . yet , this early response is thought to be one of the strongest predictors of subsequent functional and clinical outcomes in psychosis .", "a pooled analysis , published in 1992 , examined the relationship between response to antipsychotics and brain measures , such as ventricular - brain ratio , sulcal prominence , third ventricles , and other brain measures .", "the authors found that the composite effect sizes were very small and did not reach statistical significance .", "however , recent studies have lent renewed support to the notion that alterations in both gray and white matter may be associated with a poorer response to antipsychotics , possibly because they used more homogeneous populations , or in some cases , better imaging acquisition approaches and more sophisticated methods of analysis .", "for example , zipursky and colleagues examined global tissue volumes in firstepisode psychosis patients , and found that subjects with a poorer response ( defined as having a lower reduction in symptom severity from baseline ) to the antipsychotic haloperidol in the first month of treatment had smaller cortical gray matter volume than those who showed symptomatic improvement .", "relatively consistent data have been reported on the relationship between response to clozapine and brain structure .", "cortical measures , such as sulcal enlargement , particularly in the prefrontal cortex , have been frequently related to poor response to clozapine in patients with chronic schizophrenia . in a double - blind , 10-week trial of clozapine and haloperidol , arango and colleagues found that a larger prefrontal gray matter volume was associated with better treatment response in clozapine - treated patients , but with a poor response in haloperidol- treated patients .", "they also found no relationship between response to these drugs and hippocampal and caudate nucleus volumes .", "the authors suggested that the presence of larger brain volumes might characterize patients who are more likely to benefit from clozapine .", "this finding is also consistent with evidence from a study in patients with treatment - resistant schizophrenia who were treated for with clozapine 6 months .", "patients with a larger baseline volume of the dorsolateral - prefrontal cortex were more likely to show improvement in their negative symptoms .", "interestingly , amelioration of positive symptoms was related to a larger temporal cortex volume , and amelioration of disorganization symptoms was inversely related to hippocampal volume .", "another temporal area has been reported as being related to early outcome the parahippocampal gyrus .", "using voxel - based morphometry , bodnar and colleagues found that the parahippocampal gyrus was significantly smaller in first - episode psychosis patients who , after 6 months of antipsychotic treatment , had not remitted , compared with those who had achieved remission .", "furthermore , by building a classification model using parahippocampal gray matter concentration , they were able to correctly classify remission status in 79% of the cases . in another study of patients with established schizophrenia treated with olanzapine or risperidone , 3-week response to these antipsychotics", "was associated with smaller volumes of other areas , such as the insula and rectal gyrus , and larger volumes of the basal ganglia .", "white matter has also been associated with treatment response , but the evidence is considerably more scant .", "response to antipsychotics was evaluated by garver and colleagues in a small sample of patients with schizophrenia after 28 days of treatment . here , somewhat counterintuitively , the 8 patients who responded to antipsychotics showed worse white matter microstructural integrity than the 5 patients who did not respond to medication . of note , individuals , in this study , had an established illness , and therefore ,", "in contrast , studies in antipsychotic naive or minimally treated patients have the advantage that any structural brain alterations would be less likely to reflect the effect of antipsychotics on brain structure , and more likely to be a marker of illness .", "luck and colleagues analyzed a sample of first - episode psychosis patients using dti , and then reassessed them at 6 months , which is when they classified them as having either a poor or good treatment outcome .", "they specifically examined three white matter tracts connecting frontal and temporal regions : the cingulum , the superior longitudinal fasciculus , and the uncinate fasciculus .", "patients with a poor outcome showed greater alterations in white matter integrity in the superior longitudinal fasciculus and uncinate than patients with a good outcome , suggesting that abnormal fronto - temporal connectivity may represent an early marker of short - term clinical outcomes .", "our group has also recently focused on the evaluation of the relationship between neuroanatomical markers in white and gray matter at the time of presentation , and response to treatment at 12 weeks .", "this is a time when all patients would have received at least one full therapeutic course of antipsychotic medication .", "we examined white matter integrity , using dti and tractbased spatial statistics , to assess fa in a large sample of patients at their first episode of any psychosis .", "we operationalized response to treatment as having achieved symptomatic remission according to the criteria of the schizophrenia working group .", "we found that already at illness onset , patients who subsequently did not respond ( n=40 ) had lower fa than healthy controls in the uncinate , cingulum , and corpus callosum .", "furthermore , they also had lower fa in these regions than patients who responded to treatment ( n=40 ; figure 1 , tables i and ii ) .", "one of the most striking findings of this work was that the responders were indistinguishable from the healthy control subjects , suggesting that the original clinical sample was biologically rather heterogeneous .", "we rescanned these subjects at the 12-week clinical evaluation , and found that the same regional differences seen at baseline between the two patient groups were also present at 12 weeks , although the differences appeared less widespread .", "furthermore , although there was some increase in fa over time , this was in the same direction in the two patient groups , with no significant time vs group interaction .", "we interpreted these findings to indicate that white matter integrity and brain connectivity are important moderators of response to antipsychotics .", "it is intriguing to speculate whether these alterations are established early on , as a neurodevelopmental deviation .", "the presence of a neurodevelopmental alteration would find further support in our gray matter findings from this sample . in these individuals", ", we performed the first 3d evaluation of cortical gyrification , a marker potentially indicative of early neurodevelopmental disturbances , in relation to subsequent treatment response .", "gyrification defects have been associated with exposure to obstetric complications and with psychotic symptoms resistant to treatment .", "interestingly , the earlier neuroimaging studies in treatment - resistant patients that were mentioned in the previous section , found that sulcal enlargement ( which could lead to hypogyria ) was associated with a poorer response to clozapine . in our samples , we found that , already at illness onset , patients who subsequently did not respond to treatment had significant cortical folding defects ( hypogyria ) of several frontotemporal regions and the insula when compared with the responders .", "they also had widespread deficits in gyrification extending to the precuneus , angular gyrus , and lingual gyrus when compared with healthy controls ( figure 2 , \n table iii ) .", "in contrast , and similarly to what we saw for white matter , patients who subsequently responded were virtually indistinguishable from the healthy controls .", "these findings are very consistent with the evidence described earlier that only the nonresponders have reduced integrity in the white matter tracts that connect these cortical regions .", "it is also interesting that the alterations in gyrification and white matter were evident in nonresponders with both affective ( bipolar disorder or major depression with psychotic symptoms ) and nonaffective ( schizophrenia , schizophreniform disorder , schizoaffective disorder and psychosis not otherwise specified ) psychosis , suggesting that as a group , nonresponders are likely to have a more homogeneous pathophysiological process underlying psychoses than the responders .", "taken together , these findings suggest that individuals who are less likely to respond to treatment represent either a more severely affected group , or a group that has experienced a pathophysiological insult at an early neurodevelopmental stage than those who respond to treatment , possibly with a differential exposure to factors that determine axonal integrity ( genetic or molecular ) , which , in turn , influences brain connectivity and gyrification .", "although crucial to our understanding of psychosis , these group findings need to be extended if they are to inform clinical outcomes at the level of a single individual .", "structural imaging has strong potential for clinical applicability as it is : ( i ) noninvasive ; ( ii ) quick to acquire ; ( iii ) widely available ; and finally , ( iv ) cheap compared with other imaging methods such as positron emission tomography .", "therefore , what progress have we made toward its clinical applicability ? over the last few years , the application of novel approaches to the analysis of structural imaging data has made us feel closer to their potential use in clinical management . to this end ,", "machine - learning methods , such as support vector machine , have shown promise in differentiating patients or ultra - high - risk individuals from healthy controls , based on structural mri data .", "obviously , an even more interesting translational question would be : can we use structural mri data at illness onset to predict which individuals will develop worse outcomes or poorer responses to treatment ? about 2 years ago", ", our group published the first paper showing that structural brain scans at the first episode could be used to predict , with significant accuracy , outcomes at 6 years . in this study", ", we found that the mri scan obtained when patients first presented to services could be used to predict which patients developed a continuous nonremitting illness course ; distinguishing them from both healthy controls ( sensitivity , 71 ; specificity , 61 ) and from patients who had an episodic , more benign illness ( sensitivity , 71 ; specificity , 68 ) .", "consistent with our recent data , discussed in the previous section , we were not able to distinguish the patients who went on to have an episodic course from the healthy controls .", "other papers have since confirmed , in different clinical populations , the potential of machine - learning approaches to discriminate individual patients in predicting the onset and subsequent severity of psychosis in ultra - high - risk individuals . while promising , these approaches need replication in larger samples of patients at the same illness stages and treated with the same pharmacological interventions , and also need validation for scans obtained using different scanners .", "these are just some of the aims of an ongoing , large , multicenter study funded by the fp7 programme of the european commission , optimise ( optimization of treatment and management of schizophrenia in europe , www.optimisetrial.eu ) . across various centers in europe", ", we are acquiring mri scans from a very large sample of patients with first - episode schizophrenia , who are then all treated with the same antipsychotic , amisulpride , for 4 weeks , when their symptomatic response is evaluated . this study will use both univariate and multivariate image analysis approaches , ie , support vector machine , to establish whether it is possible to predict the 4-week response based on the mri scan obtained at the first episode , alone or in combination with , other biological markers , including magnetic resonance spectroscopy .", "we know that the outcome and response to available treatments for schizophrenia , and for psychosis in general , is heterogeneous .", "still , we do not have strong biological markers that could help us disentangle this heterogeneity and be useful in clinical practice .", "there are , however , structural brain alterations that , if refined , have potential for use early in the stratification of patients with psychosis . for this to be achieved", "for example , validation with very large datasets could help establish a reference group ( a databank ) , which clinical imaging centers could access for an automated classification of their patients ' mris in order to obtain an estimate of the likelihood of a specific outcome .", "for this to be achieved , it would be essential to have standardized definitions of outcomes within the clinical academic community that are relevant to clinicians , patients , and their careers .", "such a system could then be used to assign a patient to targeted assertive case management at first presentation to services , in the optimization of pharmacological treatment , or in cognitive and family interventions .", "these have all been shown to improve treatment adherence and reduce relapse rates , eventually improving outcome .", "at the same time , they could help identify those patients most likely to have a good remitting illness after their first episode , who could then avoid long - term exposure to antipsychotic medication . furthermore , the identification of gray matter and connectivity markers that characterize poor response poses the question of whether these markers could be used in patient stratification in clinical trials of new treatment strategies , or even to inform drug development of agents that can enhance and restore brain connectivity , which may elicit a better response in those who currently do not respond to available antipsychotics ." ]

[ "<S> studies that have used structural magnetic resonance imaging ( mri ) suggest that individuals with psychoses have brain alterations , particularly in frontal and temporal cortices , and in the white matter tracts that connect them . </S>", "<S> furthermore , these studies suggest that brain alterations may be particularly prominent , already at illness onset , in those individuals more likely to have poorer outcomes ( eg , higher number of hospital admissions , and poorer symptom remission , level of functioning , and response to the first treatment with antipsychotic drugs ) . </S>", "<S> the fact that , even when present , these brain alterations are subtle and distributed in nature , has limited , until now , the utility of mri in the clinical management of these disorders . more recently , mri approaches , such as machine learning , have suggested that these neuroanatomical biomarkers can be used for direct clinical benefits . </S>", "<S> for example , using support vector machine , mri data obtained at illness onset have been used to predict , with significant accuracy , whether a specific individual is likely to experience a remission of symptoms later on in the course of the illness . </S>", "<S> taken together , this evidence suggests that validated , strong neuroanatomical markers could be used not only to inform tailored intervention strategies in a single individual , but also to allow patient stratification in clinical trials for new treatments . </S>" ]

[ "Introduction", "Structural brain alterations in psychosis\n", "Brain structure and its relationship with medium- to long-term clinical and functional outcomes", "Brain structure and its relationship with early outcomes", "Moving toward using brain structure at illness onset in the individual prediction of outcome", "Conclusion" ]

[ [ "why have we not been able to use neuroimaging in the clinical management of psychoses ?", "after 30 years of using this approach , we have established that structural brain alterations are present in individuals with psychoses . while this information has advanced academic knowledge on the pathophysiology of these disorders , it has had limited utility in clinical practice .", "the main reason can be found in the nature of brain alterations in psychoses : they are subtle and spatially distributed .", "still , the time may now have come when quick and noninvasive structural neuroimaging abandons the academic solitude in which it has been operating , and brings its novel methods and findings into the clinical arena .", "this paper aims to appraise this evidence , and discuss it in the context of potential future applications of magnetic resonance imaging ( mri ) in the clinical management of psychoses .", "the first part of this review will present evidence on the presence of neuroanatomical alterations in psychoses .", "it will then discuss studies that have examined the relationship between brain alterations and various medium- to long - term clinical and functional outcomes . in the third section", ", it will discuss more recent evidence on the relationship between brain alterations and early outcomes , with a particular focus on the relationship between symptomatic responses and initial treatment with antipsychotic drugs .", "the paper will then present a discussion of more novel approaches for the analysis of mri data , such as support vector machine .", "findings from these approaches have started to suggest that mri has the potential to provide clinically meaningful information .", "finally , it will discuss the implications of having strong and valid neuroimaging markers of psychosis outcomes in developing targeted , as well as novel , treatment interventions for these disorders ." ], [ "moving from the first evidence that individuals with established schizophrenia have subtle structural brain alterations , most notably an enlargement of the lateral ventricles , we have been studying individuals earlier and earlier along the disease course .", "more than 10 years ago , the seminal work by pantelis and colleagues showed that individuals at high risk of developing psychosis , but not ill yet , already show smaller volumes of the same brain areas that have been reported as smaller in individuals with an established psychosis , namely frontal and temporal cortices .", "even more novel , however , was their report that this is particularly the case for individuals who then actually develop the illness , and that some of these alterations could be specific to the type of psychosis that these subjects developed .", "for example , they found that subjects who develop an affective type of psychosis showed reductions in the subgenual anterior cingulate cortex , an area involved in the generation of affective symptoms .", "complementary to these gray - matter changes , ultra - high - risk individuals also have white matter alterations similar to , albeit less extensive than , first - episode patients .", "these include poorer integrity of the major associative fibers that connect fronto - parieto - temporal ( superior longitudinal fasciculus ) and fronto - parieto - occipital ( inferior fronto - occipital fasciculus ) regions , commissural fibers ( corpus callosum ) , and cortico - subcortical pathways ( corona radiata , corticospinal tract , and corticopontine tract ) .", "these findings , integrated with evidence from many reviews and meta - analyses , suggest that brain alterations become more extensive at the time of a first full psychotic episode , and then possibly even more marked over the years , when the illness becomes established .", "these are indeed two main factors that have limited our ability to use this information about brain alterations in the clinical management of the individual patient .", "therefore , many studies have tried to establish whether these distributed brain changes are more likely to occur in individuals with a more severe illness course or a worse clinical and functional outcome .", "for example , a poorer clinical outcome has been variously defined as response to early treatment , number of subsequent episodes , severity of symptoms , hospitalizations , or duration of remissions ; while functional outcome has been defined as the ability to live independently , maintain employment , or be in a relationship .", "nevertheless , many studies have tried to establish if structural brain alterations are related to illness characteristics by comparing brain structure in patients and healthy controls and then evaluating the relationship with outcomes at a single time point ; or conducting repeated evaluations of brain structure , and then investigating the relationship between longitudinal brain changes and outcomes .", "most of these studies have evaluated the relationship with medium to long - term outcomes , and only more recently has the attention shifted to the evaluation of the relationship between brain alterations and early outcomes such as response to the first treatment ." ], [ "studies that have evaluated brain structure at the time of illness onset and again following treatment , have often reported smaller volumes of gray matter in association with indicators of subsequent poorer clinical outcomes ( eg , higher number of admissions or worse symptom severity ) , and with worse social or occupational outcomes .", "for example , smaller volumes of prefrontal areas have been found to predict poorer functioning ( defined as a combination of social , occupational , and psychological functioning ) 1 year later in a small sample of first - episode psychosis patients . similarly , in a sample of patients with schizophrenia , drawn from an unselected general population sample , jskelinen and colleagues found that individuals showing higher density ( an indirect measure of volume ) of frontal and limbic areas had an overall better clinical ( defined as a lower number of hospitalizations ) and functional ( defined as not being on a disability pension ) outcome in the subsequent 16 years . wassink and colleagues reported an association between smaller volumes of another brain area , the cerebellum , and longer negative and psychotic symptom duration , as well as psychosocial impairment ( eg , quality of relationships , sexual activity , enjoyment of recreation , and work performance ) in individuals with schizophrenia spectrum disorders after a 7-year follow - up .", "still , others have not found an association between brain volumes at onset and subsequent outcomes .", "for example , the prospective study by van ilaren and colleagues found no relationship between brain volume measurements and symptom severity , level of functioning ( defined as the ability to maintain a variety of social roles ) , need for care , and illness course after 2 years .", "mitelman and colleagues examined white matter integrity using diffusion tensor imaging ( dti ) , in a group of 104 patients with an average 4-year schizophrenia duration .", "they classified them as having a poor or good outcome , based on both symptom severity and level of functioning at this time point .", "they found that although both patient groups showed reduced overall white matter integrity of the prefrontal and temporal areas ( as a measure of fractional anisotropy [ fa ] ) , these alterations were more extensive in patients with a poor outcome than in those with a good outcome .", "longitudinal studies that have evaluated brain structure at multiple time points after illness onset can help clarify if brain alterations , evident at illness onset , progress differently in individuals who then developed a poorer outcome . for example , a larger decrease in the volume of the lingual gyrus , insula , and cerebellum has been reported in patients with a worse functional outcome ( a combination of social , occupational , and psychological functioning ) 4 years after the first psychotic episode . with a similar duration of follow - up , van ilaren and colleagues also reported a greater gray matter volume reduction , and a greater thinning of temporal and frontal cortices in individuals who had poorer outcomes ( including a higher number of hospitalizations and a lower level of social and personal functioning ) 5 years after illness onset .", "similarly , in a sample of children and adolescents with early onset first - episode schizophrenia , arango and colleagues showed that more weeks of hospitalization and higher severity of negative symptoms were correlated with greater left frontal gray matter volume loss and greater cerebral spinal fluid increase , respectively .", "dynamic changes in relation to outcomes were also observed by our group in a 6-year follow - up study of hippocampal volume in first - episode psychosis patients . in this sample , we specifically found that individuals who had an increase in hippocampal volume over the first 6 years of illness were the ones who showed a less severe illness course and lower symptom severity , as well as a better functional outcome at follow - up , thus suggesting an important role for brain neuroplasticity .", "taken together , these findings suggest that brain alterations are present at illness onset and may be associated with worse medium- to long - term illness outcomes and ability to function .", "still , two important considerations need to be made in the interpretation of these findings .", "first , it is not possible to establish what the casual link is between brain structure and illness course . while brain alterations could be the substrate of a more severe illness , the more severe illness itself could affect brain plasticity by increasing exposure to substance abuse , worse living conditions , or a less stimulating environment .", "it is , therefore , possible that patients with a more severe illness are exposed to longer periods of treatment or higher doses of antipsychotics , which may themselves affect brain volumes . from a clinical perspective", ", it may be more informative to study the relationship between brain alterations at illness onset and outcome measures that occur earlier in the course of illness , which is the topic of the next section ." ], [ "if brain alterations could be used to predict , as early as possible , which patients are destined to have a poorer response to the first few weeks or months of treatment with antipsychotic medications ( the main stake for the treatment of psychosis ) , we could implement interventional strategies targeted at this specific subgroup .", "currently , we treat the first psychotic episode on a trialand - error basis , and we simply have no way of saying who will respond to the first antipsychotic drug and who might benefit from longer or different interventions . in fact , we know that only -55% of patients respond to antipsychotics in the first 12 months of treatment . yet , this early response is thought to be one of the strongest predictors of subsequent functional and clinical outcomes in psychosis .", "a pooled analysis , published in 1992 , examined the relationship between response to antipsychotics and brain measures , such as ventricular - brain ratio , sulcal prominence , third ventricles , and other brain measures .", "the authors found that the composite effect sizes were very small and did not reach statistical significance .", "however , recent studies have lent renewed support to the notion that alterations in both gray and white matter may be associated with a poorer response to antipsychotics , possibly because they used more homogeneous populations , or in some cases , better imaging acquisition approaches and more sophisticated methods of analysis .", "for example , zipursky and colleagues examined global tissue volumes in firstepisode psychosis patients , and found that subjects with a poorer response ( defined as having a lower reduction in symptom severity from baseline ) to the antipsychotic haloperidol in the first month of treatment had smaller cortical gray matter volume than those who showed symptomatic improvement .", "relatively consistent data have been reported on the relationship between response to clozapine and brain structure .", "cortical measures , such as sulcal enlargement , particularly in the prefrontal cortex , have been frequently related to poor response to clozapine in patients with chronic schizophrenia . in a double - blind , 10-week trial of clozapine and haloperidol , arango and colleagues found that a larger prefrontal gray matter volume was associated with better treatment response in clozapine - treated patients , but with a poor response in haloperidol- treated patients .", "they also found no relationship between response to these drugs and hippocampal and caudate nucleus volumes .", "the authors suggested that the presence of larger brain volumes might characterize patients who are more likely to benefit from clozapine .", "this finding is also consistent with evidence from a study in patients with treatment - resistant schizophrenia who were treated for with clozapine 6 months .", "patients with a larger baseline volume of the dorsolateral - prefrontal cortex were more likely to show improvement in their negative symptoms .", "interestingly , amelioration of positive symptoms was related to a larger temporal cortex volume , and amelioration of disorganization symptoms was inversely related to hippocampal volume .", "another temporal area has been reported as being related to early outcome the parahippocampal gyrus .", "using voxel - based morphometry , bodnar and colleagues found that the parahippocampal gyrus was significantly smaller in first - episode psychosis patients who , after 6 months of antipsychotic treatment , had not remitted , compared with those who had achieved remission .", "furthermore , by building a classification model using parahippocampal gray matter concentration , they were able to correctly classify remission status in 79% of the cases . in another study of patients with established schizophrenia treated with olanzapine or risperidone , 3-week response to these antipsychotics", "was associated with smaller volumes of other areas , such as the insula and rectal gyrus , and larger volumes of the basal ganglia .", "white matter has also been associated with treatment response , but the evidence is considerably more scant .", "response to antipsychotics was evaluated by garver and colleagues in a small sample of patients with schizophrenia after 28 days of treatment . here , somewhat counterintuitively , the 8 patients who responded to antipsychotics showed worse white matter microstructural integrity than the 5 patients who did not respond to medication . of note , individuals , in this study , had an established illness , and therefore ,", "in contrast , studies in antipsychotic naive or minimally treated patients have the advantage that any structural brain alterations would be less likely to reflect the effect of antipsychotics on brain structure , and more likely to be a marker of illness .", "luck and colleagues analyzed a sample of first - episode psychosis patients using dti , and then reassessed them at 6 months , which is when they classified them as having either a poor or good treatment outcome .", "they specifically examined three white matter tracts connecting frontal and temporal regions : the cingulum , the superior longitudinal fasciculus , and the uncinate fasciculus .", "patients with a poor outcome showed greater alterations in white matter integrity in the superior longitudinal fasciculus and uncinate than patients with a good outcome , suggesting that abnormal fronto - temporal connectivity may represent an early marker of short - term clinical outcomes .", "our group has also recently focused on the evaluation of the relationship between neuroanatomical markers in white and gray matter at the time of presentation , and response to treatment at 12 weeks .", "this is a time when all patients would have received at least one full therapeutic course of antipsychotic medication .", "we examined white matter integrity , using dti and tractbased spatial statistics , to assess fa in a large sample of patients at their first episode of any psychosis .", "we operationalized response to treatment as having achieved symptomatic remission according to the criteria of the schizophrenia working group .", "we found that already at illness onset , patients who subsequently did not respond ( n=40 ) had lower fa than healthy controls in the uncinate , cingulum , and corpus callosum .", "furthermore , they also had lower fa in these regions than patients who responded to treatment ( n=40 ; figure 1 , tables i and ii ) .", "one of the most striking findings of this work was that the responders were indistinguishable from the healthy control subjects , suggesting that the original clinical sample was biologically rather heterogeneous .", "we rescanned these subjects at the 12-week clinical evaluation , and found that the same regional differences seen at baseline between the two patient groups were also present at 12 weeks , although the differences appeared less widespread .", "furthermore , although there was some increase in fa over time , this was in the same direction in the two patient groups , with no significant time vs group interaction .", "we interpreted these findings to indicate that white matter integrity and brain connectivity are important moderators of response to antipsychotics .", "it is intriguing to speculate whether these alterations are established early on , as a neurodevelopmental deviation .", "the presence of a neurodevelopmental alteration would find further support in our gray matter findings from this sample . in these individuals", ", we performed the first 3d evaluation of cortical gyrification , a marker potentially indicative of early neurodevelopmental disturbances , in relation to subsequent treatment response .", "gyrification defects have been associated with exposure to obstetric complications and with psychotic symptoms resistant to treatment .", "interestingly , the earlier neuroimaging studies in treatment - resistant patients that were mentioned in the previous section , found that sulcal enlargement ( which could lead to hypogyria ) was associated with a poorer response to clozapine . in our samples , we found that , already at illness onset , patients who subsequently did not respond to treatment had significant cortical folding defects ( hypogyria ) of several frontotemporal regions and the insula when compared with the responders .", "they also had widespread deficits in gyrification extending to the precuneus , angular gyrus , and lingual gyrus when compared with healthy controls ( figure 2 , \n table iii ) .", "in contrast , and similarly to what we saw for white matter , patients who subsequently responded were virtually indistinguishable from the healthy controls .", "these findings are very consistent with the evidence described earlier that only the nonresponders have reduced integrity in the white matter tracts that connect these cortical regions .", "it is also interesting that the alterations in gyrification and white matter were evident in nonresponders with both affective ( bipolar disorder or major depression with psychotic symptoms ) and nonaffective ( schizophrenia , schizophreniform disorder , schizoaffective disorder and psychosis not otherwise specified ) psychosis , suggesting that as a group , nonresponders are likely to have a more homogeneous pathophysiological process underlying psychoses than the responders .", "taken together , these findings suggest that individuals who are less likely to respond to treatment represent either a more severely affected group , or a group that has experienced a pathophysiological insult at an early neurodevelopmental stage than those who respond to treatment , possibly with a differential exposure to factors that determine axonal integrity ( genetic or molecular ) , which , in turn , influences brain connectivity and gyrification .", "although crucial to our understanding of psychosis , these group findings need to be extended if they are to inform clinical outcomes at the level of a single individual ." ], [ "structural imaging has strong potential for clinical applicability as it is : ( i ) noninvasive ; ( ii ) quick to acquire ; ( iii ) widely available ; and finally , ( iv ) cheap compared with other imaging methods such as positron emission tomography .", "therefore , what progress have we made toward its clinical applicability ? over the last few years , the application of novel approaches to the analysis of structural imaging data has made us feel closer to their potential use in clinical management . to this end ,", "machine - learning methods , such as support vector machine , have shown promise in differentiating patients or ultra - high - risk individuals from healthy controls , based on structural mri data .", "obviously , an even more interesting translational question would be : can we use structural mri data at illness onset to predict which individuals will develop worse outcomes or poorer responses to treatment ? about 2 years ago", ", our group published the first paper showing that structural brain scans at the first episode could be used to predict , with significant accuracy , outcomes at 6 years . in this study", ", we found that the mri scan obtained when patients first presented to services could be used to predict which patients developed a continuous nonremitting illness course ; distinguishing them from both healthy controls ( sensitivity , 71 ; specificity , 61 ) and from patients who had an episodic , more benign illness ( sensitivity , 71 ; specificity , 68 ) .", "consistent with our recent data , discussed in the previous section , we were not able to distinguish the patients who went on to have an episodic course from the healthy controls .", "other papers have since confirmed , in different clinical populations , the potential of machine - learning approaches to discriminate individual patients in predicting the onset and subsequent severity of psychosis in ultra - high - risk individuals . while promising , these approaches need replication in larger samples of patients at the same illness stages and treated with the same pharmacological interventions , and also need validation for scans obtained using different scanners .", "these are just some of the aims of an ongoing , large , multicenter study funded by the fp7 programme of the european commission , optimise ( optimization of treatment and management of schizophrenia in europe , www.optimisetrial.eu ) . across various centers in europe", ", we are acquiring mri scans from a very large sample of patients with first - episode schizophrenia , who are then all treated with the same antipsychotic , amisulpride , for 4 weeks , when their symptomatic response is evaluated . this study will use both univariate and multivariate image analysis approaches , ie , support vector machine , to establish whether it is possible to predict the 4-week response based on the mri scan obtained at the first episode , alone or in combination with , other biological markers , including magnetic resonance spectroscopy ." ], [ "we know that the outcome and response to available treatments for schizophrenia , and for psychosis in general , is heterogeneous .", "still , we do not have strong biological markers that could help us disentangle this heterogeneity and be useful in clinical practice .", "there are , however , structural brain alterations that , if refined , have potential for use early in the stratification of patients with psychosis . for this to be achieved", "for example , validation with very large datasets could help establish a reference group ( a databank ) , which clinical imaging centers could access for an automated classification of their patients ' mris in order to obtain an estimate of the likelihood of a specific outcome .", "for this to be achieved , it would be essential to have standardized definitions of outcomes within the clinical academic community that are relevant to clinicians , patients , and their careers .", "such a system could then be used to assign a patient to targeted assertive case management at first presentation to services , in the optimization of pharmacological treatment , or in cognitive and family interventions .", "these have all been shown to improve treatment adherence and reduce relapse rates , eventually improving outcome .", "at the same time , they could help identify those patients most likely to have a good remitting illness after their first episode , who could then avoid long - term exposure to antipsychotic medication . furthermore , the identification of gray matter and connectivity markers that characterize poor response poses the question of whether these markers could be used in patient stratification in clinical trials of new treatment strategies , or even to inform drug development of agents that can enhance and restore brain connectivity , which may elicit a better response in those who currently do not respond to available antipsychotics ." ] ]

[ "the treatment of patients with multiple trauma requires a different approach to that of patients with regular trauma because they are threatened not only by the injuries themselves , but also by the metabolic disruptions that follow.1 delay in surgery , blunt trauma , extensive soft tissue damage , and combined orthopedic and vascular injuries have been associated with an increased risk of amputation , while associated nerve and bone injuries with extensive soft tissue damage are risk factors for a poor outcome.2 acute renal failure is the main cause of death in patients with war wounds and trauma of the extremities", ". it would be helpful to minimize mortality in these patients by managing shock in a timely manner and taking the decision to amputate appropriately and promptly.3 severe head injury is known to be a major determinant of mortality in patients with multiple injuries , but other injuries also contribute to the clinical outcome.4,5 different mechanisms of injury , such as motor vehicle crashes , falls , or pedestrians being struck by a motor vehicle , impart varying degrees of force and energy transfer that may impact outcomes ; this was found to predict mortality and functional impairment independently at hospital discharge.6 acute lower extremity compartment syndrome is a devastating complication that often presents silently in critically injured patients.7 patients who underwent delayed fasciotomy had twice the rate of major amputation and a three - fold higher mortality rate.8 the aim of this study was to evaluate if injuries of the extremities are associated with a higher one - month mortality rate than other types of associated trauma .", "this prospective , observational , cohort study was carried out in the regional emergency center of hospital de base after prior approval by the ethics research committee of the so jos do rio preto medical school .", "the emergency department follows a systematic pathway to provide initial assistance to accident victims using the atls ( advanced trauma life support ) protocol .", "all live accident victims treated in the emergency department from july 2004 to june 2005 were included .", "patients who were dead on arrival and not submitted to any type of inhospital resuscitation procedure were not included in the study .", "accident victims were allocated to two groups , ie , those with severe injuries to the extremities or pelvis ( abbreviated injury scale [ ais ] 34 ) and those without injuries or with minor injuries to the extremities ( ais 02 ) .", "the fisher s exact test and relative risk were used for statistical analysis , and an alpha error of 5% ( p 0.05 ) was considered statistically significant .", "a total of 3489 patients were evaluated in this study ; 3244 ( 92.98% ) did not have severe trauma of the extremities ( ais 02 ) , 34 ( 1.05% ) of whom died .", "severe injuries of the extremities ( ais 34 ) occurred in 245 ( 7.02% ) of the patients , with 13 ( 5.31% ) dying ( fisher s exact test : p = 0.001 , relative risk 5.063 , 95% confidence interval [ ci ] : 2.7079.467 , table 1 ) .", "table 2 shows the age , type of injury sustained , and time until death after trauma for the patients who died .", "of the 245 patients with ais 34 , 71 ( 28.98% ) were women and 174 ( 71.02% ) were men , with the mean age of the men being 40.1 20.5 years and of the women 60.2 23.6 years .", "this study assessed whether severe injuries of the extremities affect the overall mortality rate in accident victims .", "the death rate was found to be higher for patients with ais 34 than in those without injuries or with minor injuries to the limbs .", "thus , these data serve as a warning in respect to increased risk of death in orthopedic patients compared with general trauma patients . when the severity of specific injuries in accident victims is reported in the literature ,", "head trauma is cited as one of the main causes of death.5,6 however , there are few data in the literature about the association between death and injuries to the extremities .", "one study reported that the mortality rate in accident victims with extremity injuries was higher in pedestrians struck by motor vehicles ( 20% ) , and for those with head injuries , it was higher for motor cycle crash victims ( 16%).6 the first phase of management for these patients aims to control bleeding , by surgical intervention if necessary , and to prevent further wound contamination .", "the second phase consists of resuscitation in the intensive care unit , and the third phase aims at definitive repair of the injuries sustained.9 pelvic injuries represent a thorny and stubborn therapeutic challenge .", "rapid diagnosis and effective treatment ( damage control ) of these injuries play a key role in the patient s survival , inasmuch as the mortality of multiply injured patients with pelvic ring disruption remains high ( 20%35%).10 the preclinical management of patients with multiple trauma influences the prognosis regarding mortality and morbidity .", "diagnostic overview , protection of vital functions in the special circumstance of shock , immobilization of the spine , and treatment of isolated injuries are an essential part of preclinical management.11 the type of trauma is known to influence the mortality rate.12 in this study , all the patients received specialized pre - hospital assistance , and were treated in a regional trauma reference center . in spite of all the care given at the scene of the accident through to discharge from hospital , trauma of the extremities was a significant cause of death .", "accident victims with injuries of the extremities are at higher risk for death than those with other types of trauma ." ]

[ "<S> background : the aim of this study was to evaluate one - month hospital mortality in victims with injuries of the extremities.methods:all accident victims admitted to the hospital de base in so jos do rio preto , brazil , during the period from july 2004 to june 2005 , were evaluated in an observational study . </S>", "<S> patients were classified using the abbreviated injury scale ( ais ) . </S>", "<S> patients with severe injuries of the extremities ( ais 34 ) were compared with those without injuries or with minor extremity injuries ( ais 02).results : a total of 3489 accident victims were evaluated ; 3244 ( 92.98% ) did not suffer injuries or had minor injuries of the extremities ( ais 02 ) and 245 ( 7.02% ) had severe injuries ( ais 34 ) . </S>", "<S> of the 245 patients with ais 34 extremity injuries , 13 ( 5.31% ) patients died , and of those without severe injuries to the extremities , 34 ( 1.05% ) died ( fisher s exact test p = 0.0000 , relative risk 5.063 , 95% confidence interval [ ci ] : 2.7079.467).conclusion : patients with injuries of the extremities are at greater risk of death than accident victims with other types of trauma . </S>" ]

[ "Introduction", "Methods", "Results", "Discussion", "Conclusion" ]

[ [ "the treatment of patients with multiple trauma requires a different approach to that of patients with regular trauma because they are threatened not only by the injuries themselves , but also by the metabolic disruptions that follow.1 delay in surgery , blunt trauma , extensive soft tissue damage , and combined orthopedic and vascular injuries have been associated with an increased risk of amputation , while associated nerve and bone injuries with extensive soft tissue damage are risk factors for a poor outcome.2 acute renal failure is the main cause of death in patients with war wounds and trauma of the extremities", ". it would be helpful to minimize mortality in these patients by managing shock in a timely manner and taking the decision to amputate appropriately and promptly.3 severe head injury is known to be a major determinant of mortality in patients with multiple injuries , but other injuries also contribute to the clinical outcome.4,5 different mechanisms of injury , such as motor vehicle crashes , falls , or pedestrians being struck by a motor vehicle , impart varying degrees of force and energy transfer that may impact outcomes ; this was found to predict mortality and functional impairment independently at hospital discharge.6 acute lower extremity compartment syndrome is a devastating complication that often presents silently in critically injured patients.7 patients who underwent delayed fasciotomy had twice the rate of major amputation and a three - fold higher mortality rate.8 the aim of this study was to evaluate if injuries of the extremities are associated with a higher one - month mortality rate than other types of associated trauma ." ], [ "this prospective , observational , cohort study was carried out in the regional emergency center of hospital de base after prior approval by the ethics research committee of the so jos do rio preto medical school .", "the emergency department follows a systematic pathway to provide initial assistance to accident victims using the atls ( advanced trauma life support ) protocol .", "all live accident victims treated in the emergency department from july 2004 to june 2005 were included .", "patients who were dead on arrival and not submitted to any type of inhospital resuscitation procedure were not included in the study .", "accident victims were allocated to two groups , ie , those with severe injuries to the extremities or pelvis ( abbreviated injury scale [ ais ] 34 ) and those without injuries or with minor injuries to the extremities ( ais 02 ) .", "the fisher s exact test and relative risk were used for statistical analysis , and an alpha error of 5% ( p 0.05 ) was considered statistically significant ." ], [ "a total of 3489 patients were evaluated in this study ; 3244 ( 92.98% ) did not have severe trauma of the extremities ( ais 02 ) , 34 ( 1.05% ) of whom died .", "severe injuries of the extremities ( ais 34 ) occurred in 245 ( 7.02% ) of the patients , with 13 ( 5.31% ) dying ( fisher s exact test : p = 0.001 , relative risk 5.063 , 95% confidence interval [ ci ] : 2.7079.467 , table 1 ) .", "table 2 shows the age , type of injury sustained , and time until death after trauma for the patients who died .", "of the 245 patients with ais 34 , 71 ( 28.98% ) were women and 174 ( 71.02% ) were men , with the mean age of the men being 40.1 20.5 years and of the women 60.2 23.6 years ." ], [ "this study assessed whether severe injuries of the extremities affect the overall mortality rate in accident victims .", "the death rate was found to be higher for patients with ais 34 than in those without injuries or with minor injuries to the limbs .", "thus , these data serve as a warning in respect to increased risk of death in orthopedic patients compared with general trauma patients . when the severity of specific injuries in accident victims is reported in the literature ,", "head trauma is cited as one of the main causes of death.5,6 however , there are few data in the literature about the association between death and injuries to the extremities .", "one study reported that the mortality rate in accident victims with extremity injuries was higher in pedestrians struck by motor vehicles ( 20% ) , and for those with head injuries , it was higher for motor cycle crash victims ( 16%).6 the first phase of management for these patients aims to control bleeding , by surgical intervention if necessary , and to prevent further wound contamination .", "the second phase consists of resuscitation in the intensive care unit , and the third phase aims at definitive repair of the injuries sustained.9 pelvic injuries represent a thorny and stubborn therapeutic challenge .", "rapid diagnosis and effective treatment ( damage control ) of these injuries play a key role in the patient s survival , inasmuch as the mortality of multiply injured patients with pelvic ring disruption remains high ( 20%35%).10 the preclinical management of patients with multiple trauma influences the prognosis regarding mortality and morbidity .", "diagnostic overview , protection of vital functions in the special circumstance of shock , immobilization of the spine , and treatment of isolated injuries are an essential part of preclinical management.11 the type of trauma is known to influence the mortality rate.12 in this study , all the patients received specialized pre - hospital assistance , and were treated in a regional trauma reference center . in spite of all the care given at the scene of the accident through to discharge from hospital , trauma of the extremities was a significant cause of death ." ], [ "accident victims with injuries of the extremities are at higher risk for death than those with other types of trauma ." ] ]

[ "voltage - gated ca channels are the signature feature of excitable cells , transducing electrical activity into increased intracellular [ ca ] that mediates specific cellular effects such as muscle contraction , hormone secretion , and release of neurotransmitters .", "thus , many regulatory mechanisms have evolved to fine tune ca channel activity and the resultant ca influx , mostly by protein ", "protein interactions with , or posttranslational modifications of , the pore - forming 1 subunit . some are rapid , such as ca - dependent inactivation of l - type ( cav1.2 ) channels ( budde et al . , 2002 )", "; others occur after the activation of signaling pathways , such as pka potentiation of cav1.2 channels or g protein inhibition of n - type ( cav2.2 ) channels ( catterall , 2000 ) .", "in contrast , mechanisms that result in finely graded responses to changes in the cellular environment developing over longer time scales have not been well described .", "rgk gtpases ( rad , rem , rem2 , gem / kir ) , the most recently characterized group within the ras family of gtp - binding proteins ( reynet and kahn , 1993 ; maguire et al . , 1994 ; finlin and andres , 1997 ; finlin et al . , 2000", ") , have received special attention because they are potent inhibitors of ca channels and candidates for ca channel regulators under transcriptional control that can therefore integrate the influence of multiple extracellular signals .", "experiments in a variety of cell types have shown a drastic reduction of peak current amplitude for multiple ca channels after expression of gem / kir ( beguin et al . , 2001 , 2005b ; murata et al . , 2004 ;", "ward et al . , 2004 ) , rem , rad ( finlin et al . , 2003 ; crump et al . ,", "2006 ) , and rem2 ( chen et al . , 2005 ; finlin et al . , 2005 ) . among ras family members ,", "rgks differ by having extended variable n - terminal regions and conserved c - terminal extensions lacking the caax motif for fatty acylation , and containing binding motifs for calmodulin and 14 - 3 - 3 proteins ( kelly , 2005 ) .", "individual rgks have nonoverlapping patterns of expression , and are transcriptionally induced and repressed by different factors .", "for example , gem and rem2 transcription has been reported to be stimulated by glucose in insulin - secreting pancreatic cells but follow a different time course ( ohsugi et al . , 2004 ; finlin et al . , 2005 ) ; rad is overexpressed in muscle of type ii diabetics ( reynet and kahn , 1993 ) , and rem transcription is repressed by lipopolysaccharide exposure ( finlin and andres , 1997 ) .", "gem inhibits the rho / rhoa kinase pathway ( ward et al . , 2002 ) and induces neuroblastoma morphological and ganglionic differentiation ( leone et al . , 2001 ) .", "expression of both gem and rem2 has been shown to decrease glucose - stimulated insulin secretion ( beguin et al . , 2001 ; finlin et al . , 2005 ) .", "a two - hybrid experiment identified ca channel subunits as a gem - interacting protein in the insulin - secreting min6 cell line ( beguin et al . , 2001 ) .", "since subunits have been implicated in trafficking 1 subunits to the plasma membrane , this led to the hypothesis that rgks prevent subunits from interacting with 1 subunits , thereby preventing membrane targeting and resulting in reduced channels at the cell surface ( beguin et al .", "a number of recent studies suggest instead that rgks inhibit channels already resident at the cell surface ( chen et al . , 2005 ; finlin et al . , 2005", ") . moreover , though it is their potency that has earned them interest , it is a more subtle and tunable response that likely has physiological ramifications . it has already been established that changes in ca channel currents less severe than the near complete reduction observed when rgks are expressed in heterologous systems lead to drastic pathophysiological consequences ( splawski et al . , 2004 ) .", "it is difficult to understand how rgk expression could result in a finely graded response . in this study , we provide new insights into how gem and rem2 regulate ca channels . exploiting the xenopus oocyte system to control levels of expression ( canti et al . , 2001 )", ", we found that gem and rem2 drive a dose - dependent inhibition of ca currents .", "rem2 , but not gem , also modulated both the kinetics of channel activation and inactivation in a manner that was dependent on subunit interaction with the 1 interaction domain ( aid ) .", "together , these results suggest that specific rgks contribute to the fine tuning of ca influx by different mechanisms .", "constructs for 1c ( pcardhe ) , 2 , and the 1c c - terminal deletion ( amino acids 16702171 ) , and the gst i - ii loop have been previously reported ( zhlke et al . , 2000 ;", "nm_000725 ) was cloned into the pgem - he oocyte expression vector using standard molecular biology techniques .", "bc018219 ) was obtained as an est and cloned into the pcs2 + oocyte expression vector ( gift from d. mckinnon , state university of new york , stony brook , ny ) .", "rem2 full - length ( ay916790 ) , a gift from d. andres ( university of kentucky , lexington , ky ) , was digested out of the original pcdna3.1 vector and ligated into compatible sites in pcs2 + . the 1c n - terminal deletion ( amino acids 2139 )", "the 1c3 concatemer included amino acids 12134 from 1c and the entire 3 with a valine linker between them .", "the mutant 1c and corresponding concatemer included mutations y467s and w470a created by quikchange ( stratagene ) .", "the kchip2b clone was a gift from p. pfaffinger ( baylor college of medicine , houston , tx ) . in vitro crna transcription and microinjection into xenopus oocytes", "the following amounts of crna were injected : 1c ( 1 ng ) , 2 ( 1 ng ) , 3 ( 0.22 ng ) .", "two - electrode voltage clamp recordings were performed as previously described ( kim et al . , 2004 ) .", "during recordings , oocytes were constantly superfused with a solution containing 40 mm ba(oh)2 ( or 40 mm ca(oh)2 in experiments recording ca currents ) , 50 mm naoh , 1 mm koh , and 10 mm hepes ( adjusted to ph 7.4 with methanesulfonic acid ) .", "recordings were performed with a standard two - electrode voltage clamp configuration using an oocyte clamp oc-725c amplifier ( warner instrument corp . ) connected through a digidata 1322a a / d interface ( axon instruments , inc . ) to a personal computer .", "ionic currents were filtered at 1 khz by an integral 4 pole bessel filter and sampled 10 khz and analyzed with clampfit 9.2 .", "steady - state inactivation was analyzed with a two - pulse protocol in which a 5-s conditioning pulse ( p1 ) from 60 mv to + 50 mv was followed by a 100-ms test pulse ( p2 ) at + 10 mv .", "normalized p2 values were fitted with a boltzmann equation ( i / ipeak = ( 1 io)/[1 + exp((v v1/2)/k ) ] + io ) .", "activation time constants were estimated by fitting the activating component of the current trace to the following equation : i = io + afastexp(t/fast ) + aslowexp(t/slow ) .", "bursts of pancreatic cell action potentials were simulated by a 5-s depolarization to 40 mv from 70 mv followed by a series of 26 100-ms voltage - clamp depolarizations between 40 and 0 mv at 5 hz ( kanno et al . , 2002 ) .", "all values are given as means sem , with statistical comparisons performed with a student 's t test .", "protein expression / gst pull - down assays were performed as previously described ( maltez et al . , 2005 ) .", "oocytes were injected with either 1,000 pg of gem crna , 1,000 pg gem crna with 1,000 pg crna cam , or 4,000 pg of gem crna .", "oocytes were incubated at 17c for 24 h , lysed in ice - cold oocyte extraction buffer ( 20 mm hepes , 5 mm kcl , 1.5 mm mgcl2 , 1 mm edta , 10% glycerol , 0.1% np-40 , and roche protease inhibitor tablets ) , and then solubilized in sds .", "constructs for 1c ( pcardhe ) , 2 , and the 1c c - terminal deletion ( amino acids 16702171 ) , and the gst i - ii loop have been previously reported ( zhlke et al . , 2000 ;", "nm_000725 ) was cloned into the pgem - he oocyte expression vector using standard molecular biology techniques .", "bc018219 ) was obtained as an est and cloned into the pcs2 + oocyte expression vector ( gift from d. mckinnon , state university of new york , stony brook , ny ) .", "rem2 full - length ( ay916790 ) , a gift from d. andres ( university of kentucky , lexington , ky ) , was digested out of the original pcdna3.1 vector and ligated into compatible sites in pcs2 + . the 1c n - terminal deletion ( amino acids 2139 )", "the 1c3 concatemer included amino acids 12134 from 1c and the entire 3 with a valine linker between them .", "the mutant 1c and corresponding concatemer included mutations y467s and w470a created by quikchange ( stratagene ) .", "the kchip2b clone was a gift from p. pfaffinger ( baylor college of medicine , houston , tx ) .", "in vitro crna transcription and microinjection into xenopus oocytes has been previously reported ( kim et al . , 2004 ) .", "the following amounts of crna were injected : 1c ( 1 ng ) , 2 ( 1 ng ) , 3 ( 0.22 ng ) .", "two - electrode voltage clamp recordings were performed as previously described ( kim et al . , 2004 ) .", "during recordings , oocytes were constantly superfused with a solution containing 40 mm ba(oh)2 ( or 40 mm ca(oh)2 in experiments recording ca currents ) , 50 mm naoh , 1 mm koh , and 10 mm hepes ( adjusted to ph 7.4 with methanesulfonic acid ) .", "recordings were performed with a standard two - electrode voltage clamp configuration using an oocyte clamp oc-725c amplifier ( warner instrument corp . ) connected through a digidata 1322a a / d interface ( axon instruments , inc . ) to a personal computer .", "ionic currents were filtered at 1 khz by an integral 4 pole bessel filter and sampled 10 khz and analyzed with clampfit 9.2 .", "steady - state inactivation was analyzed with a two - pulse protocol in which a 5-s conditioning pulse ( p1 ) from 60 mv to + 50 mv was followed by a 100-ms test pulse ( p2 ) at + 10 mv .", "normalized p2 values were fitted with a boltzmann equation ( i / ipeak = ( 1 io)/[1 + exp((v v1/2)/k ) ] + io ) .", "activation time constants were estimated by fitting the activating component of the current trace to the following equation : i = io + afastexp(t/fast ) + aslowexp(t/slow ) .", "bursts of pancreatic cell action potentials were simulated by a 5-s depolarization to 40 mv from 70 mv followed by a series of 26 100-ms voltage - clamp depolarizations between 40 and 0 mv at 5 hz ( kanno et al . , 2002 ) .", "all values are given as means sem , with statistical comparisons performed with a student 's t test .", "protein expression / gst pull - down assays were performed as previously described ( maltez et al . , 2005 ) .", "oocytes were injected with either 1,000 pg of gem crna , 1,000 pg gem crna with 1,000 pg crna cam , or 4,000 pg of gem crna .", "oocytes were incubated at 17c for 24 h , lysed in ice - cold oocyte extraction buffer ( 20 mm hepes , 5 mm kcl , 1.5 mm mgcl2 , 1 mm edta , 10% glycerol , 0.1% np-40 , and roche protease inhibitor tablets ) , and then solubilized in sds .", "to explore the mechanisms by which rgks inhibit ca channel currents , we expressed cav1.2 channels ( 1c , 2 , and 3 ) with gem , rem , or rem2 in xenopus oocytes and recorded the resulting currents by two - electrode voltage clamp . as observed previously , expression of any of these rgks drastically reduced the iba peak current amplitude ( fig .", "1 d ) . to distinguish among the possible models by which rgks inhibit ca currents and to explore the physiological implications , we took advantage of the xenopus oocyte system , in which it has been shown that expression levels of proteins can be accurately titrated in a monotonic fashion ( canti et al . , 2001 ) , in order to test whether inhibition were dose dependent .", "b demonstrates a monotonic increase in gem protein with increasing amounts of gem crna injected over the range that we studied .", "moreover , coinjection of crna for another unrelated protein ( calmodulin ) did not affect gem protein levels , suggesting that crna amounts in this range did not exceed the protein synthesis capacity in oocytes . with this confirmation ,", "1 c shows that gem , rem , and rem2 inhibit iba peak current amplitude at 10 mv in a dose - dependent manner .", "this was not a nonspecific effect of increasing the amounts of crna injection ; coexpression of crna for kchip2b , a protein that modulates k currents and is of similar mass to the rgks ( an et al . , 2000 ) , did not alter cav1.2 current amplitude .", "examination of individual current traces also revealed differences in the mechanisms by which gem and rem2 affect cav1.2 currents .", "while coexpression of all three rgks decreased current amplitude , coexpression of rem2 also appeared to affect kinetics of activation and inactivation ( fig .", "effects of gem and rem appeared similar , so we focused on gem in further studies .", "( a ) normalized i - v relationships of iba from oocytes injected with 1c/3/2 without or with gem ( 130 pg ) or rem2 ( 936 pg ) .", "( b ) immunoblots with anti - gem antibody . on left is shown gst and gst - gem , demonstrating specificity of the antibody .", "the right panel shows an increase in the amount of gem protein detected with an increasing amount ( as indicated ) of crna injected per oocyte .", "gem is indicated by an arrow ; a nonspecific band seen even without injection of gem crna is shown by an asterisk .", "( c ) dose response of gem- , rem- , or rem2-mediated inhibition of iba .", "( d ) exemplar traces of cav1.2 channels expressed without a rgk , with gem ( 20 pg ) , with rem ( 100 pg ) , or with rem2 ( 468 pg ) .", ". coexpression of gem appeared to inhibit cav1.2 currents by a direct scaling effect , while rem2 altered the kinetics of activation and inactivation ( fig .", "these different effects upon channel activation and inactivation are better appreciated by examining scaled traces from cav1.2 channels compared with traces from cav1.2 channels coexpressed with gem ( 26 pg ) or rem2 ( 936 pg ) .", "quantitative analysis of the kinetics of inactivation for cav1.2 channels coexpressed with rem2 was complicated because the decay phases of currents were contaminated by overlapping slow activation .", "thus , we analyzed steady - state inactivation with a two - pulse protocol in which the normalized residual peak current during a + 10-mv test pulse ( p2 ) was plotted against the voltage of a 5-s inactivating prepulse ( p1 ) and found that both gem ( 26 pg ) and rem2 ( 936 pg ) affected steady - state inactivation ( fig .", "rem2 mainly affected the pedestal from 0.23 0.02 to 0.12 0.02 , ( n = 1112 , p < 0.0001 ) but gem reduced the slope to 14.1 0.6 from 9.0 0.8", "( a ) top panels show exemplar current traces during a 2-s test pulse at + 10 mv for gem and rem2 at the indicated doses .", "bars , 1 a . the bottom panels show scaled exemplar traces of 1c/3/2 ( gray ) coinjected with gem ( 65 pg ) or rem2 ( 936 pg ) ( black ) during a 2-s test pulse to + 10 mv .", "( b ) steady - state inactivation for 1c/3/2 without or with gem ( 26 pg ) or rem2 ( 936 pg ) .", "n = 1012 ( c e ) dose response of rem2-mediated effects upon kinetics of activation ( fast , fraction aslow , and slow , respectively , at the indicated test potentials see legend in c and with the amounts of crna injected as indicated on the x - axis ) , n = 714 .", "( f ) scaled exemplar traces of activation phases at + 10 mv for 1c/3/2 coexpressed with the indicated rem2 doses .", "( g ) ca currents recorded during a simulated burst of action potentials in a pancreatic cell ( see materials and methods ) with no rgk or rem2 ( 468 pg ) .", "bars : 200 na , 1 s. rem2 also affected cav1.2 channel activation in a dose - dependent and voltage - dependent manner ( fig .", "2 c , 0 pg ) , the activating phase for currents elicited with test potentials from 0 to + 30 mv was best fitted with two exponentials ( i = afaste + aslowe + c ) and the dominant component was fast ( fraction afast was 8090% at all test potentials ; fig .", "instead , the slower activation of cav1.2 channels induced by higher doses of coexpressed rem2 could be explained by two effects upon slow : slow became longer with increasing doses of rem2 and the fraction of aslow increased .", "these effects were most prominent at test potentials near the peak of the i - v curve ( fig .", "the overall consequences of these dose - dependent effects upon activation induced by rem2 are illustrated by the overlaid traces in fig .", "2 f. since these effects upon channel kinetics suggested that rem2 could alter the temporal nature of channel responsiveness during action potentials , we tested whether rem2 altered simulated ca - dependent action potentials that underlie the rhythmic bursting of electrical activity essential for insulin secretion in pancreatic islet cells ( mears , 2004 ) .", "2 g shows the resultant ca currents from cav1.2 channels ( 1c , 3 , and 2 ) expressed in xenopus oocytes during 26 successive 100-ms depolarizations from 40 to 0 mv ( at 5 hz ) , a protocol that simulates the bursting activity during insulin secretion and has been used in isolated cells ( kanno et al . , 2002 ) .", "in the absence of rgks , the peak ca current amplitude decreased sequentially during the 26 successive depolarizations so that the current amplitude during the last depolarization was 46 4% ( n = 8) of the peak current during the first depolarization . not only were the current amplitudes smaller with coexpression of rem2 ( consistent with the effects of rem2 presented above ) , but rem2 accelerated the decrease in amplitude during the successive depolarizations so that the current amplitude during the last depolarization was 12 7% ( n = 7 ; p = 0.001 compared with no rgk ) of the peak current during the first depolarization .", "this accelerated decrement is likely due to the increased rate of inactivation observed in the presence of rem2 ( fig .", "coexpression of gem led to decreased current ( compared with no rgk ) , but did not affect the rate of decrement of the current amplitude ( unpublished data ) , consistent with the lack of effect upon channel kinetics shown above .", "thus , the presence of rem2 would decrease the integrated ca influx and alter its kinetics during a burst of action potentials such as those that drive insulin secretion in pancreatic islet cells .", "we next tested whether the 1c n or c termini were necessary for these effects by testing whether deletion constructs ( 2 - 139 in the n - terminus or 1669 - 2171 in the c terminus ) were modulated by gem or rem2 .", "these experiments were prompted in part by a recent report suggesting that rem , another rgk member , required the 1c c terminus , particularly the pka phosphorylation site at ser , for inhibition of ca channel currents ( crump et al .", "in contrast , we found that gem ( 250 pg ) and rem2 ( 936 pg ) consistently reduced peak current amplitude for channels containing intact or truncated 1c subunits ( fig .", "3 , a and b ) . rem2 also maintained its effects upon kinetics of activation and inactivation in the truncated channels , as shown in the scaled exemplar current traces ( fig .", "the ca channel subunit , but not the 1c n and c termini , are required for gem- or rem2-mediated effects .", "( a and b ) normalized iba for the indicated combinations of 1c ( wt ) , the n - terminal truncation ( n ) , or the c - terminal truncation ( c ) , with or with gem or rem2 , as indicated .", "all combinations were coexpressed with 3 and 2. scaled exemplar current for each pair with ( black ) or without ( gray ) gem or rem2 are shown on right .", "( c and d ) normalized iba for the indicated combinations of c and 2 with or without 3 and gem or rem2 , as indicated . for the pair without 3 , scaled exemplar currents with ( black ) or without ( gray ) gem or rem2 are shown on right .", "we also used the 1c c - terminal deletion to analyze whether subunits were necessary for gem or rem2 modulation .", "truncation of the 1c c terminus produces increased current amplitude in the absence of subunits ( wei et al .", ", 1994 ; klckner et al . , 1995 ; gerhardstein et al . , 2000 ; ivanina et al . , 2000 ) , thereby providing a larger baseline current from which to assess rgk inhibition .", "3 ( c and d ) shows that , in the absence of a coexpressed subunit , neither gem nor rem2 inhibited channel currents .", "further , rem2 modulated neither activation nor inactivation in the absence of a coexpressed subunit .", "these results show that rgk modulation is independent of the 1c n or c terminus , but requires subunits .", "recent models have suggested that rgks inhibit ca channels by direct competition with subunits for this high affinity interaction site on the 1 subunit ( sasaki et al . , 2005 ) . although subunit interaction with the aid is not required for all aspects of subunit modulation of ca channel function ( maltez et al . , 2005 ) ,", "aid mutations that block subunit binding render channel currents too small to accurately assess an inhibitory effect of rgks ( singer et al . , 1991 ) .", "building upon a previous hypothesis that the aid interaction serves mainly to secure subunits to 1 so as to allow other , lower affinity regulatory interactions , we covalently tethered subunits directly to the 1c c terminus after amino acid 2134 ( 1c3 ) .", "currents from this concatemer ( expressed with 2 ) were similar to currents from untethered channels ( 1c + 3 with 2 ) , except that the peak of the i - v curve shifted to more depolarized potentials ( fig .", "4 a ) as previously reported ( dalton et al . , 2005 ) . to prevent 3 interaction with aid we made an 1c with two mutations in aid , y467s and w470a ( 1c ) ,", "either of which has been shown to singly disrupt subunit interaction and block subunit modulation ( van petegem et al .", "confirmation of abolished subunit binding to the mutant 1c i - ii loop is shown in a gst pull - down assay ( fig . 4 b ) .", "current amplitudes from an 1c subunit with the same mutations coexpressed with 3 ( 1c + 3 ) were very small ( fig .", "4 , a , c , and d ) and indistinguishable from currents from an 1c expressed without 3 ( not depicted ) , which is consistent with an absence of 3 interaction .", "when 3 was tethered to the aid mutant ( 1c3 ) however , the resulting current amplitude was significantly larger than from 1c coexpressed with untethered 3 ( fig .", "4 , a , c , and d ) since the requirement for -aid interaction could be at least partially circumvented by tethering the subunit to 1 , this supported the hypothesis that other interactions between 1 and are important for -dependent modulation .", "having generated an 1c subunit that was modulated by a subunit independent of its aid interaction , we therefore could test whether -aid was required for rgk inhibition .", "currents from channels containing 1c3 coexpressed with either gem ( 250 pg ) or rem2 ( 628 pg ) crna showed that they both produced a similar reduction of current as for 1c + 3 ( compare fig .", "4 , d and e ) . although coexpression of gem or rem2 also reduced currents from channels containing 1c3 , the reduction was much more modest ( fig .", "4 , e and f ) . moreover , although the effects of rem2 on activation and inactivation were preserved when coexpressed with 1c3 , rem2 did not affect activation or inactivation when coexpressed with 1c3 ( fig . 4 e ) .", "these results show that rem2-mediated effects upon activation and inactivation require the -aid interaction while gem- or rem2-induced inhibition of current amplitude does not .", "interaction between the ca channel subunits and the aid influence gem- and rem2-mediated effects . ( a ) normalized i - v relationships of iba from oocytes injected with the indicated constructs or combinations , all coinjected with 2. n = 3134 .", "( b ) a hisx6 immunoblot of a pull - down experiment for purified 2 sh3-gk core ( maltez et al . , 2005 ) using a gst-1c i - ii or i - ii mutant loop .", "( d f ) normalized iba for the indicated concatemers or combinations ( all expressed with 2 ) , with or without gem or rem2 . scaled exemplar traces are shown on right .", "bars : 1 s ( d and f ) and 50 ms ( e ) .", "heterologous overexpression of several rgks in a variety of systems produces almost complete inhibition of coexpressed or endogenous ca channel current ( beguin et al . , 2001 , 2005b ; finlin et al . , 2003 ; murata et al . , 2004 ;", "ward et al . , 2004 ; chen et al . , 2005 ; crump et al . ,", "2006 ) and rgk inhibition of ca influx has been proposed as a mechanism for physiologic control of ca channel activity for responses such as regulation of insulin secretion from pancreatic islet cells ( beguin et al . , 2001 ; finlin et al . , 2005 ) or control of cardiac rhythm ( murata et al . , 2004 ) .", "lacking a detailed molecular understanding of how rgks could fine tune ca influx eclipses how this mode of regulation could shape a specific physiological response ; for example , up - regulation of an rgk ( ohsugi et al . , 2004 ; finlin et al . , 2005 ) and subsequent channel inhibition ( beguin et al .", ", 2001 ; finlin et al . , 2005 ) after glucose stimulation might protect islet cells from excessive ca influx during chronic hyperglycemia , but how would cells retain their ability to secrete insulin with the almost complete loss of ca currents observed in previous overexpression experiments ? in this study we describe two unexpected means by which rgks regulate ca channels , providing a framework for understanding how a wide array of ca signaling events can be precisely regulated .", "exploiting the xenopus oocyte system to control protein expression levels , we found that rgk inhibition of ca channel current was dose dependent .", "the mechanism(s ) by which rgks lead to current amplitude reduction , previously a source of controversy , is not revealed by these experiments ; direct effects upon channels resident at the cell surface ( chen et al .", ", 2005 ; finlin et al . , 2005 ) or effects upon channel trafficking / assembly ( beguin et al . , 2001 ) can not be easily distinguished by the experiments presented here .", "the significant finding , however , is that the suppression of current depends upon the level of rgk expression , thus promising a predictable and titratable attenuation of ca current by specific rgks .", "thus , our results suggest that the high glucose - stimulated induction of a specific rgk and the resultant down - regulation of the ca current in pancreatic islet cells contribute to a protective mechanism against the detrimental effects of an enhanced ca signal resulting from chronic glucose exposure ( juntti - berggren et al . , 1993 ) ; conversely , a reduction of rgk protein in response to elevated glucose would serve to compensate hyperglycemia acutely by increasing ca channel activity and consequent insulin secretion .", "furthermore we found that rem2 , but not gem or rem , also altered cav1.2 gating kinetics , slowing activation and enhancing inactivation .", "these effects upon kinetics suggest that rem2 must act at least in part upon channels resident at the cell surface .", "not only could rem2 decrease peak ca influx , it could also impart profound influence on the ca - dependent action potentials that underlie the rhythmic bursting of electrical activity essential for insulin secretion in pancreatic islet cells ( mears , 2004 ) , by shaping the temporal nature of channel responsiveness as suggested by our experiments in fig . 2 g.", "although the molecular basis for the effects of rem2 upon channel kinetics is not clear , we speculate that its extended n terminus and c terminus that flank the ras core may be responsible ; neither extension is found in rem or gem ( fig .", "the kinetic actions of rem2 could result from an additional contact between rem2 and the channel within these nonconserved regions .", "clustal w ( 1.83 ) multiple sequence alignment ( thompson et al . , 1994 )", "the gray - boxed areas highlight the extended n and c termini in rem2 that flank the conserved ras - like core .", "these different modes of regulation by gem and rem2 , in conjunction with their differential temporal patterns of expression , may yield an integrated response to oppose effects of hyperglycemia .", "gem , up - regulated in min6 cells within 45 min after exposure to glucose ( ohsugi et al . , 2004 ) , would diminish ca influx during acute hyperglycemia ; rem2 , induced after 16 h of high glucose ( finlin et al . , 2005 ) , would serve to shape ca responsiveness during chronic hyperglycemia .", "although our experiments were not designed to address the relative potency of gem vs. rem2since their comparative levels in pancreatic cells have not yet been determined it is intriguing that rem2 appears to have a broader dose response range , which supports the proposed role in fine tuning ca responsiveness over time .", "our study provides several new insights that help clarify the molecular mechanisms by which rgks inhibit ca channels .", "first , we demonstrated that subunits are necessary for rgk inhibition , corroborating previous reports of subunit dependence ( beguin et al . , 2001 ) .", "by using 1c subunits with deletions in either the n or c terminus in order to increase basal current amplitude , we avoided the difficulties of accurately measuring the inhibition of an already small signal , which may explain the contrasting result obtained with rem ( crump et al . , 2006 ) .", "second , our experiments with the truncated 1c subunits demonstrated that neither the 1c n terminus nor c terminus were required for gem- or rem2-mediated inhibition or alteration of channel gating , also in contrast to a recent report ( crump et al . , 2006 ) . while these differences may be attributed to rem- vs. rem2-specific effects , failure of the c - terminal deletion ( 1733 ) in that report to augment ca currents compared with those from intact 1c subunits , as has been reported previously ( wei et al .", "our results also help clarify a conflict between two recent biochemical studies concerning whether the aid competes with cav for gem binding ( sasaki et al . , 2005 ) or is present as a complex with cav and rem ( finlin et al . , 2006 ) .", "our studies support the latter , where the rgks function only when the 1 subunit is in association with a subunit through its high affinity interaction site aid . in this context ,", "our findings offer additional insights into mechanisms by which subunits modulate ca channel currents .", "coexpression of subunits with 1 subunits increases current amplitude and affects kinetics of activation and inactivation ( dolphin , 2003 ) . within 1 subunits ,", "the major interaction site for subunits is the aid ( pragnell et al . , 1994 ) .", "the aid , however , is not absolutely required for all aspects of subunit modulation as 2a still modulated channel activation and inactivation for an 1a ( cav2.1 ) subunit in which the aid had been deleted ( maltez et al . , 2005 ) . because the wa mutation in the aid completely blocks subunit interaction ( leroy et al . , 2005 ) ,", "our experiments showing that the subunit dependent augmentation of current amplitude is partially preserved with the 1c3 concatemers demonstrate clearly that subunits can still modulate ca channels through interactions exclusive of the aid ( walker et al . , 1998 ; leroy et al . , 2005 ;", "utilization of these concatemers also elucidated the mechanism of rgk modulation of ca channels : the partial preservation of the gem- or rem2-mediated decrease in current amplitude with the 1c3 concatemers rules out models in which rgks compete with subunits for aid interaction ( beguin et al .", "in contrast , the complete loss of rem2-mediated effects upon activation and inactivation suggest that the aid interaction is necessary only for rem2-mediated effects upon channel gating ." ]

[ "<S> although inhibition of voltage - gated calcium channels by rgk gtpases ( rgks ) represents an important mode of regulation to control ca2 + influx in excitable cells , their exact mechanism of inhibition remains controversial . </S>", "<S> this has prevented an understanding of how rgk regulation can be significant in a physiological context . </S>", "<S> here we show that rgks gem , rem , and rem2decreased cav1.2 ca2 + current amplitude in a dose - dependent manner . </S>", "<S> moreover , rem2 , but not rem or gem , produced dose - dependent alterations on gating kinetics , uncovering a new mode by which certain rgks can precisely modulate ca2 + currents and affect ca2 + influx during action potentials . to explore how rgks influence gating kinetics , we separated the roles mediated by the ca2 + channel accessory subunit 's interaction with its high affinity binding site in the pore - forming 1c subunit ( aid ) from its other putative contact sites by utilizing an 1c3 concatemer in which the aid was mutated to prevent subunit interaction . </S>", "<S> this mutant concatemer generated currents with all the hallmarks of subunit modulation , demonstrating that aid-independent interactions are sufficient for subunit modulation . using this construct we found that although inhibition of current amplitude was still partially sensitive to rgks , rem2 no longer altered gating kinetics , implicating different determinants for this specific mode of rem2-mediated regulation . </S>", "<S> together , these results offer new insights into the molecular mechanism of rgk - mediated ca2 + channel current modulation . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Construction of cDNA Plasmids", "Electrophysiological Recordings and Analysis", "Protein Expression/GST Pull-Down Assays", "Immunoblotting", "RESULTS", "DISCUSSION" ]

[ [ "voltage - gated ca channels are the signature feature of excitable cells , transducing electrical activity into increased intracellular [ ca ] that mediates specific cellular effects such as muscle contraction , hormone secretion , and release of neurotransmitters .", "thus , many regulatory mechanisms have evolved to fine tune ca channel activity and the resultant ca influx , mostly by protein ", "protein interactions with , or posttranslational modifications of , the pore - forming 1 subunit . some are rapid , such as ca - dependent inactivation of l - type ( cav1.2 ) channels ( budde et al . , 2002 )", "; others occur after the activation of signaling pathways , such as pka potentiation of cav1.2 channels or g protein inhibition of n - type ( cav2.2 ) channels ( catterall , 2000 ) .", "in contrast , mechanisms that result in finely graded responses to changes in the cellular environment developing over longer time scales have not been well described .", "rgk gtpases ( rad , rem , rem2 , gem / kir ) , the most recently characterized group within the ras family of gtp - binding proteins ( reynet and kahn , 1993 ; maguire et al . , 1994 ; finlin and andres , 1997 ; finlin et al . , 2000", ") , have received special attention because they are potent inhibitors of ca channels and candidates for ca channel regulators under transcriptional control that can therefore integrate the influence of multiple extracellular signals .", "experiments in a variety of cell types have shown a drastic reduction of peak current amplitude for multiple ca channels after expression of gem / kir ( beguin et al . , 2001 , 2005b ; murata et al . , 2004 ;", "ward et al . , 2004 ) , rem , rad ( finlin et al . , 2003 ; crump et al . ,", "2006 ) , and rem2 ( chen et al . , 2005 ; finlin et al . , 2005 ) . among ras family members ,", "rgks differ by having extended variable n - terminal regions and conserved c - terminal extensions lacking the caax motif for fatty acylation , and containing binding motifs for calmodulin and 14 - 3 - 3 proteins ( kelly , 2005 ) .", "individual rgks have nonoverlapping patterns of expression , and are transcriptionally induced and repressed by different factors .", "for example , gem and rem2 transcription has been reported to be stimulated by glucose in insulin - secreting pancreatic cells but follow a different time course ( ohsugi et al . , 2004 ; finlin et al . , 2005 ) ; rad is overexpressed in muscle of type ii diabetics ( reynet and kahn , 1993 ) , and rem transcription is repressed by lipopolysaccharide exposure ( finlin and andres , 1997 ) .", "gem inhibits the rho / rhoa kinase pathway ( ward et al . , 2002 ) and induces neuroblastoma morphological and ganglionic differentiation ( leone et al . , 2001 ) .", "expression of both gem and rem2 has been shown to decrease glucose - stimulated insulin secretion ( beguin et al . , 2001 ; finlin et al . , 2005 ) .", "a two - hybrid experiment identified ca channel subunits as a gem - interacting protein in the insulin - secreting min6 cell line ( beguin et al . , 2001 ) .", "since subunits have been implicated in trafficking 1 subunits to the plasma membrane , this led to the hypothesis that rgks prevent subunits from interacting with 1 subunits , thereby preventing membrane targeting and resulting in reduced channels at the cell surface ( beguin et al .", "a number of recent studies suggest instead that rgks inhibit channels already resident at the cell surface ( chen et al . , 2005 ; finlin et al . , 2005", ") . moreover , though it is their potency that has earned them interest , it is a more subtle and tunable response that likely has physiological ramifications . it has already been established that changes in ca channel currents less severe than the near complete reduction observed when rgks are expressed in heterologous systems lead to drastic pathophysiological consequences ( splawski et al . , 2004 ) .", "it is difficult to understand how rgk expression could result in a finely graded response . in this study , we provide new insights into how gem and rem2 regulate ca channels . exploiting the xenopus oocyte system to control levels of expression ( canti et al . , 2001 )", ", we found that gem and rem2 drive a dose - dependent inhibition of ca currents .", "rem2 , but not gem , also modulated both the kinetics of channel activation and inactivation in a manner that was dependent on subunit interaction with the 1 interaction domain ( aid ) .", "together , these results suggest that specific rgks contribute to the fine tuning of ca influx by different mechanisms ." ], [ "constructs for 1c ( pcardhe ) , 2 , and the 1c c - terminal deletion ( amino acids 16702171 ) , and the gst i - ii loop have been previously reported ( zhlke et al . , 2000 ;", "nm_000725 ) was cloned into the pgem - he oocyte expression vector using standard molecular biology techniques .", "bc018219 ) was obtained as an est and cloned into the pcs2 + oocyte expression vector ( gift from d. mckinnon , state university of new york , stony brook , ny ) .", "rem2 full - length ( ay916790 ) , a gift from d. andres ( university of kentucky , lexington , ky ) , was digested out of the original pcdna3.1 vector and ligated into compatible sites in pcs2 + . the 1c n - terminal deletion ( amino acids 2139 )", "the 1c3 concatemer included amino acids 12134 from 1c and the entire 3 with a valine linker between them .", "the mutant 1c and corresponding concatemer included mutations y467s and w470a created by quikchange ( stratagene ) .", "the kchip2b clone was a gift from p. pfaffinger ( baylor college of medicine , houston , tx ) . in vitro crna transcription and microinjection into xenopus oocytes", "the following amounts of crna were injected : 1c ( 1 ng ) , 2 ( 1 ng ) , 3 ( 0.22 ng ) .", "two - electrode voltage clamp recordings were performed as previously described ( kim et al . , 2004 ) .", "during recordings , oocytes were constantly superfused with a solution containing 40 mm ba(oh)2 ( or 40 mm ca(oh)2 in experiments recording ca currents ) , 50 mm naoh , 1 mm koh , and 10 mm hepes ( adjusted to ph 7.4 with methanesulfonic acid ) .", "recordings were performed with a standard two - electrode voltage clamp configuration using an oocyte clamp oc-725c amplifier ( warner instrument corp . ) connected through a digidata 1322a a / d interface ( axon instruments , inc . ) to a personal computer .", "ionic currents were filtered at 1 khz by an integral 4 pole bessel filter and sampled 10 khz and analyzed with clampfit 9.2 .", "steady - state inactivation was analyzed with a two - pulse protocol in which a 5-s conditioning pulse ( p1 ) from 60 mv to + 50 mv was followed by a 100-ms test pulse ( p2 ) at + 10 mv .", "normalized p2 values were fitted with a boltzmann equation ( i / ipeak = ( 1 io)/[1 + exp((v v1/2)/k ) ] + io ) .", "activation time constants were estimated by fitting the activating component of the current trace to the following equation : i = io + afastexp(t/fast ) + aslowexp(t/slow ) .", "bursts of pancreatic cell action potentials were simulated by a 5-s depolarization to 40 mv from 70 mv followed by a series of 26 100-ms voltage - clamp depolarizations between 40 and 0 mv at 5 hz ( kanno et al . , 2002 ) .", "all values are given as means sem , with statistical comparisons performed with a student 's t test .", "protein expression / gst pull - down assays were performed as previously described ( maltez et al . , 2005 ) .", "oocytes were injected with either 1,000 pg of gem crna , 1,000 pg gem crna with 1,000 pg crna cam , or 4,000 pg of gem crna .", "oocytes were incubated at 17c for 24 h , lysed in ice - cold oocyte extraction buffer ( 20 mm hepes , 5 mm kcl , 1.5 mm mgcl2 , 1 mm edta , 10% glycerol , 0.1% np-40 , and roche protease inhibitor tablets ) , and then solubilized in sds ." ], [ "constructs for 1c ( pcardhe ) , 2 , and the 1c c - terminal deletion ( amino acids 16702171 ) , and the gst i - ii loop have been previously reported ( zhlke et al . , 2000 ;", "nm_000725 ) was cloned into the pgem - he oocyte expression vector using standard molecular biology techniques .", "bc018219 ) was obtained as an est and cloned into the pcs2 + oocyte expression vector ( gift from d. mckinnon , state university of new york , stony brook , ny ) .", "rem2 full - length ( ay916790 ) , a gift from d. andres ( university of kentucky , lexington , ky ) , was digested out of the original pcdna3.1 vector and ligated into compatible sites in pcs2 + . the 1c n - terminal deletion ( amino acids 2139 )", "the 1c3 concatemer included amino acids 12134 from 1c and the entire 3 with a valine linker between them .", "the mutant 1c and corresponding concatemer included mutations y467s and w470a created by quikchange ( stratagene ) .", "the kchip2b clone was a gift from p. pfaffinger ( baylor college of medicine , houston , tx ) ." ], [ "in vitro crna transcription and microinjection into xenopus oocytes has been previously reported ( kim et al . , 2004 ) .", "the following amounts of crna were injected : 1c ( 1 ng ) , 2 ( 1 ng ) , 3 ( 0.22 ng ) .", "two - electrode voltage clamp recordings were performed as previously described ( kim et al . , 2004 ) .", "during recordings , oocytes were constantly superfused with a solution containing 40 mm ba(oh)2 ( or 40 mm ca(oh)2 in experiments recording ca currents ) , 50 mm naoh , 1 mm koh , and 10 mm hepes ( adjusted to ph 7.4 with methanesulfonic acid ) .", "recordings were performed with a standard two - electrode voltage clamp configuration using an oocyte clamp oc-725c amplifier ( warner instrument corp . ) connected through a digidata 1322a a / d interface ( axon instruments , inc . ) to a personal computer .", "ionic currents were filtered at 1 khz by an integral 4 pole bessel filter and sampled 10 khz and analyzed with clampfit 9.2 .", "steady - state inactivation was analyzed with a two - pulse protocol in which a 5-s conditioning pulse ( p1 ) from 60 mv to + 50 mv was followed by a 100-ms test pulse ( p2 ) at + 10 mv .", "normalized p2 values were fitted with a boltzmann equation ( i / ipeak = ( 1 io)/[1 + exp((v v1/2)/k ) ] + io ) .", "activation time constants were estimated by fitting the activating component of the current trace to the following equation : i = io + afastexp(t/fast ) + aslowexp(t/slow ) .", "bursts of pancreatic cell action potentials were simulated by a 5-s depolarization to 40 mv from 70 mv followed by a series of 26 100-ms voltage - clamp depolarizations between 40 and 0 mv at 5 hz ( kanno et al . , 2002 ) .", "all values are given as means sem , with statistical comparisons performed with a student 's t test ." ], [ "protein expression / gst pull - down assays were performed as previously described ( maltez et al . , 2005 ) ." ], [ "oocytes were injected with either 1,000 pg of gem crna , 1,000 pg gem crna with 1,000 pg crna cam , or 4,000 pg of gem crna .", "oocytes were incubated at 17c for 24 h , lysed in ice - cold oocyte extraction buffer ( 20 mm hepes , 5 mm kcl , 1.5 mm mgcl2 , 1 mm edta , 10% glycerol , 0.1% np-40 , and roche protease inhibitor tablets ) , and then solubilized in sds ." ], [ "to explore the mechanisms by which rgks inhibit ca channel currents , we expressed cav1.2 channels ( 1c , 2 , and 3 ) with gem , rem , or rem2 in xenopus oocytes and recorded the resulting currents by two - electrode voltage clamp . as observed previously , expression of any of these rgks drastically reduced the iba peak current amplitude ( fig .", "1 d ) . to distinguish among the possible models by which rgks inhibit ca currents and to explore the physiological implications , we took advantage of the xenopus oocyte system , in which it has been shown that expression levels of proteins can be accurately titrated in a monotonic fashion ( canti et al . , 2001 ) , in order to test whether inhibition were dose dependent .", "b demonstrates a monotonic increase in gem protein with increasing amounts of gem crna injected over the range that we studied .", "moreover , coinjection of crna for another unrelated protein ( calmodulin ) did not affect gem protein levels , suggesting that crna amounts in this range did not exceed the protein synthesis capacity in oocytes . with this confirmation ,", "1 c shows that gem , rem , and rem2 inhibit iba peak current amplitude at 10 mv in a dose - dependent manner .", "this was not a nonspecific effect of increasing the amounts of crna injection ; coexpression of crna for kchip2b , a protein that modulates k currents and is of similar mass to the rgks ( an et al . , 2000 ) , did not alter cav1.2 current amplitude .", "examination of individual current traces also revealed differences in the mechanisms by which gem and rem2 affect cav1.2 currents .", "while coexpression of all three rgks decreased current amplitude , coexpression of rem2 also appeared to affect kinetics of activation and inactivation ( fig .", "effects of gem and rem appeared similar , so we focused on gem in further studies .", "( a ) normalized i - v relationships of iba from oocytes injected with 1c/3/2 without or with gem ( 130 pg ) or rem2 ( 936 pg ) .", "( b ) immunoblots with anti - gem antibody . on left is shown gst and gst - gem , demonstrating specificity of the antibody .", "the right panel shows an increase in the amount of gem protein detected with an increasing amount ( as indicated ) of crna injected per oocyte .", "gem is indicated by an arrow ; a nonspecific band seen even without injection of gem crna is shown by an asterisk .", "( c ) dose response of gem- , rem- , or rem2-mediated inhibition of iba .", "( d ) exemplar traces of cav1.2 channels expressed without a rgk , with gem ( 20 pg ) , with rem ( 100 pg ) , or with rem2 ( 468 pg ) .", ". coexpression of gem appeared to inhibit cav1.2 currents by a direct scaling effect , while rem2 altered the kinetics of activation and inactivation ( fig .", "these different effects upon channel activation and inactivation are better appreciated by examining scaled traces from cav1.2 channels compared with traces from cav1.2 channels coexpressed with gem ( 26 pg ) or rem2 ( 936 pg ) .", "quantitative analysis of the kinetics of inactivation for cav1.2 channels coexpressed with rem2 was complicated because the decay phases of currents were contaminated by overlapping slow activation .", "thus , we analyzed steady - state inactivation with a two - pulse protocol in which the normalized residual peak current during a + 10-mv test pulse ( p2 ) was plotted against the voltage of a 5-s inactivating prepulse ( p1 ) and found that both gem ( 26 pg ) and rem2 ( 936 pg ) affected steady - state inactivation ( fig .", "rem2 mainly affected the pedestal from 0.23 0.02 to 0.12 0.02 , ( n = 1112 , p < 0.0001 ) but gem reduced the slope to 14.1 0.6 from 9.0 0.8", "( a ) top panels show exemplar current traces during a 2-s test pulse at + 10 mv for gem and rem2 at the indicated doses .", "bars , 1 a . the bottom panels show scaled exemplar traces of 1c/3/2 ( gray ) coinjected with gem ( 65 pg ) or rem2 ( 936 pg ) ( black ) during a 2-s test pulse to + 10 mv .", "( b ) steady - state inactivation for 1c/3/2 without or with gem ( 26 pg ) or rem2 ( 936 pg ) .", "n = 1012 ( c e ) dose response of rem2-mediated effects upon kinetics of activation ( fast , fraction aslow , and slow , respectively , at the indicated test potentials see legend in c and with the amounts of crna injected as indicated on the x - axis ) , n = 714 .", "( f ) scaled exemplar traces of activation phases at + 10 mv for 1c/3/2 coexpressed with the indicated rem2 doses .", "( g ) ca currents recorded during a simulated burst of action potentials in a pancreatic cell ( see materials and methods ) with no rgk or rem2 ( 468 pg ) .", "bars : 200 na , 1 s. rem2 also affected cav1.2 channel activation in a dose - dependent and voltage - dependent manner ( fig .", "2 c , 0 pg ) , the activating phase for currents elicited with test potentials from 0 to + 30 mv was best fitted with two exponentials ( i = afaste + aslowe + c ) and the dominant component was fast ( fraction afast was 8090% at all test potentials ; fig .", "instead , the slower activation of cav1.2 channels induced by higher doses of coexpressed rem2 could be explained by two effects upon slow : slow became longer with increasing doses of rem2 and the fraction of aslow increased .", "these effects were most prominent at test potentials near the peak of the i - v curve ( fig .", "the overall consequences of these dose - dependent effects upon activation induced by rem2 are illustrated by the overlaid traces in fig .", "2 f. since these effects upon channel kinetics suggested that rem2 could alter the temporal nature of channel responsiveness during action potentials , we tested whether rem2 altered simulated ca - dependent action potentials that underlie the rhythmic bursting of electrical activity essential for insulin secretion in pancreatic islet cells ( mears , 2004 ) .", "2 g shows the resultant ca currents from cav1.2 channels ( 1c , 3 , and 2 ) expressed in xenopus oocytes during 26 successive 100-ms depolarizations from 40 to 0 mv ( at 5 hz ) , a protocol that simulates the bursting activity during insulin secretion and has been used in isolated cells ( kanno et al . , 2002 ) .", "in the absence of rgks , the peak ca current amplitude decreased sequentially during the 26 successive depolarizations so that the current amplitude during the last depolarization was 46 4% ( n = 8) of the peak current during the first depolarization . not only were the current amplitudes smaller with coexpression of rem2 ( consistent with the effects of rem2 presented above ) , but rem2 accelerated the decrease in amplitude during the successive depolarizations so that the current amplitude during the last depolarization was 12 7% ( n = 7 ; p = 0.001 compared with no rgk ) of the peak current during the first depolarization .", "this accelerated decrement is likely due to the increased rate of inactivation observed in the presence of rem2 ( fig .", "coexpression of gem led to decreased current ( compared with no rgk ) , but did not affect the rate of decrement of the current amplitude ( unpublished data ) , consistent with the lack of effect upon channel kinetics shown above .", "thus , the presence of rem2 would decrease the integrated ca influx and alter its kinetics during a burst of action potentials such as those that drive insulin secretion in pancreatic islet cells .", "we next tested whether the 1c n or c termini were necessary for these effects by testing whether deletion constructs ( 2 - 139 in the n - terminus or 1669 - 2171 in the c terminus ) were modulated by gem or rem2 .", "these experiments were prompted in part by a recent report suggesting that rem , another rgk member , required the 1c c terminus , particularly the pka phosphorylation site at ser , for inhibition of ca channel currents ( crump et al .", "in contrast , we found that gem ( 250 pg ) and rem2 ( 936 pg ) consistently reduced peak current amplitude for channels containing intact or truncated 1c subunits ( fig .", "3 , a and b ) . rem2 also maintained its effects upon kinetics of activation and inactivation in the truncated channels , as shown in the scaled exemplar current traces ( fig .", "the ca channel subunit , but not the 1c n and c termini , are required for gem- or rem2-mediated effects .", "( a and b ) normalized iba for the indicated combinations of 1c ( wt ) , the n - terminal truncation ( n ) , or the c - terminal truncation ( c ) , with or with gem or rem2 , as indicated .", "all combinations were coexpressed with 3 and 2. scaled exemplar current for each pair with ( black ) or without ( gray ) gem or rem2 are shown on right .", "( c and d ) normalized iba for the indicated combinations of c and 2 with or without 3 and gem or rem2 , as indicated . for the pair without 3 , scaled exemplar currents with ( black ) or without ( gray ) gem or rem2 are shown on right .", "we also used the 1c c - terminal deletion to analyze whether subunits were necessary for gem or rem2 modulation .", "truncation of the 1c c terminus produces increased current amplitude in the absence of subunits ( wei et al .", ", 1994 ; klckner et al . , 1995 ; gerhardstein et al . , 2000 ; ivanina et al . , 2000 ) , thereby providing a larger baseline current from which to assess rgk inhibition .", "3 ( c and d ) shows that , in the absence of a coexpressed subunit , neither gem nor rem2 inhibited channel currents .", "further , rem2 modulated neither activation nor inactivation in the absence of a coexpressed subunit .", "these results show that rgk modulation is independent of the 1c n or c terminus , but requires subunits .", "recent models have suggested that rgks inhibit ca channels by direct competition with subunits for this high affinity interaction site on the 1 subunit ( sasaki et al . , 2005 ) . although subunit interaction with the aid is not required for all aspects of subunit modulation of ca channel function ( maltez et al . , 2005 ) ,", "aid mutations that block subunit binding render channel currents too small to accurately assess an inhibitory effect of rgks ( singer et al . , 1991 ) .", "building upon a previous hypothesis that the aid interaction serves mainly to secure subunits to 1 so as to allow other , lower affinity regulatory interactions , we covalently tethered subunits directly to the 1c c terminus after amino acid 2134 ( 1c3 ) .", "currents from this concatemer ( expressed with 2 ) were similar to currents from untethered channels ( 1c + 3 with 2 ) , except that the peak of the i - v curve shifted to more depolarized potentials ( fig .", "4 a ) as previously reported ( dalton et al . , 2005 ) . to prevent 3 interaction with aid we made an 1c with two mutations in aid , y467s and w470a ( 1c ) ,", "either of which has been shown to singly disrupt subunit interaction and block subunit modulation ( van petegem et al .", "confirmation of abolished subunit binding to the mutant 1c i - ii loop is shown in a gst pull - down assay ( fig . 4 b ) .", "current amplitudes from an 1c subunit with the same mutations coexpressed with 3 ( 1c + 3 ) were very small ( fig .", "4 , a , c , and d ) and indistinguishable from currents from an 1c expressed without 3 ( not depicted ) , which is consistent with an absence of 3 interaction .", "when 3 was tethered to the aid mutant ( 1c3 ) however , the resulting current amplitude was significantly larger than from 1c coexpressed with untethered 3 ( fig .", "4 , a , c , and d ) since the requirement for -aid interaction could be at least partially circumvented by tethering the subunit to 1 , this supported the hypothesis that other interactions between 1 and are important for -dependent modulation .", "having generated an 1c subunit that was modulated by a subunit independent of its aid interaction , we therefore could test whether -aid was required for rgk inhibition .", "currents from channels containing 1c3 coexpressed with either gem ( 250 pg ) or rem2 ( 628 pg ) crna showed that they both produced a similar reduction of current as for 1c + 3 ( compare fig .", "4 , d and e ) . although coexpression of gem or rem2 also reduced currents from channels containing 1c3 , the reduction was much more modest ( fig .", "4 , e and f ) . moreover , although the effects of rem2 on activation and inactivation were preserved when coexpressed with 1c3 , rem2 did not affect activation or inactivation when coexpressed with 1c3 ( fig . 4 e ) .", "these results show that rem2-mediated effects upon activation and inactivation require the -aid interaction while gem- or rem2-induced inhibition of current amplitude does not .", "interaction between the ca channel subunits and the aid influence gem- and rem2-mediated effects . ( a ) normalized i - v relationships of iba from oocytes injected with the indicated constructs or combinations , all coinjected with 2. n = 3134 .", "( b ) a hisx6 immunoblot of a pull - down experiment for purified 2 sh3-gk core ( maltez et al . , 2005 ) using a gst-1c i - ii or i - ii mutant loop .", "( d f ) normalized iba for the indicated concatemers or combinations ( all expressed with 2 ) , with or without gem or rem2 . scaled exemplar traces are shown on right .", "bars : 1 s ( d and f ) and 50 ms ( e ) ." ], [ "heterologous overexpression of several rgks in a variety of systems produces almost complete inhibition of coexpressed or endogenous ca channel current ( beguin et al . , 2001 , 2005b ; finlin et al . , 2003 ; murata et al . , 2004 ;", "ward et al . , 2004 ; chen et al . , 2005 ; crump et al . ,", "2006 ) and rgk inhibition of ca influx has been proposed as a mechanism for physiologic control of ca channel activity for responses such as regulation of insulin secretion from pancreatic islet cells ( beguin et al . , 2001 ; finlin et al . , 2005 ) or control of cardiac rhythm ( murata et al . , 2004 ) .", "lacking a detailed molecular understanding of how rgks could fine tune ca influx eclipses how this mode of regulation could shape a specific physiological response ; for example , up - regulation of an rgk ( ohsugi et al . , 2004 ; finlin et al . , 2005 ) and subsequent channel inhibition ( beguin et al .", ", 2001 ; finlin et al . , 2005 ) after glucose stimulation might protect islet cells from excessive ca influx during chronic hyperglycemia , but how would cells retain their ability to secrete insulin with the almost complete loss of ca currents observed in previous overexpression experiments ? in this study we describe two unexpected means by which rgks regulate ca channels , providing a framework for understanding how a wide array of ca signaling events can be precisely regulated .", "exploiting the xenopus oocyte system to control protein expression levels , we found that rgk inhibition of ca channel current was dose dependent .", "the mechanism(s ) by which rgks lead to current amplitude reduction , previously a source of controversy , is not revealed by these experiments ; direct effects upon channels resident at the cell surface ( chen et al .", ", 2005 ; finlin et al . , 2005 ) or effects upon channel trafficking / assembly ( beguin et al . , 2001 ) can not be easily distinguished by the experiments presented here .", "the significant finding , however , is that the suppression of current depends upon the level of rgk expression , thus promising a predictable and titratable attenuation of ca current by specific rgks .", "thus , our results suggest that the high glucose - stimulated induction of a specific rgk and the resultant down - regulation of the ca current in pancreatic islet cells contribute to a protective mechanism against the detrimental effects of an enhanced ca signal resulting from chronic glucose exposure ( juntti - berggren et al . , 1993 ) ; conversely , a reduction of rgk protein in response to elevated glucose would serve to compensate hyperglycemia acutely by increasing ca channel activity and consequent insulin secretion .", "furthermore we found that rem2 , but not gem or rem , also altered cav1.2 gating kinetics , slowing activation and enhancing inactivation .", "these effects upon kinetics suggest that rem2 must act at least in part upon channels resident at the cell surface .", "not only could rem2 decrease peak ca influx , it could also impart profound influence on the ca - dependent action potentials that underlie the rhythmic bursting of electrical activity essential for insulin secretion in pancreatic islet cells ( mears , 2004 ) , by shaping the temporal nature of channel responsiveness as suggested by our experiments in fig . 2 g.", "although the molecular basis for the effects of rem2 upon channel kinetics is not clear , we speculate that its extended n terminus and c terminus that flank the ras core may be responsible ; neither extension is found in rem or gem ( fig .", "the kinetic actions of rem2 could result from an additional contact between rem2 and the channel within these nonconserved regions .", "clustal w ( 1.83 ) multiple sequence alignment ( thompson et al . , 1994 )", "the gray - boxed areas highlight the extended n and c termini in rem2 that flank the conserved ras - like core .", "these different modes of regulation by gem and rem2 , in conjunction with their differential temporal patterns of expression , may yield an integrated response to oppose effects of hyperglycemia .", "gem , up - regulated in min6 cells within 45 min after exposure to glucose ( ohsugi et al . , 2004 ) , would diminish ca influx during acute hyperglycemia ; rem2 , induced after 16 h of high glucose ( finlin et al . , 2005 ) , would serve to shape ca responsiveness during chronic hyperglycemia .", "although our experiments were not designed to address the relative potency of gem vs. rem2since their comparative levels in pancreatic cells have not yet been determined it is intriguing that rem2 appears to have a broader dose response range , which supports the proposed role in fine tuning ca responsiveness over time .", "our study provides several new insights that help clarify the molecular mechanisms by which rgks inhibit ca channels .", "first , we demonstrated that subunits are necessary for rgk inhibition , corroborating previous reports of subunit dependence ( beguin et al . , 2001 ) .", "by using 1c subunits with deletions in either the n or c terminus in order to increase basal current amplitude , we avoided the difficulties of accurately measuring the inhibition of an already small signal , which may explain the contrasting result obtained with rem ( crump et al . , 2006 ) .", "second , our experiments with the truncated 1c subunits demonstrated that neither the 1c n terminus nor c terminus were required for gem- or rem2-mediated inhibition or alteration of channel gating , also in contrast to a recent report ( crump et al . , 2006 ) . while these differences may be attributed to rem- vs. rem2-specific effects , failure of the c - terminal deletion ( 1733 ) in that report to augment ca currents compared with those from intact 1c subunits , as has been reported previously ( wei et al .", "our results also help clarify a conflict between two recent biochemical studies concerning whether the aid competes with cav for gem binding ( sasaki et al . , 2005 ) or is present as a complex with cav and rem ( finlin et al . , 2006 ) .", "our studies support the latter , where the rgks function only when the 1 subunit is in association with a subunit through its high affinity interaction site aid . in this context ,", "our findings offer additional insights into mechanisms by which subunits modulate ca channel currents .", "coexpression of subunits with 1 subunits increases current amplitude and affects kinetics of activation and inactivation ( dolphin , 2003 ) . within 1 subunits ,", "the major interaction site for subunits is the aid ( pragnell et al . , 1994 ) .", "the aid , however , is not absolutely required for all aspects of subunit modulation as 2a still modulated channel activation and inactivation for an 1a ( cav2.1 ) subunit in which the aid had been deleted ( maltez et al . , 2005 ) . because the wa mutation in the aid completely blocks subunit interaction ( leroy et al . , 2005 ) ,", "our experiments showing that the subunit dependent augmentation of current amplitude is partially preserved with the 1c3 concatemers demonstrate clearly that subunits can still modulate ca channels through interactions exclusive of the aid ( walker et al . , 1998 ; leroy et al . , 2005 ;", "utilization of these concatemers also elucidated the mechanism of rgk modulation of ca channels : the partial preservation of the gem- or rem2-mediated decrease in current amplitude with the 1c3 concatemers rules out models in which rgks compete with subunits for aid interaction ( beguin et al .", "in contrast , the complete loss of rem2-mediated effects upon activation and inactivation suggest that the aid interaction is necessary only for rem2-mediated effects upon channel gating ." ] ]

[ "the incidence of endometrial carcinoma in korea has increased rapidly in the last ten years possibly due to an increasingly westernized life style , decreased number of pregnancy , and increase of postmenopausal hormone therapy ( 1 ) .", "surgery is the treatment of choice and total hysterectomy with bilateral salpingo - oophorectomy is the cornerstone of both the staging and treatment of endometrial carcinoma .", "however , although the international federation of gynecology and obstetrics ( figo ) adopted surgical staging in 1988 , and the national comprehensive cancer network ( 2 ) also recommended standard surgical procedures , there is a significant variety in the actual surgical procedures employed in the treatment of patients with endometrial cancer .", "the management of patients with cervical involvement remains controversial and treatment options range from extrafascial hysterectomy ( eh ) to radical hysterectomy ( rh ) , with or without radiation ( 3 , 4 ) .", "however , the literature regarding the treatment of these patients is limited by its low frequency .", "cervical extension of endometrial carcinoma represents about 10% to 15% of cases at the time of diagnosis ( 5 ) .", "such cervical invasion is considered to increase the risk of nodal metastasis ; and those patients are reported to have a worse prognosis than those without it ( 6 - 9 ) .", "the rationale for performing rh in patients with cervical involvement is to eradicate all possible parametrial involvement ( pmi ) .", "however , the preoperative detection of cervical involvement or parametrial disease is not entirely reliable despite the progress in imaging techniques such as magnetic resonance imaging ( 10 , 11 ) .", "thus , although the only definitive method of ensuring the status and extent of disease in the parametria is through radical excision and a histological study of the operative specimen , the unreliability of preoperative prediction , and the relative rarity of pmi may make it unwise to resort to rh as a matter of routine in patients with cervical invasion ( 12 ) .", "we reviewed the medical records of endometrial cancer patients with cervical involvement to determine the relation of parametrial spread with other histopathological variables and to evaluate whether the type of primary surgery using eh or rh alters the patients ' outcome from a histological perspective .", "the cancer database from five different institutes were reviewed ; patients surgically diagnosed with stage ii and a part of stage iii or iva endometrial cancer with cervical involvement during the period of 1993 - 2005 were identified .", "the recorded data were then extracted by a medically qualified gynecology trainee in each institute .", "the original pathology reports and operation reports of 133 patients were reviewed for the following information : age at diagnosis , pre - operative data , operative procedure , adjuvant treatment , follow - up data including date of recurrence , the patient status at last visit . a pathologic data such as figo stage , histologic type , tumor grade , depth of myometrial invasion , and the status of parametrium , and retroperitoneal lymph node were also reviewed .", "patients with mixed mllerian tumor , including carcinosarcoma or endometrial stromal sarcoma , were excluded from the review . when the tumor grade varied between pre - operative biopsy and final pathologic reports , the higher grade was recorded .", "confirmation of cervical invasion and staging was made by reviewing pathology reports , although pathology slides review was not performed .", "the term ' radical hysterectomy ' included modified hysterectomy ( mrh ) that could not be exactly differentiated in operating records .", "patients with surgically staged iia cancer who had undergone eh were selectively treated with adjuvant radiation according to the degree of depth of myometrial invasion . otherwise , most patients with stage iib disease or above underwent post - operative pelvic irradiation or brachytherapy .", "if sufficient follow - up information regarding survival and recurrence was unavailable through medical records , we referred to death certificates to obtain the required information .", "one hundred thirty - three patients with endometrial cancer stage iia to iva were found to have cervical invasion .", "ninety - three percent ( 124/133 ) of the patients underwent complete surgical staging with or without paraaortic lymph node dissection .", "we found no significant difference in age , body mass index ( bmi ) , distribution of figo stage , histology , and tumor grade between the eh and rh groups .", "preoperative evaluations for disease extent including cervical involvement were mostly done by magnetic resonance imaging ( mri ) and partly by computed tomography ( ct ) or endocervical curettage .", "pre - operative mri was performed in 63.9% ( 85/133 ) of the patients and sensitivity for cervical invasion was 44.7% ( 38/85 ) .", "when the cases with stromal invasion were analyzed , the sensitivity was 53.3% ( 24/45 ) .", "table 2 presents the data for the choice of the extent of hysterectomy according to the results of preoperative evaluation .", "eh was performed in 12/62 ( 19.4% ) patients who were preoperatively suspected of cervical involvement , and more than half ( 37/71 , 52.1% ) of the patients who underwent rh had no preoperative evidence of cervical invasion .", "the results show that many surgeons did not strictly follow the results of preoperative evaluation in choosing the type of hysterectomy and the decision regarding eh or rh was made at their own discretion . fig .", "1 shows the relationship between the depth of myometrial invasion and that of cervical stromal invasion .", "a total of 121 cases had available information about both about the depth of myometrial and presence of cervical stromal invasion . in patients with disease limited to the endometrium , no case showed cervical stromal invasion . on the other hand , a majority of patients with disease involving cervical stroma had deep myometrial invasion ( p=0.001 ) .", "table 3 showed the relationship between prognostic factors and parametrial involvement in 71 patients undergoing rh .", "eight of ten patients ( 80.0% ) had pelvic node metastasis , 1 patient had grossly positive adnexal involvement , and 3 had tumor spread outside the pelvis ( 2 of 3 had coexisting pelvic lymph node metastasis ) .", "depth of myometrial invasion and pelvic or paraaortic lymph node positivity were significantly correlated with paramatrial involvement .", "of the 19 patients with pelvic lymph node metastasis , 8 patients ( 42.1% ) had concomitant pmi .", "conversely , of the 10 patients with pmi , 8 ( 80.0% ) had lymph node metastasis .", "in 74 patients with surgical stage ii , i.e. , disease showing no demonstrable evidence of tumor spread in the pelvic cavity , rh was performed in 41 cases and no patients showed parametrial involvement .", "sixty six of 74 ( 89% ) patients underwent adjuvant radiation therapy ( 35 whole pelvic irradiation , 7 vaginal brachytherapy , 23 both , 1 unknown ) and there were 3 patients who developed recurrent disease in the rh and none in the eh group ( mean follow - up : 51 months , table 4 ) .", "our results show that a large number of korean gynecologic surgeons have a tendency to choose the surgical extent of hysterectomy through their own disposition and do not strictly adhere the results of pre - operative evaluation .", "actually , the type of hysterectomy procedure selected for endometrial cancer varies in each nation and in each institution .", "watanabe et al . ( 13 ) reported the results of a survey of a japanese group showing that more than 70% of institutes never perform rh without regarding the preoperative status of cervical involvement .", "moreover , according to a survey in north america , nearly 30% of centers never performed class ii or iii extended hysterectomy for the treatment of endometrial cancer ( 14 ) .", "as shown in table 2 , surgeons who overuse rh without regarding the pre - operative results may believe that the current pre - operative evaluation method is not sensitive enough to detect cervical invasion and thus , worry about possible pmi or post - operative stump recurrence . on the other hand , those who perform only eh despite the positive findings from pre - operative evaluation may believe that cervical stromal invasion should be followed by adjuvant radiotherapy and thus , the prognosis would not be changed by performing a ' high morbidity producing surgery ' considering the low incidence of pmi .", "this inconsistent treatment policy , in fact , necessitates the need for an evidenced - based reformed guideline to which most clinicians can conform , although the definition of rh or eh is not standardized among surgeons and thus , the actual extent of surgery investigated as rh or eh by medical records might be different for each patient and this is one of the limitations of this study .", "our results also showed that the pmi rate in patients with cervical invasion is about 14% in the rh group .", "this is within the range reported by other investigators , and is not significantly lower than reported values ( 15 - 17 ) .", "however , parametrial spread was not found in any of the patients with figo stage ii , i.e. , patients showing no intraoperative tumor spread , but was identified in 8 patients with positive pelvic lymph nodes , and two showed grossly frank extrauterine spread around the adnexa and another site in the pelvic cavity .", "these findings indicate that a risk of leaving an occult metastasis in parametrial tissue when performing only eh in patients without evidence of extrauterine tumor spread seems to be very low , even though all lymph node metastases may not be completely screened by intraoperative frozen section .", "although they reported a relatively high rate of parametrial involvement , the points we focused on is not the crude value of the pmi itself , but the necessity of parametrial resection .", "undoubtedly , cervical stromal invasion is known to increase the risk of pmi . however , the pattern of spread in the invaded cervix of endometrial cancer is quite different from that of primary cervical carcinoma . as shown in fig .", "1 , the pattern of cervical spread of endometrial cancer seems to follow myometrial invasion and originate from the endocervical glandular region , and as it advances , it invades the stroma .", "therefore , we can speculate that before the tumor of the endometrium reaches the deep myometrium , a chance to detect direct pmi in the patients with suspicious cervical invasion seems to be low .", "however , pmi in endometrial cancer can not be completely excluded only by the absence of cervical stromal invasion because pmi is reported to consist histologically of 2 patterns of spread : direct invasion of cancer cells to the parametrial connective tissues like that seen in cervical cancer , and parametrial lymph - vascular space invasion frequently seen in patients with deep myometrial involvement without cervical involvement ( 17 - 19 ) .", "current guidelines for endometrial cancer show that cervical stromal invasion is regarded as one of the major indications of adjuvant radiotherapy .", "we might guess that adjuvant radiation is required due to the implication that cervical stromal invasion raises the risk of recurrence not just by direct pmi from the cervix , but also by increasing the risk of occult parametrial involvement from the preceding deep myometrial invasion or myometrial lymph - vascular space invasion .", "suffice it to say , this suggest that performing rh only in patients with cervical involvement can not guarantee the complete eradication of possible parametrial involvement . in addition", ", it is very likely that we may have ' cut through ' the invaded parametrium in stage ii patients who underwent eh even though it was not revealed by pathology because there was no direct connection between the invaded cervical stroma and parametrium itself . even acknowledging that occult parametrial spread had existed", ", our data showed that no recurrences occurred in patients who only underwent eh followed by adjuvant radiation and thus , it suggests that occult parametrial metastasis in patients with no gross extrauterine spread can be successfully controlled by radiation therapy .", "we could not evaluate the value of rh in patients with stage iii or over because their survival outcome is much likely to be influenced by variables other than parametrial involvement , i.e. , lymph node status , extent of tumor spread , different treatment strategy to the recurred patients , etc .", "we did not overcome the limitations of retrospective analysis , i.e. , lack of verifying the actually performed surgical extent , lack of centralization of pathologic review , and limited number of cases .", "nonetheless , we cautiously suggest that routine rh in patients with cervical invasion and having no grossly positive extrauterine spread should be reappraised and that these patients are worthy of consideration for less radical surgery performed in conjunction with pelvic and paraaortic lymphadenectomy considering the low rate of parametrial involvement , inaccurate preoperative prediction , high post operative morbidity , and successful control through adjuvant radiation . since this is an area of continued debate and a principal shift from our current practice and attitudes , a randomized study would be required to define the low - risk population that would have a modification of the extent of parametrial resection performed . in this", "regards , we can conclude that the real value of rh in endometrial cancer patients with suspicious cervical involvement can be evaluated by performing a large scaled randomized controlled study comparing the survival and quality of life between the group who underwent rh only or followed by tailored adjuvant radiation and the group with eh and routine adjuvant radiation therapy ." ]

[ "<S> to determine whether radical hysterectomy is necessary in the treatment of endometrial cancer patients with cervical involvement , we reviewed the medical records of women who underwent primary surgical treatment for endometrial carcinoma and selected patients with pathologically proven cervical invasion . among 133 patients </S>", "<S> , 62 patients underwent extrafascial hysterectomy ( eh ) and 71 radical or modified radical hysterectomy ( rh ) . the decision regarding eh or rh </S>", "<S> was made at the discretion of the attending surgeon . </S>", "<S> the sensitivity of pre - operative magnetic resonance imaging for cervical invasion was 44.7% ( 38/85 ) . </S>", "<S> in rh patients , 10/71 ( 14.1% ) patients had frankly histologic parametrial involvement ( pmi ) . </S>", "<S> all were stage iii or over . </S>", "<S> eight of 10 patients had pelvic / paraaortic node metastasis and two showed extrauterine spread . in 74 </S>", "<S> patients with stage ii cancer , rh was performed in 41 and pmi was not seen . </S>", "<S> sixty - six ( 89.2% ) patients had adjuvant radiation therapy and there were 3 patients who had developed recurrent disease in the rh group and none in the eh group ( mean follow - up : 51 months ) . although these findings can not conclusively refute or support the necessity of radical hysterectomy in patients with cervical extension , it is noteworthy that the risk of pmi seems to be minimal in patients with a tumor confined to the uterus without evidence of extrauterine spread . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "the incidence of endometrial carcinoma in korea has increased rapidly in the last ten years possibly due to an increasingly westernized life style , decreased number of pregnancy , and increase of postmenopausal hormone therapy ( 1 ) .", "surgery is the treatment of choice and total hysterectomy with bilateral salpingo - oophorectomy is the cornerstone of both the staging and treatment of endometrial carcinoma .", "however , although the international federation of gynecology and obstetrics ( figo ) adopted surgical staging in 1988 , and the national comprehensive cancer network ( 2 ) also recommended standard surgical procedures , there is a significant variety in the actual surgical procedures employed in the treatment of patients with endometrial cancer .", "the management of patients with cervical involvement remains controversial and treatment options range from extrafascial hysterectomy ( eh ) to radical hysterectomy ( rh ) , with or without radiation ( 3 , 4 ) .", "however , the literature regarding the treatment of these patients is limited by its low frequency .", "cervical extension of endometrial carcinoma represents about 10% to 15% of cases at the time of diagnosis ( 5 ) .", "such cervical invasion is considered to increase the risk of nodal metastasis ; and those patients are reported to have a worse prognosis than those without it ( 6 - 9 ) .", "the rationale for performing rh in patients with cervical involvement is to eradicate all possible parametrial involvement ( pmi ) .", "however , the preoperative detection of cervical involvement or parametrial disease is not entirely reliable despite the progress in imaging techniques such as magnetic resonance imaging ( 10 , 11 ) .", "thus , although the only definitive method of ensuring the status and extent of disease in the parametria is through radical excision and a histological study of the operative specimen , the unreliability of preoperative prediction , and the relative rarity of pmi may make it unwise to resort to rh as a matter of routine in patients with cervical invasion ( 12 ) .", "we reviewed the medical records of endometrial cancer patients with cervical involvement to determine the relation of parametrial spread with other histopathological variables and to evaluate whether the type of primary surgery using eh or rh alters the patients ' outcome from a histological perspective ." ], [ "the cancer database from five different institutes were reviewed ; patients surgically diagnosed with stage ii and a part of stage iii or iva endometrial cancer with cervical involvement during the period of 1993 - 2005 were identified .", "the recorded data were then extracted by a medically qualified gynecology trainee in each institute .", "the original pathology reports and operation reports of 133 patients were reviewed for the following information : age at diagnosis , pre - operative data , operative procedure , adjuvant treatment , follow - up data including date of recurrence , the patient status at last visit . a pathologic data such as figo stage , histologic type , tumor grade , depth of myometrial invasion , and the status of parametrium , and retroperitoneal lymph node were also reviewed .", "patients with mixed mllerian tumor , including carcinosarcoma or endometrial stromal sarcoma , were excluded from the review . when the tumor grade varied between pre - operative biopsy and final pathologic reports , the higher grade was recorded .", "confirmation of cervical invasion and staging was made by reviewing pathology reports , although pathology slides review was not performed .", "the term ' radical hysterectomy ' included modified hysterectomy ( mrh ) that could not be exactly differentiated in operating records .", "patients with surgically staged iia cancer who had undergone eh were selectively treated with adjuvant radiation according to the degree of depth of myometrial invasion . otherwise , most patients with stage iib disease or above underwent post - operative pelvic irradiation or brachytherapy .", "if sufficient follow - up information regarding survival and recurrence was unavailable through medical records , we referred to death certificates to obtain the required information ." ], [ "one hundred thirty - three patients with endometrial cancer stage iia to iva were found to have cervical invasion .", "ninety - three percent ( 124/133 ) of the patients underwent complete surgical staging with or without paraaortic lymph node dissection .", "we found no significant difference in age , body mass index ( bmi ) , distribution of figo stage , histology , and tumor grade between the eh and rh groups .", "preoperative evaluations for disease extent including cervical involvement were mostly done by magnetic resonance imaging ( mri ) and partly by computed tomography ( ct ) or endocervical curettage .", "pre - operative mri was performed in 63.9% ( 85/133 ) of the patients and sensitivity for cervical invasion was 44.7% ( 38/85 ) .", "when the cases with stromal invasion were analyzed , the sensitivity was 53.3% ( 24/45 ) .", "table 2 presents the data for the choice of the extent of hysterectomy according to the results of preoperative evaluation .", "eh was performed in 12/62 ( 19.4% ) patients who were preoperatively suspected of cervical involvement , and more than half ( 37/71 , 52.1% ) of the patients who underwent rh had no preoperative evidence of cervical invasion .", "the results show that many surgeons did not strictly follow the results of preoperative evaluation in choosing the type of hysterectomy and the decision regarding eh or rh was made at their own discretion . fig .", "1 shows the relationship between the depth of myometrial invasion and that of cervical stromal invasion .", "a total of 121 cases had available information about both about the depth of myometrial and presence of cervical stromal invasion . in patients with disease limited to the endometrium , no case showed cervical stromal invasion . on the other hand , a majority of patients with disease involving cervical stroma had deep myometrial invasion ( p=0.001 ) .", "table 3 showed the relationship between prognostic factors and parametrial involvement in 71 patients undergoing rh .", "eight of ten patients ( 80.0% ) had pelvic node metastasis , 1 patient had grossly positive adnexal involvement , and 3 had tumor spread outside the pelvis ( 2 of 3 had coexisting pelvic lymph node metastasis ) .", "depth of myometrial invasion and pelvic or paraaortic lymph node positivity were significantly correlated with paramatrial involvement .", "of the 19 patients with pelvic lymph node metastasis , 8 patients ( 42.1% ) had concomitant pmi .", "conversely , of the 10 patients with pmi , 8 ( 80.0% ) had lymph node metastasis .", "in 74 patients with surgical stage ii , i.e. , disease showing no demonstrable evidence of tumor spread in the pelvic cavity , rh was performed in 41 cases and no patients showed parametrial involvement .", "sixty six of 74 ( 89% ) patients underwent adjuvant radiation therapy ( 35 whole pelvic irradiation , 7 vaginal brachytherapy , 23 both , 1 unknown ) and there were 3 patients who developed recurrent disease in the rh and none in the eh group ( mean follow - up : 51 months , table 4 ) ." ], [ "our results show that a large number of korean gynecologic surgeons have a tendency to choose the surgical extent of hysterectomy through their own disposition and do not strictly adhere the results of pre - operative evaluation .", "actually , the type of hysterectomy procedure selected for endometrial cancer varies in each nation and in each institution .", "watanabe et al . ( 13 ) reported the results of a survey of a japanese group showing that more than 70% of institutes never perform rh without regarding the preoperative status of cervical involvement .", "moreover , according to a survey in north america , nearly 30% of centers never performed class ii or iii extended hysterectomy for the treatment of endometrial cancer ( 14 ) .", "as shown in table 2 , surgeons who overuse rh without regarding the pre - operative results may believe that the current pre - operative evaluation method is not sensitive enough to detect cervical invasion and thus , worry about possible pmi or post - operative stump recurrence . on the other hand , those who perform only eh despite the positive findings from pre - operative evaluation may believe that cervical stromal invasion should be followed by adjuvant radiotherapy and thus , the prognosis would not be changed by performing a ' high morbidity producing surgery ' considering the low incidence of pmi .", "this inconsistent treatment policy , in fact , necessitates the need for an evidenced - based reformed guideline to which most clinicians can conform , although the definition of rh or eh is not standardized among surgeons and thus , the actual extent of surgery investigated as rh or eh by medical records might be different for each patient and this is one of the limitations of this study .", "our results also showed that the pmi rate in patients with cervical invasion is about 14% in the rh group .", "this is within the range reported by other investigators , and is not significantly lower than reported values ( 15 - 17 ) .", "however , parametrial spread was not found in any of the patients with figo stage ii , i.e. , patients showing no intraoperative tumor spread , but was identified in 8 patients with positive pelvic lymph nodes , and two showed grossly frank extrauterine spread around the adnexa and another site in the pelvic cavity .", "these findings indicate that a risk of leaving an occult metastasis in parametrial tissue when performing only eh in patients without evidence of extrauterine tumor spread seems to be very low , even though all lymph node metastases may not be completely screened by intraoperative frozen section .", "although they reported a relatively high rate of parametrial involvement , the points we focused on is not the crude value of the pmi itself , but the necessity of parametrial resection .", "undoubtedly , cervical stromal invasion is known to increase the risk of pmi . however , the pattern of spread in the invaded cervix of endometrial cancer is quite different from that of primary cervical carcinoma . as shown in fig .", "1 , the pattern of cervical spread of endometrial cancer seems to follow myometrial invasion and originate from the endocervical glandular region , and as it advances , it invades the stroma .", "therefore , we can speculate that before the tumor of the endometrium reaches the deep myometrium , a chance to detect direct pmi in the patients with suspicious cervical invasion seems to be low .", "however , pmi in endometrial cancer can not be completely excluded only by the absence of cervical stromal invasion because pmi is reported to consist histologically of 2 patterns of spread : direct invasion of cancer cells to the parametrial connective tissues like that seen in cervical cancer , and parametrial lymph - vascular space invasion frequently seen in patients with deep myometrial involvement without cervical involvement ( 17 - 19 ) .", "current guidelines for endometrial cancer show that cervical stromal invasion is regarded as one of the major indications of adjuvant radiotherapy .", "we might guess that adjuvant radiation is required due to the implication that cervical stromal invasion raises the risk of recurrence not just by direct pmi from the cervix , but also by increasing the risk of occult parametrial involvement from the preceding deep myometrial invasion or myometrial lymph - vascular space invasion .", "suffice it to say , this suggest that performing rh only in patients with cervical involvement can not guarantee the complete eradication of possible parametrial involvement . in addition", ", it is very likely that we may have ' cut through ' the invaded parametrium in stage ii patients who underwent eh even though it was not revealed by pathology because there was no direct connection between the invaded cervical stroma and parametrium itself . even acknowledging that occult parametrial spread had existed", ", our data showed that no recurrences occurred in patients who only underwent eh followed by adjuvant radiation and thus , it suggests that occult parametrial metastasis in patients with no gross extrauterine spread can be successfully controlled by radiation therapy .", "we could not evaluate the value of rh in patients with stage iii or over because their survival outcome is much likely to be influenced by variables other than parametrial involvement , i.e. , lymph node status , extent of tumor spread , different treatment strategy to the recurred patients , etc .", "we did not overcome the limitations of retrospective analysis , i.e. , lack of verifying the actually performed surgical extent , lack of centralization of pathologic review , and limited number of cases .", "nonetheless , we cautiously suggest that routine rh in patients with cervical invasion and having no grossly positive extrauterine spread should be reappraised and that these patients are worthy of consideration for less radical surgery performed in conjunction with pelvic and paraaortic lymphadenectomy considering the low rate of parametrial involvement , inaccurate preoperative prediction , high post operative morbidity , and successful control through adjuvant radiation . since this is an area of continued debate and a principal shift from our current practice and attitudes , a randomized study would be required to define the low - risk population that would have a modification of the extent of parametrial resection performed . in this", "regards , we can conclude that the real value of rh in endometrial cancer patients with suspicious cervical involvement can be evaluated by performing a large scaled randomized controlled study comparing the survival and quality of life between the group who underwent rh only or followed by tailored adjuvant radiation and the group with eh and routine adjuvant radiation therapy ." ] ]

[ "pyrazolines are nitrogen - containing heterocyclic compounds , well known for their pronounced biological activity .", "it was demonstrated that the combination of pyrazole with azole ring , linked to each by one sigma bond , led to more biologically active targets ; for example , pyrazolylthiazoles showed excellent antimicrobial activities . continuing our work in this research field [ 69 ] and in an attempt to identify new and potent antimicrobial agents", ", we tried here to generate new benzofuryl 2-pyrazolin-1-ylthiazoles as antimicrobial agents using simple methods .", "the starting pyrazoline-1-carbothioamide 5 was prepared by treatment of 2-acetylbenzofuran 1 with equivalent of 4-flurobenzaldehyde 2 in the presence of 10% alcoholic naoh in 90% ethanol with stirring at room temperature to give chaconne 3 .", "reaction of chalcone 3 with equivalent amount of thiosemicarbazide was performed in ethanol in the presence of 2.5 equivalent of sodium hydroxide to the target precursor 5 .", "the resulting pyrazoline-1-carbothioamide 5 was cyclized to the corresponding 2-(3-(benzofuran-2-yl)-5-(4-fluorophenyl)-4,5-dihydro-1h - pyrazol-1-yl)-4-methyl-5-(p - subs.phenyldiazenyl)thiazole derivatives 8a d by reaction with hydrazonoyl halides 6a d in anhydrous ethanol and in the presence of an equivalent of triethylamine ( scheme 1 ) .", "the reaction product structures were elucidated by means of nmr , ms spectroscopy , and elemental analyses ( table 1 ) .", "h nmr spectra of 8a d contained two doublet - doublet and one triplet signals due to the presence of ch2 adjacent asymmetric carbon .", "the mass spectra of 8a d showed the molecular ion peaks at m / z 481 , 499 , 515 and 561 , respectively in agreement with the calculated masses .", "the reaction between pyrazoline-1-carbothioamide 5 and the equivalent amount of -haloketones , for example , phenacyl bromides derivatives , 2-bromoacetylbenzofuran , and 3-bromoacetylcoumarin , was performed in refluxing ethanol to yield pyrazol-1-ylthiazoles 1315 in good yields via the intermediates a ( scheme 2 ) .", "the h nmr of compounds 1316 indicate the disappearance of nh2 signal due to blocking of nh2 with thiazole nucleus .", "the mass spectra of 13a , b16 showed the molecular ion peaks at m / z 473 , 518 , 479 , 507 , and 465 , respectively in agreement with the calculated masses .", "all the new synthesized compounds were screened for their antibacterial and antifungal activities at 100 g / ml concentration against four gram - positive bacteria ( staphylococcus aureus atcc 29213 ; b. subtilis atcc6633 ; b. megaterium atcc 9885 ; sarcina lutea ) , three gram - negative bacteria ( klebsiella pneumoniae atcc13883 ; pseudomonas aeruginosa atcc27953 ; e. coli atcc 25922 ) , and two yeast ( saccharomyces cerevisiae and candida albicans nrrl y-477 ) .", "most of the newly synthesized compounds showed good antimicrobial activities with respect to the control drugs .", "compound 8c exhibited the highest potency against all tested organisms with respect to reference drugs .", "compounds 8d and 13b inhibited the growth of staphylococcus aureus atcc 29213 with inhibition zones 23 , 22 mm , respectively , while compound 5 showed excellent activities against klebsiella pneumoniae atcc13883 ; pseudomonas aeruginosa atcc27953 ; and e. coli atcc 25922 with inhibition zone about 24 mm . also , compound 8c showed the highest activity against staphylococcus aureus atcc 29213 , saccharomyces cerevisiae , and candida albicans nrrl y-477 with inhibition zone about 23 mm .", "the minimum inhibitory concentration ( mic ) of the synthesized compounds against highly inhibited organisms is reported in table 3 .", "compounds 5 revealed low mic ( 200 g / ml ) against staphylococcus aureus atcc 29213 , b. megaterium atcc 9885 , and candida albicans nrrl", ", compound 8a exhibited high mic ( 16 g / ml ) against b. subtilis atcc6633 ( table 3 ) .", "elemental analytical data were carried from the microanalytical unit , cairo university , giza , egypt .", "the ir spectra were recorded in potassium bromide disks on a jasco ft / ir-6100 .", "h nmr spectra were run on joel - eca 500 mhz in deuterateddimethyl sulphoxide ( dmso - d6 ) .", "chemical shifts values ( ) are given in parts per million ( ppm ) .", "the mass spectra were performed using mass varian mat ch-5 spectrometer at 70 ev .", "( e)-1-(benzofuran-2-yl)-3-(4-fluorophenyl)prop-2-en-1-one 3 ; hydrazonoyl halides ; 1-(benzofuran-2-yl)-2-bromoethanone 9 ; 3-(2-bromoacetyl)-2h - chromen-2-one 10 ; and 2,3-dichloroquinoxaline were prepared according to the literature . to a suspension of chalcone 3", "( 10 mmol , 2.66 g ) and sodium hydroxide ( 25 mmol , 1.0 g ) in ethanol ( 50 ml ) , thiosemicarbazide ( 12 mmol , 1.1 g ) was added .", "the mixture was refluxed for 12 h , then left to cool ; the solid product was filtered off , washed with ethanol , and dried . yield 52% ; m.p .", ") max / cm 3460 , 3335 ( nh2 ) ; h nmr ( dmso - d6 ) 3.09 , 3.14 ( dd , 1h , ch , j = 3.05 hz , j = 3.05 hz ) , 3.94 , 4.06 ( dd , 1h , ch , j = 11.45 hz , j = 11.5 hz ) , 5.88 ( t , 1h , ch , j = 3.05 hz , j = 7.65 hz ) , 7.137.43 ( m , 9h , ar - h ) , 9.44 ( s , 2h , nh2 , d2o - exchangeable ) ; ms m / z ( % ) : 339 ( m , 75 ) , 60 ( 100 ) . to a suspension of compound 5 ( 1 mmol , 0.34 g ) in ethanol ( 20 ml ) , the 1 mmol of appropriate reagent { ( appropriate hydrozonoyl chlorides 6 + et3n ) ; ( appropriate phenacyl bromides 9 ) ; ( 2-bromoacetylbenzofuran 10 ) ; ( 3-bromoacetylcoumarin 11 ) ; or ( 3,4-dichloroquinoxaline 12 ) } was added and heated under reflux for 4 h. after cooling , the precipitate was collected by suction filtration . yield 58% ; m.p.180 - 2c ; h nmr ( dmso - d6 ) 2.54 ( s , 3h , ch3 ) , 4.09 , 4.11 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.87 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.187.73 ( m , 14h , ar - h ) ; ms m / z ( % ) : 481 ( m , 75 ) , 95 ( 100 ) . yield 66% ; m.p . 201 - 3c ; h nmr ( dmso - d6 ) 2.54 ( s , 3h , ch3 ) , 4.07 , 4.12 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.89 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.73 ( m , 13h , ar - h ) ; ms m / z ( % ) : 499 ( m , 80 ) , 95 ( 100 ) . yield 72% ; m.p . 206 - 8c ; h nmr ( dmso - d6 ) 2.53 ( s , 3h , ch3 ) , 4.10 , 4.34 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.89 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.73 ( m , 13h , ar - h ) ; ms m / z ( % ) : 515 ( m , 70 ) , 95 ( 100 ) .", "128 - 30c ; h nmr ( dmso - d6 ) 2.55 ( s , 3h , ch3 ) , 4.10 , 4.34 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.87 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.72 ( m , 13h , ar - h ) ; ms m / z ( % ) : 561 ( m , 62 ) , 95 ( 100 ) . yield 49% ; m.p .", "4.014.05 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.73 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.177.92 ( m , 14h , ar - h ) ; ms m / z ( % ) : 473 ( m , 80 ) , 91 ( 100 ) . yield 58% ; m.p . 208 - 10c ; h nmr ( dmso - d6 ) 4.014.08 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.71 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.177.92 ( m , 14h , ar - h ) ; ms m / z ( % ) : 518 ( m , 49 ) , 91 ( 100 ) . yield 63% ; m.p . 252 - 4c ; h nmr ( dmso - d6 ) 4.044.07 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.73 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 6.88 ( s , 2h , benzofuryl - h ) , 7.197.70 ( m , 13h , ar - h ) ; ms m / z ( % ) : 479 ( m , 100 ) .", "233 - 4c ; h nmr ( dmso - d6 ) 4.05 , 4.08 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.72 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.227.72 ( m , 14h , ar - h ) , 8.23 ( s , 1h , coumarinyl - h ) ; ms m / z ( % ) : 507 ( m , 100 ) .", "; h nmr ( dmso - d6 ) 4.02 , 4.06 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.70 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.227.72 ( m , 13h , ar - h ) ; ms m / z ( % ) : 465 ( m , 100 ) .", "chemical compounds were individually tested against a panel of gram - positive and gram - negative bacterial pathogens and yeast .", "antimicrobial tests were carried out by the agar well diffusion method using 100 l of suspension containing 1 10 cfu / ml of pathological tested bacteria and 1 10 cfu / ml of yeast spread on nutrient agar ( na ) and sabourund dextrose agar ( sda ) , respectively . after the media had cooled and solidified , wells ( 10 mm in diameter )", "were made in the solidified agar and loaded with 100 l of tested compound solution prepared by dissolving 100 mg of the chemical compound in one ml of dimethyl sulfoxide ( dmso ) .", "the inculcated plates were then incubated for 24 h at 37c for bacteria and at 28c for yeast .", "ciprofloxacin ( 50 g / ml ) and ketoconazole ( 50 g / ml ) were used as standard for antibacterial and antifungal activities respectively .", "after incubation time , antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms and compared with that of the standard .", "the experiment was carried out in triplicate , and the average zone of inhibition was calculated .", "the bacteriostatic activity of the active compounds ( having inhibition zones ( iz ) 18 mm ) was then evaluated using the twofold serial dilution technique .", "twofold serial dilutions of the tested compounds solutions were prepared using the proper nutrient broth .", "the final concentration of the solutions was 200 , 100 , 50 , and 25 g / ml .", "the tubes were then inoculated with the test organisms , grown in their suitable broth at 37c for 24 hours for bacteria ( about 1 10 cfu / ml and 1 10 cfu / ml of yeast ) , and each 5 ml received 0.1 ml of the above inoculum and incubated at 37c for 24 hours . the lowest concentration showing no growth was taken as the minimum inhibitory concentration ( mic ) .", "this precursor reacted with thiosemicarbazide in strong basic medium to afford the n - pyrazoline thioamide 5 .", "the new compounds were tested for their antimicrobial activities and significant activities due to presence of three nucleuses : benzofuran , pyrazole , and thiazole .", "also , some substituent increases the antimicrobial activities such as chloro substituent in compounds 8c and 13a ." ]

[ "<S> a new series of 2-pyrazolin-1-ylthiazoles 8a d and 1316 was synthesized by cyclization of n - thiocarboxamide-2-pyrazoline with different haloketones and 2,3-dichloroquinoxaline . the structures of the new compounds were confirmed by elemental analyses as well as nmr , ir , and mass spectral data . </S>", "<S> the newly synthesized compounds were evaluated for their antimicrobial activities , and also their minimum inhibitory concentration ( mic ) against most of test organisms was performed . amongst the tested ones , </S>", "<S> compound 8c displayed excellent antimicrobial activity . </S>" ]

[ "1. Introduction", "2. Results and Discussion", "3. Experimental", "4. Conclusion" ]

[ [ "pyrazolines are nitrogen - containing heterocyclic compounds , well known for their pronounced biological activity .", "it was demonstrated that the combination of pyrazole with azole ring , linked to each by one sigma bond , led to more biologically active targets ; for example , pyrazolylthiazoles showed excellent antimicrobial activities . continuing our work in this research field [ 69 ] and in an attempt to identify new and potent antimicrobial agents", ", we tried here to generate new benzofuryl 2-pyrazolin-1-ylthiazoles as antimicrobial agents using simple methods ." ], [ "the starting pyrazoline-1-carbothioamide 5 was prepared by treatment of 2-acetylbenzofuran 1 with equivalent of 4-flurobenzaldehyde 2 in the presence of 10% alcoholic naoh in 90% ethanol with stirring at room temperature to give chaconne 3 .", "reaction of chalcone 3 with equivalent amount of thiosemicarbazide was performed in ethanol in the presence of 2.5 equivalent of sodium hydroxide to the target precursor 5 .", "the resulting pyrazoline-1-carbothioamide 5 was cyclized to the corresponding 2-(3-(benzofuran-2-yl)-5-(4-fluorophenyl)-4,5-dihydro-1h - pyrazol-1-yl)-4-methyl-5-(p - subs.phenyldiazenyl)thiazole derivatives 8a d by reaction with hydrazonoyl halides 6a d in anhydrous ethanol and in the presence of an equivalent of triethylamine ( scheme 1 ) .", "the reaction product structures were elucidated by means of nmr , ms spectroscopy , and elemental analyses ( table 1 ) .", "h nmr spectra of 8a d contained two doublet - doublet and one triplet signals due to the presence of ch2 adjacent asymmetric carbon .", "the mass spectra of 8a d showed the molecular ion peaks at m / z 481 , 499 , 515 and 561 , respectively in agreement with the calculated masses .", "the reaction between pyrazoline-1-carbothioamide 5 and the equivalent amount of -haloketones , for example , phenacyl bromides derivatives , 2-bromoacetylbenzofuran , and 3-bromoacetylcoumarin , was performed in refluxing ethanol to yield pyrazol-1-ylthiazoles 1315 in good yields via the intermediates a ( scheme 2 ) .", "the h nmr of compounds 1316 indicate the disappearance of nh2 signal due to blocking of nh2 with thiazole nucleus .", "the mass spectra of 13a , b16 showed the molecular ion peaks at m / z 473 , 518 , 479 , 507 , and 465 , respectively in agreement with the calculated masses .", "all the new synthesized compounds were screened for their antibacterial and antifungal activities at 100 g / ml concentration against four gram - positive bacteria ( staphylococcus aureus atcc 29213 ; b. subtilis atcc6633 ; b. megaterium atcc 9885 ; sarcina lutea ) , three gram - negative bacteria ( klebsiella pneumoniae atcc13883 ; pseudomonas aeruginosa atcc27953 ; e. coli atcc 25922 ) , and two yeast ( saccharomyces cerevisiae and candida albicans nrrl y-477 ) .", "most of the newly synthesized compounds showed good antimicrobial activities with respect to the control drugs .", "compound 8c exhibited the highest potency against all tested organisms with respect to reference drugs .", "compounds 8d and 13b inhibited the growth of staphylococcus aureus atcc 29213 with inhibition zones 23 , 22 mm , respectively , while compound 5 showed excellent activities against klebsiella pneumoniae atcc13883 ; pseudomonas aeruginosa atcc27953 ; and e. coli atcc 25922 with inhibition zone about 24 mm . also , compound 8c showed the highest activity against staphylococcus aureus atcc 29213 , saccharomyces cerevisiae , and candida albicans nrrl y-477 with inhibition zone about 23 mm .", "the minimum inhibitory concentration ( mic ) of the synthesized compounds against highly inhibited organisms is reported in table 3 .", "compounds 5 revealed low mic ( 200 g / ml ) against staphylococcus aureus atcc 29213 , b. megaterium atcc 9885 , and candida albicans nrrl", ", compound 8a exhibited high mic ( 16 g / ml ) against b. subtilis atcc6633 ( table 3 ) ." ], [ "elemental analytical data were carried from the microanalytical unit , cairo university , giza , egypt .", "the ir spectra were recorded in potassium bromide disks on a jasco ft / ir-6100 .", "h nmr spectra were run on joel - eca 500 mhz in deuterateddimethyl sulphoxide ( dmso - d6 ) .", "chemical shifts values ( ) are given in parts per million ( ppm ) .", "the mass spectra were performed using mass varian mat ch-5 spectrometer at 70 ev .", "( e)-1-(benzofuran-2-yl)-3-(4-fluorophenyl)prop-2-en-1-one 3 ; hydrazonoyl halides ; 1-(benzofuran-2-yl)-2-bromoethanone 9 ; 3-(2-bromoacetyl)-2h - chromen-2-one 10 ; and 2,3-dichloroquinoxaline were prepared according to the literature . to a suspension of chalcone 3", "( 10 mmol , 2.66 g ) and sodium hydroxide ( 25 mmol , 1.0 g ) in ethanol ( 50 ml ) , thiosemicarbazide ( 12 mmol , 1.1 g ) was added .", "the mixture was refluxed for 12 h , then left to cool ; the solid product was filtered off , washed with ethanol , and dried . yield 52% ; m.p .", ") max / cm 3460 , 3335 ( nh2 ) ; h nmr ( dmso - d6 ) 3.09 , 3.14 ( dd , 1h , ch , j = 3.05 hz , j = 3.05 hz ) , 3.94 , 4.06 ( dd , 1h , ch , j = 11.45 hz , j = 11.5 hz ) , 5.88 ( t , 1h , ch , j = 3.05 hz , j = 7.65 hz ) , 7.137.43 ( m , 9h , ar - h ) , 9.44 ( s , 2h , nh2 , d2o - exchangeable ) ; ms m / z ( % ) : 339 ( m , 75 ) , 60 ( 100 ) . to a suspension of compound 5 ( 1 mmol , 0.34 g ) in ethanol ( 20 ml ) , the 1 mmol of appropriate reagent { ( appropriate hydrozonoyl chlorides 6 + et3n ) ; ( appropriate phenacyl bromides 9 ) ; ( 2-bromoacetylbenzofuran 10 ) ; ( 3-bromoacetylcoumarin 11 ) ; or ( 3,4-dichloroquinoxaline 12 ) } was added and heated under reflux for 4 h. after cooling , the precipitate was collected by suction filtration . yield 58% ; m.p.180 - 2c ; h nmr ( dmso - d6 ) 2.54 ( s , 3h , ch3 ) , 4.09 , 4.11 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.87 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.187.73 ( m , 14h , ar - h ) ; ms m / z ( % ) : 481 ( m , 75 ) , 95 ( 100 ) . yield 66% ; m.p . 201 - 3c ; h nmr ( dmso - d6 ) 2.54 ( s , 3h , ch3 ) , 4.07 , 4.12 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.89 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.73 ( m , 13h , ar - h ) ; ms m / z ( % ) : 499 ( m , 80 ) , 95 ( 100 ) . yield 72% ; m.p . 206 - 8c ; h nmr ( dmso - d6 ) 2.53 ( s , 3h , ch3 ) , 4.10 , 4.34 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.89 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.73 ( m , 13h , ar - h ) ; ms m / z ( % ) : 515 ( m , 70 ) , 95 ( 100 ) .", "128 - 30c ; h nmr ( dmso - d6 ) 2.55 ( s , 3h , ch3 ) , 4.10 , 4.34 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.87 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.72 ( m , 13h , ar - h ) ; ms m / z ( % ) : 561 ( m , 62 ) , 95 ( 100 ) . yield 49% ; m.p .", "4.014.05 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.73 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.177.92 ( m , 14h , ar - h ) ; ms m / z ( % ) : 473 ( m , 80 ) , 91 ( 100 ) . yield 58% ; m.p . 208 - 10c ; h nmr ( dmso - d6 ) 4.014.08 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.71 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.177.92 ( m , 14h , ar - h ) ; ms m / z ( % ) : 518 ( m , 49 ) , 91 ( 100 ) . yield 63% ; m.p . 252 - 4c ; h nmr ( dmso - d6 ) 4.044.07 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.73 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 6.88 ( s , 2h , benzofuryl - h ) , 7.197.70 ( m , 13h , ar - h ) ; ms m / z ( % ) : 479 ( m , 100 ) .", "233 - 4c ; h nmr ( dmso - d6 ) 4.05 , 4.08 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.72 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.227.72 ( m , 14h , ar - h ) , 8.23 ( s , 1h , coumarinyl - h ) ; ms m / z ( % ) : 507 ( m , 100 ) .", "; h nmr ( dmso - d6 ) 4.02 , 4.06 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.70 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.227.72 ( m , 13h , ar - h ) ; ms m / z ( % ) : 465 ( m , 100 ) .", "chemical compounds were individually tested against a panel of gram - positive and gram - negative bacterial pathogens and yeast .", "antimicrobial tests were carried out by the agar well diffusion method using 100 l of suspension containing 1 10 cfu / ml of pathological tested bacteria and 1 10 cfu / ml of yeast spread on nutrient agar ( na ) and sabourund dextrose agar ( sda ) , respectively . after the media had cooled and solidified , wells ( 10 mm in diameter )", "were made in the solidified agar and loaded with 100 l of tested compound solution prepared by dissolving 100 mg of the chemical compound in one ml of dimethyl sulfoxide ( dmso ) .", "the inculcated plates were then incubated for 24 h at 37c for bacteria and at 28c for yeast .", "ciprofloxacin ( 50 g / ml ) and ketoconazole ( 50 g / ml ) were used as standard for antibacterial and antifungal activities respectively .", "after incubation time , antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms and compared with that of the standard .", "the experiment was carried out in triplicate , and the average zone of inhibition was calculated .", "the bacteriostatic activity of the active compounds ( having inhibition zones ( iz ) 18 mm ) was then evaluated using the twofold serial dilution technique .", "twofold serial dilutions of the tested compounds solutions were prepared using the proper nutrient broth .", "the final concentration of the solutions was 200 , 100 , 50 , and 25 g / ml .", "the tubes were then inoculated with the test organisms , grown in their suitable broth at 37c for 24 hours for bacteria ( about 1 10 cfu / ml and 1 10 cfu / ml of yeast ) , and each 5 ml received 0.1 ml of the above inoculum and incubated at 37c for 24 hours . the lowest concentration showing no growth was taken as the minimum inhibitory concentration ( mic ) ." ], [ "this precursor reacted with thiosemicarbazide in strong basic medium to afford the n - pyrazoline thioamide 5 .", "the new compounds were tested for their antimicrobial activities and significant activities due to presence of three nucleuses : benzofuran , pyrazole , and thiazole .", "also , some substituent increases the antimicrobial activities such as chloro substituent in compounds 8c and 13a ." ] ]

[ "there is increasing evidence linking endoplasmic reticulum ( er ) stress coping responses with the development of diseases .", "this is not surprising , given that the er is a multifunctional essential organelle in eukaryotic cells ; it functions as the critical entrance to the secretory pathways , and is also a regulator of ca homeostasis , as well as protein and lipid synthesis , all of which are highly sensitive to a variety of environmental , physiological , and pathophysiological conditions .", "the er is also closely associated with all cellular organelles , and therefore , capable of driving sophisticated mechanisms of intracellular signaling , including activation of transcriptional processes , ca mobilization , and influencing energy metabolism . in this review", ", we briefly summarize the er stress coping responses , particularly the unfolded protein response ( upr ) , and discuss the potential role of upr as a physiologically relevant pathway in supporting and controlling proper embryonic development .", "it impairs er functions such as protein folding and the heat shock response , and leads to altered ca levels and nutrient starvation .", "activation of er stress can result in the buildup of misfolded proteins in the er and activation of the upr as a coping strategy ( figure 1 ) .", "the upr involves distinct components designed to reestablish the protein synthesis machinery ( figure 1 ) .", "these include ( 1 ) translational attenuation to arrest the entry of new proteins into the er ; ( 2 ) transcriptional activation of genes encoding proteins involved in protein folding to assist the maturation of proteins ; ( 3 ) transcriptional activation of genes for components of the er - associated protein degradation ( erad ) system to decrease the number of misfolded proteins ; and if these are not successful , ( 4 ) activation of apoptotic pathways to eliminate defective cells .", "three er transmembrane proteins : dsrna - activated protein kinase - like er kinase ( perk ) , activating transcription factor 6 ( atf6 ) , and inositol - requiring enzyme 1 ( ire1 ) , in combination with the er molecular chaperone immunoglobulin binding protein ( bip , also known as glucose - regulated protein [ grp ] 78 ) , compose the upr , an er stress coping response .", "it is a monomeric , globular protein that functionally sorts and releases terminally misfolded substrates to the erad pathway . under non - stressed conditions grp78/bip interacts with ire , perk , and atf6 , but upon an increase in misfolded proteins", ", grp78/bip is sequestered away from these inducers , leading to the activation of the upr .", "release of grp78/bip from perk triggers its dimerization and autophosphorylation , followed by phosphorylation of eif2 , at ser , which inhibits protein synthesis by sequestering the trnamet responsible for initiating the translation of nascent protein ( figure 1 ) . during this period ,", "atf4 transcriptional activity induces both pro - survival ( early ) and pro - apoptotic ( late ) transcriptional programs .", "atf6 is an er transmembrane protein cleaved in the golgi . under non - stress conditions", "when bip is sequestered away from atf6 , the atf6 is translocated to the golgi , where it undergoes cleavage by site-1 and site-2 proteases ( s1p and s2p ) ( figure 1 ) .", "cleavage of atf6 produces a soluble basic leucine zipper ( bzip ) transcription factor ( cleaved atf6 ) that binds to er stress response elements ( erse - i and ii ) to induce transcriptional activation of er stress response genes .", "atf6 can induce the expression upr - induced genes including grp78/bip , proteins involved in erad , and xbp1 mrna .", "interestingly , er stress triggered by ca depletion induces the formation of a nascent , partially glycosylated form of atf6 with reduced interaction with calreticulin , and a faster rate of traversing the golgi , resulting in higher transactivation of atf6 gene targets . upon release of grp78/bip under stress conditions , ire1 homodimerizes , causing a conformational change that is transmitted across the membrane , leading to activation of its cytoplasmic kinase activity , autophosphorylation , and activation of its endoribonuclease activity ( figure 1 ) .", "ire1 endoribonuclease activity cleaves 28s rrna and specific micrornas , inhibits protein synthesis , and also splices xbp1", "the protein product from the sxbp1 variant binds to the specific promoter elements , erse , and upre , triggering transactivation upr responsive genes such as grp78/bip , calreticulin , and others , which are part of the protein folding quality control and degradation machinery .", "one part of the cellular recovery system that is turned on by the upr is er - associated degradation ( erad ) .", "there is tight regulation of the three arms of the upr with respect to the timing and response amplitude .", "the activation of each arm of the upr is specific to the source of er stress and governs cell fate , supporting either an adaptive response ( cell survival ) or a maladaptive response ( cell death ) . under prolonged and irreversible er stress ,", ", upr activates autophagy , a controlled self - degradation process that can promote cell survival by eliminating damaged cellular components .", "the intensity , timing , and duration of the upr appear to be important in determining cell fate .", "these observations suggest that er stress coping responses may also play an important role in the maintenance of physiological events associated with organ function and/or development .", "although there is emerging evidence that activation of er stress coping responses , including upr , may play an important role in diverse human diseases , it is conceivable that these coping responses may also be critical under normal physiological conditions and during development .", "interestingly , there exists a group of small molecules that may stabilize protein folding , prevent protein aggregation , and consequently affect the severity of upr activation .", "the most studied of these molecules are synthetic - fatty acid derivative 4-phenylbutyric acid and the natural bile acid tauroursodeoxycholic acid ( tudca ) . although the precise mechanisms of their action are not clear at present , these compounds have a profound impact on many pathologies including obesity pathways , brain ischemic - reperfusion , photoreceptor pathology , and steatosis , to name a few .", "these findings suggest that targeting er physiology and er stress signaling and stress - induced coping responses has therapeutic potential for treating diseases related to er stress .", "the development of preimplantation embryos is under the influence of various hormones and growth factors that originate from maternal tissues and/or the embryo proper .", "preimplantation embryos synthesize and secrete a wide range of hormones and growth factors that promote embryonic survival ; in the blastocyst stage , they increase in cell number via activation of transcription as well as protein synthesis . during this synthetic activity", ", the embryos may experience an inherent level of er stress and consequently activate specific coping responses to recover homeostasis and support development or death .", "for example , in the mouse , the upr contributes to preimplantation embryo death in the ddk syndrome , suggesting that the er stress coping mechanism counters the stresses intrinsic to embryos .", "mammalian early embryos placed in vitro are vulnerable to a variety of physicochemical stresses such as shearing , temperature changes , altered ph , and higher oxygen pressure , all of which are known inducers of er stress coping responses .", "these stresses alter gene expression , epigenetic mechanisms , and metabolism , reducing the embryonic development and viability .", "although the influence of er stress coping responses on cellular physiology in adult tissues has been widely studied , their effects on the oocytes and early embryos of mammals remains unclear .", "in a porcine model , the functional abundance of sxbp1 is low in mature oocytes and at the 1- , 2- , and 8-cell stages of embryos , but it is highly abundant at the germinal vesicle , 4-cell , morula , and blastocyst stages . in mouse cumulus - oocyte complexes , fatty acid - induced er stress impairs protein secretion , mitochondrial membrane potential , and embryo development .", "er stress is a key mechanism mediating the developmental potential of oocytes . in mouse oocytes ,", "the cancer drug doxorubicin affects the mitochondria , perk , and er - associated caspase-12 , while it inactivates parp , resulting in the activation of apoptotic signals .", "this suggests that activation of er stress coping responses may contribute , at least in part , to ovarian failure in women treated for cancer . in mouse preimplantation embryos , er stress - induced coping response molecules such as perk , ask1 , bip , chop , gadd34 , ire1 , atf4 , atf6 , and xbp1 are expressed . taking into account that perk , ire1 , and atf6 are the three major transducers of the upr , the expression of the major transducers of the upr suggests that preimplantation embryos use the upr coping response as one mechanism to deal with preimplantation stress . in mice lacking upr - associated genes such as grp78 and ppp1r15a / ppp1r15b", ", the embryos do not survive beyond preimplantation development , suggesting the indispensable role of these er chaperones and er stress - induced coping responses in preimplantation embryos .", "xbp1 is an important transcription factor and regulator of a subset of genes activated during er stress - induced upr . in drosophila , xenopus , and mice , xbp1 is essential for early embryonic development . in mice ,", "functional sxbp1 is abundant in the germinal vesicle of immature oocytes and on spindles in mii oocytes but is solely detected in the cytoplasm at the one - cell stage when sxbp1 mrna is scarce .", "this indicates that sxbp1 acts as an important regulator of er stress - induced coping responses during specific developmental stages from oocyte to blastocyst . in mouse embryos ,", "upr inducers such as tunicamycin and sorbitol increased nuclear sxbp1 at the one- and two - cell stages and activate xbp1 \n mrna splicing at the 8-cell , morula , and blastocyst stages , indicating that the ire arm of the upr is activated as an important coping response and adaptation to er stress in preimplantation embryos . in early mouse embryos ,", "grp78/bip ( hspa5 ) , a central regulator for the er stress - induced upr coping response , is essential for cell proliferation and protecting the inner cell mass from apoptosis . considering the importance of stress - activated protein kinase mitogen - activated protein kinase kinase ( sapk - map3k ) activation during cellular stress and the early stage of er stress , as well as the indispensable role of stress - activated proteins and map3k for preimplantation embryonic development ,", "er stress manifested by sapk - map3k is a normal event critical during preimplantation development .", "the coupling of autophagy and upr has been implicated in a variety of biological processes .", "induction of autophagy promotes preattachment development of in vitro - produced bovine embryos by reducing er stress , suggesting that autophagy / er stress balance is important for the developmental competence of early embryos .", "the inhibition of er stress - induced upr responses by tudca was found to enhance the maturation and developmental potential of porcine oocytes by preventing er stress - mediated apoptosis in vitro . in mouse cumulus - oocyte complexes , fatty acid - induced er stress", "coping responses are normalized by the upr inhibitor salubrinal , demonstrating that er stress is a key mechanism affecting oocyte developmental potential .", "preimplantation embryos , under in vitro conditions , experience an increase in reactive oxygen species ( ros ) , oxidative stress , heat - shock stress , and/or the culture stress due to insufficiency of maternally - derived embryotropic factors .", "growth factors and ros scavengers have been successfully used as a medium supplement in assisted reproductive technology ( art ) programs to prevent poor outcomes for embryo survival under in vitro conditions .", "tudca attenuates xbp1 mrna splicing in embryos at the two - cell stage and abolishes dna fragmentation induced by tunicamycin or sorbitol in blastocysts and recovered nuclear localization of the sxbp1 protein .", "in addition , tudca inhibits hyperosmolar - induced er stress coping responses as well as the er stress - induced apoptotic coping response during preimplantation embryo development .", "er stress - induced coping responses may also play a critical role for embryo development after implantation . indeed , increased er stress in decidual tissue in pregnancy has been shown to be implicated in fetal growth restriction with and without pre - eclampsia .", "sustained er stress acts as a cofactor of oxidative stress in decidual cells from patients with early pregnancy loss .", "in decidual cells , excessive oxidative stress influences upr pathways to activate erad by decreasing valosin - containing protein , which is a type ii er - associated protein and a member of the aaaatpase family that facilitates delivery of misfolded proteins to the proteasome , which results in cell damage , inhibition of cell growth , and activation of apoptosis . among upr constituents , p - eif2 , grp94 , and", "c / ebp homologous protein ( chop ) are increased in the placenta from intrauterine growth restriction and pre - eclampsia , indicating that er stress responses are also inducible under diseased conditions .", "inositol - requiring enzyme 1 ( ire1 ) or xbp1 null mice are unable to produce functional placentas , which is lethal to the embryo , indicating that the ire1 arm of the upr coping response is essential for placental development and embryonic viability . in mice ,", "cadmium - induced teratogenicity is associated with ros - mediated induction of er stress coping responses in the placenta .", "maternal exposure to cadmium resulted in activation of perk , phosphorylation of placental eif2 , and an increase in chop , indicating that upr signaling is activated in the placenta due to cadmium - induced toxicity .", "this further supports the notion that er stress - induced coping responses are important mechanisms in embryos and extraembryonic tissues following exposure to environmental toxins .", "furthermore , er stress - induced coping responses in the maternal decidua and placenta may counteract developmental problems during implantation and postimplantation development .", "stress coping mechanisms are critical to minimizing or overcoming damage caused by ever changing environmental conditions .", "the upr pathway is mobilized in response to the accumulation of unfolded proteins and to ultimately regain er homeostasis .", "various arms of the upr , an er stress coping response , are expressed in oocytes and preimplantation embryos , suggesting that er stress as a normal physiological adaptive / coping mechanism plays a pivotal role in the development of preimplantation embryos . as such , upr - associated molecules and pathways such as sxbp1 mrna may become useful markers for the potential diagnosis of stress conditions for preimplantation embryos .", "most importantly , upr inhibitors , such as tudca , were found to effectively support the in vitro development of preimplantation embryos under stress conditions .", "further identification of er stress - induced coping responses in preimplantation embryos would be helpful for a comprehensive understanding of development from preimplantation through early postimplantation ." ]

[ "<S> stress coping mechanisms are critical to minimize or overcome damage caused by ever changing environmental conditions . </S>", "<S> they are designed to promote cell survival . the unfolded protein response ( upr ) pathway is mobilized in response to the accumulation of unfolded proteins , ultimately in order to regain endoplasmic reticulum ( er ) homeostasis . </S>", "<S> various elements of coping responses to er stress including perk , ask1 , bip , chop , gadd34 , ire1 , atf4 , atf6 , and xbp1 have been identified and were found to be inducible in oocytes and preimplantation embryos , suggesting that , as a normal part of the cellular adaptive mechanism , these coping responses , including the upr , play a pivotal role in the development of preimplantation embryos . as such </S>", "<S> , the upr - associated molecules and pathways may become useful markers for the potential diagnosis of stress conditions for preimplantation embryos . </S>", "<S> after implantation , er stress - induced coping responses become physiologically important for a normal decidual response , placentation , and early organogenesis . </S>", "<S> attenuation of er stress coping responses by tauroursodeoxycholate and salubrinal was effective for prevention of cell death of cultured embryos . </S>", "<S> further elucidation of new and relevant er stress coping responses in periimplantation embryos might contribute to a comprehensive understanding of the regulation of normal development of embryonic development and potentiation of embryonic development in vitro . </S>" ]

[ "Introduction", "ER stress and UPR signaling", "Targeting UPR, relief of ER stress", "ER stress is intrinsic as well as inducible in oocytes and preimplantation embryos", "Key regulators of the ER stress pathway in oocytes and preimplantation embryos", "Effect of UPR inhibitors in oocytes and preimplantation embryos", "ER stress signaling in post-implantation embryos", "Conclusions" ]

[ [ "there is increasing evidence linking endoplasmic reticulum ( er ) stress coping responses with the development of diseases .", "this is not surprising , given that the er is a multifunctional essential organelle in eukaryotic cells ; it functions as the critical entrance to the secretory pathways , and is also a regulator of ca homeostasis , as well as protein and lipid synthesis , all of which are highly sensitive to a variety of environmental , physiological , and pathophysiological conditions .", "the er is also closely associated with all cellular organelles , and therefore , capable of driving sophisticated mechanisms of intracellular signaling , including activation of transcriptional processes , ca mobilization , and influencing energy metabolism . in this review", ", we briefly summarize the er stress coping responses , particularly the unfolded protein response ( upr ) , and discuss the potential role of upr as a physiologically relevant pathway in supporting and controlling proper embryonic development ." ], [ "it impairs er functions such as protein folding and the heat shock response , and leads to altered ca levels and nutrient starvation .", "activation of er stress can result in the buildup of misfolded proteins in the er and activation of the upr as a coping strategy ( figure 1 ) .", "the upr involves distinct components designed to reestablish the protein synthesis machinery ( figure 1 ) .", "these include ( 1 ) translational attenuation to arrest the entry of new proteins into the er ; ( 2 ) transcriptional activation of genes encoding proteins involved in protein folding to assist the maturation of proteins ; ( 3 ) transcriptional activation of genes for components of the er - associated protein degradation ( erad ) system to decrease the number of misfolded proteins ; and if these are not successful , ( 4 ) activation of apoptotic pathways to eliminate defective cells .", "three er transmembrane proteins : dsrna - activated protein kinase - like er kinase ( perk ) , activating transcription factor 6 ( atf6 ) , and inositol - requiring enzyme 1 ( ire1 ) , in combination with the er molecular chaperone immunoglobulin binding protein ( bip , also known as glucose - regulated protein [ grp ] 78 ) , compose the upr , an er stress coping response .", "it is a monomeric , globular protein that functionally sorts and releases terminally misfolded substrates to the erad pathway . under non - stressed conditions grp78/bip interacts with ire , perk , and atf6 , but upon an increase in misfolded proteins", ", grp78/bip is sequestered away from these inducers , leading to the activation of the upr .", "release of grp78/bip from perk triggers its dimerization and autophosphorylation , followed by phosphorylation of eif2 , at ser , which inhibits protein synthesis by sequestering the trnamet responsible for initiating the translation of nascent protein ( figure 1 ) . during this period ,", "atf4 transcriptional activity induces both pro - survival ( early ) and pro - apoptotic ( late ) transcriptional programs .", "atf6 is an er transmembrane protein cleaved in the golgi . under non - stress conditions", "when bip is sequestered away from atf6 , the atf6 is translocated to the golgi , where it undergoes cleavage by site-1 and site-2 proteases ( s1p and s2p ) ( figure 1 ) .", "cleavage of atf6 produces a soluble basic leucine zipper ( bzip ) transcription factor ( cleaved atf6 ) that binds to er stress response elements ( erse - i and ii ) to induce transcriptional activation of er stress response genes .", "atf6 can induce the expression upr - induced genes including grp78/bip , proteins involved in erad , and xbp1 mrna .", "interestingly , er stress triggered by ca depletion induces the formation of a nascent , partially glycosylated form of atf6 with reduced interaction with calreticulin , and a faster rate of traversing the golgi , resulting in higher transactivation of atf6 gene targets . upon release of grp78/bip under stress conditions , ire1 homodimerizes , causing a conformational change that is transmitted across the membrane , leading to activation of its cytoplasmic kinase activity , autophosphorylation , and activation of its endoribonuclease activity ( figure 1 ) .", "ire1 endoribonuclease activity cleaves 28s rrna and specific micrornas , inhibits protein synthesis , and also splices xbp1", "the protein product from the sxbp1 variant binds to the specific promoter elements , erse , and upre , triggering transactivation upr responsive genes such as grp78/bip , calreticulin , and others , which are part of the protein folding quality control and degradation machinery .", "one part of the cellular recovery system that is turned on by the upr is er - associated degradation ( erad ) .", "there is tight regulation of the three arms of the upr with respect to the timing and response amplitude .", "the activation of each arm of the upr is specific to the source of er stress and governs cell fate , supporting either an adaptive response ( cell survival ) or a maladaptive response ( cell death ) . under prolonged and irreversible er stress ,", ", upr activates autophagy , a controlled self - degradation process that can promote cell survival by eliminating damaged cellular components .", "the intensity , timing , and duration of the upr appear to be important in determining cell fate .", "these observations suggest that er stress coping responses may also play an important role in the maintenance of physiological events associated with organ function and/or development ." ], [ "although there is emerging evidence that activation of er stress coping responses , including upr , may play an important role in diverse human diseases , it is conceivable that these coping responses may also be critical under normal physiological conditions and during development .", "interestingly , there exists a group of small molecules that may stabilize protein folding , prevent protein aggregation , and consequently affect the severity of upr activation .", "the most studied of these molecules are synthetic - fatty acid derivative 4-phenylbutyric acid and the natural bile acid tauroursodeoxycholic acid ( tudca ) . although the precise mechanisms of their action are not clear at present , these compounds have a profound impact on many pathologies including obesity pathways , brain ischemic - reperfusion , photoreceptor pathology , and steatosis , to name a few .", "these findings suggest that targeting er physiology and er stress signaling and stress - induced coping responses has therapeutic potential for treating diseases related to er stress ." ], [ "the development of preimplantation embryos is under the influence of various hormones and growth factors that originate from maternal tissues and/or the embryo proper .", "preimplantation embryos synthesize and secrete a wide range of hormones and growth factors that promote embryonic survival ; in the blastocyst stage , they increase in cell number via activation of transcription as well as protein synthesis . during this synthetic activity", ", the embryos may experience an inherent level of er stress and consequently activate specific coping responses to recover homeostasis and support development or death .", "for example , in the mouse , the upr contributes to preimplantation embryo death in the ddk syndrome , suggesting that the er stress coping mechanism counters the stresses intrinsic to embryos .", "mammalian early embryos placed in vitro are vulnerable to a variety of physicochemical stresses such as shearing , temperature changes , altered ph , and higher oxygen pressure , all of which are known inducers of er stress coping responses .", "these stresses alter gene expression , epigenetic mechanisms , and metabolism , reducing the embryonic development and viability .", "although the influence of er stress coping responses on cellular physiology in adult tissues has been widely studied , their effects on the oocytes and early embryos of mammals remains unclear ." ], [ "in a porcine model , the functional abundance of sxbp1 is low in mature oocytes and at the 1- , 2- , and 8-cell stages of embryos , but it is highly abundant at the germinal vesicle , 4-cell , morula , and blastocyst stages . in mouse cumulus - oocyte complexes , fatty acid - induced er stress impairs protein secretion , mitochondrial membrane potential , and embryo development .", "er stress is a key mechanism mediating the developmental potential of oocytes . in mouse oocytes ,", "the cancer drug doxorubicin affects the mitochondria , perk , and er - associated caspase-12 , while it inactivates parp , resulting in the activation of apoptotic signals .", "this suggests that activation of er stress coping responses may contribute , at least in part , to ovarian failure in women treated for cancer . in mouse preimplantation embryos , er stress - induced coping response molecules such as perk , ask1 , bip , chop , gadd34 , ire1 , atf4 , atf6 , and xbp1 are expressed . taking into account that perk , ire1 , and atf6 are the three major transducers of the upr , the expression of the major transducers of the upr suggests that preimplantation embryos use the upr coping response as one mechanism to deal with preimplantation stress . in mice lacking upr - associated genes such as grp78 and ppp1r15a / ppp1r15b", ", the embryos do not survive beyond preimplantation development , suggesting the indispensable role of these er chaperones and er stress - induced coping responses in preimplantation embryos .", "xbp1 is an important transcription factor and regulator of a subset of genes activated during er stress - induced upr . in drosophila , xenopus , and mice , xbp1 is essential for early embryonic development . in mice ,", "functional sxbp1 is abundant in the germinal vesicle of immature oocytes and on spindles in mii oocytes but is solely detected in the cytoplasm at the one - cell stage when sxbp1 mrna is scarce .", "this indicates that sxbp1 acts as an important regulator of er stress - induced coping responses during specific developmental stages from oocyte to blastocyst . in mouse embryos ,", "upr inducers such as tunicamycin and sorbitol increased nuclear sxbp1 at the one- and two - cell stages and activate xbp1 \n mrna splicing at the 8-cell , morula , and blastocyst stages , indicating that the ire arm of the upr is activated as an important coping response and adaptation to er stress in preimplantation embryos . in early mouse embryos ,", "grp78/bip ( hspa5 ) , a central regulator for the er stress - induced upr coping response , is essential for cell proliferation and protecting the inner cell mass from apoptosis . considering the importance of stress - activated protein kinase mitogen - activated protein kinase kinase ( sapk - map3k ) activation during cellular stress and the early stage of er stress , as well as the indispensable role of stress - activated proteins and map3k for preimplantation embryonic development ,", "er stress manifested by sapk - map3k is a normal event critical during preimplantation development .", "the coupling of autophagy and upr has been implicated in a variety of biological processes .", "induction of autophagy promotes preattachment development of in vitro - produced bovine embryos by reducing er stress , suggesting that autophagy / er stress balance is important for the developmental competence of early embryos ." ], [ "the inhibition of er stress - induced upr responses by tudca was found to enhance the maturation and developmental potential of porcine oocytes by preventing er stress - mediated apoptosis in vitro . in mouse cumulus - oocyte complexes , fatty acid - induced er stress", "coping responses are normalized by the upr inhibitor salubrinal , demonstrating that er stress is a key mechanism affecting oocyte developmental potential .", "preimplantation embryos , under in vitro conditions , experience an increase in reactive oxygen species ( ros ) , oxidative stress , heat - shock stress , and/or the culture stress due to insufficiency of maternally - derived embryotropic factors .", "growth factors and ros scavengers have been successfully used as a medium supplement in assisted reproductive technology ( art ) programs to prevent poor outcomes for embryo survival under in vitro conditions .", "tudca attenuates xbp1 mrna splicing in embryos at the two - cell stage and abolishes dna fragmentation induced by tunicamycin or sorbitol in blastocysts and recovered nuclear localization of the sxbp1 protein .", "in addition , tudca inhibits hyperosmolar - induced er stress coping responses as well as the er stress - induced apoptotic coping response during preimplantation embryo development ." ], [ "er stress - induced coping responses may also play a critical role for embryo development after implantation . indeed , increased er stress in decidual tissue in pregnancy has been shown to be implicated in fetal growth restriction with and without pre - eclampsia .", "sustained er stress acts as a cofactor of oxidative stress in decidual cells from patients with early pregnancy loss .", "in decidual cells , excessive oxidative stress influences upr pathways to activate erad by decreasing valosin - containing protein , which is a type ii er - associated protein and a member of the aaaatpase family that facilitates delivery of misfolded proteins to the proteasome , which results in cell damage , inhibition of cell growth , and activation of apoptosis . among upr constituents , p - eif2 , grp94 , and", "c / ebp homologous protein ( chop ) are increased in the placenta from intrauterine growth restriction and pre - eclampsia , indicating that er stress responses are also inducible under diseased conditions .", "inositol - requiring enzyme 1 ( ire1 ) or xbp1 null mice are unable to produce functional placentas , which is lethal to the embryo , indicating that the ire1 arm of the upr coping response is essential for placental development and embryonic viability . in mice ,", "cadmium - induced teratogenicity is associated with ros - mediated induction of er stress coping responses in the placenta .", "maternal exposure to cadmium resulted in activation of perk , phosphorylation of placental eif2 , and an increase in chop , indicating that upr signaling is activated in the placenta due to cadmium - induced toxicity .", "this further supports the notion that er stress - induced coping responses are important mechanisms in embryos and extraembryonic tissues following exposure to environmental toxins .", "furthermore , er stress - induced coping responses in the maternal decidua and placenta may counteract developmental problems during implantation and postimplantation development ." ], [ "stress coping mechanisms are critical to minimizing or overcoming damage caused by ever changing environmental conditions .", "the upr pathway is mobilized in response to the accumulation of unfolded proteins and to ultimately regain er homeostasis .", "various arms of the upr , an er stress coping response , are expressed in oocytes and preimplantation embryos , suggesting that er stress as a normal physiological adaptive / coping mechanism plays a pivotal role in the development of preimplantation embryos . as such , upr - associated molecules and pathways such as sxbp1 mrna may become useful markers for the potential diagnosis of stress conditions for preimplantation embryos .", "most importantly , upr inhibitors , such as tudca , were found to effectively support the in vitro development of preimplantation embryos under stress conditions .", "further identification of er stress - induced coping responses in preimplantation embryos would be helpful for a comprehensive understanding of development from preimplantation through early postimplantation ." ] ]

[ "osmotic demyelination syndrome ( ods ) including central pontine myelinolysis ( cpm ) and extrapontine myelinolysis ( epm ) following overly rapid correction of hyponatremia has been well recognized515 ) .", "although ods occurring after transsphenoidal resection of pituitary adenoma has been reported occasionally111618 ) , to our knowledge , few preoperative epm cases with pituitary adenoma have been reported14 ) .", "unfortunately , the outcomes of patients with ods are often disappointing with high rate of mortality and irreversible neurological deficits5 ) . here", ", we report a case of preoperative epm with nonfunctional pituitary macroadenoma who made a great clinical recovery after supportive therapy and transsphenoidal adenoma resection .", "a 38-year - old man was admitted to our hospital in december 2012 because of nausea , malaise , altered mental status , behavioral disturbances , dysarthria and dysphagia .", "the patient had been well until 2 months before admission , when nausea and malaise developed .", "his wife also reported that the patient had two episodes of transient loss of consciousness .", "he was sent to the emergency department of an outside hospital . at presentation , the patient had no sign of dehydration like poor skin turgor , orthostasis or decreased urine output , nor sign of volume overload including peripheral edema , ascites or elevated jugular vein pressure .", "serum sodium of 102 mmol / l , potassium of 3.9 mmol / l and phosphate of 1.08", "mmol / l were reported . the complete blood count and the remainder of metabolic panel including", "intravenous hypertonic saline was given with an average correction rate of 8 mmol/24 h. despite slow rate of correction , neurologic symptoms including restlessness and irritability developed . in the following days , the patient developed apathy , confusion and obtundation , followed by dysarthria , dysphagia , paraparesis , behavioral disturbances and disorientation .", "endocrine studies revealed a serum cortisol concentration ( 8 : 00 a.m. ) of 62.4 nmol / l ( normal , 138635 nmol / l ) , plasma adrenocorticotrophin ( acth ) of 1.03 pmol / l ( normal , 2.217.6 pmol / l ) .", "miu / l ) ; serum free thyroxine ( ft4 ) was 6.91 pmol / l ( normal , 11.522.7 pmol / l ) , and serum total thyroxine ( t4 ) was 43.0 nmol / l ( normal , 65155", "cranial magnetic resonance imaging ( mri ) was performed and a pituitary macroadenoma ( fig .", "one week before admission , mri was repeated and epm was detected at bilateral basal ganglion and thalamus ( fig .", "the patient was referred to our hospital because of persistent neurologic deficits . on physical exam , he was unresponsive to verbal or pain stimuli .", "hormone studies showed normal atch , cortisol , tsh , t4 , and ft4 , decreased growth hormone and insulin - like growth factor 1 , normal prolactin , decreased gonadotrophin ( follicle - stimulating hormone and luteinizing hormone ) , estrogen and testosterone .", "the patient had several episodes of restlessness and agitation with shouting and fighting lasting 10 minutes every day .", "the decision of surgery was made then based on the improved general state of the patient and the intent to remove the underlying cause of hypopituitarism and hyponatremia .", "pathologically , the mass was confirmed to be nonfunctional adenoma by hematoxylin and eosin ( h&e ) staining and immunohistochemical examination .", "pseudorosette patterns were found on h&e staining and nuclear division was less than one per 10 high power field .", "the tumor cells were scattered positive for p53 and ki-67 . and ki-67 index was < 1% .", "the immunostainings for acth , fsh , lh , prl , tsh and gh were negative .", "recent hormone studies showed normal level of acth , cortisol , tsh , ft4 and t4 .", "the patient was able to resume his work as a bank accountant three months after the surgery .", "the latest follow - up mri was obtained 7 months postoperatively , and these images showed demyelination at bilateral basal ganglions and thalamus resolved and pituitary adenoma resected ( fig .", "among various etiologies of hyponatremia , endocrine disorders , including adrenal insufficiency and hypothyroidism , are uncommon causes which may be overlooked1712 ) .", "while hyponatremia can be caused by hypopituitarism , severe hyponatremia occurring as the presenting manifestation of pituitary adenoma is rare8 ) . at the onset of hyponatremia ,", "given the endocrine abnormality and histology - confirmed pituitary adenoma , we propose that secondary adrenal insufficiency and hypothyroidism resulting from hypopituitarism was the underlying cause of his hyponatremia .", "however , demyelination can occur even with this correction rate ( as in our case ) , especially in those with other ods risk factors5 ) .", "except for the rate of correction , serum sodium concentration at presentation and the duration of the hyponatremia are also important risk factors . on presentation ,", "our patient had been symptomatic for 2 months and the serum sodium level was as low as 102", "therefore , patients with more than one risk factors for ods may still be at risk for development ods at \" normal \" rates of correction and may require slower rates . if ods does develop despite the steps taken to avoid overly rapid correction , effective management is rare .", "although reintroduction of hyponatremia , plasmapheresis and corticosteroids has been used , most evidence comes from animal models and data in humans are limited to case reports2513 ) .", "a more recent review of 32 german patients with ods showed a much better outcome with only 30% having irreversible and incapacitating neurological deficits9 ) .", "this review suggests that patients with ods can survive if the nonspecific secondary complications of transient illnesses such as aspiration pneumonia , deep venous thrombosis , and pulmonary embolism can be avoided .", "some patients with ods can recover function after prolonged periods of severe neurologic impairment59 ) .", "as in our case , the patient had developed progressive neurologic symptoms for two months on admission to our hospital .", "the patient was able to resume the job as a banking account after surgery , indicating complete recovery from neurologic and cognitive deficits .", "therefore , supportive therapy should be continued for at least six to eight weeks before concluding that the deficits are irreversible .", "surgery is the ultimate effective treatment for nonfunctioning adenoma , which not only removes the underlying cause of hypopituitarism , but also reverses or prevents vision impairment . according to a systemic review and meta - analysis evaluating the outcome of surgical treatment for nonfunctioning pituitary adenomas , pituitary hypofunction", "improved in as many as 30 percent after surgery10 ) . while for patients with epm , appropriate surgical timing of adenoma resection is of great importance .", "neurologic deficits of preoperative epm such as decreased level of consciousness , paraparesis , dysarthria and dysphagia , posed great challenge to the transsphenoidal operation of pituitary adenoma .", "risk of complications such as aspiration , respiratory failure , and deep vein thrombosis is much higher than patients without neurologic deficits49 ) . to minimize the risk of surgery and to remove the underlying cause of hyponatremia", ", we selectively arrange the operation after dysphagia and dysarthria resolved , and the patient became ambulant and able to obey simple commands .", "we report a case of preoperative epm with nonfunctional pituitary macroadenoma who made a great clinical recovery after supportive therapy and transsphenoidal adenoma resection .", "more cases are required to clarify the prognosis of ods and identify the factors associated with better outcome ." ]

[ "<S> few preoperative extrapontine myelinolysis ( epm ) cases with pituitary adenoma have been reported . </S>", "<S> no such case had long follow - up to see the outcome of epm . </S>", "<S> we reported a 38-year - old man complaining of nausea , malaise and transient loss of consciousness who was found to have severe hyponatremia . </S>", "<S> neurologic deficits including altered mental status , behavioral disturbances , dysarthria and dysphagia developed despite slow correction of hyponatremia . </S>", "<S> endocrine and imaging studies revealed hypopituitarism , nonfunctional pituitary macroadenoma and extrapontine myelinolysis . </S>", "<S> transsphenoidal surgery was performed after three weeks of supportive therapy , when neurological symptoms improved significantly . </S>", "<S> the patient recovered function completely 3 months after surgery . </S>", "<S> our case indicates that outcome of epm can be good even with prolonged periods of severe neurologic impairment . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION" ]

[ [ "osmotic demyelination syndrome ( ods ) including central pontine myelinolysis ( cpm ) and extrapontine myelinolysis ( epm ) following overly rapid correction of hyponatremia has been well recognized515 ) .", "although ods occurring after transsphenoidal resection of pituitary adenoma has been reported occasionally111618 ) , to our knowledge , few preoperative epm cases with pituitary adenoma have been reported14 ) .", "unfortunately , the outcomes of patients with ods are often disappointing with high rate of mortality and irreversible neurological deficits5 ) . here", ", we report a case of preoperative epm with nonfunctional pituitary macroadenoma who made a great clinical recovery after supportive therapy and transsphenoidal adenoma resection ." ], [ "a 38-year - old man was admitted to our hospital in december 2012 because of nausea , malaise , altered mental status , behavioral disturbances , dysarthria and dysphagia .", "the patient had been well until 2 months before admission , when nausea and malaise developed .", "his wife also reported that the patient had two episodes of transient loss of consciousness .", "he was sent to the emergency department of an outside hospital . at presentation , the patient had no sign of dehydration like poor skin turgor , orthostasis or decreased urine output , nor sign of volume overload including peripheral edema , ascites or elevated jugular vein pressure .", "serum sodium of 102 mmol / l , potassium of 3.9 mmol / l and phosphate of 1.08", "mmol / l were reported . the complete blood count and the remainder of metabolic panel including", "intravenous hypertonic saline was given with an average correction rate of 8 mmol/24 h. despite slow rate of correction , neurologic symptoms including restlessness and irritability developed . in the following days , the patient developed apathy , confusion and obtundation , followed by dysarthria , dysphagia , paraparesis , behavioral disturbances and disorientation .", "endocrine studies revealed a serum cortisol concentration ( 8 : 00 a.m. ) of 62.4 nmol / l ( normal , 138635 nmol / l ) , plasma adrenocorticotrophin ( acth ) of 1.03 pmol / l ( normal , 2.217.6 pmol / l ) .", "miu / l ) ; serum free thyroxine ( ft4 ) was 6.91 pmol / l ( normal , 11.522.7 pmol / l ) , and serum total thyroxine ( t4 ) was 43.0 nmol / l ( normal , 65155", "cranial magnetic resonance imaging ( mri ) was performed and a pituitary macroadenoma ( fig .", "one week before admission , mri was repeated and epm was detected at bilateral basal ganglion and thalamus ( fig .", "the patient was referred to our hospital because of persistent neurologic deficits . on physical exam , he was unresponsive to verbal or pain stimuli .", "hormone studies showed normal atch , cortisol , tsh , t4 , and ft4 , decreased growth hormone and insulin - like growth factor 1 , normal prolactin , decreased gonadotrophin ( follicle - stimulating hormone and luteinizing hormone ) , estrogen and testosterone .", "the patient had several episodes of restlessness and agitation with shouting and fighting lasting 10 minutes every day .", "the decision of surgery was made then based on the improved general state of the patient and the intent to remove the underlying cause of hypopituitarism and hyponatremia .", "pathologically , the mass was confirmed to be nonfunctional adenoma by hematoxylin and eosin ( h&e ) staining and immunohistochemical examination .", "pseudorosette patterns were found on h&e staining and nuclear division was less than one per 10 high power field .", "the tumor cells were scattered positive for p53 and ki-67 . and ki-67 index was < 1% .", "the immunostainings for acth , fsh , lh , prl , tsh and gh were negative .", "recent hormone studies showed normal level of acth , cortisol , tsh , ft4 and t4 .", "the patient was able to resume his work as a bank accountant three months after the surgery .", "the latest follow - up mri was obtained 7 months postoperatively , and these images showed demyelination at bilateral basal ganglions and thalamus resolved and pituitary adenoma resected ( fig ." ], [ "among various etiologies of hyponatremia , endocrine disorders , including adrenal insufficiency and hypothyroidism , are uncommon causes which may be overlooked1712 ) .", "while hyponatremia can be caused by hypopituitarism , severe hyponatremia occurring as the presenting manifestation of pituitary adenoma is rare8 ) . at the onset of hyponatremia ,", "given the endocrine abnormality and histology - confirmed pituitary adenoma , we propose that secondary adrenal insufficiency and hypothyroidism resulting from hypopituitarism was the underlying cause of his hyponatremia .", "however , demyelination can occur even with this correction rate ( as in our case ) , especially in those with other ods risk factors5 ) .", "except for the rate of correction , serum sodium concentration at presentation and the duration of the hyponatremia are also important risk factors . on presentation ,", "our patient had been symptomatic for 2 months and the serum sodium level was as low as 102", "therefore , patients with more than one risk factors for ods may still be at risk for development ods at \" normal \" rates of correction and may require slower rates . if ods does develop despite the steps taken to avoid overly rapid correction , effective management is rare .", "although reintroduction of hyponatremia , plasmapheresis and corticosteroids has been used , most evidence comes from animal models and data in humans are limited to case reports2513 ) .", "a more recent review of 32 german patients with ods showed a much better outcome with only 30% having irreversible and incapacitating neurological deficits9 ) .", "this review suggests that patients with ods can survive if the nonspecific secondary complications of transient illnesses such as aspiration pneumonia , deep venous thrombosis , and pulmonary embolism can be avoided .", "some patients with ods can recover function after prolonged periods of severe neurologic impairment59 ) .", "as in our case , the patient had developed progressive neurologic symptoms for two months on admission to our hospital .", "the patient was able to resume the job as a banking account after surgery , indicating complete recovery from neurologic and cognitive deficits .", "therefore , supportive therapy should be continued for at least six to eight weeks before concluding that the deficits are irreversible .", "surgery is the ultimate effective treatment for nonfunctioning adenoma , which not only removes the underlying cause of hypopituitarism , but also reverses or prevents vision impairment . according to a systemic review and meta - analysis evaluating the outcome of surgical treatment for nonfunctioning pituitary adenomas , pituitary hypofunction", "improved in as many as 30 percent after surgery10 ) . while for patients with epm , appropriate surgical timing of adenoma resection is of great importance .", "neurologic deficits of preoperative epm such as decreased level of consciousness , paraparesis , dysarthria and dysphagia , posed great challenge to the transsphenoidal operation of pituitary adenoma .", "risk of complications such as aspiration , respiratory failure , and deep vein thrombosis is much higher than patients without neurologic deficits49 ) . to minimize the risk of surgery and to remove the underlying cause of hyponatremia", ", we selectively arrange the operation after dysphagia and dysarthria resolved , and the patient became ambulant and able to obey simple commands ." ], [ "we report a case of preoperative epm with nonfunctional pituitary macroadenoma who made a great clinical recovery after supportive therapy and transsphenoidal adenoma resection .", "more cases are required to clarify the prognosis of ods and identify the factors associated with better outcome ." ] ]

[ "mood disorders ( md ) such as depression and bipolar disorders are one of the most disabling types of diseases . in 2004 , depression was the leading cause of disability as measured by years lost due to disability ( yld ) and the 3rd leading contributor to the global burden of disease assessed using the disability - adjusted life year ( daly ) , a time - based measure that combines years of life lost due to premature mortality and years of life lost due to time lived in states of less than full health .", "bipolar disorder was one of the top 10 leading global causes of yld in 2004 .", "presence of risk factors ( e.g. , excessive nicotine use and alcohol and other drug use ) , cooccurring anxiety disorders , and eating disorders can lead to the early development of severe medical conditions .", "also personality disorders ( pd ) are a class of disorders that can significantly worsen a patient 's quality of life .", "in fact , psychosocial impairment is one of the diagnostic criteria for personality disorders according to the dsm iv and there is empirical evidence that the most severe personality disorders ( e.g. , schizotypal personality disorder and borderline personality disorder ) are a major cause of psychosocial disability compared to unipolar depression without personality disorders .", "both clinical practice and empirical studies show that there is often interdependence between md and pd [ 69 ] .", "data from national epidemiologic survey on alcohol and related conditions showed cooccurrence rates of lifetime prevalence of three pd ( borderline , narcissistic , and schizotypal ) with any md ranging from 17.2% to 10.3% [ 1012 ]", "one of the reasons for the interest in this issue is to contribute to improve treatments .", "empirical evidence reports a tendency of poor outcome in case of cooccurrence of depression and pd compared to patients with a diagnosis of depression only .", "this has been attributed either to worse compliance of pharmacological therapy or to the difficulty of maintaining an active and efficient social support , which could protect against relapses [ 15 , 16 ] .", "moreover , there is some evidence that the presence of pd , especially of the avoidant type , interferes with treatment response at interpersonal psychotherapy of depression . ", "several hypotheses have been suggested to understand md - dp relationship . among others , lewinsohn and colleagues proposed that low levels of mood could have a", " scar effect on individuals : pd could develop probably by one 's modification of coping and appraisal styles . from another perspective , it is argued that some maladaptive personality traits could be seen as risk factors of developing both an md and also a true pd . ", "recently it has been suggested that the age of onset of md could be a more efficacious criterion of classification than polarity .", "unfortunately , the studies that have explored the age of onset of md are not many and , with the exception cited above , have investigated bipolar or unipolar samples separately , with an implicit assumption of the polarity criterion .", "moreover , the common denominator of these studies was a priori determined thresholds that could explain the clinical difference in terms of severity or comorbidity found between patients who have developed the disorder at different ages .", "fava and colleagues found a higher prevalence of pd in patients who had earlier onset of major depressive disorder ( below 18 years ) compared to patients with later onset .", "this result was not replicated by skodol and colleagues who instead found that severity and recurrence of major depressive disorder were predictors of borderline personality disorder .", "perlis and colleagues have compared bipolar disorder patients with very early onset with patients whose onset was after the age of 18 finding a poor outcome in the first group in terms of fewer days of euthymia and greater impairment in functioning and quality of life . in a recent study , bukh and", "colleagues found higher comorbidity of personality disorder between patients with relative early onset of md .", "the main goal of the present study is to evaluate the association between the age of onset of md and the complexity of the personality traits of the patient .", "secondly , we will be interested in identifying an age threshold able to maximize the differences between early and later onsets in terms of the complexity of the personality traits .", "the patients for the study were recruited in three psychiatric wards in piedmont ( italy ) .", "patients consecutively admitted for major depressive episodes or manic / hypomanic episodes between april 2006 and april 2007 were considered .", "inclusion criteria were age over 18 and an agreement to participate in the study with informed consent , whereas patients diagnosed with schizophrenia and other psychotic disorders , chronic substance abuse , severe medical illnesses , or cognitive disorders were excluded from the study .", "the study was approved by the ethic committees of the hospitals involved in the study , and written informed consent was obtained from all participants .", "axis i diagnoses were evaluated with the structured clinical interview ( scid-1 ) for dsm - iv .", "results of a recent study showed that scid - i validity was high and that interrater reliability ranged from .60 to .83 .", "age at onset of depression or mania / hypomania , number of episodes of each type , average duration of each phase of illness , number of admissions for mood disorders , and family history of psychiatric illness were assessed by an anamnestic interview controlled whenever possible with corroborating family reports and medical records . ", "the severity of the mood disorder was analyzed in terms of age of onset , duration of episodes , and frequency of episodes .", "all the participants included in the study were administered the millon clinical multiaxial inventory - iii [ 27 , 28 ] .", "the mcmi - iii is a 175-item true / false self - report instrument that assesses axis i and ii psychopathology .", "the mcmi - iii raw scores are transformed and reported as weighted base rate ( br ) scores .", "good internal consistency ( = .66.90 ) and stability ( test - retest r = .84.96 ) have generally been found for the mcmi - iii scales . ", "a recent study ( zennaro , in press ) , carried out on the italian version of mcmi - iii , shows that the inventory falls short in assigning pd categorical attributions to patients .", "the reason of such results can be found in br cutoffs used to determine the presence of traits versus the presence of pd disorders .", "otherwise the cited study shows how mcmi - iii can correctly and reliably distinguish between pathological and not - pathological individuals . even for this reason ,", "mcmi - iii was used with the most elevated anchor point with the purpose of exploring personality traits rather than assigning a diagnosis .", "the severity of the personality traits was indeed analyzed in terms of complexity of the pd , with reference to the number of dimensions of the mcmi - iii with a br score of 85 or above . according to mcmi - iii scoring guidelines , patients with br scores of 85 or above on any of the mcmi - iii personality scales ( i.e. , schizoid , avoidant , depressive , dependent , histrionic , narcissistic , antisocial , aggressive , compulsive , passive aggressive , self - defeating , schizotypal , borderline , paranoid ) are to be considered personality disorder elevated .", "the sample was thus divided in three groups : participants with none , one ( i.e. , simple pd ) , or with more than one ( i.e. , complex pd ) elevations on the mcmi - iii .", "mcm - iii was administered just before discharge and after the patients had recovered from the affective episode . to have 90% power to detect an effect size of 0.30 in the comparison of complexity of pd with six hypothetical nodes produced by recursive partitioning with two - sided significance level alpha of 0.05 , we required about 200 patients .", "the shape of the distribution of the continuous variables was evaluated , and comparisons amongst the three groups defined by the mcmi - iii scores were done . after excluding patients with a single major depressive episode , who could not be analysed in terms of duration and number of episodes , a recursive partitioning analysis was used to find the most characterizing variables for the different complexity distributions of the pd .", "the recursive partitioning analysis was realized with the party procedure ( ( for a methodological description of procedure ) , ) of the system for statistical computation and graphicsr . to study the individual contribution of each variable to the prediction of the complexity of the personality disorder , a factorial analysis of variance", "after a graphical inspection , a logarithmic transformation was applied to the continuous variables which had a nonnormal distribution ( i.e. , the age of onset of the md and the average number of episodes per year ) . in the model ,", "the independent variables were gender , duration of episodes ( divided into less than 1 month , 1 to 3 months and over 3 months ) , the age of onset of the md ( after logarithmic transformation ) , the average number of episodes per year ( after logarithmic transformation ) , and the age of testing .", "finally , an analysis of the profiles of the mcmi - iii scales was performed with a repeated measures anova , encompassing the cluster found by the recursive partitioning as a grouping variable and the elevations of the mcmi - iii scales as repeating measures .", "all the other statistical analyses were performed with the spss for windows , release version 17.0 , ( spss , inc .", "the final sample encompassed 209 patients ( 66 males and 143 females ; mean age 55.48 sd 13.04 ) .", "sample characteristics and clinical data relative to the md are shown in table 1 and also allow comparison with the complexity of the pd .", "regarding axis i diagnoses , 10.5% of the patients had unipolar depression - single episode , 52.2% unipolar depression recurrent , 22.5% bipolar type ii disorder , and 14.8% bipolar type i disorder .", "regarding personality disorders , 17.2% of patients had no elevated mcmi - iii scores , 45.9% had one peak , and 36.9% had two or more elevated values .", "the prevalence of elevated scores ( i.e. , at least a scale with a score of 85 or above ) on the mcmi - iii is 83% in the group of patients with mood disorders .", "this value drops down to 77% if one considers only patients with depressive disorder . from the recursive partitioning analysis , the age of onset of the md was the most explicative variable with a threshold of 29 years .", "later on , the analysis found a further significant classification : the group of participants over 29 years of age was divided according to the duration of the episodes ( under 3 months versus over 3 months ) .", "consequently the type of md was not significant in the explanation of the different patterns of the personality complexity ( figure 1 ) .", "the three patterns differed from each other on all of the three levels of complexity of pd .", "in particular , node 2 is characterized by more than half the patients with high complexity of pd ( absolute majority of the node ) .", "the other two nodes show for patients with later onset of the md a cluster ( node 5 ) with high presence of pd ( only 8% of patients do not have a pd ) represented by patients with longer episodes ( over 3 months ) and a cluster of patients ( node 4 ) with lower presence of pd and , more importantly , less complexity ( only 16% have a complex personality disorder ) .", "the analysis was applied to the participants with at least one mcmi - iii elevated value once more excluding patients diagnosed with a single major depressive episode . in table 2 , the results of the comparison between the two groups are presented .", "the only statistically significant predictor emerging from multivariate analysis was the age of onset of the md ( f = 8.945 ; df = 1 ; p = .003 ; = .058 ) .", "the significance of the age of testing disappears : its connection to the complexity of the pd was probably influenced by the age of onset of the disorder itself .", "the duration of the episodes between simple and complex disorders was not statistically significant , and this difference did not improve its significance in the multivariate analysis ( f = 1.230 ; df = 2 ; p = .295 ; = .017 ) .", "this led us to think it is not a mere type ii error but more likely a variable with heterogeneous distributions in both personality disorder conditions .", "the profile comparisons ( figure 2 ) show statistically significant differences between the three clusters based on pd complexity ( f = 9.346 ; ( df = 2 ) ; p < .01 ; = .092 ) and so did the interaction between cluster and mcmi - iii profile ( correction of huynh - feldt ( f = 3.640 ; ( df = 14.357 ) ; p < .001 ; = .038 ) ) . from the multiple comparisons with bonferroni corrections , the cluster of patients with early onset of md significantly differed from the cluster with later onset and longer episodes , but it did not differ from the one with shorter episodes . on the other hand , the cluster with later md onset and longer episodes significantly differed from the other clusters .", "in particular , there was a clear difference between the cluster with early onset and the other two .", "the difference is seen in the elevation of the three last scales representing , according to millon , severe personality disorders with more impaired functioning .", "the clusters with later onset differed in the three scales : avoidant , depressive , and dependent , partially differed from each other in the paranoid scale , and remained very close to each other in all the other scales .", "the aim of the present study was to explore the relationship between the age of onset of md and the complexity of the personality traits of the patients . moreover , we were interested in identifying an age threshold able to maximize the difference of personality traits between early and later onsets and investigating the association between mood disorder severity and personality traits . to start with , the prevalence of the personality disorders ( pd ) evaluated with the mcmi - iii in patients admitted for a depressive / hypomaniac episode through a dimensional evaluation was higher than that in previous studies , regarding both outpatients with a depression diagnosis and dimensional evaluation and patients with unipolar and bipolar disorders [ 6 , 16 ] .", "these results can be in part linked to the fact that all the patients recruited for the present study had at least one hospital admission , a strong indicator of worse severity of the disorder . in the recursive partitioning analysis , the age of onset was the most significant predicting factor .", "interestingly , the type of mood disorder was not a significant predicting factor , as previously shown .", "the threshold of onset was higher compared to previous studies [ 21 , 23 , 33 ] while it overlaps with what was recently highlighted about depressive disorders .", "it is interesting to underline the relationship identified between the severity of md in terms of duration of episodes and of pd , particularly in the cluster with later onset .", "the presence of maladaptive personality makes more likely both the onset of an md and the creation of a more complex personality disorder .", "this interpretation could explain the independence between the complexity of the pd ( with the presence of the elevations on the scales more often associated with severe cases ) and the severity of the md .", "conversely , in the later onsets , the development of the particularly severe md can increase the personality symptomatology and elevate the mcmi - iii scale scores .", "this is what can be found in the three scales : avoidant , depressive , and dependent .", "in fact , between the patients with later md onset , the ones with longer episodes have more elevations on these three scales , while , on the other scales , except partially for the paranoid scale , they are practically identical .", "in particular , avoidant and dependent are two scales whose elevations are expected in the presence of depressive disorder . to date , it is not possible to highlight a clear direction of causality for the examined relationship and state whether the presence of a more complex personality disorder condition is caused by the onset of a more severe md or influences the severity of the md itself ( e.g. , by reducing the presence of social support around the patient ) .", "thus , further studies are necessary to clarify this relevant issue , as the presence of an md as an inclusion criterion and the difficulty to accurately establish an age of onset for an md do not allow any definitive conclusion .", "firstly , the cross - sectional design did not allow us to detect precisely how the pd and md developed .", "secondly , as the assessment was conducted at the end of admission period , we could have found more pd than in the euthymic phase .", "finally , the use of a self - administered tool for the detection of the pd may have led to an overestimation of the presence and complexity of the personality traits , particularly in keeping with the psychometric characteristics of the mcmi - iii , as described in a recent study on the topic ( zennaro , in press ) even if a conservative cutoff criterion was adopted .", "however , mcmi - iii ( as other self - report inventories , e.g. , minnesota multiphasic personality inventory-2 ) has the advantage of including validity scales that can detect possible patterns of response sets , biases , and distortions that might compromise the validity of the clinical assessment assuring specific quality of data at the study .", "this study has introduced the issue of disorder severity aspects in the investigation of the relationship between md and pd .", "our findings suggest that it is important to design prospective studies able to evaluate the level of comorbidity also on later md onset patients and to include variables about the severity of the disorders . from a clinical point of view", ", the results suggest the need to assess maladaptive personality traits not only during adolescence but also in young adults too in order to prevent and treat potentially severe md ." ]

[ "<S> \n objective . </S>", "<S> the aim of the present study is to evaluate the link between the age of onset of mood disorders and the complexity of the personality traits </S>", "<S> . methods . </S>", "<S> 209 patients with major depressive or manic / hypomanic episodes were assessed using the structured clinical interview for dsm axis i diagnoses and the millon clinical multiaxial inventory - iii ( mcmi - iii ) . results . </S>", "<S> 17.2% of the patients had no elevated mcmi - iii scores , 45.9% had one peak , and 36.9% had a complex personality disorder with two or more elevated scores . </S>", "<S> mood disorders onset of 29 years or less was the variable most related to the complexity of personality disorders as indicated from a recursive partitioning analysis . </S>", "<S> conclusions . </S>", "<S> the relationship between mood disorders and personality traits differ in reference to age of onset of the mood disorder . in younger patients </S>", "<S> , maladaptive personality traits can evolve both in a mood disorder onset and in a complex personality disorder , while the later development of a severe mood disorder can increase the personality symptomatology . </S>", "<S> our results suggest a threshold of mood disorder onset higher compared to previous studies . </S>", "<S> maladaptive personality traits should be assessed not only during adolescence but also in young adults to identify and treat potential severe mood disorders . </S>" ]

[ "1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Discussion", "5. Conclusions" ]

[ [ "mood disorders ( md ) such as depression and bipolar disorders are one of the most disabling types of diseases . in 2004 , depression was the leading cause of disability as measured by years lost due to disability ( yld ) and the 3rd leading contributor to the global burden of disease assessed using the disability - adjusted life year ( daly ) , a time - based measure that combines years of life lost due to premature mortality and years of life lost due to time lived in states of less than full health .", "bipolar disorder was one of the top 10 leading global causes of yld in 2004 .", "presence of risk factors ( e.g. , excessive nicotine use and alcohol and other drug use ) , cooccurring anxiety disorders , and eating disorders can lead to the early development of severe medical conditions .", "also personality disorders ( pd ) are a class of disorders that can significantly worsen a patient 's quality of life .", "in fact , psychosocial impairment is one of the diagnostic criteria for personality disorders according to the dsm iv and there is empirical evidence that the most severe personality disorders ( e.g. , schizotypal personality disorder and borderline personality disorder ) are a major cause of psychosocial disability compared to unipolar depression without personality disorders .", "both clinical practice and empirical studies show that there is often interdependence between md and pd [ 69 ] .", "data from national epidemiologic survey on alcohol and related conditions showed cooccurrence rates of lifetime prevalence of three pd ( borderline , narcissistic , and schizotypal ) with any md ranging from 17.2% to 10.3% [ 1012 ]", "one of the reasons for the interest in this issue is to contribute to improve treatments .", "empirical evidence reports a tendency of poor outcome in case of cooccurrence of depression and pd compared to patients with a diagnosis of depression only .", "this has been attributed either to worse compliance of pharmacological therapy or to the difficulty of maintaining an active and efficient social support , which could protect against relapses [ 15 , 16 ] .", "moreover , there is some evidence that the presence of pd , especially of the avoidant type , interferes with treatment response at interpersonal psychotherapy of depression . ", "several hypotheses have been suggested to understand md - dp relationship . among others , lewinsohn and colleagues proposed that low levels of mood could have a", " scar effect on individuals : pd could develop probably by one 's modification of coping and appraisal styles . from another perspective , it is argued that some maladaptive personality traits could be seen as risk factors of developing both an md and also a true pd . ", "recently it has been suggested that the age of onset of md could be a more efficacious criterion of classification than polarity .", "unfortunately , the studies that have explored the age of onset of md are not many and , with the exception cited above , have investigated bipolar or unipolar samples separately , with an implicit assumption of the polarity criterion .", "moreover , the common denominator of these studies was a priori determined thresholds that could explain the clinical difference in terms of severity or comorbidity found between patients who have developed the disorder at different ages .", "fava and colleagues found a higher prevalence of pd in patients who had earlier onset of major depressive disorder ( below 18 years ) compared to patients with later onset .", "this result was not replicated by skodol and colleagues who instead found that severity and recurrence of major depressive disorder were predictors of borderline personality disorder .", "perlis and colleagues have compared bipolar disorder patients with very early onset with patients whose onset was after the age of 18 finding a poor outcome in the first group in terms of fewer days of euthymia and greater impairment in functioning and quality of life . in a recent study , bukh and", "colleagues found higher comorbidity of personality disorder between patients with relative early onset of md .", "the main goal of the present study is to evaluate the association between the age of onset of md and the complexity of the personality traits of the patient .", "secondly , we will be interested in identifying an age threshold able to maximize the differences between early and later onsets in terms of the complexity of the personality traits ." ], [ "the patients for the study were recruited in three psychiatric wards in piedmont ( italy ) .", "patients consecutively admitted for major depressive episodes or manic / hypomanic episodes between april 2006 and april 2007 were considered .", "inclusion criteria were age over 18 and an agreement to participate in the study with informed consent , whereas patients diagnosed with schizophrenia and other psychotic disorders , chronic substance abuse , severe medical illnesses , or cognitive disorders were excluded from the study .", "the study was approved by the ethic committees of the hospitals involved in the study , and written informed consent was obtained from all participants .", "axis i diagnoses were evaluated with the structured clinical interview ( scid-1 ) for dsm - iv .", "results of a recent study showed that scid - i validity was high and that interrater reliability ranged from .60 to .83 .", "age at onset of depression or mania / hypomania , number of episodes of each type , average duration of each phase of illness , number of admissions for mood disorders , and family history of psychiatric illness were assessed by an anamnestic interview controlled whenever possible with corroborating family reports and medical records . ", "the severity of the mood disorder was analyzed in terms of age of onset , duration of episodes , and frequency of episodes .", "all the participants included in the study were administered the millon clinical multiaxial inventory - iii [ 27 , 28 ] .", "the mcmi - iii is a 175-item true / false self - report instrument that assesses axis i and ii psychopathology .", "the mcmi - iii raw scores are transformed and reported as weighted base rate ( br ) scores .", "good internal consistency ( = .66.90 ) and stability ( test - retest r = .84.96 ) have generally been found for the mcmi - iii scales . ", "a recent study ( zennaro , in press ) , carried out on the italian version of mcmi - iii , shows that the inventory falls short in assigning pd categorical attributions to patients .", "the reason of such results can be found in br cutoffs used to determine the presence of traits versus the presence of pd disorders .", "otherwise the cited study shows how mcmi - iii can correctly and reliably distinguish between pathological and not - pathological individuals . even for this reason ,", "mcmi - iii was used with the most elevated anchor point with the purpose of exploring personality traits rather than assigning a diagnosis .", "the severity of the personality traits was indeed analyzed in terms of complexity of the pd , with reference to the number of dimensions of the mcmi - iii with a br score of 85 or above . according to mcmi - iii scoring guidelines , patients with br scores of 85 or above on any of the mcmi - iii personality scales ( i.e. , schizoid , avoidant , depressive , dependent , histrionic , narcissistic , antisocial , aggressive , compulsive , passive aggressive , self - defeating , schizotypal , borderline , paranoid ) are to be considered personality disorder elevated .", "the sample was thus divided in three groups : participants with none , one ( i.e. , simple pd ) , or with more than one ( i.e. , complex pd ) elevations on the mcmi - iii .", "mcm - iii was administered just before discharge and after the patients had recovered from the affective episode . to have 90% power to detect an effect size of 0.30 in the comparison of complexity of pd with six hypothetical nodes produced by recursive partitioning with two - sided significance level alpha of 0.05 , we required about 200 patients .", "the shape of the distribution of the continuous variables was evaluated , and comparisons amongst the three groups defined by the mcmi - iii scores were done . after excluding patients with a single major depressive episode , who could not be analysed in terms of duration and number of episodes , a recursive partitioning analysis was used to find the most characterizing variables for the different complexity distributions of the pd .", "the recursive partitioning analysis was realized with the party procedure ( ( for a methodological description of procedure ) , ) of the system for statistical computation and graphicsr . to study the individual contribution of each variable to the prediction of the complexity of the personality disorder , a factorial analysis of variance", "after a graphical inspection , a logarithmic transformation was applied to the continuous variables which had a nonnormal distribution ( i.e. , the age of onset of the md and the average number of episodes per year ) . in the model ,", "the independent variables were gender , duration of episodes ( divided into less than 1 month , 1 to 3 months and over 3 months ) , the age of onset of the md ( after logarithmic transformation ) , the average number of episodes per year ( after logarithmic transformation ) , and the age of testing .", "finally , an analysis of the profiles of the mcmi - iii scales was performed with a repeated measures anova , encompassing the cluster found by the recursive partitioning as a grouping variable and the elevations of the mcmi - iii scales as repeating measures .", "all the other statistical analyses were performed with the spss for windows , release version 17.0 , ( spss , inc ." ], [ "the final sample encompassed 209 patients ( 66 males and 143 females ; mean age 55.48 sd 13.04 ) .", "sample characteristics and clinical data relative to the md are shown in table 1 and also allow comparison with the complexity of the pd .", "regarding axis i diagnoses , 10.5% of the patients had unipolar depression - single episode , 52.2% unipolar depression recurrent , 22.5% bipolar type ii disorder , and 14.8% bipolar type i disorder .", "regarding personality disorders , 17.2% of patients had no elevated mcmi - iii scores , 45.9% had one peak , and 36.9% had two or more elevated values .", "the prevalence of elevated scores ( i.e. , at least a scale with a score of 85 or above ) on the mcmi - iii is 83% in the group of patients with mood disorders .", "this value drops down to 77% if one considers only patients with depressive disorder . from the recursive partitioning analysis , the age of onset of the md was the most explicative variable with a threshold of 29 years .", "later on , the analysis found a further significant classification : the group of participants over 29 years of age was divided according to the duration of the episodes ( under 3 months versus over 3 months ) .", "consequently the type of md was not significant in the explanation of the different patterns of the personality complexity ( figure 1 ) .", "the three patterns differed from each other on all of the three levels of complexity of pd .", "in particular , node 2 is characterized by more than half the patients with high complexity of pd ( absolute majority of the node ) .", "the other two nodes show for patients with later onset of the md a cluster ( node 5 ) with high presence of pd ( only 8% of patients do not have a pd ) represented by patients with longer episodes ( over 3 months ) and a cluster of patients ( node 4 ) with lower presence of pd and , more importantly , less complexity ( only 16% have a complex personality disorder ) .", "the analysis was applied to the participants with at least one mcmi - iii elevated value once more excluding patients diagnosed with a single major depressive episode . in table 2 , the results of the comparison between the two groups are presented .", "the only statistically significant predictor emerging from multivariate analysis was the age of onset of the md ( f = 8.945 ; df = 1 ; p = .003 ; = .058 ) .", "the significance of the age of testing disappears : its connection to the complexity of the pd was probably influenced by the age of onset of the disorder itself .", "the duration of the episodes between simple and complex disorders was not statistically significant , and this difference did not improve its significance in the multivariate analysis ( f = 1.230 ; df = 2 ; p = .295 ; = .017 ) .", "this led us to think it is not a mere type ii error but more likely a variable with heterogeneous distributions in both personality disorder conditions .", "the profile comparisons ( figure 2 ) show statistically significant differences between the three clusters based on pd complexity ( f = 9.346 ; ( df = 2 ) ; p < .01 ; = .092 ) and so did the interaction between cluster and mcmi - iii profile ( correction of huynh - feldt ( f = 3.640 ; ( df = 14.357 ) ; p < .001 ; = .038 ) ) . from the multiple comparisons with bonferroni corrections , the cluster of patients with early onset of md significantly differed from the cluster with later onset and longer episodes , but it did not differ from the one with shorter episodes . on the other hand , the cluster with later md onset and longer episodes significantly differed from the other clusters .", "in particular , there was a clear difference between the cluster with early onset and the other two .", "the difference is seen in the elevation of the three last scales representing , according to millon , severe personality disorders with more impaired functioning .", "the clusters with later onset differed in the three scales : avoidant , depressive , and dependent , partially differed from each other in the paranoid scale , and remained very close to each other in all the other scales ." ], [ "the aim of the present study was to explore the relationship between the age of onset of md and the complexity of the personality traits of the patients . moreover , we were interested in identifying an age threshold able to maximize the difference of personality traits between early and later onsets and investigating the association between mood disorder severity and personality traits . to start with , the prevalence of the personality disorders ( pd ) evaluated with the mcmi - iii in patients admitted for a depressive / hypomaniac episode through a dimensional evaluation was higher than that in previous studies , regarding both outpatients with a depression diagnosis and dimensional evaluation and patients with unipolar and bipolar disorders [ 6 , 16 ] .", "these results can be in part linked to the fact that all the patients recruited for the present study had at least one hospital admission , a strong indicator of worse severity of the disorder . in the recursive partitioning analysis , the age of onset was the most significant predicting factor .", "interestingly , the type of mood disorder was not a significant predicting factor , as previously shown .", "the threshold of onset was higher compared to previous studies [ 21 , 23 , 33 ] while it overlaps with what was recently highlighted about depressive disorders .", "it is interesting to underline the relationship identified between the severity of md in terms of duration of episodes and of pd , particularly in the cluster with later onset .", "the presence of maladaptive personality makes more likely both the onset of an md and the creation of a more complex personality disorder .", "this interpretation could explain the independence between the complexity of the pd ( with the presence of the elevations on the scales more often associated with severe cases ) and the severity of the md .", "conversely , in the later onsets , the development of the particularly severe md can increase the personality symptomatology and elevate the mcmi - iii scale scores .", "this is what can be found in the three scales : avoidant , depressive , and dependent .", "in fact , between the patients with later md onset , the ones with longer episodes have more elevations on these three scales , while , on the other scales , except partially for the paranoid scale , they are practically identical .", "in particular , avoidant and dependent are two scales whose elevations are expected in the presence of depressive disorder . to date , it is not possible to highlight a clear direction of causality for the examined relationship and state whether the presence of a more complex personality disorder condition is caused by the onset of a more severe md or influences the severity of the md itself ( e.g. , by reducing the presence of social support around the patient ) .", "thus , further studies are necessary to clarify this relevant issue , as the presence of an md as an inclusion criterion and the difficulty to accurately establish an age of onset for an md do not allow any definitive conclusion .", "firstly , the cross - sectional design did not allow us to detect precisely how the pd and md developed .", "secondly , as the assessment was conducted at the end of admission period , we could have found more pd than in the euthymic phase .", "finally , the use of a self - administered tool for the detection of the pd may have led to an overestimation of the presence and complexity of the personality traits , particularly in keeping with the psychometric characteristics of the mcmi - iii , as described in a recent study on the topic ( zennaro , in press ) even if a conservative cutoff criterion was adopted .", "however , mcmi - iii ( as other self - report inventories , e.g. , minnesota multiphasic personality inventory-2 ) has the advantage of including validity scales that can detect possible patterns of response sets , biases , and distortions that might compromise the validity of the clinical assessment assuring specific quality of data at the study ." ], [ "this study has introduced the issue of disorder severity aspects in the investigation of the relationship between md and pd .", "our findings suggest that it is important to design prospective studies able to evaluate the level of comorbidity also on later md onset patients and to include variables about the severity of the disorders . from a clinical point of view", ", the results suggest the need to assess maladaptive personality traits not only during adolescence but also in young adults too in order to prevent and treat potentially severe md ." ] ]

[ "cognitive decline in older adulthood remains an area of great concern as the population ages .", "some changes in cognitive function , such as decreased processing speed , are considered normative aspects of the aging process .", "however , the impact of even mild cognitive impairment on functional capacity highlights the importance of maintaining cognitive function for as long as possible .", "sometimes summarized by the adage use it or lose it , current evidence suggests that leading an active lifestyle using it ", "may buffer the effects of age - related cognitive decline losing it [ 35 ] .", "the mechanisms by which an active and engaged lifestyle may be related to better or preserved cognitive function in older adulthood remain to be fully elucidated .", "however , the cognitive reserve hypothesis predicts that some individuals are better able to withstand the physiological insults to the brain without measurable cognitive deficits because they had greater capacity to begin with . individuals may be able to actively increase their reserve through engaging in cognitively stimulating activities . ", "social activities are considered part of what constitutes an active and engaged lifestyle , alongside cognitive and physical activities [ 3 , 4 , 79 ] . however , the evidence for a relationship between social activity participation and cognitive function is mixed .", "some studies have found a relationship between social activities and cognitive function , while others have failed to do so . in an intervention study ,", "older adults living in residential care with normal cognitive function , after participation in daily short duration social and physical activity sessions , performed better cognitively than they had during their baseline assessment .", "conversely , aartsen et al . found no relationship between social activities and cognitive function six years later using a cross - lagged regression approach that attempted to elucidate the strongest causal pathway .", "similarly , green et al . did not find support for the hypothesis that social contact is protective against later cognitive decline .", "the inconclusiveness of results has been acknowledged previously and suggested to relate to differences in statistical techniques [ 4 , 8 ] .", "however , among studies that have used similar analytical methods , such as logistic regression , results are still mixed ( e.g. , [ 1417 ] ) .", "research examining whether social engagement can predict cognitive function and change in cognitive function over time , using growth modeling techniques , has also lacked a consistent finding .", "james and colleagues found that social activity at baseline was associated with a higher baseline level of global cognitive function and lower rate of decline .", "ertel et al . found that baseline social integration was associated with a slower rate of memory decline , but not baseline memory performance . in a study of danish twins , mcgue and", "christensen found that social activity at baseline was related to level of cognitive function but not to change in cognitive function over time .", "they also found that , within monozygotic same - sex twin pairs , there was no evidence that the more socially active twin was less likely to experience cognitive decline than the less active cotwin .", "overall , these studies demonstrate that many questions remain about the relationship between social activity and cognitive function [ 4 , 7 ] .", "it is not clear whether it is a lifetime of social engagement ( the effects of social activity accrued over time ) that is protective , or if changes in social activity are related to cognitive function .", "small and colleagues examined the relationship between changes in social activity and changes in cognitive function and found stronger evidence for changes in cognitive function predicting changes in social activity than the reverse .", "changes in social activity at any point in time may correlate with changes in cognitive functioning after controlling for its overall trend . ", "the current paper builds on previous work by exploring whether including social activity as a variable that changes over time can clarify the relationship between social activity and cognitive function in older adulthood . using multilevel growth modeling with social activity as a time - varying covariate allows an examination of the relations between time - specific changes in cognition and social activity .", "this allows for a detailed test of whether the impact of social activity on cognitive function is accrued over time or whether changing social activity levels might relatively quickly impact cognitive function .", "examining the temporal relationship between social engagement and cognitive function is needed to inform theories of possible mechanisms . ", "the current analysis examines the relationship between social activity , change in social activity , and four domains of cognition including : reasoning , memory , fluency , and semantic knowledge in four different populations .", "the same models are tested with data from four different longitudinal studies : the long beach longitudinal study ( lbls ) , the seattle longitudinal study ( sls ) , the victoria longitudinal study ( vls ) , and the origins of variance in the oldest - old : octogenarian twins study ( octo - twin ) .", "this addresses the possibility that differing analytical methods may produce differing results and provides the opportunity for immediate replication and direct comparison of results .", "the diversity of the samples , two american , one canadian , and one swedish , increases the generalizability of the results and decreases the possibility that findings might be due to the particular features of one country or community .", "this research , initiated as a partnership between the advanced psychometric methods workshop series ( mungas et al .", ", nia conference grant ) and the integrative analysis of longitudinal studies on aging ( ialsa ) network , brought workshop participants together with researchers from four ialsa member studies .", "these studies were specifically selected based on their collection of cognitive , physical , and social activity data along with a range of cognitive functioning measures over multiple occasions held in common across the four studies . while the activity and cognitive functioning variables are not always identical , the subsets of variables in each study were chosen based on the rationale that they tapped similar domains at the construct level ( e.g. , fluid reasoning ( gf ) , crystallized knowledge ( gc ) , short - term memory ( gsm ) , and long - term storage and retrieval ( glr ; e.g. , category fluency ) ) . in some cases", "the measures are the same , but more often they differ , providing opportunities for both strict and conceptual replication .", "an exception to this is the octo - twin dataset , for which a fluency measure was not available . ", "the octo - twin study is based on the oldest cohort of the swedish twin registry and includes 702 participants aged 80 years and older at the time of the first assessment . beginning in 19911993 , the longitudinal", "all individuals with a dementia diagnosis at baseline were excluded from the analyses ( n = 98 ) .", "the total sample includes 604 individuals , of whom 524 had the social activity measure and at least one of the cognitive measures .", "approximately 20% of the sample was lost to followup at each wave ( 10% per year ) , but most of this attrition was due to death .", "the ratios for gender , education , socioeconomic status , marital status , and housing of the octo - twin sample correspond to population statistics for this age range of the swedish population .", "octo - twin cognitive measures \n reasoning was assessed using koh 's block design test . in this task , participants are presented with red and white blocks and several patterns on cards and asked to construct the design on the card with the blocks .", "memory was assessed using the prose recall test in which participants are asked for immediate free recall of a brief ( 100 words ) story that has a humorous point .", "responses are coded for the amount of information recalled in a manner similar to the wechsler memory scale .", "semantic knowledge was assessed using the swedish version of the information task , which includes questions of general knowledge . \n \n", "reasoning was assessed using koh 's block design test . in this task , participants are presented with red and white blocks and several patterns on cards and asked to construct the design on the card with the blocks .", "memory was assessed using the prose recall test in which participants are asked for immediate free recall of a brief ( 100 words ) story that has a humorous point .", "responses are coded for the amount of information recalled in a manner similar to the wechsler memory scale .", "semantic knowledge was assessed using the swedish version of the information task , which includes questions of general knowledge . \n ", "octo - twin social activityparticipants were asked at each wave : how many people do you see ? the possible response was : none ( 0 ) ; 1 - 2 ( 1 ) ; 35 ( 2 ) ; 610 ( 3 ) , or 11 or more ( 4 )", ". \n participants were asked at each wave : how many people do you see ? the possible response was : none ( 0 ) ; 1 - 2 ( 1 ) ; 35 ( 2 ) ; 610 ( 3 ) , or 11 or more ( 4 ) .", "the lbls was initiated in 1978 when participants were recruited from the family health plan health maintenance organization ( hmo ) , including mainly residents of long beach and orange county .", "the ethnic composition of the older group ( 98% caucasian ) was similar to the 65 + population for the area based on the 1970 census .", "panel 2 , initiated in 1992 , included 633 individuals contacted from the same hmo ( 64 were excluded due to frank dementia or serious sensory or neurological problems ) . in order to include the same measures as those in the seattle longitudinal study ,", "lbls panel 1 ( n = 106 ) and panel 2 ( n = 631 ) data from 1994 to 2003 were used in the current analysis , excluding individuals younger than age 55 in 1994 ( baseline n = 565 ) . during this period ,", "additional information on the lbls design , measures , and participants can be found elsewhere [ 29 , 30 ] . \n", "lbls cognitive measures \n reasoning was assessed using a composite score of the schaie - thurstone adult mental abilities test ( stamat ; schaie , ) letter and number series tests . in letter series ,", "participants view a series of letters ( e.g. , a b c c b a d e f f ) and are asked to discover the rule that governs the series by identifying the letter that should come next in the series .", "participants were to complete as many of the 30 items as possible within six minutes .", "word series was a parallel test to letter series but the letters were replaced with months ( e.g. , january ) and days of the week ( e.g. , monday ) .", "memory involved immediate written recall of a list of 20 concrete high - frequency nouns studied for 3.5 minutes .", "fluency was assessed by a word fluency task where participants were instructed to write down as many words as possible in five minutes that begin with the letter ", "s. participants were instructed that they could not use proper nouns or create words by changing endings of other listed words ( e.g. , if the letter was w and you already said want , you should not also say wants , wanting , or wanted ) . semantic knowledge consisted of the stamat recognition vocabulary test .", "participants were given a word and asked to circle a synonym of that word from four possible alternatives .", "reasoning was assessed using a composite score of the schaie - thurstone adult mental abilities test ( stamat ; schaie , ) letter and number series tests . in letter series ,", "participants view a series of letters ( e.g. , a b c c b a d e f f ) and are asked to discover the rule that governs the series by identifying the letter that should come next in the series .", "participants were to complete as many of the 30 items as possible within six minutes .", "word series was a parallel test to letter series but the letters were replaced with months ( e.g. , january ) and days of the week ( e.g. , monday ) .", "memory involved immediate written recall of a list of 20 concrete high - frequency nouns studied for 3.5 minutes .", "fluency was assessed by a word fluency task where participants were instructed to write down as many words as possible in five minutes that begin with the letter ", "s. participants were instructed that they could not use proper nouns or create words by changing endings of other listed words ( e.g. , if the letter was w and you already said want , you should not also say wants , wanting , or wanted ) . semantic knowledge consisted of the stamat recognition vocabulary test .", "participants were given a word and asked to circle a synonym of that word from four possible alternatives .", "lbls social activity measurea measure of social activity was derived from a modified version of the life complexity scale that was originally developed for the seattle longitudinal study .", "participants were asked to record the number of hours per week on average they spent doing various activities ( e.g. , going to parties ) . the lbls version of the scale included 34 specific activities , 7 of which were considered social . in the current study , these activity measures were dichotomized in order to distinguish those who reported no activity ( coded as 0 ) from those who reported one or more hours of activity per week ( coded as 1 ) .", "this was done because the range of scores varied greatly within and between measures , and because some scores were highly deviant ( skewed ) from expected values ( e.g. , reporting more than 100 hours of reading per week ) .", "the social activity variable was created by selecting questions from the life complexity scale that fit the social activity construct .", "that is , a composite score was formed by summing up the number of social activity items that were endorsed as having one or more hours of activity per week .", "the measure consisted of seven questions including : phone conversations , voluntary activities , going to parties , going to dances , playing cards , visiting others , and attending church .", "a social activity change variable was computed by subtracting the social activity measure in 1994 from social activity in 1994 , 1997 , 2000 , and 2003 .", "a measure of social activity was derived from a modified version of the life complexity scale that was originally developed for the seattle longitudinal study .", "participants were asked to record the number of hours per week on average they spent doing various activities ( e.g. , going to parties ) . the lbls version of the scale included 34 specific activities , 7 of which were considered social . in the current study", ", these activity measures were dichotomized in order to distinguish those who reported no activity ( coded as 0 ) from those who reported one or more hours of activity per week ( coded as 1 ) . this", "was done because the range of scores varied greatly within and between measures , and because some scores were highly deviant ( skewed ) from expected values ( e.g. , reporting more than 100 hours of reading per week ) . the social activity variable was created by selecting questions from the life complexity scale that fit the social activity construct .", "that is , a composite score was formed by summing up the number of social activity items that were endorsed as having one or more hours of activity per week .", "the measure consisted of seven questions including : phone conversations , voluntary activities , going to parties , going to dances , playing cards , visiting others , and attending church .", "a social activity change variable was computed by subtracting the social activity measure in 1994 from social activity in 1994 , 1997 , 2000 , and 2003 .", "the sls is a very long - running longitudinal study initiated by schaie , who first recruited members of a local health maintenance organization in 1956 .", "current analyses used up to four waves of sls data from 1984 to 2005 , which include an expanded set of measures that also overlap with the lbls . only participants of 55 years and older at baseline were included in the analysis .", "baseline was defined as each participant 's first study visit , and time was measured in all analyses as years in study ( coded as 0 , 7 , 14 , and 21 ) .", "see table 3 for sls participant characteristics over the four waves of data analyzed here . \n", "sls cognitive measuresin order to model roughly equivalent cognitive outcomes across the four studies included in this coordinated effort , our analysis included measures of reasoning , fluency , memory , and semantic knowledge from a larger battery of tests .", "reasoning was assessed with the word series test from the schaie - thurstone adult mental abilities test ( stamat ) , in which participants were asked to determine a rule that governs a series of words ( months or days of the week ) by identifying what word should come next in a given series .", "participants were provided with a printed word series and instructed to choose the next word in the series in multiple - choice format .", "the test consists of 30 items and total score is based on number of correct responses completed in 6 minutes .", "as in lbls , fluency was assessed with the word fluency test from the primary mental abilities test .", "memory was assessed with a task in which participants were asked to study a list of 20 printed words for 3.5 minutes and provide immediate written recall of the items .", "semantic knowledge was assessed with the educational testing service ( ets ) test of advanced vocabulary , in which participants were asked to identify synonyms for printed words from 5 choices .", "total score was based on number of correctly identified synonyms out of 36 test items completed within 4 minutes . \n in order to model", "roughly equivalent cognitive outcomes across the four studies included in this coordinated effort , our analysis included measures of reasoning , fluency , memory , and semantic knowledge from a larger battery of tests .", "reasoning was assessed with the word series test from the schaie - thurstone adult mental abilities test ( stamat ) , in which participants were asked to determine a rule that governs a series of words ( months or days of the week ) by identifying what word should come next in a given series .", "participants were provided with a printed word series and instructed to choose the next word in the series in multiple - choice format .", "the test consists of 30 items and total score is based on number of correct responses completed in 6 minutes .", "as in lbls , fluency was assessed with the word fluency test from the primary mental abilities test .", "memory was assessed with a task in which participants were asked to study a list of 20 printed words for 3.5 minutes and provide immediate written recall of the items .", "semantic knowledge was assessed with the educational testing service ( ets ) test of advanced vocabulary , in which participants were asked to identify synonyms for printed words from 5 choices .", "total score was based on number of correctly identified synonyms out of 36 test items completed within 4 minutes . \n ", "sls social activity measurewe followed the methodology described in the lbls method portion of this paper in order to generate a roughly equivalent index of social activity ( see lbls section for a detailed description ) . following this methodology", ", we created a composite activity measure by summing dichotomized responses from a modified version of the life complexity scale creating a seven - item social activity composite ( volunteering , playing cards , phone conversations , visiting others , attending church , dancing , and partying ) .", "social activity change was computed by subtracting baseline activity from each follow - up activity measure . \n ", "we followed the methodology described in the lbls method portion of this paper in order to generate a roughly equivalent index of social activity ( see lbls section for a detailed description ) . following this methodology", ", we created a composite activity measure by summing dichotomized responses from a modified version of the life complexity scale creating a seven - item social activity composite ( volunteering , playing cards , phone conversations , visiting others , attending church , dancing , and partying ) .", "social activity change was computed by subtracting baseline activity from each follow - up activity measure . ", "the victoria longitudinal study began in 1986 - 1987 with a sample of 484 community residing volunteers and three - year retest intervals .", "using a longitudinal sequential design , second and third independent samples began in 1992 - 1993 ( n = 530 ) and 2001 - 2002 ( n = 550 ) .", "each sample is tested at three - year intervals . to date , sample 1 has been tested on seven occasions ( over 18 years ) , sample 2 on five ( over 12 years ) , and sample 3 on two occasions ( over 6 years ) .", "participants in all three samples were recruited between the ages of 55 and 85 years . ", "data from seven waves of sample 1 and five waves of sample 2 were included in the current investigation .", "approximately 25% of the sample was lost to followup at each wave , or 8% per year .", "further detail on the vls design , measures , and participants can be found elsewhere . \n ", "vls cognitive ability measures \n fluency was measured by performance on a similarities task . in this timed task , participants were presented with target words and asked to write as many words as possible with the same or nearly the same meaning within six minutes .", "memory was indexed using a 30 item , noun list learning task comprised of five semantic categories .", "participants studied the word list for two minutes followed by a five minute free recall task .", "reasoning was indexed by letter series in which participants were presented with a series of letters and asked to identify the next letter in the sequence that was consistent with the sequence rule .", "fluency was measured by performance on a similarities task . in this timed task , participants were presented with target words and asked to write as many words as possible with the same or nearly the same meaning within six minutes .", "memory was indexed using a 30 item , noun list learning task comprised of five semantic categories .", "participants studied the word list for two minutes followed by a five minute free recall task .", "reasoning was indexed by letter series in which participants were presented with a series of letters and asked to identify the next letter in the sequence that was consistent with the sequence rule .", "vls social activity / lifestyle measurethe social activity / lifestyle measure used in the presented investigation included a subset of items from the vls activity lifestyle questionnaire .", "individual item distributions were reviewed and a small number of poorly distributed items were eliminated .", "seven items were selected due to their social nature ( eat at restaurants , visit friend / relative , give dinner party , attend church , meetings of service organizations , meetings of clubs , and do volunteer work ) . for each item , participants indicated the frequency of engagement in that activity over the past two years on a scale from 0 to 9 ( i.e. , never , less than once a year , about once a year , 2 or 3 times a year , about once a month , 2 or 3 times a month , about once a week , 2 or 3 times a week , daily ) . \n ", "the social activity / lifestyle measure used in the presented investigation included a subset of items from the vls activity lifestyle questionnaire .", "individual item distributions were reviewed and a small number of poorly distributed items were eliminated .", "seven items were selected due to their social nature ( eat at restaurants , visit friend / relative , give dinner party , attend church , meetings of service organizations , meetings of clubs , and do volunteer work ) . for each item , participants indicated the frequency of engagement in that activity over the past two years on a scale from 0 to 9 ( i.e. , never , less than once a year , about once a year , 2 or 3 times a year , about once a month , 2 or 3 times a month , about once a week , 2 or 3 times a week , daily ) . ", "the current analysis was conducted as part of a larger effort to examine the effects of lifestyle activities on cognitive function using the same analytic approach across studies from the integrative analysis of longitudinal studies on aging ( ialsa ) network .", "thus , final models were selected in part to maintain consistency across lifestyle activities . in order to improve ease of interpretation of our results , age ,", "the means for baseline age , education , and social activity were subtracted from their baseline values for each individual .", "this centered the covariates so that the intercept and linear slope terms would be interpreted as the expected value for an individual at the mean age and with the mean level of education for the study .", "the reference category for sex was male . in order to examine the effects of social activity on cognition , a series of multilevel models was fit with social activity as a baseline and a time varying covariate , with time specified as time since baseline , using multilevel mixed - effects regression in statacorp , the restricted maximum likelihood estimator ( reml ) , and an unstructured covariance matrix . in the octo - twin study , participants were nested within their twin pair . in the vls , we controlled for enrolment cohort .", "separate models were fit for each of the four cognitive measures ( reasoning , fluency , memory , and semantic knowledge ) resulting in four reported models for each study . while the familywise alpha rate within each individual study may be somewhat liberal , given our use of p < .05 as significance criterion , our focus on repetition of findings across studies imposes a strict limit to any reliance on chance findings .", "formal meta - analytic methods , which would require identical measures and a larger number of studies , were not used .", "an initial 19-term model included the following terms : ( 1 ) baseline age , ( 2 ) sex , ( 3 ) education , ( 4 ) baseline social activity , ( 5 ) baseline social activity age , ( 6 ) baseline social activity sex , ( 7 ) baseline social activity education , ( 8) individually defined time since baseline , ( 9 ) time baseline age , ( 10 ) time sex , ( 11 ) time education , ( 12 ) time baseline social activity , ( 13 ) time baseline social activity baseline age , ( 14 ) time baseline social activity sex , ( 15 ) time baseline social activity education , ( 16 ) change in social activity from baseline ( activity change ) , ( 17 ) social activity change baseline age , ( 18 ) social activity change sex , and ( 19 ) social activity change education .", "however , several terms were not significant for most of the studies and outcomes and so were trimmed to facilitate model interpretation .", "this process eliminated 7 of the 19 terms , first the 3-way interactions were eliminated , then the interactions with change in social activity .", "this resulted in a 12-term final model , presented in table 5 for separate cognitive constructs of reasoning , memory , semantic knowledge , and fluency .", "significant between person age differences were seen at the first occasion of measurement for all memory , reasoning , and fluency tests , with older adults performing less well than their younger counterparts ( all p < .01 ) .", "semantic knowledge results were less consistent , with sls showing no age differences , b = 0.02 , p = .35 , lbls and octo - twin suggesting the older individuals score worse , b = 0.28 , p < .01 , and b = 0.55 , p < .01 , respectively , and vls finding that older individuals performed slightly better , b = 0.08 , p = .02 . ", "at baseline , individuals with more years of education had significantly higher cognitive performance on all tasks , across all studies ( all p < .01 ) .", "women at the mean baseline age had higher memory scores than did same aged men across all studies ( all p < .01 ) .", "lbls and sls women scored higher than men on all measures ( reasoning : p = .02 and p < .01 ; fluency : both p < .01 ) , except for lbls semantic knowledge ( p = .13 , sls p = .02 ) .", "octo - twin women had significantly lower information scores , considered semantic knowledge , at baseline than octo - twin men ( b = 4.20 , p < .01 ) .", "significant within person declines were seen over time in study in all cognitive abilities and all studies ( all ps < .01 ) .", "a significant timeage interaction indicated that within each sample , those who were older at baseline declined significantly faster than the younger participants on all vls , sls , and lbls measures except lbls immediate memory . except for the information test , evidence for differential decline in older individuals", "was not seen in octo - twin , which has a much narrower age range .", "women showed less decline than men in the sls data only , and only for the fluency and semantic knowledge measures .", "evidence for differential decline related to education was seen only for the lbls reasoning measure .", "higher social activity levels at baseline , for individuals of average age and education , were associated with higher scores on reasoning within the vls and octo - twin studies , b = 0.05 , p = .03 , and b = 1.01 , p < .01 , respectively .", "the most consistent finding was that individuals with higher social activity levels at baseline also had higher memory scores .", "this was true for the sls , vls , and octo - twin studies , b = 0.14 , p = .01 , b = 0.06 , p < .01 , b = 0.34 , p < .01 , respectively .", "baseline social activity was also positively related to fluency in the lbls ( though the same measure was not significant in sls sample ) .", "social activity at baseline and semantic knowledge was positively related in the octo - twin sample ( operationalized as information ; b = 0.87 , p = .03 ) , but negatively related in the sls sample ( operationalized as vocabulary ; b = 0.22 , p = .02 ) .", "the octo - twin sample was the only one where all cognitive measures considered had a positive association with baseline social activity levels . ", "the baseline agesocial activity interaction terms were significant and positive for lbls reasoning ( b = 0.08 , p < .01 ) and semantic knowledge ( b = 0.08 , p < .01 ) , indicating a stronger effect of social activity for older participants than for younger participants .", "however , the interaction of age and social activity was not significant for any other studies or cognitive measures ( ps > .05 ) .", "the interaction between education and social activity at baseline was not significantly associated with any cognitive measure within any of the samples . in terms of within person", "age changes , social activity at baseline was significantly related to the slope of semantic knowledge for sls participants such that higher social activity at baseline was associated with less semantic knowledge decline ( p = .01 ) .", "there were no other significant relationships between social activity at baseline and rate of change for any cognitive measure within any of the samples ( all p > .05 ) . in examining associations with change in social activity as a time - varying covariate in each of the models ,", "was positively associated with performance on the fluency measures in all studies except sls ( lbls p < .01 , vls p < .01 , sls p = .21 ) , such that individuals who increased their level of social activity from their own baseline level exhibited higher occasion - specific fluency performance relative to their expected linear trajectory over time . in vls and sls , a significant relationship was also found between change in social activity and the reasoning ( vls p = .02 , sls p = .04 ) and memory ( vls p < .01 , sls p = .03 ) measure , indicating that participants who increased their social activity level also scored higher relative to their expected trajectory on the reasoning or memory measure .", "changes in social activity were related to all cognitive measures in the octo - twin study ( all p < .05 ) , such that octo - twin participants who increased their social activity level from baseline had higher occasion - specific cognitive scores relative to their own linear trajectory in all domains tested .", "comparing results of the same statistical model across four longitudinal studies of aging , we observed relatively few consistent associations between social activity and cognitive functioning . looking at within person change in social activity and the four cognitive measures , the most consistent finding was an association with fluency in two of the three studies measuring it , after adjusting for linear effects of time and other covariates . change in social activity was associated with memory performance in only half of the four studies ( sls and vls ) . in only one study , octo - twin ,", "change in social activity was significantly related to performance in all three of the cognitive domains considered ( fluency measure not available ) .", "the octo - twin sample was the oldest and had the narrowest range of ages .", "social activity in the octo - twin study was defined by the number of people with whom participants had contact , whereas the other studies included a range of activities with a social component . given that within person decreases in performance were seen across all cognitive measures in all studies , the positive relationships generally represent attenuated decline , rather than improvement in cognitive performance . ", "the use of within person change in social activity as a time - varying predictor of cognitive function is somewhat unique .", "however , in another study examining the temporal relations of social activity change and cognitive function , small and colleagues found significant coupling of social activity change and three cognitive domains : semantic decision speed ( similar to our fluency measure ) , episodic memory , and semantic memory .", "they , however , found greater support for models where cognitive measures predicted changes in social activity levels , than the reverse", ". there was little evidence that initial levels of social activity were related to within person rate of decline in cognitive function .", "although higher initial levels of social activity were associated with less decline on the sls semantic knowledge measure , this was not evident in any other samples .", "sls has the widest interval between measurements and so correspondingly greater attrition between waves ( though a similar yearly rate ) , but this does not suggest an obvious reason for the difference .", "the findings from the other studies are consistent with mcgue and christensen , who found that social activity at the first assessment was related to initial level of cognitive function but not change in cognitive function .", "however , other groups have found that social activity levels are associated with both baseline cognitive function and a reduced rate of decline over time .", ". found that social integration was related to a slower rate of memory decline .", "we did not include a composite measure of general cognitive function , but our results , and the results of others who have examined specific facets of cognition , suggest that it is important to examine cognitive domains separately . ", "specifically , individuals with higher initial levels of social activity performed better on the memory measures in three of the four samples .", "interestingly , the sample that did not show the effect was the lbls , which used the same measure as the sls study .", "this suggests the discrepancy is not a function of different memory measures being used , or how social activity was characterized in the study .", "it is perhaps related to differences in samples , although both were similar in average years of education and were american , although from different regions .", "this , and the accessibility of the activities , may influence the association with cognitive function", "the mean age of lbls participants was about six years older and the first wave of lbls assessment included in the present study was conducted ten years later than the first assessment of the sls .", "the age differences are not an obvious explanation , because the mean age of lbls participants is only slightly older than sls and vls participants and is nearly ten years younger than octo - twin participants .", "the lbls is the only study for which the majority of participants are not female , though this difference is slim ( 49% female in the lbls versus 52% in sls at baseline ) .", "the lbls does have the highest yearly rate of attrition ( 17% versus 10% , 7% , and 8% ) , although the loss at each measurement wave is similar to that of sls .", "it is unclear how this would affect the association between baseline social activity and memory performance ; however , particularly considering that the mean social activity and memory measure scores for the lbls and sls were similar . it would be interesting in future work to consider general health and its age gradient in each of the studies .", "the lack of obvious reasons for discrepant findings , however , supports the importance of considering the reproducibility of results in coordinated , rather than pooled , analyses .", "social integration has been posited to influence general health through multiple pathways that may overlap with those influencing cognition .", "unfortunately , our findings do not strongly suggest that increased engagement in social activities confers immediate benefit in terms of cognitive function . nor do they suggest that social participation reduces risk of cognitive decline in any domain apart from fluency .", "the cognitive reserve hypothesis suggests that , over time , reserve can be built up through stimulating activities and that individuals with high reserve can withstand more physiological deterioration before cognitive decline is observable . that social participation does not reduce the risk of cognitive decline in a broad range of measures suggests that it is not conferring reserve .", " one possible mechanism through which social engagement and cognitive function are related may be the cognitively stimulating nature of social activities .", "the cognitive training literature has found that training in specific tasks ( e.g. , memory tasks ) does not necessarily transfer to other cognitive domains .", "this may be similarly true of social activity participation , whereby a relationship is only seen in cognitive domains that are being challenged by the social activity .", "social interactions do typically involve verbal communication , and thus fluency , likely specifically verbal fluency , may be the cognitive domain most similar to our participants ' social activities . ", "discrepancies in which activities are considered social may contribute to the lack of consistent association between social activity and cognitive function .", "for example , some activities ( e.g. , card games ) may generally be more cognitively demanding than others ( e.g. , visits from family members ) , or primarily tax different cognitive skills , but this is likely to differ across individuals and situations .", "although plausible , further research would need to confirm the validity of such hypothesis . in the context of physical health ,", "others have suggested that different social factors ( e.g. , social influence , social engagement , and social support ) may act primarily through different behavioral , psychological , and physiological pathways .", "similarly , cognitive function may be differentially influenced depending on the particular combination of social factors and pathways . in this way ,", "how social activity is conceptualized and measured may be related to the mechanistic pathway and thus differentially related to various domains of cognitive function .", "these effects may have contributed to the discrepancies between octo - twin and the other three studies in the current analysis , as octo - twin focused on frequency of contact with people as the social activity measure , whereas the other three encompassed a variety of activities .", "however , the lack of consistency between the two studies employing the same measures suggests that conceptualization of social activity does not fully explain the findings .", "a clue to the source of inconsistency across lbls and sls may be the very small average change in the lbls social activities measure , though its variance is comparable to those of sls and octo - twin .", "the association between cognition and social activity in lbls in particular also seems to vary by age more than in the other studies .", "a considerable strength of our analysis is the replication of the models across four longitudinal studies from geographically separate regions .", "this limits the possibility of spurious findings taking on undue importance and provides an opportunity to examine consistencies and inconsistencies between sample groups . in terms of potential methodological limitations ,", "( for slope covariances ; for variance components ) suggest that the power to detect correlated change is extremely low .", "however , analysis of longitudinal studies on aging , including those used in the current paper , has consistently reported statistically significant variances and covariances in rates of change in cognitive outcomes ( [ 4354 ] , for summary see ) . in addition , the tests used by hertzog et al .", "are based on significance tests for variance components whereas change was evaluated in our paper by examining fixed effects which will generally have greater power .", "lending support to the argument that analyses in the current paper were adequately powered are the findings of significant associations between the identical set of cognitive outcomes in these same studies and physical and cognitive activities [ 57 , 58 ] .", "another limitation to our analysis is that while the study samples were restricted to initially healthy older adults , efforts were not made to exclude individuals who developed dementia over the course of the study periods ( dementia diagnosis was available only in the octo - twin study ) .", "we can not assume that any protective effects of social activity on cognitive function would be equivalent across healthy and dementing older adults .", "including individuals whose cognitive decline may have been driven by the dementia process may impact the associations between social activity and cognitive function over time in nondementing individuals , and this may also contribute to inconsistencies in the literature . finally , it is difficult to rule out the possibility that individuals decrease their social activities because their declining cognitive abilities make it more difficult for them to maintain social ties .", "the difficulty of determining the directionality of the relationship has been well acknowledged in the literature and it is similarly difficult to determine causal pathways in the current analysis [ 4 , 5 , 10 ] .", "some attempts have been made to determine the most likely direction of the relationship , but the evidence is limited .", "the longitudinal nature of the studies considered here , and allowing social activity to vary across time , have narrowed the temporal distance between the social activity and cognitive performance , at least partially disentangling whether the relationship is primarily based on historical activity levels or whether the two tracks over time . across four studies in three countries ,", "baseline social activity levels failed to predict rate of decline in most cognitive abilities , and changes in social activity were not consistently associated with within person fluctuations in cognitive functioning .", "our findings leave little support for the hypothesis that changes in social activity correspond to immediate benefits in cognitive functioning , except perhaps for fluency ." ]

[ "<S> social activity is typically viewed as part of an engaged lifestyle that may help mitigate the deleterious effects of advanced age on cognitive function . as such </S>", "<S> , social activity has been examined in relation to cognitive abilities later in life . </S>", "<S> however , longitudinal evidence for this hypothesis thus far remains inconclusive . </S>", "<S> the current study sought to clarify the relationship between social activity and cognitive function over time using a coordinated data analysis approach across four longitudinal studies . </S>", "<S> a series of multilevel growth models with social activity included as a covariate is presented . </S>", "<S> four domains of cognitive function were assessed : reasoning , memory , fluency , and semantic knowledge . </S>", "<S> results suggest that baseline social activity is related to some , but not all , cognitive functions . </S>", "<S> baseline social activity levels failed to predict rate of decline in most cognitive abilities . </S>", "<S> changes in social activity were not consistently associated with cognitive functioning . </S>", "<S> our findings do not provide consistent evidence that changes in social activity correspond to immediate benefits in cognitive functioning , except perhaps for verbal fluency . </S>" ]

[ "1. Introduction", "2. Method", "3. Results", "4. Discussion" ]

[ [ "cognitive decline in older adulthood remains an area of great concern as the population ages .", "some changes in cognitive function , such as decreased processing speed , are considered normative aspects of the aging process .", "however , the impact of even mild cognitive impairment on functional capacity highlights the importance of maintaining cognitive function for as long as possible .", "sometimes summarized by the adage use it or lose it , current evidence suggests that leading an active lifestyle using it ", "may buffer the effects of age - related cognitive decline losing it [ 35 ] .", "the mechanisms by which an active and engaged lifestyle may be related to better or preserved cognitive function in older adulthood remain to be fully elucidated .", "however , the cognitive reserve hypothesis predicts that some individuals are better able to withstand the physiological insults to the brain without measurable cognitive deficits because they had greater capacity to begin with . individuals may be able to actively increase their reserve through engaging in cognitively stimulating activities . ", "social activities are considered part of what constitutes an active and engaged lifestyle , alongside cognitive and physical activities [ 3 , 4 , 79 ] . however , the evidence for a relationship between social activity participation and cognitive function is mixed .", "some studies have found a relationship between social activities and cognitive function , while others have failed to do so . in an intervention study ,", "older adults living in residential care with normal cognitive function , after participation in daily short duration social and physical activity sessions , performed better cognitively than they had during their baseline assessment .", "conversely , aartsen et al . found no relationship between social activities and cognitive function six years later using a cross - lagged regression approach that attempted to elucidate the strongest causal pathway .", "similarly , green et al . did not find support for the hypothesis that social contact is protective against later cognitive decline .", "the inconclusiveness of results has been acknowledged previously and suggested to relate to differences in statistical techniques [ 4 , 8 ] .", "however , among studies that have used similar analytical methods , such as logistic regression , results are still mixed ( e.g. , [ 1417 ] ) .", "research examining whether social engagement can predict cognitive function and change in cognitive function over time , using growth modeling techniques , has also lacked a consistent finding .", "james and colleagues found that social activity at baseline was associated with a higher baseline level of global cognitive function and lower rate of decline .", "ertel et al . found that baseline social integration was associated with a slower rate of memory decline , but not baseline memory performance . in a study of danish twins , mcgue and", "christensen found that social activity at baseline was related to level of cognitive function but not to change in cognitive function over time .", "they also found that , within monozygotic same - sex twin pairs , there was no evidence that the more socially active twin was less likely to experience cognitive decline than the less active cotwin .", "overall , these studies demonstrate that many questions remain about the relationship between social activity and cognitive function [ 4 , 7 ] .", "it is not clear whether it is a lifetime of social engagement ( the effects of social activity accrued over time ) that is protective , or if changes in social activity are related to cognitive function .", "small and colleagues examined the relationship between changes in social activity and changes in cognitive function and found stronger evidence for changes in cognitive function predicting changes in social activity than the reverse .", "changes in social activity at any point in time may correlate with changes in cognitive functioning after controlling for its overall trend . ", "the current paper builds on previous work by exploring whether including social activity as a variable that changes over time can clarify the relationship between social activity and cognitive function in older adulthood . using multilevel growth modeling with social activity as a time - varying covariate allows an examination of the relations between time - specific changes in cognition and social activity .", "this allows for a detailed test of whether the impact of social activity on cognitive function is accrued over time or whether changing social activity levels might relatively quickly impact cognitive function .", "examining the temporal relationship between social engagement and cognitive function is needed to inform theories of possible mechanisms . ", "the current analysis examines the relationship between social activity , change in social activity , and four domains of cognition including : reasoning , memory , fluency , and semantic knowledge in four different populations .", "the same models are tested with data from four different longitudinal studies : the long beach longitudinal study ( lbls ) , the seattle longitudinal study ( sls ) , the victoria longitudinal study ( vls ) , and the origins of variance in the oldest - old : octogenarian twins study ( octo - twin ) .", "this addresses the possibility that differing analytical methods may produce differing results and provides the opportunity for immediate replication and direct comparison of results .", "the diversity of the samples , two american , one canadian , and one swedish , increases the generalizability of the results and decreases the possibility that findings might be due to the particular features of one country or community ." ], [ "this research , initiated as a partnership between the advanced psychometric methods workshop series ( mungas et al .", ", nia conference grant ) and the integrative analysis of longitudinal studies on aging ( ialsa ) network , brought workshop participants together with researchers from four ialsa member studies .", "these studies were specifically selected based on their collection of cognitive , physical , and social activity data along with a range of cognitive functioning measures over multiple occasions held in common across the four studies . while the activity and cognitive functioning variables are not always identical , the subsets of variables in each study were chosen based on the rationale that they tapped similar domains at the construct level ( e.g. , fluid reasoning ( gf ) , crystallized knowledge ( gc ) , short - term memory ( gsm ) , and long - term storage and retrieval ( glr ; e.g. , category fluency ) ) . in some cases", "the measures are the same , but more often they differ , providing opportunities for both strict and conceptual replication .", "an exception to this is the octo - twin dataset , for which a fluency measure was not available . ", "the octo - twin study is based on the oldest cohort of the swedish twin registry and includes 702 participants aged 80 years and older at the time of the first assessment . beginning in 19911993 , the longitudinal", "all individuals with a dementia diagnosis at baseline were excluded from the analyses ( n = 98 ) .", "the total sample includes 604 individuals , of whom 524 had the social activity measure and at least one of the cognitive measures .", "approximately 20% of the sample was lost to followup at each wave ( 10% per year ) , but most of this attrition was due to death .", "the ratios for gender , education , socioeconomic status , marital status , and housing of the octo - twin sample correspond to population statistics for this age range of the swedish population .", "octo - twin cognitive measures \n reasoning was assessed using koh 's block design test . in this task , participants are presented with red and white blocks and several patterns on cards and asked to construct the design on the card with the blocks .", "memory was assessed using the prose recall test in which participants are asked for immediate free recall of a brief ( 100 words ) story that has a humorous point .", "responses are coded for the amount of information recalled in a manner similar to the wechsler memory scale .", "semantic knowledge was assessed using the swedish version of the information task , which includes questions of general knowledge . \n \n", "reasoning was assessed using koh 's block design test . in this task , participants are presented with red and white blocks and several patterns on cards and asked to construct the design on the card with the blocks .", "memory was assessed using the prose recall test in which participants are asked for immediate free recall of a brief ( 100 words ) story that has a humorous point .", "responses are coded for the amount of information recalled in a manner similar to the wechsler memory scale .", "semantic knowledge was assessed using the swedish version of the information task , which includes questions of general knowledge . \n ", "octo - twin social activityparticipants were asked at each wave : how many people do you see ? the possible response was : none ( 0 ) ; 1 - 2 ( 1 ) ; 35 ( 2 ) ; 610 ( 3 ) , or 11 or more ( 4 )", ". \n participants were asked at each wave : how many people do you see ? the possible response was : none ( 0 ) ; 1 - 2 ( 1 ) ; 35 ( 2 ) ; 610 ( 3 ) , or 11 or more ( 4 ) .", "the lbls was initiated in 1978 when participants were recruited from the family health plan health maintenance organization ( hmo ) , including mainly residents of long beach and orange county .", "the ethnic composition of the older group ( 98% caucasian ) was similar to the 65 + population for the area based on the 1970 census .", "panel 2 , initiated in 1992 , included 633 individuals contacted from the same hmo ( 64 were excluded due to frank dementia or serious sensory or neurological problems ) . in order to include the same measures as those in the seattle longitudinal study ,", "lbls panel 1 ( n = 106 ) and panel 2 ( n = 631 ) data from 1994 to 2003 were used in the current analysis , excluding individuals younger than age 55 in 1994 ( baseline n = 565 ) . during this period ,", "additional information on the lbls design , measures , and participants can be found elsewhere [ 29 , 30 ] . \n", "lbls cognitive measures \n reasoning was assessed using a composite score of the schaie - thurstone adult mental abilities test ( stamat ; schaie , ) letter and number series tests . in letter series ,", "participants view a series of letters ( e.g. , a b c c b a d e f f ) and are asked to discover the rule that governs the series by identifying the letter that should come next in the series .", "participants were to complete as many of the 30 items as possible within six minutes .", "word series was a parallel test to letter series but the letters were replaced with months ( e.g. , january ) and days of the week ( e.g. , monday ) .", "memory involved immediate written recall of a list of 20 concrete high - frequency nouns studied for 3.5 minutes .", "fluency was assessed by a word fluency task where participants were instructed to write down as many words as possible in five minutes that begin with the letter ", "s. participants were instructed that they could not use proper nouns or create words by changing endings of other listed words ( e.g. , if the letter was w and you already said want , you should not also say wants , wanting , or wanted ) . semantic knowledge consisted of the stamat recognition vocabulary test .", "participants were given a word and asked to circle a synonym of that word from four possible alternatives .", "reasoning was assessed using a composite score of the schaie - thurstone adult mental abilities test ( stamat ; schaie , ) letter and number series tests . in letter series ,", "participants view a series of letters ( e.g. , a b c c b a d e f f ) and are asked to discover the rule that governs the series by identifying the letter that should come next in the series .", "participants were to complete as many of the 30 items as possible within six minutes .", "word series was a parallel test to letter series but the letters were replaced with months ( e.g. , january ) and days of the week ( e.g. , monday ) .", "memory involved immediate written recall of a list of 20 concrete high - frequency nouns studied for 3.5 minutes .", "fluency was assessed by a word fluency task where participants were instructed to write down as many words as possible in five minutes that begin with the letter ", "s. participants were instructed that they could not use proper nouns or create words by changing endings of other listed words ( e.g. , if the letter was w and you already said want , you should not also say wants , wanting , or wanted ) . semantic knowledge consisted of the stamat recognition vocabulary test .", "participants were given a word and asked to circle a synonym of that word from four possible alternatives .", "lbls social activity measurea measure of social activity was derived from a modified version of the life complexity scale that was originally developed for the seattle longitudinal study .", "participants were asked to record the number of hours per week on average they spent doing various activities ( e.g. , going to parties ) . the lbls version of the scale included 34 specific activities , 7 of which were considered social . in the current study , these activity measures were dichotomized in order to distinguish those who reported no activity ( coded as 0 ) from those who reported one or more hours of activity per week ( coded as 1 ) .", "this was done because the range of scores varied greatly within and between measures , and because some scores were highly deviant ( skewed ) from expected values ( e.g. , reporting more than 100 hours of reading per week ) .", "the social activity variable was created by selecting questions from the life complexity scale that fit the social activity construct .", "that is , a composite score was formed by summing up the number of social activity items that were endorsed as having one or more hours of activity per week .", "the measure consisted of seven questions including : phone conversations , voluntary activities , going to parties , going to dances , playing cards , visiting others , and attending church .", "a social activity change variable was computed by subtracting the social activity measure in 1994 from social activity in 1994 , 1997 , 2000 , and 2003 .", "a measure of social activity was derived from a modified version of the life complexity scale that was originally developed for the seattle longitudinal study .", "participants were asked to record the number of hours per week on average they spent doing various activities ( e.g. , going to parties ) . the lbls version of the scale included 34 specific activities , 7 of which were considered social . in the current study", ", these activity measures were dichotomized in order to distinguish those who reported no activity ( coded as 0 ) from those who reported one or more hours of activity per week ( coded as 1 ) . this", "was done because the range of scores varied greatly within and between measures , and because some scores were highly deviant ( skewed ) from expected values ( e.g. , reporting more than 100 hours of reading per week ) . the social activity variable was created by selecting questions from the life complexity scale that fit the social activity construct .", "that is , a composite score was formed by summing up the number of social activity items that were endorsed as having one or more hours of activity per week .", "the measure consisted of seven questions including : phone conversations , voluntary activities , going to parties , going to dances , playing cards , visiting others , and attending church .", "a social activity change variable was computed by subtracting the social activity measure in 1994 from social activity in 1994 , 1997 , 2000 , and 2003 .", "the sls is a very long - running longitudinal study initiated by schaie , who first recruited members of a local health maintenance organization in 1956 .", "current analyses used up to four waves of sls data from 1984 to 2005 , which include an expanded set of measures that also overlap with the lbls . only participants of 55 years and older at baseline were included in the analysis .", "baseline was defined as each participant 's first study visit , and time was measured in all analyses as years in study ( coded as 0 , 7 , 14 , and 21 ) .", "see table 3 for sls participant characteristics over the four waves of data analyzed here . \n", "sls cognitive measuresin order to model roughly equivalent cognitive outcomes across the four studies included in this coordinated effort , our analysis included measures of reasoning , fluency , memory , and semantic knowledge from a larger battery of tests .", "reasoning was assessed with the word series test from the schaie - thurstone adult mental abilities test ( stamat ) , in which participants were asked to determine a rule that governs a series of words ( months or days of the week ) by identifying what word should come next in a given series .", "participants were provided with a printed word series and instructed to choose the next word in the series in multiple - choice format .", "the test consists of 30 items and total score is based on number of correct responses completed in 6 minutes .", "as in lbls , fluency was assessed with the word fluency test from the primary mental abilities test .", "memory was assessed with a task in which participants were asked to study a list of 20 printed words for 3.5 minutes and provide immediate written recall of the items .", "semantic knowledge was assessed with the educational testing service ( ets ) test of advanced vocabulary , in which participants were asked to identify synonyms for printed words from 5 choices .", "total score was based on number of correctly identified synonyms out of 36 test items completed within 4 minutes . \n in order to model", "roughly equivalent cognitive outcomes across the four studies included in this coordinated effort , our analysis included measures of reasoning , fluency , memory , and semantic knowledge from a larger battery of tests .", "reasoning was assessed with the word series test from the schaie - thurstone adult mental abilities test ( stamat ) , in which participants were asked to determine a rule that governs a series of words ( months or days of the week ) by identifying what word should come next in a given series .", "participants were provided with a printed word series and instructed to choose the next word in the series in multiple - choice format .", "the test consists of 30 items and total score is based on number of correct responses completed in 6 minutes .", "as in lbls , fluency was assessed with the word fluency test from the primary mental abilities test .", "memory was assessed with a task in which participants were asked to study a list of 20 printed words for 3.5 minutes and provide immediate written recall of the items .", "semantic knowledge was assessed with the educational testing service ( ets ) test of advanced vocabulary , in which participants were asked to identify synonyms for printed words from 5 choices .", "total score was based on number of correctly identified synonyms out of 36 test items completed within 4 minutes . \n ", "sls social activity measurewe followed the methodology described in the lbls method portion of this paper in order to generate a roughly equivalent index of social activity ( see lbls section for a detailed description ) . following this methodology", ", we created a composite activity measure by summing dichotomized responses from a modified version of the life complexity scale creating a seven - item social activity composite ( volunteering , playing cards , phone conversations , visiting others , attending church , dancing , and partying ) .", "social activity change was computed by subtracting baseline activity from each follow - up activity measure . \n ", "we followed the methodology described in the lbls method portion of this paper in order to generate a roughly equivalent index of social activity ( see lbls section for a detailed description ) . following this methodology", ", we created a composite activity measure by summing dichotomized responses from a modified version of the life complexity scale creating a seven - item social activity composite ( volunteering , playing cards , phone conversations , visiting others , attending church , dancing , and partying ) .", "social activity change was computed by subtracting baseline activity from each follow - up activity measure . ", "the victoria longitudinal study began in 1986 - 1987 with a sample of 484 community residing volunteers and three - year retest intervals .", "using a longitudinal sequential design , second and third independent samples began in 1992 - 1993 ( n = 530 ) and 2001 - 2002 ( n = 550 ) .", "each sample is tested at three - year intervals . to date , sample 1 has been tested on seven occasions ( over 18 years ) , sample 2 on five ( over 12 years ) , and sample 3 on two occasions ( over 6 years ) .", "participants in all three samples were recruited between the ages of 55 and 85 years . ", "data from seven waves of sample 1 and five waves of sample 2 were included in the current investigation .", "approximately 25% of the sample was lost to followup at each wave , or 8% per year .", "further detail on the vls design , measures , and participants can be found elsewhere . \n ", "vls cognitive ability measures \n fluency was measured by performance on a similarities task . in this timed task , participants were presented with target words and asked to write as many words as possible with the same or nearly the same meaning within six minutes .", "memory was indexed using a 30 item , noun list learning task comprised of five semantic categories .", "participants studied the word list for two minutes followed by a five minute free recall task .", "reasoning was indexed by letter series in which participants were presented with a series of letters and asked to identify the next letter in the sequence that was consistent with the sequence rule .", "fluency was measured by performance on a similarities task . in this timed task , participants were presented with target words and asked to write as many words as possible with the same or nearly the same meaning within six minutes .", "memory was indexed using a 30 item , noun list learning task comprised of five semantic categories .", "participants studied the word list for two minutes followed by a five minute free recall task .", "reasoning was indexed by letter series in which participants were presented with a series of letters and asked to identify the next letter in the sequence that was consistent with the sequence rule .", "vls social activity / lifestyle measurethe social activity / lifestyle measure used in the presented investigation included a subset of items from the vls activity lifestyle questionnaire .", "individual item distributions were reviewed and a small number of poorly distributed items were eliminated .", "seven items were selected due to their social nature ( eat at restaurants , visit friend / relative , give dinner party , attend church , meetings of service organizations , meetings of clubs , and do volunteer work ) . for each item , participants indicated the frequency of engagement in that activity over the past two years on a scale from 0 to 9 ( i.e. , never , less than once a year , about once a year , 2 or 3 times a year , about once a month , 2 or 3 times a month , about once a week , 2 or 3 times a week , daily ) . \n ", "the social activity / lifestyle measure used in the presented investigation included a subset of items from the vls activity lifestyle questionnaire .", "individual item distributions were reviewed and a small number of poorly distributed items were eliminated .", "seven items were selected due to their social nature ( eat at restaurants , visit friend / relative , give dinner party , attend church , meetings of service organizations , meetings of clubs , and do volunteer work ) . for each item , participants indicated the frequency of engagement in that activity over the past two years on a scale from 0 to 9 ( i.e. , never , less than once a year , about once a year , 2 or 3 times a year , about once a month , 2 or 3 times a month , about once a week , 2 or 3 times a week , daily ) . ", "the current analysis was conducted as part of a larger effort to examine the effects of lifestyle activities on cognitive function using the same analytic approach across studies from the integrative analysis of longitudinal studies on aging ( ialsa ) network .", "thus , final models were selected in part to maintain consistency across lifestyle activities . in order to improve ease of interpretation of our results , age ,", "the means for baseline age , education , and social activity were subtracted from their baseline values for each individual .", "this centered the covariates so that the intercept and linear slope terms would be interpreted as the expected value for an individual at the mean age and with the mean level of education for the study .", "the reference category for sex was male . in order to examine the effects of social activity on cognition , a series of multilevel models was fit with social activity as a baseline and a time varying covariate , with time specified as time since baseline , using multilevel mixed - effects regression in statacorp , the restricted maximum likelihood estimator ( reml ) , and an unstructured covariance matrix . in the octo - twin study , participants were nested within their twin pair . in the vls , we controlled for enrolment cohort .", "separate models were fit for each of the four cognitive measures ( reasoning , fluency , memory , and semantic knowledge ) resulting in four reported models for each study . while the familywise alpha rate within each individual study may be somewhat liberal , given our use of p < .05 as significance criterion , our focus on repetition of findings across studies imposes a strict limit to any reliance on chance findings .", "formal meta - analytic methods , which would require identical measures and a larger number of studies , were not used .", "an initial 19-term model included the following terms : ( 1 ) baseline age , ( 2 ) sex , ( 3 ) education , ( 4 ) baseline social activity , ( 5 ) baseline social activity age , ( 6 ) baseline social activity sex , ( 7 ) baseline social activity education , ( 8) individually defined time since baseline , ( 9 ) time baseline age , ( 10 ) time sex , ( 11 ) time education , ( 12 ) time baseline social activity , ( 13 ) time baseline social activity baseline age , ( 14 ) time baseline social activity sex , ( 15 ) time baseline social activity education , ( 16 ) change in social activity from baseline ( activity change ) , ( 17 ) social activity change baseline age , ( 18 ) social activity change sex , and ( 19 ) social activity change education .", "however , several terms were not significant for most of the studies and outcomes and so were trimmed to facilitate model interpretation .", "this process eliminated 7 of the 19 terms , first the 3-way interactions were eliminated , then the interactions with change in social activity .", "this resulted in a 12-term final model , presented in table 5 for separate cognitive constructs of reasoning , memory , semantic knowledge , and fluency ." ], [ "significant between person age differences were seen at the first occasion of measurement for all memory , reasoning , and fluency tests , with older adults performing less well than their younger counterparts ( all p < .01 ) .", "semantic knowledge results were less consistent , with sls showing no age differences , b = 0.02 , p = .35 , lbls and octo - twin suggesting the older individuals score worse , b = 0.28 , p < .01 , and b = 0.55 , p < .01 , respectively , and vls finding that older individuals performed slightly better , b = 0.08 , p = .02 . ", "at baseline , individuals with more years of education had significantly higher cognitive performance on all tasks , across all studies ( all p < .01 ) .", "women at the mean baseline age had higher memory scores than did same aged men across all studies ( all p < .01 ) .", "lbls and sls women scored higher than men on all measures ( reasoning : p = .02 and p < .01 ; fluency : both p < .01 ) , except for lbls semantic knowledge ( p = .13 , sls p = .02 ) .", "octo - twin women had significantly lower information scores , considered semantic knowledge , at baseline than octo - twin men ( b = 4.20 , p < .01 ) .", "significant within person declines were seen over time in study in all cognitive abilities and all studies ( all ps < .01 ) .", "a significant timeage interaction indicated that within each sample , those who were older at baseline declined significantly faster than the younger participants on all vls , sls , and lbls measures except lbls immediate memory . except for the information test , evidence for differential decline in older individuals", "was not seen in octo - twin , which has a much narrower age range .", "women showed less decline than men in the sls data only , and only for the fluency and semantic knowledge measures .", "evidence for differential decline related to education was seen only for the lbls reasoning measure .", "higher social activity levels at baseline , for individuals of average age and education , were associated with higher scores on reasoning within the vls and octo - twin studies , b = 0.05 , p = .03 , and b = 1.01 , p < .01 , respectively .", "the most consistent finding was that individuals with higher social activity levels at baseline also had higher memory scores .", "this was true for the sls , vls , and octo - twin studies , b = 0.14 , p = .01 , b = 0.06 , p < .01 , b = 0.34 , p < .01 , respectively .", "baseline social activity was also positively related to fluency in the lbls ( though the same measure was not significant in sls sample ) .", "social activity at baseline and semantic knowledge was positively related in the octo - twin sample ( operationalized as information ; b = 0.87 , p = .03 ) , but negatively related in the sls sample ( operationalized as vocabulary ; b = 0.22 , p = .02 ) .", "the octo - twin sample was the only one where all cognitive measures considered had a positive association with baseline social activity levels . ", "the baseline agesocial activity interaction terms were significant and positive for lbls reasoning ( b = 0.08 , p < .01 ) and semantic knowledge ( b = 0.08 , p < .01 ) , indicating a stronger effect of social activity for older participants than for younger participants .", "however , the interaction of age and social activity was not significant for any other studies or cognitive measures ( ps > .05 ) .", "the interaction between education and social activity at baseline was not significantly associated with any cognitive measure within any of the samples . in terms of within person", "age changes , social activity at baseline was significantly related to the slope of semantic knowledge for sls participants such that higher social activity at baseline was associated with less semantic knowledge decline ( p = .01 ) .", "there were no other significant relationships between social activity at baseline and rate of change for any cognitive measure within any of the samples ( all p > .05 ) . in examining associations with change in social activity as a time - varying covariate in each of the models ,", "was positively associated with performance on the fluency measures in all studies except sls ( lbls p < .01 , vls p < .01 , sls p = .21 ) , such that individuals who increased their level of social activity from their own baseline level exhibited higher occasion - specific fluency performance relative to their expected linear trajectory over time . in vls and sls , a significant relationship was also found between change in social activity and the reasoning ( vls p = .02 , sls p = .04 ) and memory ( vls p < .01 , sls p = .03 ) measure , indicating that participants who increased their social activity level also scored higher relative to their expected trajectory on the reasoning or memory measure .", "changes in social activity were related to all cognitive measures in the octo - twin study ( all p < .05 ) , such that octo - twin participants who increased their social activity level from baseline had higher occasion - specific cognitive scores relative to their own linear trajectory in all domains tested ." ], [ "comparing results of the same statistical model across four longitudinal studies of aging , we observed relatively few consistent associations between social activity and cognitive functioning . looking at within person change in social activity and the four cognitive measures , the most consistent finding was an association with fluency in two of the three studies measuring it , after adjusting for linear effects of time and other covariates . change in social activity was associated with memory performance in only half of the four studies ( sls and vls ) . in only one study , octo - twin ,", "change in social activity was significantly related to performance in all three of the cognitive domains considered ( fluency measure not available ) .", "the octo - twin sample was the oldest and had the narrowest range of ages .", "social activity in the octo - twin study was defined by the number of people with whom participants had contact , whereas the other studies included a range of activities with a social component . given that within person decreases in performance were seen across all cognitive measures in all studies , the positive relationships generally represent attenuated decline , rather than improvement in cognitive performance . ", "the use of within person change in social activity as a time - varying predictor of cognitive function is somewhat unique .", "however , in another study examining the temporal relations of social activity change and cognitive function , small and colleagues found significant coupling of social activity change and three cognitive domains : semantic decision speed ( similar to our fluency measure ) , episodic memory , and semantic memory .", "they , however , found greater support for models where cognitive measures predicted changes in social activity levels , than the reverse", ". there was little evidence that initial levels of social activity were related to within person rate of decline in cognitive function .", "although higher initial levels of social activity were associated with less decline on the sls semantic knowledge measure , this was not evident in any other samples .", "sls has the widest interval between measurements and so correspondingly greater attrition between waves ( though a similar yearly rate ) , but this does not suggest an obvious reason for the difference .", "the findings from the other studies are consistent with mcgue and christensen , who found that social activity at the first assessment was related to initial level of cognitive function but not change in cognitive function .", "however , other groups have found that social activity levels are associated with both baseline cognitive function and a reduced rate of decline over time .", ". found that social integration was related to a slower rate of memory decline .", "we did not include a composite measure of general cognitive function , but our results , and the results of others who have examined specific facets of cognition , suggest that it is important to examine cognitive domains separately . ", "specifically , individuals with higher initial levels of social activity performed better on the memory measures in three of the four samples .", "interestingly , the sample that did not show the effect was the lbls , which used the same measure as the sls study .", "this suggests the discrepancy is not a function of different memory measures being used , or how social activity was characterized in the study .", "it is perhaps related to differences in samples , although both were similar in average years of education and were american , although from different regions .", "this , and the accessibility of the activities , may influence the association with cognitive function", "the mean age of lbls participants was about six years older and the first wave of lbls assessment included in the present study was conducted ten years later than the first assessment of the sls .", "the age differences are not an obvious explanation , because the mean age of lbls participants is only slightly older than sls and vls participants and is nearly ten years younger than octo - twin participants .", "the lbls is the only study for which the majority of participants are not female , though this difference is slim ( 49% female in the lbls versus 52% in sls at baseline ) .", "the lbls does have the highest yearly rate of attrition ( 17% versus 10% , 7% , and 8% ) , although the loss at each measurement wave is similar to that of sls .", "it is unclear how this would affect the association between baseline social activity and memory performance ; however , particularly considering that the mean social activity and memory measure scores for the lbls and sls were similar . it would be interesting in future work to consider general health and its age gradient in each of the studies .", "the lack of obvious reasons for discrepant findings , however , supports the importance of considering the reproducibility of results in coordinated , rather than pooled , analyses .", "social integration has been posited to influence general health through multiple pathways that may overlap with those influencing cognition .", "unfortunately , our findings do not strongly suggest that increased engagement in social activities confers immediate benefit in terms of cognitive function . nor do they suggest that social participation reduces risk of cognitive decline in any domain apart from fluency .", "the cognitive reserve hypothesis suggests that , over time , reserve can be built up through stimulating activities and that individuals with high reserve can withstand more physiological deterioration before cognitive decline is observable . that social participation does not reduce the risk of cognitive decline in a broad range of measures suggests that it is not conferring reserve .", " one possible mechanism through which social engagement and cognitive function are related may be the cognitively stimulating nature of social activities .", "the cognitive training literature has found that training in specific tasks ( e.g. , memory tasks ) does not necessarily transfer to other cognitive domains .", "this may be similarly true of social activity participation , whereby a relationship is only seen in cognitive domains that are being challenged by the social activity .", "social interactions do typically involve verbal communication , and thus fluency , likely specifically verbal fluency , may be the cognitive domain most similar to our participants ' social activities . ", "discrepancies in which activities are considered social may contribute to the lack of consistent association between social activity and cognitive function .", "for example , some activities ( e.g. , card games ) may generally be more cognitively demanding than others ( e.g. , visits from family members ) , or primarily tax different cognitive skills , but this is likely to differ across individuals and situations .", "although plausible , further research would need to confirm the validity of such hypothesis . in the context of physical health ,", "others have suggested that different social factors ( e.g. , social influence , social engagement , and social support ) may act primarily through different behavioral , psychological , and physiological pathways .", "similarly , cognitive function may be differentially influenced depending on the particular combination of social factors and pathways . in this way ,", "how social activity is conceptualized and measured may be related to the mechanistic pathway and thus differentially related to various domains of cognitive function .", "these effects may have contributed to the discrepancies between octo - twin and the other three studies in the current analysis , as octo - twin focused on frequency of contact with people as the social activity measure , whereas the other three encompassed a variety of activities .", "however , the lack of consistency between the two studies employing the same measures suggests that conceptualization of social activity does not fully explain the findings .", "a clue to the source of inconsistency across lbls and sls may be the very small average change in the lbls social activities measure , though its variance is comparable to those of sls and octo - twin .", "the association between cognition and social activity in lbls in particular also seems to vary by age more than in the other studies .", "a considerable strength of our analysis is the replication of the models across four longitudinal studies from geographically separate regions .", "this limits the possibility of spurious findings taking on undue importance and provides an opportunity to examine consistencies and inconsistencies between sample groups . in terms of potential methodological limitations ,", "( for slope covariances ; for variance components ) suggest that the power to detect correlated change is extremely low .", "however , analysis of longitudinal studies on aging , including those used in the current paper , has consistently reported statistically significant variances and covariances in rates of change in cognitive outcomes ( [ 4354 ] , for summary see ) . in addition , the tests used by hertzog et al .", "are based on significance tests for variance components whereas change was evaluated in our paper by examining fixed effects which will generally have greater power .", "lending support to the argument that analyses in the current paper were adequately powered are the findings of significant associations between the identical set of cognitive outcomes in these same studies and physical and cognitive activities [ 57 , 58 ] .", "another limitation to our analysis is that while the study samples were restricted to initially healthy older adults , efforts were not made to exclude individuals who developed dementia over the course of the study periods ( dementia diagnosis was available only in the octo - twin study ) .", "we can not assume that any protective effects of social activity on cognitive function would be equivalent across healthy and dementing older adults .", "including individuals whose cognitive decline may have been driven by the dementia process may impact the associations between social activity and cognitive function over time in nondementing individuals , and this may also contribute to inconsistencies in the literature . finally , it is difficult to rule out the possibility that individuals decrease their social activities because their declining cognitive abilities make it more difficult for them to maintain social ties .", "the difficulty of determining the directionality of the relationship has been well acknowledged in the literature and it is similarly difficult to determine causal pathways in the current analysis [ 4 , 5 , 10 ] .", "some attempts have been made to determine the most likely direction of the relationship , but the evidence is limited .", "the longitudinal nature of the studies considered here , and allowing social activity to vary across time , have narrowed the temporal distance between the social activity and cognitive performance , at least partially disentangling whether the relationship is primarily based on historical activity levels or whether the two tracks over time . across four studies in three countries ,", "baseline social activity levels failed to predict rate of decline in most cognitive abilities , and changes in social activity were not consistently associated with within person fluctuations in cognitive functioning .", "our findings leave little support for the hypothesis that changes in social activity correspond to immediate benefits in cognitive functioning , except perhaps for fluency ." ] ]

[ "the pt mice , tcr- mice , and tcr- mice have been described ( 6 , 17 , 18 ) .", "tcr- pt mice were bred in the animal colony of the basel institute for immunology .", "breeding of tcr- pt mice was done in the animal facilities at the hpital necker ( paris , france ) .", "the tcr- transgenic mice , with a transgenic tcr specific for the male antigen ( h - y ) in the context of h-2d mhc molecules , have been described previously and were crossed on the c57bl/6 ( b6 ) background ( 19 ) .", "tcr- transgenic pt mice were bred in the animal colony of the basel institute for immunology .", "the following mabs were used for staining : anti - cd4 ( h129.19 , pe - conjugated ; gibco brl , gaithersburg , md ; or h129.19 , fitc - conjugated ; gibco brl ) , anti - cd8 ( ly-2 , fitc - conjugated ; pharmingen , san diego , ca ; or 53 - 6.7 , biotinylated ; gibco brl ; or 53 - 6.7 , red613conjugated ; gibco brl ) , anti - cd25 ( 3c7 , pe - conjugated ; pharmingen ) , anti - cd44 ( biotinylated km81 ; american type culture collection , rockville , md ) , anti - pantcr- ( h57 - 597 , fitc - conjugated ) , anti - tcr- ( gl3 , fitc - conjugated ; pharmingen ) , t3.70 ( specific for the tcr- chain of the hyreactive tcr , fitc - conjugated ) , and f23.1 ( specific for the tcr- chain of hy - reactive tcr , fluos - conjugated ) .", "two- and three - color stainings were performed with fitc- , pe- , and biotin - labeled antibodies at optimal concentrations . ", "biotin - conjugated antibodies were revealed by either streptavidin - pe ( southern biotechnology , birmingham , al ) or streptavidin - tricolor ( caltag laboratories , san francisco , ca ) .", "all stainings were done in 96-well plates ( 0.5 10 cells per well ) in 20 l of mab in pbs plus 2% fcs plus 0.1% sodium azide for 20 min on ice . between first and second step reagents cells were washed in pbs plus 2% fcs plus 0.1% sodium azide as was done after the last step .", "data were analyzed on a facscan ( beckton dickinson , mountain view , ca ) , using lysis ii software ( beckton dickinson ) .", "for intracellular / extracellular double staining of thymocytes cells were first incubated with culture supernatant of mab 2.4g2 to block fcrii / iii .", "cells were then stained for surface markers as described above . after washing in pbs ,", "cells were fixed in pbs plus 1% paraformaldehyde for 15 min at room temperature , followed by two washing steps in pbs .", "cells were then permeabilized in 0.5% saponin ( sigma , heidelberg , germany ) for 10 min at room temperature and washed in pbs .", "intracellular staining with fitc - conjugated antibodies diluted in pbs plus 0.5% saponin was performed for 20 min at room temperature , followed by two washing steps in pbs and 2 15 min on a rocking platform in pbs plus 2% fcs plus 0.5% saponin on ice .", "finally , cells were washed in pbs plus 2% fcs and analyzed on a facscan , using lysis ii software .", "the pt mice , tcr- mice , and tcr- mice have been described ( 6 , 17 , 18 ) .", "tcr- pt mice were bred in the animal colony of the basel institute for immunology .", "breeding of tcr- pt mice was done in the animal facilities at the hpital necker ( paris , france ) .", "the tcr- transgenic mice , with a transgenic tcr specific for the male antigen ( h - y ) in the context of h-2d mhc molecules , have been described previously and were crossed on the c57bl/6 ( b6 ) background ( 19 ) .", "tcr- transgenic pt mice were bred in the animal colony of the basel institute for immunology .", "the following mabs were used for staining : anti - cd4 ( h129.19 , pe - conjugated ; gibco brl , gaithersburg , md ; or h129.19 , fitc - conjugated ; gibco brl ) , anti - cd8 ( ly-2 , fitc - conjugated ; pharmingen , san diego , ca ; or 53 - 6.7 , biotinylated ; gibco brl ; or 53 - 6.7 , red613conjugated ; gibco brl ) , anti - cd25 ( 3c7 , pe - conjugated ; pharmingen ) , anti - cd44 ( biotinylated km81 ; american type culture collection , rockville , md ) , anti - pantcr- ( h57 - 597 , fitc - conjugated ) , anti - tcr- ( gl3 , fitc - conjugated ; pharmingen ) , t3.70 ( specific for the tcr- chain of the hyreactive tcr , fitc - conjugated ) , and f23.1 ( specific for the tcr- chain of hy - reactive tcr , fluos - conjugated ) .", "two- and three - color stainings were performed with fitc- , pe- , and biotin - labeled antibodies at optimal concentrations . ", "biotin - conjugated antibodies were revealed by either streptavidin - pe ( southern biotechnology , birmingham , al ) or streptavidin - tricolor ( caltag laboratories , san francisco , ca ) .", "stainings were done in 96-well plates ( 0.5 10 cells per well ) in 20 l of mab in pbs plus 2% fcs plus 0.1% sodium azide for 20 min on ice . between first and second step reagents cells", "were washed in pbs plus 2% fcs plus 0.1% sodium azide as was done after the last step .", "data were analyzed on a facscan ( beckton dickinson , mountain view , ca ) , using lysis ii software ( beckton dickinson ) .", "for intracellular / extracellular double staining of thymocytes cells were first incubated with culture supernatant of mab 2.4g2 to block fcrii / iii .", "cells were then stained for surface markers as described above . after washing in pbs ,", "cells were fixed in pbs plus 1% paraformaldehyde for 15 min at room temperature , followed by two washing steps in pbs .", "cells were then permeabilized in 0.5% saponin ( sigma , heidelberg , germany ) for 10 min at room temperature and washed in pbs .", "intracellular staining with fitc - conjugated antibodies diluted in pbs plus 0.5% saponin was performed for 20 min at room temperature , followed by two washing steps in pbs and 2 15 min on a rocking platform in pbs plus 2% fcs plus 0.5% saponin on ice .", "finally , cells were washed in pbs plus 2% fcs and analyzed on a facscan , using lysis ii software .", "in initial experiments , it was determined whether either the tcr- or the tcr- could be responsible for the production of cd48 t cells in pt mice by analyzing the cellular composition of thymuses from either pt tcr- or pt tcr- double mutant mice that can only produce the and the tcr- , respectively . ", "as shown in table 1 both types of mutant mice contained cd48 t cells that were further analyzed by cytoplasmic staining with antibodies specific for tcr- and tcr- chains .", "for this purpose cells were double stained for surface expression of cd4 and cd8 molecules as well as either for cytoplasmic tcr- or tcr- chains by double fluorescence using cd4 and cd8 antibodies in one color ( green ) and tcr- or tcr- antibodies in another color ( red ) . in this analysis", "single positive cd48 and cd48 cells show an intermediate fluorescence between that of cd48 and cd48 thymocytes and cells were gated accordingly into double negative , double positive ( dp ) , and single positive cells ( fig .", "1 shows that 64% of cd48 cells in wild - type mice expressed tcr- chains , and that due to tcr- selection by the pre - tcr ( 22 ) the vast majority of cd48 cells contained tcr- chains in their cytoplasm . on the other hand ,", "the picture was different in pt/ mice where , due to the diminution of rapidly cycling tcr-selected cd484425 cells ( 6 ) , only 21% of the cd48 cells were tcr- positive . in addition , only 39% of the cd48 cells contained tcr- chains in their cytoplasm indicating that in the pt/ mice the majority of the cd48 cells were generated by a mechanism that did not involve tcr- selection . the fact that not all single positive cells in these mice were tcr- is due to the fact that these cells are in part immature tcr- single positive cells , on their way from cd48 to cd48 cells .", "the tcr- single positive cells had a mature cd48 phenotype as confirmed by independent three - color stainings indicating also that these cells expressed tcr- receptors on the cell surface .", "these cells were present in a higher number in pt mice consistent with the notion that the pre - tcr may have a role in regulating rearrangement and/or expression ( 23 and unpublished observations ) .", "in pt tcr- mice the proportion of tcr- cd48 and tcr- single positive cells was even further reduced . when looking at the absolute numbers of various cell subsets ( table 1 and fig .", "1 ) it is clear that there was a very marked reduction in cell numbers of cd48 thymocytes and more mature cells in pt and pt tcr- mice , whereas the numbers of cd48 cells were within the same range .", "pt tcr- mice also had reduced numbers of dp cells but here the picture differed from that in pt and pt tcr- mice in that all of the cd48 cells were tcr- positive , i.e. , were exclusively generated through a mechanism that involved tcr- selection .", "the single positive tcr- cells in pt tcr- mice were exported from the thymi and cd48 as well as cd48 cells could be detected in lymphnodes of these mice ( not shown ) .", "this excludes the possibility that these cells belong exclusively to the nk1.1cd4 subset that exhibits an unusual phenotype ( 24 ) .", "the above results were reproducible in the different mice with marginal deviations in either the percentage of cells or absolute cell numbers and are schematically presented in fig .", "the main message from this analysis is that the tcr- can generate cd48 cells through tcr- selection , i.e. , by intracellular or cell - autonomous signaling only .", "in contrast , the tcr- can generate cd48 cells that are either tcr- or tcr- but all tcr- through a mechanism that may involve intercellular communication of unknown nature . if the tcr- would generate a significant number of dp cells by cell - autonomous signaling one might expect to find some tcr- expression in these cells .", "however , the fact that the cd48 cells are tcr- negative suggests that these cells are not selected by cell - autonomous signaling by the tcr- even though it can not be entirely excluded that tcr- expression is abruptly switched off in cd48 cells .", "the notion of intercellular communication is in line with experiments that involved transfer of t cells into thymuses of rearrangement - deficient mice that resulted in generation of cd48 cells of host origin ( 25 ) and also with earlier data by shores et al .", "our experiments suggest that in the latter experiments but not t cells promoted the development of cd48 thymocytes and make the additional point that the generation of dp cells was not due to an artefact caused by adoptive transfer of cells . ", "the fact that in the absence of the pre - tcr the generation of cd48 cells by the tcr- is rather inefficient , i.e. , 240 10 versus 2,880 10 in pt tcr- versus wild - type mice , could depend on the fact that the tcr- is inefficiently formed in cd48 cells due to the late tcr- rearrangement and/or the fact that tcr- can only inefficiently replace the pre - tcr . to analyze this question in some more detail we studied mice that express a transgenic tcr- early in development on cd48 cells , i.e. , tcr- transgenic pt mice .", "the transgenic tcr- could indeed overcome the cellular deficiency in the cd48 compartment as tcr- transgenic pt mice contained approximately one - half the number of thymocytes found in tcr- transgenic pt mice and many more than the number found in nontransgenic pt mice ( fig .", "however , there was a subtle difference between tcr- transgenic pt and tcr- transgenic pt mice in that the latter , but not the former , contained a discrete subset of cd25 cells , indicating that in spite of the presence of the transgenic tcr- , the pretcr had its role in the exit from this compartment .", "this could be due to the lack of expression of the transgenic tcr- in a fraction of cells in the cd25 compartment of the tcr- transgenic , pt mice .", "this was in fact confirmed by cytoplasmic staining : while only nine percent of cd25 cells in tcr- transgenic pt mice expressed the transgenic tcr- chain the majority of these cells expressed the transgenic tcr- chain suggesting that expression of the two transgenes is differentially regulated ( fig .", "in tcr- transgenic pt mice it is the combined action of the pre - tcr and the tcr- ( mice that have only a tcr- transgene still exhibit a significantly larger cd25 compartment than tcr- transgenic mice , not shown ) that reduce the number of cd25 cells while in tcr- transgenic pt mice this compartment is bigger in size because of the absence of the pretcr . from these data", "it would appear that the tcr- can at least partially mimic the function of the pre - tcr and that in normal mice the contribution of the tcr- to the generation of the cd48 compartment is limited due to relatively late expression of most tcr- chains ( 1 , 2 ) . thus , all of the three known tcrs can have a role in promoting the development of pre t cells : the tcr- most likely by intercellular communication that furthers the development of cd48 cells irrespective of whether or not they have succeeded in tcr- rearrangement , the tcr- that depends strictly on intracellular , cell - autonomous signals generated by the tcr- chains and the pre - tcr that operates by a similar mechanism as the tcr- but is much more efficient because of the early and abundant expression of the pt gene during the phase of tcr- rearrangement . therefore , only mice that can not produce any of these receptors will exhibit complete arrest at the cd48 stage of development as evident in rag mice or mice that are deficient in both tcr- and tcr- chains and therefore , can make neither tcr- , pre - tcr , nor tcr- ( 14 ) . in normal mice , the contribution of the tcr- in development of cells of the lineage appears to be limited based on the fact that the vast majority of cd48 cells are tcr- and thus are tcr- selected .", "likewise , in normal mice , the contribution of the tcr- to the transition of dn to dp cells may be limited because of the small number of dp cells in pt tcr- mice .", "however , in the absence of pt these receptors avoid a severe immunodeficiency by enabling the formation of a significant number of mature t cells .", "it would appear that both the pretcr and the tcr- do not only mediate maturation but also proliferation since in wild - type mice and pt tcr- mice the proportion of large cd48 blasts that are derived from dividing cd48 precursors ( 15 ) is very similar ( table 2 ) .", "there are only slightly fewer blasts in pt tcr- mice indicating that also the tcr- generates dividing cd48 cells . with regard to", "the role of the src kinases in early development , our data is consistent with the notion that signaling through the pre - tcr involves both lck and fyn kinases but is equally consistent with the idea that the fyn kinase is involved only in signaling through the tcr- or - , and thereby responsible for the incomplete developmental arrest observed in lck mice .", "the fact that the tcr- promotes development much in the same way as the pretcr , i.e. , by cell - autonomous signaling and thereby tcr- selection , suggests that t cell development may have proceeded in this way before the advent of the pre ", "tcr- chain in evolution and that the pre - tcr had simply the advantage of making the pairing of a single tcr- chain with different tcr- chains more effective .", "cd48 thymocyte subsets of wild - type and mutant mice mean values were obtained of four ( two for pt/ tcr-/ ) different mice of each genotype from 68-wk - old litter .", "intracytoplasmic staining for tcr- ( tcr-ic ) and tcr- ( tcr-ic ) within thymocyte subsets from c57bl/6 ( wt ) , pt mice ( a ) , and pt tcr- , pt tcr- mice ( b ) .", "total thymocytes were surface stained with pe - conjugated cd4 antibodies , biotinylated cd8 antibodies followed by pe - streptavidin ; cytoplasmic staining was performed with anti - pantcr- or anti tcr- antibodies .", "the percentages of cells and absolute numbers ( in brackets ) are indicated . a schematic overview of various gene - deficient mice and the corresponding defects in t cell development .", "percentages indicate the proportion of cells with cytoplasmic tcr-. the thickness of the bars is meant to correlate with the numbers of cells within the various subsets .", "comparision of surface phenotype of thymocytes from tcr- pt vs. tcr- transgenic mice .", "( top ) total thymocytes were double stained for cd4 ( fitc - conjugated anti - cd4 ) and cd8 ( red613-conjugated anti - cd8 ) surface antigens as described .", "tcr- pt transgenic mice contained approximately one - half of the number of thymocytes found in tcr- transgenic mice ( 2,870 10 vs. 4,720 10 cells ) .", "( bottom ) cells were stained with fitc - conjugated cd4 and cd8 antibodies in combination with biotinylated cd44 and peconjugated anti - cd25 antibodies .", "the expression of cd25 and cd44 was analyzed by three - color flow cytometry , using electronic gating to exclude fitc - positive cells .", "assessment of transgenic tcr- and tcr- expression by intracytoplasmic staining . for intracellular / extracellular double staining ,", "thymocytes isolated from transgenic tcr- mice and transgenic tcr- pt mice were stained with pe - conjugated cd25 antibodies and then with fitc - conjugated t3.70 antibodies specific for the transgenic tcr- chain of the hy - reactive tcr or fluos - conjugated f23.1 antibodies , specific for the transgenic tcr- chain of the hy - reactive tcr .", "proportion of cd48 lymphoblasts in wild type and mutant mice percentages of cd48 blasts were determined by facscan using forward scatter as an index of size .", "the various mice were analyzed on the same day in the same experiment . " ]

[ "<S> the development of pre t cells with productive tcr- rearrangements can be mediated by each the pre </S>", "<S> t cell receptor ( pre - tcr ) , the tcr- as well as the tcr- , albeit by distinct mechanisms . </S>", "<S> although the tcr- affects cd48 precursor cells irrespective of their rearrangement status by tcr- mechanisms not involving tcr- selection , both the pretcr and the tcr- select only cells with productive tcr- genes for expansion and maturation . the tcr- appears to be much less effective than the pre - tcr because of the paucity of tcr- proteins in tcr-positive precursors since an early expressed transgenic tcr- can largely substitute for the pre - tcr . </S>", "<S> thus , the tcr- can assume a role not only in the rescue from programmed cell death of cd4 + 8 + but also of cd48 thymocytes . in evolution </S>", "<S> this double function of the tcr- may have been responsible for the maturation of t cells before the advent of the pre tcr- chain . </S>" ]

[ "Materials and Methods", "Mice.", "Antibodies and Flow Cytometry.", "Results and Discussion", "Figures and Tables" ]

[ [ "the pt mice , tcr- mice , and tcr- mice have been described ( 6 , 17 , 18 ) .", "tcr- pt mice were bred in the animal colony of the basel institute for immunology .", "breeding of tcr- pt mice was done in the animal facilities at the hpital necker ( paris , france ) .", "the tcr- transgenic mice , with a transgenic tcr specific for the male antigen ( h - y ) in the context of h-2d mhc molecules , have been described previously and were crossed on the c57bl/6 ( b6 ) background ( 19 ) .", "tcr- transgenic pt mice were bred in the animal colony of the basel institute for immunology .", "the following mabs were used for staining : anti - cd4 ( h129.19 , pe - conjugated ; gibco brl , gaithersburg , md ; or h129.19 , fitc - conjugated ; gibco brl ) , anti - cd8 ( ly-2 , fitc - conjugated ; pharmingen , san diego , ca ; or 53 - 6.7 , biotinylated ; gibco brl ; or 53 - 6.7 , red613conjugated ; gibco brl ) , anti - cd25 ( 3c7 , pe - conjugated ; pharmingen ) , anti - cd44 ( biotinylated km81 ; american type culture collection , rockville , md ) , anti - pantcr- ( h57 - 597 , fitc - conjugated ) , anti - tcr- ( gl3 , fitc - conjugated ; pharmingen ) , t3.70 ( specific for the tcr- chain of the hyreactive tcr , fitc - conjugated ) , and f23.1 ( specific for the tcr- chain of hy - reactive tcr , fluos - conjugated ) .", "two- and three - color stainings were performed with fitc- , pe- , and biotin - labeled antibodies at optimal concentrations . ", "biotin - conjugated antibodies were revealed by either streptavidin - pe ( southern biotechnology , birmingham , al ) or streptavidin - tricolor ( caltag laboratories , san francisco , ca ) .", "all stainings were done in 96-well plates ( 0.5 10 cells per well ) in 20 l of mab in pbs plus 2% fcs plus 0.1% sodium azide for 20 min on ice . between first and second step reagents cells were washed in pbs plus 2% fcs plus 0.1% sodium azide as was done after the last step .", "data were analyzed on a facscan ( beckton dickinson , mountain view , ca ) , using lysis ii software ( beckton dickinson ) .", "for intracellular / extracellular double staining of thymocytes cells were first incubated with culture supernatant of mab 2.4g2 to block fcrii / iii .", "cells were then stained for surface markers as described above . after washing in pbs ,", "cells were fixed in pbs plus 1% paraformaldehyde for 15 min at room temperature , followed by two washing steps in pbs .", "cells were then permeabilized in 0.5% saponin ( sigma , heidelberg , germany ) for 10 min at room temperature and washed in pbs .", "intracellular staining with fitc - conjugated antibodies diluted in pbs plus 0.5% saponin was performed for 20 min at room temperature , followed by two washing steps in pbs and 2 15 min on a rocking platform in pbs plus 2% fcs plus 0.5% saponin on ice .", "finally , cells were washed in pbs plus 2% fcs and analyzed on a facscan , using lysis ii software ." ], [ "the pt mice , tcr- mice , and tcr- mice have been described ( 6 , 17 , 18 ) .", "tcr- pt mice were bred in the animal colony of the basel institute for immunology .", "breeding of tcr- pt mice was done in the animal facilities at the hpital necker ( paris , france ) .", "the tcr- transgenic mice , with a transgenic tcr specific for the male antigen ( h - y ) in the context of h-2d mhc molecules , have been described previously and were crossed on the c57bl/6 ( b6 ) background ( 19 ) .", "tcr- transgenic pt mice were bred in the animal colony of the basel institute for immunology ." ], [ "the following mabs were used for staining : anti - cd4 ( h129.19 , pe - conjugated ; gibco brl , gaithersburg , md ; or h129.19 , fitc - conjugated ; gibco brl ) , anti - cd8 ( ly-2 , fitc - conjugated ; pharmingen , san diego , ca ; or 53 - 6.7 , biotinylated ; gibco brl ; or 53 - 6.7 , red613conjugated ; gibco brl ) , anti - cd25 ( 3c7 , pe - conjugated ; pharmingen ) , anti - cd44 ( biotinylated km81 ; american type culture collection , rockville , md ) , anti - pantcr- ( h57 - 597 , fitc - conjugated ) , anti - tcr- ( gl3 , fitc - conjugated ; pharmingen ) , t3.70 ( specific for the tcr- chain of the hyreactive tcr , fitc - conjugated ) , and f23.1 ( specific for the tcr- chain of hy - reactive tcr , fluos - conjugated ) .", "two- and three - color stainings were performed with fitc- , pe- , and biotin - labeled antibodies at optimal concentrations . ", "biotin - conjugated antibodies were revealed by either streptavidin - pe ( southern biotechnology , birmingham , al ) or streptavidin - tricolor ( caltag laboratories , san francisco , ca ) .", "stainings were done in 96-well plates ( 0.5 10 cells per well ) in 20 l of mab in pbs plus 2% fcs plus 0.1% sodium azide for 20 min on ice . between first and second step reagents cells", "were washed in pbs plus 2% fcs plus 0.1% sodium azide as was done after the last step .", "data were analyzed on a facscan ( beckton dickinson , mountain view , ca ) , using lysis ii software ( beckton dickinson ) .", "for intracellular / extracellular double staining of thymocytes cells were first incubated with culture supernatant of mab 2.4g2 to block fcrii / iii .", "cells were then stained for surface markers as described above . after washing in pbs ,", "cells were fixed in pbs plus 1% paraformaldehyde for 15 min at room temperature , followed by two washing steps in pbs .", "cells were then permeabilized in 0.5% saponin ( sigma , heidelberg , germany ) for 10 min at room temperature and washed in pbs .", "intracellular staining with fitc - conjugated antibodies diluted in pbs plus 0.5% saponin was performed for 20 min at room temperature , followed by two washing steps in pbs and 2 15 min on a rocking platform in pbs plus 2% fcs plus 0.5% saponin on ice .", "finally , cells were washed in pbs plus 2% fcs and analyzed on a facscan , using lysis ii software ." ], [ "in initial experiments , it was determined whether either the tcr- or the tcr- could be responsible for the production of cd48 t cells in pt mice by analyzing the cellular composition of thymuses from either pt tcr- or pt tcr- double mutant mice that can only produce the and the tcr- , respectively . ", "as shown in table 1 both types of mutant mice contained cd48 t cells that were further analyzed by cytoplasmic staining with antibodies specific for tcr- and tcr- chains .", "for this purpose cells were double stained for surface expression of cd4 and cd8 molecules as well as either for cytoplasmic tcr- or tcr- chains by double fluorescence using cd4 and cd8 antibodies in one color ( green ) and tcr- or tcr- antibodies in another color ( red ) . in this analysis", "single positive cd48 and cd48 cells show an intermediate fluorescence between that of cd48 and cd48 thymocytes and cells were gated accordingly into double negative , double positive ( dp ) , and single positive cells ( fig .", "1 shows that 64% of cd48 cells in wild - type mice expressed tcr- chains , and that due to tcr- selection by the pre - tcr ( 22 ) the vast majority of cd48 cells contained tcr- chains in their cytoplasm . on the other hand ,", "the picture was different in pt/ mice where , due to the diminution of rapidly cycling tcr-selected cd484425 cells ( 6 ) , only 21% of the cd48 cells were tcr- positive . in addition , only 39% of the cd48 cells contained tcr- chains in their cytoplasm indicating that in the pt/ mice the majority of the cd48 cells were generated by a mechanism that did not involve tcr- selection . the fact that not all single positive cells in these mice were tcr- is due to the fact that these cells are in part immature tcr- single positive cells , on their way from cd48 to cd48 cells .", "the tcr- single positive cells had a mature cd48 phenotype as confirmed by independent three - color stainings indicating also that these cells expressed tcr- receptors on the cell surface .", "these cells were present in a higher number in pt mice consistent with the notion that the pre - tcr may have a role in regulating rearrangement and/or expression ( 23 and unpublished observations ) .", "in pt tcr- mice the proportion of tcr- cd48 and tcr- single positive cells was even further reduced . when looking at the absolute numbers of various cell subsets ( table 1 and fig .", "1 ) it is clear that there was a very marked reduction in cell numbers of cd48 thymocytes and more mature cells in pt and pt tcr- mice , whereas the numbers of cd48 cells were within the same range .", "pt tcr- mice also had reduced numbers of dp cells but here the picture differed from that in pt and pt tcr- mice in that all of the cd48 cells were tcr- positive , i.e. , were exclusively generated through a mechanism that involved tcr- selection .", "the single positive tcr- cells in pt tcr- mice were exported from the thymi and cd48 as well as cd48 cells could be detected in lymphnodes of these mice ( not shown ) .", "this excludes the possibility that these cells belong exclusively to the nk1.1cd4 subset that exhibits an unusual phenotype ( 24 ) .", "the above results were reproducible in the different mice with marginal deviations in either the percentage of cells or absolute cell numbers and are schematically presented in fig .", "the main message from this analysis is that the tcr- can generate cd48 cells through tcr- selection , i.e. , by intracellular or cell - autonomous signaling only .", "in contrast , the tcr- can generate cd48 cells that are either tcr- or tcr- but all tcr- through a mechanism that may involve intercellular communication of unknown nature . if the tcr- would generate a significant number of dp cells by cell - autonomous signaling one might expect to find some tcr- expression in these cells .", "however , the fact that the cd48 cells are tcr- negative suggests that these cells are not selected by cell - autonomous signaling by the tcr- even though it can not be entirely excluded that tcr- expression is abruptly switched off in cd48 cells .", "the notion of intercellular communication is in line with experiments that involved transfer of t cells into thymuses of rearrangement - deficient mice that resulted in generation of cd48 cells of host origin ( 25 ) and also with earlier data by shores et al .", "our experiments suggest that in the latter experiments but not t cells promoted the development of cd48 thymocytes and make the additional point that the generation of dp cells was not due to an artefact caused by adoptive transfer of cells . ", "the fact that in the absence of the pre - tcr the generation of cd48 cells by the tcr- is rather inefficient , i.e. , 240 10 versus 2,880 10 in pt tcr- versus wild - type mice , could depend on the fact that the tcr- is inefficiently formed in cd48 cells due to the late tcr- rearrangement and/or the fact that tcr- can only inefficiently replace the pre - tcr . to analyze this question in some more detail we studied mice that express a transgenic tcr- early in development on cd48 cells , i.e. , tcr- transgenic pt mice .", "the transgenic tcr- could indeed overcome the cellular deficiency in the cd48 compartment as tcr- transgenic pt mice contained approximately one - half the number of thymocytes found in tcr- transgenic pt mice and many more than the number found in nontransgenic pt mice ( fig .", "however , there was a subtle difference between tcr- transgenic pt and tcr- transgenic pt mice in that the latter , but not the former , contained a discrete subset of cd25 cells , indicating that in spite of the presence of the transgenic tcr- , the pretcr had its role in the exit from this compartment .", "this could be due to the lack of expression of the transgenic tcr- in a fraction of cells in the cd25 compartment of the tcr- transgenic , pt mice .", "this was in fact confirmed by cytoplasmic staining : while only nine percent of cd25 cells in tcr- transgenic pt mice expressed the transgenic tcr- chain the majority of these cells expressed the transgenic tcr- chain suggesting that expression of the two transgenes is differentially regulated ( fig .", "in tcr- transgenic pt mice it is the combined action of the pre - tcr and the tcr- ( mice that have only a tcr- transgene still exhibit a significantly larger cd25 compartment than tcr- transgenic mice , not shown ) that reduce the number of cd25 cells while in tcr- transgenic pt mice this compartment is bigger in size because of the absence of the pretcr . from these data", "it would appear that the tcr- can at least partially mimic the function of the pre - tcr and that in normal mice the contribution of the tcr- to the generation of the cd48 compartment is limited due to relatively late expression of most tcr- chains ( 1 , 2 ) . thus , all of the three known tcrs can have a role in promoting the development of pre t cells : the tcr- most likely by intercellular communication that furthers the development of cd48 cells irrespective of whether or not they have succeeded in tcr- rearrangement , the tcr- that depends strictly on intracellular , cell - autonomous signals generated by the tcr- chains and the pre - tcr that operates by a similar mechanism as the tcr- but is much more efficient because of the early and abundant expression of the pt gene during the phase of tcr- rearrangement . therefore , only mice that can not produce any of these receptors will exhibit complete arrest at the cd48 stage of development as evident in rag mice or mice that are deficient in both tcr- and tcr- chains and therefore , can make neither tcr- , pre - tcr , nor tcr- ( 14 ) . in normal mice , the contribution of the tcr- in development of cells of the lineage appears to be limited based on the fact that the vast majority of cd48 cells are tcr- and thus are tcr- selected .", "likewise , in normal mice , the contribution of the tcr- to the transition of dn to dp cells may be limited because of the small number of dp cells in pt tcr- mice .", "however , in the absence of pt these receptors avoid a severe immunodeficiency by enabling the formation of a significant number of mature t cells .", "it would appear that both the pretcr and the tcr- do not only mediate maturation but also proliferation since in wild - type mice and pt tcr- mice the proportion of large cd48 blasts that are derived from dividing cd48 precursors ( 15 ) is very similar ( table 2 ) .", "there are only slightly fewer blasts in pt tcr- mice indicating that also the tcr- generates dividing cd48 cells . with regard to", "the role of the src kinases in early development , our data is consistent with the notion that signaling through the pre - tcr involves both lck and fyn kinases but is equally consistent with the idea that the fyn kinase is involved only in signaling through the tcr- or - , and thereby responsible for the incomplete developmental arrest observed in lck mice .", "the fact that the tcr- promotes development much in the same way as the pretcr , i.e. , by cell - autonomous signaling and thereby tcr- selection , suggests that t cell development may have proceeded in this way before the advent of the pre ", "tcr- chain in evolution and that the pre - tcr had simply the advantage of making the pairing of a single tcr- chain with different tcr- chains more effective ." ], [ "cd48 thymocyte subsets of wild - type and mutant mice mean values were obtained of four ( two for pt/ tcr-/ ) different mice of each genotype from 68-wk - old litter .", "intracytoplasmic staining for tcr- ( tcr-ic ) and tcr- ( tcr-ic ) within thymocyte subsets from c57bl/6 ( wt ) , pt mice ( a ) , and pt tcr- , pt tcr- mice ( b ) .", "total thymocytes were surface stained with pe - conjugated cd4 antibodies , biotinylated cd8 antibodies followed by pe - streptavidin ; cytoplasmic staining was performed with anti - pantcr- or anti tcr- antibodies .", "the percentages of cells and absolute numbers ( in brackets ) are indicated . a schematic overview of various gene - deficient mice and the corresponding defects in t cell development .", "percentages indicate the proportion of cells with cytoplasmic tcr-. the thickness of the bars is meant to correlate with the numbers of cells within the various subsets .", "comparision of surface phenotype of thymocytes from tcr- pt vs. tcr- transgenic mice .", "( top ) total thymocytes were double stained for cd4 ( fitc - conjugated anti - cd4 ) and cd8 ( red613-conjugated anti - cd8 ) surface antigens as described .", "tcr- pt transgenic mice contained approximately one - half of the number of thymocytes found in tcr- transgenic mice ( 2,870 10 vs. 4,720 10 cells ) .", "( bottom ) cells were stained with fitc - conjugated cd4 and cd8 antibodies in combination with biotinylated cd44 and peconjugated anti - cd25 antibodies .", "the expression of cd25 and cd44 was analyzed by three - color flow cytometry , using electronic gating to exclude fitc - positive cells .", "assessment of transgenic tcr- and tcr- expression by intracytoplasmic staining . for intracellular / extracellular double staining ,", "thymocytes isolated from transgenic tcr- mice and transgenic tcr- pt mice were stained with pe - conjugated cd25 antibodies and then with fitc - conjugated t3.70 antibodies specific for the transgenic tcr- chain of the hy - reactive tcr or fluos - conjugated f23.1 antibodies , specific for the transgenic tcr- chain of the hy - reactive tcr .", "proportion of cd48 lymphoblasts in wild type and mutant mice percentages of cd48 blasts were determined by facscan using forward scatter as an index of size .", "the various mice were analyzed on the same day in the same experiment . " ] ]

[ "in recent years , coupling of theoretical and experimental approaches in the study of protein folding has resulted in providing fruitful clues .", "experimental and computational protein design provides vital clues to understand the protein folding process , and it is of considerable interest in the area of protein science to engineer proteins with novel folds and desired functions .", "the field of protein design has a unique history where researchers from diverse discipline come together to explore novel catalytic , pharmaceutical , structural , and sensing properties of amino acids in proteins .", "interestingly , a designed eleven amino acid sequence folded as a helix in one position and as a sheet in another position in the protein sequence .", "this work has enabled to explore the role of nonlocal interactions in the formation of secondary structure .", "subsequently , helices were transmuted into sheets to understand the conformation change phenomenon and illustrate that not all the amino acids play an equal role in specifying a fold . utilizing the knowledge offered by several protein design groups , kuhlman et al . in 2003", "have computationally designed a 93-residue / protein called top7 and found that the protein could be experimentally folded and extremely stable .", "this pioneering work has enabled further research to understand the contribution of each amino acid residue in a protein to adopt a certain fold .", "hence , emphasizing that protein design could be a powerful experiment to understand the processes that underlie conformational plasticity in proteins .", "explored how two proteins with almost similar amino acid sequences change their fold and function . following the contribution of various theoretical and experimental protein science research groups , several such engineered proteins with selective", "nevertheless , the design of such a pair of proteins with high sequence identity with completely different topologies can be viewed as a challenge to the well - accepted paradigm that similar sequences always tend to fold into similar three - dimensional structures .", "an analysis of the literature reveals that the design of two highly identical proteins with different folds and functions is challenging and time bound as shown in [ table 1 ] .", "streptococcus protein g contains two types of domains ( ga and gb ) that bind to serum proteins in blood .", "the natural versions of ga and gb domains share no significant sequence homology and have different folds , 3 and 4 + , respectively . from the above two parent proteins , high - identity versions of ga and gb were synthesized .", "interestingly , small and critical differences in the sequences of the two proteins determine the topology of the protein early on the folding pathway .", "in addition , two proteins named ga88 ( pdb i d : 2jws ) and gb88 ( pdb", "i d : 2jwu ) by mutation experiments from the streptococcus protein g with 88% sequence identity adopt different structures and functions and these proteins are valuable tools to understand the contribution of residues to adopt a particular fold .", "these two proteins vary only at seven positions out of 56 amino acids , which are shown in [ figure 1 ] .", "this design has made a breakthrough in the field of protein science and contradicts the general statement that ", " following this , we have carried out computational sequence and structural analysis on these two designed proteins .", "we have performed secondary structure prediction of these two proteins and observed that the methods such as multivariate linear regression combiner can predict some regions as extended structures for the helical protein sequence ga , which gave us a clue that there may be structural plasticity at the region of first 15 residues , which are identical in the both proteins .", "we also discovered some patterns in the nonidentical positions of two proteins with a rare combination of residues that are not present in any publicly available sequence databases . by analyzing the structures of the two designed proteins , we predicted nucleation sites at various positions in the sequence , which may start or terminate secondary structural elements ( helix , sheet , and coil ) .", "we also observed drastic difference in the surrounding environment of nonidentical residues ( 7 out of 56 ) and difference in interaction energy . by observing the structural plasticity at the amino and carboxyl terminal of the sequences of two designed proteins and the influence of surrounding environment of each residue", ", we concluded that early on during the process of folding , both proteins may choose different energetically favorable pathways to attain the different folds .", "literature review of design of two proteins with high sequence identity adopting different folds sequence , dictionary of secondary structure of proteins assigned secondary structures and tertiary structures of pair of homologous heteromorphs ( the seven residues that vary in both sequences are indicated in rectangular boxes ) other researchers have characterized the folding of these two proteins using biophysical and computational experiments .", "they also indicated that the final native structures of these proteins were dictated very early along the folding pathway by performing equilibrium unfolding of ga88 and gb88 , folding and unfolding kinetics and molecular dynamics simulations experiments .", "concurrently , energy calculations were performed on the two designed proteins in a vacuum , which indicated that current computer modeling / simulations experiments can not explain why two highly similar sequences fold into different structures .", "however , it was suggested that improved modeling / simulations tools should be developed to predict the pair of sequences with different structures , which differ , by only few residues . in a recent study , folding and unfolding kinetics experiments performed on these two designed proteins indicated a detectable residual structure in the denatured state of gb88 whereas the denatured state of ga88 is unstructured .", "interestingly , they explored these two proteins by value analysis based on 132 site directed mutants and concluded that the protein 's topology is committed very early along the folding pathway . based on the above studies , we suggest that , along with the suitable protein design experiments , better theoretical models including folding simulations coupled with structure prediction and sequence search in databases can shed light on the phenomenon of protein folding and conformation switching which may ultimately lead us to understand the contribution of each amino acid in these proteins to adopt a specific fold ." ]

[ "<S> in our study , we have concluded that two proteins with 88% homology choose different energetically favorable pathways in the very early stage of the folding process to attain their native folds . </S>", "<S> subsequent reports from other investigators by performing folding and unfolding kinetics experiments concur with our findings . </S>", "<S> we herewith discuss the key papers revealing computational and experimental analysis of two designed proteins with similar sequence distant folds . </S>", "<S> further we suggest that the theoretical / computational analysis of protein sequences and structures along with the relevant experiments provide a better understanding of the relationship between protein sequence , folding , and structure . </S>" ]

[ "CASE REPORT" ]

[ [ "in recent years , coupling of theoretical and experimental approaches in the study of protein folding has resulted in providing fruitful clues .", "experimental and computational protein design provides vital clues to understand the protein folding process , and it is of considerable interest in the area of protein science to engineer proteins with novel folds and desired functions .", "the field of protein design has a unique history where researchers from diverse discipline come together to explore novel catalytic , pharmaceutical , structural , and sensing properties of amino acids in proteins .", "interestingly , a designed eleven amino acid sequence folded as a helix in one position and as a sheet in another position in the protein sequence .", "this work has enabled to explore the role of nonlocal interactions in the formation of secondary structure .", "subsequently , helices were transmuted into sheets to understand the conformation change phenomenon and illustrate that not all the amino acids play an equal role in specifying a fold . utilizing the knowledge offered by several protein design groups , kuhlman et al . in 2003", "have computationally designed a 93-residue / protein called top7 and found that the protein could be experimentally folded and extremely stable .", "this pioneering work has enabled further research to understand the contribution of each amino acid residue in a protein to adopt a certain fold .", "hence , emphasizing that protein design could be a powerful experiment to understand the processes that underlie conformational plasticity in proteins .", "explored how two proteins with almost similar amino acid sequences change their fold and function . following the contribution of various theoretical and experimental protein science research groups , several such engineered proteins with selective", "nevertheless , the design of such a pair of proteins with high sequence identity with completely different topologies can be viewed as a challenge to the well - accepted paradigm that similar sequences always tend to fold into similar three - dimensional structures .", "an analysis of the literature reveals that the design of two highly identical proteins with different folds and functions is challenging and time bound as shown in [ table 1 ] .", "streptococcus protein g contains two types of domains ( ga and gb ) that bind to serum proteins in blood .", "the natural versions of ga and gb domains share no significant sequence homology and have different folds , 3 and 4 + , respectively . from the above two parent proteins , high - identity versions of ga and gb were synthesized .", "interestingly , small and critical differences in the sequences of the two proteins determine the topology of the protein early on the folding pathway .", "in addition , two proteins named ga88 ( pdb i d : 2jws ) and gb88 ( pdb", "i d : 2jwu ) by mutation experiments from the streptococcus protein g with 88% sequence identity adopt different structures and functions and these proteins are valuable tools to understand the contribution of residues to adopt a particular fold .", "these two proteins vary only at seven positions out of 56 amino acids , which are shown in [ figure 1 ] .", "this design has made a breakthrough in the field of protein science and contradicts the general statement that ", " following this , we have carried out computational sequence and structural analysis on these two designed proteins .", "we have performed secondary structure prediction of these two proteins and observed that the methods such as multivariate linear regression combiner can predict some regions as extended structures for the helical protein sequence ga , which gave us a clue that there may be structural plasticity at the region of first 15 residues , which are identical in the both proteins .", "we also discovered some patterns in the nonidentical positions of two proteins with a rare combination of residues that are not present in any publicly available sequence databases . by analyzing the structures of the two designed proteins , we predicted nucleation sites at various positions in the sequence , which may start or terminate secondary structural elements ( helix , sheet , and coil ) .", "we also observed drastic difference in the surrounding environment of nonidentical residues ( 7 out of 56 ) and difference in interaction energy . by observing the structural plasticity at the amino and carboxyl terminal of the sequences of two designed proteins and the influence of surrounding environment of each residue", ", we concluded that early on during the process of folding , both proteins may choose different energetically favorable pathways to attain the different folds .", "literature review of design of two proteins with high sequence identity adopting different folds sequence , dictionary of secondary structure of proteins assigned secondary structures and tertiary structures of pair of homologous heteromorphs ( the seven residues that vary in both sequences are indicated in rectangular boxes ) other researchers have characterized the folding of these two proteins using biophysical and computational experiments .", "they also indicated that the final native structures of these proteins were dictated very early along the folding pathway by performing equilibrium unfolding of ga88 and gb88 , folding and unfolding kinetics and molecular dynamics simulations experiments .", "concurrently , energy calculations were performed on the two designed proteins in a vacuum , which indicated that current computer modeling / simulations experiments can not explain why two highly similar sequences fold into different structures .", "however , it was suggested that improved modeling / simulations tools should be developed to predict the pair of sequences with different structures , which differ , by only few residues . in a recent study , folding and unfolding kinetics experiments performed on these two designed proteins indicated a detectable residual structure in the denatured state of gb88 whereas the denatured state of ga88 is unstructured .", "interestingly , they explored these two proteins by value analysis based on 132 site directed mutants and concluded that the protein 's topology is committed very early along the folding pathway . based on the above studies , we suggest that , along with the suitable protein design experiments , better theoretical models including folding simulations coupled with structure prediction and sequence search in databases can shed light on the phenomenon of protein folding and conformation switching which may ultimately lead us to understand the contribution of each amino acid in these proteins to adopt a specific fold ." ] ]

[ "in this case report , we describe a case of asherman 's syndrome treated with adult autologous stem cells for endometrial regeneration that resulted in conception after in vitro fertilization - embryo transfer ( ivf - et ) .", "the basis of this case is that endometrium is a dynamic , cyclically regenerating tissue , a unique model of physiological angiogenesis in adults .", "angiogenesis results either from sprouting of new vessels through recruitment of local endothelial cells from neighbouring blood vessels and/or by endothelial progenitor cells circulating in the peripheral blood after release from the bone marrow.[14 ] bone marrow stem cells also contribute to regeneration of the endometrium . on the basis of these facts ,", "her past treatment included a dilatation and curettage ( d and c ) in february 2005 .", "she presented to us with infertility and scanty menstruation since her d and c. her first transvaginal ultrasound scan on day 3 of the menstrual cycle revealed normal size retroverted uterus with homogenous myometrium and thin single line endometrium , but intact endometriomyometrial junction .", "the left ovary measured 2.52 2.51 3.04 cm , had two antral follicles and a hemorrhagic cyst , and was adherent to uterus posteriorly .", "right ovary measured 2.55 1.22 1.84 cm and had only one antral follicle .", "doppler studies showed very poorly vascularised ovaries , even with minimum wall filter , pulse repetition frequency of 0.3 , and gains -0.8 .", "a repeat scan on day 14 revealed that hemorrhagic cyst in left ovary had regressed partially .", "right ovary showed a follicle of 20 mm which on color doppler showed vascularity covering more than three - fourth of the follicular circumference with resistance index ( ri ) of 0.47 and peak systolic velocity ( psv ) of 11.23 cm / s , but the endometrium was only 3.2 mm with branches of spiral vessels seen only up to endometrio - myometrial junction .", "follow - up scan after three days still showed the same endometrial picture though follicle had ruptured .", "midluteal ultrasound scan ( ninth day post ovulation ) showed that endometrium had failed to grow even during secretory phase , despite corpus luteum with vascular ring covering more than half of the corpus luteal circumference with ri of 0.43 and psv of 10.43 cm / s on right side .", "severe endometrial adhesions were seen , which were cut [ figures 1 and 2 ] .", "hysteroscopic picture - endometrial adhesions postadhesiolysis hysteroscopic picture iucd - cu t was placed to maintain surgically established patency of the endometrial cavity .", "she was treated with cyclical estrogen and progesterones with ethinyloestradiol 0.05 mg from fifth to 25 day of the cycle and with medroxy progesterone acetate 10 mg from 20 to 25 day for 6 months to obtain a functional endometrium . during this period , she had withdrawal bleeding , which was scanty .", "ultrasound assessment of the endometrium in the following cycle showed no growth of the endometrium in the periovulatory and secretory phase of the menstrual cycle despite normal follicular development , rupture , and corpus luteum formation .", "thin endometrium after removal of iucd in preovulatory period due to poor endometrial development , she was advised surrogacy with ivf .", "her fsh on day 3 of cycle was 19.70 iu / ml with an antral follicle count of 2 . in view of poor ovarian reserve ,", "she was given oral oestradiol valerate tablets in increasing doses from 4 mg daily for 3 days , followed by 6 mg daily for another 3 days , and then 8 mg daily for a total of 25 days along with aspirin 75 mg daily for endometrial preparation .", "ultrasound scans were done intermittently to assess the endometrium , but it never reached a thickness more than 3.6 mm .", "this hormone replacement therapy cycle was repeated for 6 months without improvement of the endometrium . based on reports of adult autologous stem cells applications for regeneration of", "injured cartilage and cardiomyocytes in cardiac infarction , it was thought that use of stem cells for regeneration of endometrium was worth trying , especially because endometrium naturally has a regenerating capacity .", "if the basal layer of the endometrium is repaired and further stimulated , it should increase in thickness .", "on june 15 , 2009 , her bone marrow aspiration was done from the iliac crest under local anesthesia maintaining strict asepsis .", "aspiration was done using bone marrow biopsy needle and 10 ml syringe prewashed with heparin .", "collection was done in cpda ( citrate - phosphate dextrose anticoagulant ) medium using 1 ml of medium for 7 ml of bone marrow .", "it was sent to a dedicated stem cell laboratory working as per clinical gmp / gtp standard and having a working area with laminar airflow to class 100 .", "bone marrow was centrifuged using histopaque density gradient at 1000 rpm for 10 mins and 102 million mononuclear cells were separated .", "these cells were further treated by column separation technique and customized cocktail of cd9 , cd90 , and cd133 antibodies was used for immunomagnetic isolation of endometrial angiogenic stem cells .", "gene expression study for cd9 , cd44 , and cd90 using rt - pcr technique was done for differentiated cells .", "total 39 million marker - positive endometrial angiogenic cells were supplied in 0.7 ml of pbs ( phosphate buffer saline ) with 2% autologous ( patient 's own ) heat - inactivated serum on the next day for transplant.[616 ] patient was called with partially filled bladder on june 16 , 2009 , second day of her menstrual cycle .", "curettage was done under anesthesia . with patient in lithotomy position , sim 's speculum in place ,", "anterior retractor was used to retract the anterior vaginal wall , and volsellum was used to hold the anterior lip of cervix .", "france ) attached with 1-ml syringe , filled with 0.7-ml stem cell suspension , was advanced through cervix upto the fundal end of the endometrium under transabdominal ultrasound guidance .", "when the tip of the catheter was 0.5 cm below the fundus , piston was slowly advanced to allow slow steady flow of cell suspension in the uterine cavity . after instilling 0.3 ml of stem cell suspension at the fundus , injection was continued when cannula was gradually withdrawn out , till the tip reached mid cavity of the uterus .", "it was further very gently and slowly withdrawn out of the internal os and then external os , maintaining continuous pressure on the piston to prevent any back flow .", "speculum and volsellum were removed , and patient was shifted when she recovered from anesthesia .", "she was given oestradiol valerate 6 mg daily , starting on the same day for 25 days .", "ultrasound was done on 14 day and 19 day of the cycle , which showed endometrial thickness of 5.0 and 5.2 mm , respectively , and color doppler showed spiral vessels reaching subendometrial zone .", "ultrasound scans done at midcycle showed improvement in endometrial thickness , morphology , and vascularity [ figure 4 ] .", "well - developed endometrium with low - resistance vascularity reaching zone 4 on november 2 , 2009 , endometrial thickness at ultrasound was 6.9 mm , with vascularity reaching intraendometrial region .", "there were dominant follicles in either ovary , which excludes the possibility of any endogenous luteinizing hormone surge . on november 6 , 2009 ,", "three 4 - 6 cell grade i donor oocyte ivf embryos were transferred . at this time , her endometrium was multilayered , with thickness of 7.1 mm and intraendometrial vascularity .", "she was given progesterone vaginal gel ( crinone 8% , manufactured by fleet laboratories , uk , marketed by merck serono ) twice a day after et , as luteal support and ethinyl oestradiol was continued in a dose of 6 mg daily along with aspirin 75 mg daily .", "once -hcg was positive , the dose of oestradiol valerate was increased to 8 mg daily .", "progesterone vaginal gel 8% was continued twice a day along with aspirin 75 mg daily .", "a single gestational sac of 12 mm , yolk sac of 2.9 mm , embryonic pole of 2.4 mm , with m - mode showing embryonic heart rate of 112/mt were seen at ultrasound on november 30 , 2009 .", "follow - up scan was done at 8 weeks of pregnancy which showed a healthy fetus [ figures 57 ] .", "gestational sac , yolk sac , and embryonic pole after embryo transfer and positive -hcg test m - mode of cardiac activity of embryo 3d picture of 8 weeks scan", "this was a case of infertility with severe asherman 's syndrome and bilateral cornual tubal block with low ovarian reserve . anti - mullerian hormone , which is now considered to be the most reliable marker for ovarian reserve , was not routinely used at that time ( 2007 ) and was not easily available locally , and therefore was not done .", "low ovarian reserve with blocked fallopian tubes had left her with an option of oocyte donation .", "placement of iucd after surgery and cyclical hormonal therapy in association with low - dose aspirin or nitroglycerine is an established protocol for development of functional endometrium .", "placing iucd after curettage , followed by cyclical oestrogen progesterone therapy with aspirin , could not improve her endometrium .", "stem cells derived from tissues such as bone marrow , cord blood , adipose tissue , or the amniotic fluid have demonstrated regenerative potential in a variety of diseases and degenerative disorders . therefore , we tried to regenerate the endometrium with adult autologous stem cells after curettage , which was further supported with cyclical estrogen , progesterone , and aspirin for improving vascularization .", "bone marrow aspiration is fairly safe procedure with the risk of bleeding or infection , which are extremely rare when done meticulously in the hands of the expert .", "currettage was done to evoke injury - induced inflammatory reaction and hyperemia as homing induction , which in turn would enhance the response of endometrium to cyclical hormones .", "we would like to qoute a study that has shown regeneration of injured endometrium using adult bone marrow cells . ", "donor - derived bone marrow cells have been identified in human uterine endometrium. recent evidence has implicated bone marrow - derived cells as possible endometrial progenitors .", "it is unknown whether these cells originate from bone marrow mesenchymal stem cells or , alternatively , are circulating endometrial cells originally derived from the endometrium and harbored in bone marrow .", "these cells , regardless of their origin , may serve as a source of reparative cells for the reproductive tract .", "these data show the potential for stem cells to have a role in the regeneration or repair of this tissue after injury .", "significant engraftment of endometrium by bone marrow is likely to occur after endometrial injury or inflammatory insult .", "additionally , the proliferation and development of endometrium are entirely regulated by hormonal stimuli . ovarian estrogen and progesterone drive endometrial growth and apoptosis . in the case described in this study ,", "we tried to regenerate the endometrium by endometrial angiogenic stem cells isolated from autologous adult stem cells and transplanting them in the endometrium immediately after curettage .", "this was based on a study that describes that mononuclear cells collected from the menstrual blood contains a subpopulation of adherent cells and retains expression of the markers cd9 , cd29 , cd41a , cd44 , cd59 , cd73 , cd90 , and cd105 ; the markers used were cd9 , cd44 , and cd90 to isolate the desired cells .", "there is a minimal risk of developed cells presenting certain other markers during the process , but immunomagnetic isolation with specific marker antibodies reduces this possibility .", "this experimental therapy may be tried on larger scale when the basal layer of endometrium is also damaged by surgical insult .", "the risk for malignancies after the use of adult autologous stem cells may be a concern in view of several reports , but we would like to draw attention to the fact that most of these reports discuss the cases where adult autologous stem cells were used to treat malignancies .", "the total cost of the therapy in an indian set up comes to approximately rs .", "50 000 ( rupees fifty thousand ) , excluding the expense of ivf - et .", "moreover , it has an emotional and social advantage that the patient can bear her own child as against surrogacy .", "to the best of our knowledge , no case of asherman 's syndrome conceived after endometrial regeneration with adult autologous stem cells after failure of all other conventional modes of treatment has been reported in literature .", "this therapy can be used as an alternative to surrogacy in females with severe asherman 's syndrome , though larger trials may be needed to establish this as proved line of treatment ." ]

[ "<S> in a woman with severe asherman 's syndrome , curettage followed by placement of intrauterine contraceptive device ( iucd ) ( iucd with cyclical hormonal therapy ) was tried for 6 months , for development of the endometrium . </S>", "<S> when this failed , autologous stem cells were tried as an alternative therapy . from adult autologous stem cells isolated from patient 's own bone marrow </S>", "<S> , endometrial angiogenic stem cells were separated using immunomagnetic isolation . </S>", "<S> these cells were placed in the endometrial cavity under ultrasound guidance after curettage . </S>", "<S> patient was then given cyclical hormonal therapy . </S>", "<S> endometrium was assessed intermittently on ultrasound . on development of endometrium with a thickness of 8 mm and good vascularity , in vitro </S>", "<S> fertilization and embryo transfer was done . </S>", "<S> this resulted in positive biochemical pregnancy followed by confirmation of gestational sac , yolk sac , and embryonic pole with cardiac activity on ultrasound . </S>", "<S> endometrial angiogenic stem cells isolated from autologous adult stem cells could regenerate injured endometrium not responding to conventional treatment for asherman 's syndrome . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION" ]

[ [ "in this case report , we describe a case of asherman 's syndrome treated with adult autologous stem cells for endometrial regeneration that resulted in conception after in vitro fertilization - embryo transfer ( ivf - et ) .", "the basis of this case is that endometrium is a dynamic , cyclically regenerating tissue , a unique model of physiological angiogenesis in adults .", "angiogenesis results either from sprouting of new vessels through recruitment of local endothelial cells from neighbouring blood vessels and/or by endothelial progenitor cells circulating in the peripheral blood after release from the bone marrow.[14 ] bone marrow stem cells also contribute to regeneration of the endometrium . on the basis of these facts ," ], [ "her past treatment included a dilatation and curettage ( d and c ) in february 2005 .", "she presented to us with infertility and scanty menstruation since her d and c. her first transvaginal ultrasound scan on day 3 of the menstrual cycle revealed normal size retroverted uterus with homogenous myometrium and thin single line endometrium , but intact endometriomyometrial junction .", "the left ovary measured 2.52 2.51 3.04 cm , had two antral follicles and a hemorrhagic cyst , and was adherent to uterus posteriorly .", "right ovary measured 2.55 1.22 1.84 cm and had only one antral follicle .", "doppler studies showed very poorly vascularised ovaries , even with minimum wall filter , pulse repetition frequency of 0.3 , and gains -0.8 .", "a repeat scan on day 14 revealed that hemorrhagic cyst in left ovary had regressed partially .", "right ovary showed a follicle of 20 mm which on color doppler showed vascularity covering more than three - fourth of the follicular circumference with resistance index ( ri ) of 0.47 and peak systolic velocity ( psv ) of 11.23 cm / s , but the endometrium was only 3.2 mm with branches of spiral vessels seen only up to endometrio - myometrial junction .", "follow - up scan after three days still showed the same endometrial picture though follicle had ruptured .", "midluteal ultrasound scan ( ninth day post ovulation ) showed that endometrium had failed to grow even during secretory phase , despite corpus luteum with vascular ring covering more than half of the corpus luteal circumference with ri of 0.43 and psv of 10.43 cm / s on right side .", "severe endometrial adhesions were seen , which were cut [ figures 1 and 2 ] .", "hysteroscopic picture - endometrial adhesions postadhesiolysis hysteroscopic picture iucd - cu t was placed to maintain surgically established patency of the endometrial cavity .", "she was treated with cyclical estrogen and progesterones with ethinyloestradiol 0.05 mg from fifth to 25 day of the cycle and with medroxy progesterone acetate 10 mg from 20 to 25 day for 6 months to obtain a functional endometrium . during this period , she had withdrawal bleeding , which was scanty .", "ultrasound assessment of the endometrium in the following cycle showed no growth of the endometrium in the periovulatory and secretory phase of the menstrual cycle despite normal follicular development , rupture , and corpus luteum formation .", "thin endometrium after removal of iucd in preovulatory period due to poor endometrial development , she was advised surrogacy with ivf .", "her fsh on day 3 of cycle was 19.70 iu / ml with an antral follicle count of 2 . in view of poor ovarian reserve ,", "she was given oral oestradiol valerate tablets in increasing doses from 4 mg daily for 3 days , followed by 6 mg daily for another 3 days , and then 8 mg daily for a total of 25 days along with aspirin 75 mg daily for endometrial preparation .", "ultrasound scans were done intermittently to assess the endometrium , but it never reached a thickness more than 3.6 mm .", "this hormone replacement therapy cycle was repeated for 6 months without improvement of the endometrium . based on reports of adult autologous stem cells applications for regeneration of", "injured cartilage and cardiomyocytes in cardiac infarction , it was thought that use of stem cells for regeneration of endometrium was worth trying , especially because endometrium naturally has a regenerating capacity .", "if the basal layer of the endometrium is repaired and further stimulated , it should increase in thickness .", "on june 15 , 2009 , her bone marrow aspiration was done from the iliac crest under local anesthesia maintaining strict asepsis .", "aspiration was done using bone marrow biopsy needle and 10 ml syringe prewashed with heparin .", "collection was done in cpda ( citrate - phosphate dextrose anticoagulant ) medium using 1 ml of medium for 7 ml of bone marrow .", "it was sent to a dedicated stem cell laboratory working as per clinical gmp / gtp standard and having a working area with laminar airflow to class 100 .", "bone marrow was centrifuged using histopaque density gradient at 1000 rpm for 10 mins and 102 million mononuclear cells were separated .", "these cells were further treated by column separation technique and customized cocktail of cd9 , cd90 , and cd133 antibodies was used for immunomagnetic isolation of endometrial angiogenic stem cells .", "gene expression study for cd9 , cd44 , and cd90 using rt - pcr technique was done for differentiated cells .", "total 39 million marker - positive endometrial angiogenic cells were supplied in 0.7 ml of pbs ( phosphate buffer saline ) with 2% autologous ( patient 's own ) heat - inactivated serum on the next day for transplant.[616 ] patient was called with partially filled bladder on june 16 , 2009 , second day of her menstrual cycle .", "curettage was done under anesthesia . with patient in lithotomy position , sim 's speculum in place ,", "anterior retractor was used to retract the anterior vaginal wall , and volsellum was used to hold the anterior lip of cervix .", "france ) attached with 1-ml syringe , filled with 0.7-ml stem cell suspension , was advanced through cervix upto the fundal end of the endometrium under transabdominal ultrasound guidance .", "when the tip of the catheter was 0.5 cm below the fundus , piston was slowly advanced to allow slow steady flow of cell suspension in the uterine cavity . after instilling 0.3 ml of stem cell suspension at the fundus , injection was continued when cannula was gradually withdrawn out , till the tip reached mid cavity of the uterus .", "it was further very gently and slowly withdrawn out of the internal os and then external os , maintaining continuous pressure on the piston to prevent any back flow .", "speculum and volsellum were removed , and patient was shifted when she recovered from anesthesia .", "she was given oestradiol valerate 6 mg daily , starting on the same day for 25 days .", "ultrasound was done on 14 day and 19 day of the cycle , which showed endometrial thickness of 5.0 and 5.2 mm , respectively , and color doppler showed spiral vessels reaching subendometrial zone .", "ultrasound scans done at midcycle showed improvement in endometrial thickness , morphology , and vascularity [ figure 4 ] .", "well - developed endometrium with low - resistance vascularity reaching zone 4 on november 2 , 2009 , endometrial thickness at ultrasound was 6.9 mm , with vascularity reaching intraendometrial region .", "there were dominant follicles in either ovary , which excludes the possibility of any endogenous luteinizing hormone surge . on november 6 , 2009 ,", "three 4 - 6 cell grade i donor oocyte ivf embryos were transferred . at this time , her endometrium was multilayered , with thickness of 7.1 mm and intraendometrial vascularity .", "she was given progesterone vaginal gel ( crinone 8% , manufactured by fleet laboratories , uk , marketed by merck serono ) twice a day after et , as luteal support and ethinyl oestradiol was continued in a dose of 6 mg daily along with aspirin 75 mg daily .", "once -hcg was positive , the dose of oestradiol valerate was increased to 8 mg daily .", "progesterone vaginal gel 8% was continued twice a day along with aspirin 75 mg daily .", "a single gestational sac of 12 mm , yolk sac of 2.9 mm , embryonic pole of 2.4 mm , with m - mode showing embryonic heart rate of 112/mt were seen at ultrasound on november 30 , 2009 .", "follow - up scan was done at 8 weeks of pregnancy which showed a healthy fetus [ figures 57 ] .", "gestational sac , yolk sac , and embryonic pole after embryo transfer and positive -hcg test m - mode of cardiac activity of embryo 3d picture of 8 weeks scan" ], [ "this was a case of infertility with severe asherman 's syndrome and bilateral cornual tubal block with low ovarian reserve . anti - mullerian hormone , which is now considered to be the most reliable marker for ovarian reserve , was not routinely used at that time ( 2007 ) and was not easily available locally , and therefore was not done .", "low ovarian reserve with blocked fallopian tubes had left her with an option of oocyte donation .", "placement of iucd after surgery and cyclical hormonal therapy in association with low - dose aspirin or nitroglycerine is an established protocol for development of functional endometrium .", "placing iucd after curettage , followed by cyclical oestrogen progesterone therapy with aspirin , could not improve her endometrium .", "stem cells derived from tissues such as bone marrow , cord blood , adipose tissue , or the amniotic fluid have demonstrated regenerative potential in a variety of diseases and degenerative disorders . therefore , we tried to regenerate the endometrium with adult autologous stem cells after curettage , which was further supported with cyclical estrogen , progesterone , and aspirin for improving vascularization .", "bone marrow aspiration is fairly safe procedure with the risk of bleeding or infection , which are extremely rare when done meticulously in the hands of the expert .", "currettage was done to evoke injury - induced inflammatory reaction and hyperemia as homing induction , which in turn would enhance the response of endometrium to cyclical hormones .", "we would like to qoute a study that has shown regeneration of injured endometrium using adult bone marrow cells . ", "donor - derived bone marrow cells have been identified in human uterine endometrium. recent evidence has implicated bone marrow - derived cells as possible endometrial progenitors .", "it is unknown whether these cells originate from bone marrow mesenchymal stem cells or , alternatively , are circulating endometrial cells originally derived from the endometrium and harbored in bone marrow .", "these cells , regardless of their origin , may serve as a source of reparative cells for the reproductive tract .", "these data show the potential for stem cells to have a role in the regeneration or repair of this tissue after injury .", "significant engraftment of endometrium by bone marrow is likely to occur after endometrial injury or inflammatory insult .", "additionally , the proliferation and development of endometrium are entirely regulated by hormonal stimuli . ovarian estrogen and progesterone drive endometrial growth and apoptosis . in the case described in this study ,", "we tried to regenerate the endometrium by endometrial angiogenic stem cells isolated from autologous adult stem cells and transplanting them in the endometrium immediately after curettage .", "this was based on a study that describes that mononuclear cells collected from the menstrual blood contains a subpopulation of adherent cells and retains expression of the markers cd9 , cd29 , cd41a , cd44 , cd59 , cd73 , cd90 , and cd105 ; the markers used were cd9 , cd44 , and cd90 to isolate the desired cells .", "there is a minimal risk of developed cells presenting certain other markers during the process , but immunomagnetic isolation with specific marker antibodies reduces this possibility .", "this experimental therapy may be tried on larger scale when the basal layer of endometrium is also damaged by surgical insult .", "the risk for malignancies after the use of adult autologous stem cells may be a concern in view of several reports , but we would like to draw attention to the fact that most of these reports discuss the cases where adult autologous stem cells were used to treat malignancies .", "the total cost of the therapy in an indian set up comes to approximately rs .", "50 000 ( rupees fifty thousand ) , excluding the expense of ivf - et .", "moreover , it has an emotional and social advantage that the patient can bear her own child as against surrogacy .", "to the best of our knowledge , no case of asherman 's syndrome conceived after endometrial regeneration with adult autologous stem cells after failure of all other conventional modes of treatment has been reported in literature .", "this therapy can be used as an alternative to surrogacy in females with severe asherman 's syndrome , though larger trials may be needed to establish this as proved line of treatment ." ] ]

[ "a total of 1380 eyes were treated with prp for the indication \n of pdr from march 2001 to september 2006.the modified air \n lie house classification was followed .", "cases included pdr of \n varying severity such as flat new vessels , with raised new \n vessels , subhyaloid hemorrhage , vitreous hemorrhage , trd \n and macular edema with and without vitreous traction .", "thus in \n some of these cases the clinical evidence of vitreous contraction27 \n was already present before laser treatment was applied . in \n order to study the course of changes in the vitreous tissue over \n long term , we selected 100 eyes photocoagulated for pure pdr \n without clinically evident vitreous traction ( as defined below ) . out of these 100 eyes", "74 eyes showing complete regression of \n new blood vessels following prp were studied over a follow - up \n period of one to four years .", "criteria for pure pdr ( without clinically evident vitreous \n traction ) : selection ( 1 to 5 ) and follow - up ( 6 to 7 ) . \n", "proliferative diabetic retinopathy with flat new vessels \n on the disc ( nvd ) and /or flat new vessels elsewhere \n ( nve ) .", "27,28neither hemorrhage in the vitreous nor subhyaloid \n hemorrhageno tractional edema of maculano previous ocular surgeryabsence of systemic hypertension and renal diseasefundus fluorescein angiography ( ffa ) proven regression \n of new vessels aft er prp and no recurrence of new vessels \n during a follow - up of at least one year.presence of other signs of involution in addition to regression \n of new vessels such as decrease in venous dilatation , disc \n pallor , disappearance of retinal hemorrhages .", "29 \n proliferative diabetic retinopathy with flat new vessels \n on the disc ( nvd ) and /or flat new vessels elsewhere \n ( nve ) .", "27,28 neither hemorrhage in the vitreous nor subhyaloid \n hemorrhage no tractional edema of macula no previous ocular surgery absence of systemic hypertension and renal disease fundus fluorescein angiography ( ffa ) proven regression \n of new vessels aft er prp and no recurrence of new vessels \n during a follow - up of at least one year .", "presence of other signs of involution in addition to regression \n of new vessels such as decrease in venous dilatation , disc \n pallor , disappearance of retinal hemorrhages .", "29 careful history was obtained regarding duration of diabetes , \n type of diabetes and any other systemic illness ( hypertension , renal \n failure ) contributing to retinopathy , concurrent ocular disease \n and any previous ocular surgery .", "cases with such concomitant \n pathology were not included in the study [ table 1 ] .", "detailed ocular examination including indirect \n ophthalmoscopy , slit - lamp biomicroscopy , macular examination \n with three mirror contact lens / 90 d lens and ffa was done . \n", "optical coherence tomography ( oct ) was done in 32 eyes \n with csme out of 57 eyes ( 2004 onwards ) to rule out vitreous \n traction .", "eyes with any evidence of vitreous traction before prp \n were not included in the study .", "pan retinal laser photocoagulation with or without focal \n /grid macular photocoagulation was done in all the 100 eyes . \n", "green 532 and red 810 diode laser delivered through slit - lamp , \n were used .", "diode laser was preferentially used for macular \n photocoagulation in the presence of lenticular opacities.30 a \n mild grey whitening of retina was the end point of treatment \n with spot size 200 to 300 and exposure duration of 0.1 to 0.25 \n secs , with power level adjusted to produce the desired reaction . \n", "the prp was \n completed in three to four sittings at intervals of four to seven \n days each ; mainster 165 panfundoscopic lens was used .", "follow - up was available in terms of visual acuity and ocular \n examination at the first week in all cases .", "the ffa was done \n at six weeks ( 18 cases ) , eight weeks ( 64 cases ) and 11 weeks \n ( 18 cases ) .", "seventy - four out of 100 eyes responded by way of complete \n regression of new vessels with laser photocoagulation [ table \n 2 ] .", "twenty - six eyes required further prp or other adjunctive \n treatment . out of 57 eyes with macular edema visual \n improvement of two lines or more was observed in 34 eyes , \n stabilization of visual acuity in 11 eyes and drop of visual acuity \n by one line or more in 12 eyes . sixteen out of these 74 eyes \n required to undergo vitreo retinal surgery within a period of one \n to four years after complete regression of new vessels following \n laser photocoagulation [ table 3 ] .", "other signs of regression of \n pdr such as disc pallor , ghost vessels and reduced venous \n dilatation were present in only 39 out of 74 cases .", "incidence of pdr was significantly high in type i dm ( p \n 0.001 ) and was observed to increase with duration of dm ( p \n 0.001 ) [ table 1 ] .", "incidence of pdr with and without csme was \n significantly high in the age group 41 to 55 years ( p 0.05 ) and \n also regression of pdr with prp ( p 0.001 ) [ table 2 ] .", "indications \n for vitreo retinal surgery were significantly higher in type i dm \n ( p 0.01 ) , irrespective of age and duration of dm [ table 3 ] .", "diabetes induces pathology throughout the body and also \n in the vitreous via non - enzymatic glycation of proteins.7,10 \n advanced glycosylation end products ( ages ) have been \n found to be elevated in the vitreous of diabetics along with \n aggregation of collagen fibers and alterations in the cortex and \n hyalocytes.8,31 the vitreous in diabetics shows glycated collagen \n and increased amount of other proteins .", "7 - 9,32 degenerative \n vitreous changes occurring in diabetics at a much younger age \n produce anomalous pvd , which has been said to help formation \n of new retinal vessels.9,33 structural changes at the vitreoretinal \n interface promote migration and proliferation of vasogenic cells \n in the vitreous , consequent contraction can produce vitreous \n hemorrhage and macular edema.12,34 advanced glycation end products correlate with glycemic \n control and these reactive compounds form on dna , lipids and \n proteins where they represent pathophysiological modifications \n that precipitate dysfunction at a cellular and molecular level \n in diabetics.10,34 though the term diabetic vitreopathy exists in the \n literature it has not been used to address and identify the \n pathological complex of diabetic retinopathy in any of the \n existing classifications of diabetic retinopathy .", "19 - 22 we suggest \n that changes in the vitreous primarily due to diabetes mellitus \n and occurring independent of diabetic retinopathy can be \n called primary diabetic vitreopathy. the methods currently \n available for examination of the vitreous in vivo including oct \n give good information about the vitreo retinal interface but \n not the vitreous body , therefore such changes in the vitreous \n may not be easily detected clinically", ". we may therefore also \n call primary diabetic vitreopathy as subclinical diabetic \n vitreopathy.33 the hallmark of pdr is development of new vessels .", "the \n growing vascular endothelium combines with the collagen of the \n vitreous and gives it a contractile property.2,13,14 simultaneously \n fibrous tissue developing along the new vessels lines up on the \n posterior hyaloid , this also imparts contractile property to the \n vitreous.11 contraction of the vitreous can pull new retinal vessels \n to produce a bleeding in the subhyloid space to begin with [ fig . \n 1 ] .", "if the bleeding is forceful or the vitreous largely liquefied this \n blood can break into the vitreous tissue concurrently or later .", "it \n should be noted here that bleeding outside the tissue confines \n of retina ( internal limiting membrane ) is produced by vitreous \n contraction and not proliferative retinopathy per se and thus \n any hemorrhage outside the retinal tissue whether subhyloid \n or in the vitreous should be considered a sign of vitreopathy \n and not retinopathy .", "bleeding further augments the contractile \n property of the vitreous by inclusion of vasogenic and fibrogenic \n elements and such recurrent bleeding may result in the \n formation of fibrovascular tissue in the vitreous cavity .", "2 ] , secondary \n rhegmatogenous retinal detachment , persistent macular edema , \n premacular hemorrhage in addition to recurrent vitreous \n hemorrhage.35,36 we propose to call the changes induced in the \n vitreous tissue by proliferative retinopathy secondary diabetic \n vitreopathy or clinical diabetic vitreopathy. these are mainly \n in the form of an increase in the contractility and detachment \n of the vitreous .", "these changes are different from the changes \n of primary diabetic vitreopathy. if total vitreous detachment was present before development \n of pdr no bleeding or tractional retinal detachment may occur . \n", "similarly , if there was no separation of vitreous subsequent \n to development of retinal new vessels the above pathological \n events may not occur .", "there have been several attempts in \n the past for pharmacological vitreolysis so as to abort any \n complications of retinal neovascularization by pull of vitreous \n and there is a continuous suggestion in the literature for early \n vitrectomy in pdr cases with good vision so as to forestall \n the complications produced by vitreopathy .", "37 - 39 however , the \n changes in the vitreous have never been included in any of the \n classifications of diabetic retinopathy .", "laser ablation of the retina induces regression of new vessels \n in about two - thirds of cases .", "1 - 3 this results in the disappearance \n of a ready source of bleeding i.e. new vessels . with laser \n treatment the incidence of non - resolving massive vitreous \n hemorrhage has drastically reduced .", "but the other indications \n for vitreo retinal surgery such as trd , rhegmatogenous retinal \n detachment [ fig .", "3 ] , tractional macular edema , premacular \n fibrosis , small recurrent vitreous hemorrhages , retinal wrinkling , \n macular heterotropia and dense premacular hemorrhage have \n persisted .", "4 - 6 , 15 - 18 these are all produced by vitreopathy , vitreous \n traction and not by pdr alone . in our series , out of 74 eyes showing complete regression \n of new vessels with prp , 16 eyes ( 23% ) required to undergo \n vitreous surgery for the indications of recurrent vitreous \n hemorrhage ( eight ) , tractional retinal detachment ( four ) \n secondary rhegmatogenous retinal detachment(one ) and \n tractional macular edema ( three cases ) [ figs.4 a , b , c ] .", "in other \n words 23% of cases showed a continued contraction of vitreous \n strong enough to produce the indications for surgery despite \n successful regression of new vessels .", "we invited data from \n leading retina centers in the country on incidence of vitreous \n surgery in cases of pdr fully regressed with prp , over a period \n of one to four years .", "it is logical to say that in these cases the changes of primary \n /secondary diabetic vitreopathy were relatively independent of \n the effect of laser treatment and the regressed status of pdr . in \n the remaining 77% cases", "there are several factors which explain \n the absence of complications produced by diabetic vitreopathy . \n", "regression of new vessels is withdrawal of a ready source of \n bleeding , multiple chorioretinal adhesions produced by laser \n protect against trd and rhegmatogenous retinal detachment . \n", "laser treatment in cases with csme helps resolution of edema , \n it is effective in tractional macular edema also if the vitreous \n traction is not very strong .", "in addition to the above , prp induces \n posterior vitreous detachment in 50% cases and in the large \n majority this is eventless .", "a small percentage of these cases \n may show vitreous bleeding or even trd during completion \n of prp if it is too aggressive and spaced closely.40 we therefore \n understand that in the large majority of cases ( 77% in our \n series ) a balance is established between the beneficial effect of \n laser on retina and the damaging effect of diabetic vitreopathy . \n in the remaining cases ( 23% in our series )", "this balance might \n not be established and ongoing vitreous contraction might \n have produced indications for vitreo retinal surgery .", "another \n aspect of the effect of laser photocoagulation is that though the \n new vessels disappear the fibrous tissue does not and a slow , \n late cicatrization of fibrous tissue can produce indications like \n retinal wrinkling , macular heterotropia41 and shallow trd ; \n we had one such case in our series with retinal wrinkling and \n trd occurring in regressed retinopathy after a quiet period of \n three years [ fig .", "we suggest diabetic vitreopathy as a separate subdivision \n in the existing classification of diabetic retinopathy , identifying \n the changes induced by diabetes mellitus as primary diabetic \n vitreopathy and the changes induced by pdr as secondary \n diabetic vitreopathy. it is the changes of secondary diabetic \n vitreopathy which can be clinically observed and which \n produce the indications for vitreo retinal surgery , these can \n be called diabetic vitreopathy as such or clinical diabetic \n vitreopathy. we further observe that certain cases of clinical \n diabetic vitreopathy might not be operable because of extensive \n neovascularization both in the anterior and posterior segment \n or many complex fibrovascular membranes in the vitreous . in \n other words", "we can have cases which are operable and may \n benefit by vitreous surgery i.e. surgical vitreopathy and cases \n which are inoperable or may not benefit by vitreous surgery \n i.e. non surgical vitreopathy. recently , intravitreal drugs have been used to alter the course \n of pdr .", "first is \n purified ovine hyaluronidase ( vitrase , ista pharmaceuticals ) \n which produces vitreous liquefaction .", "the second group are \n anti - vegf drugs , which reduce neovascularization , contraction \n of fibrovascular proliferations when prp is applied and \n bleeding during surgery .", "avastin ( genetech pharmaceuticals ) , \n the commonest drug , has been used as a preoperative adjunct \n for pdr , trd with severe pdr , for iris rubeosis , florid disc \n neovascularization and for treatment of pdr complicated by \n vitreous hemorrhage .", "23 - 26 this would mean that anti - vegf drugs can be used \n in cases which are either inoperable because of extensive \n neovascularization , massive vitreous hemorrhage or are risky \n to operate due to the possibility of intraoperative bleeding \n and other complications .", "use of these drugs may render such \n cases suitable for surgery . in order to identify such cases in a \n logical manner", "we suggest a separate class in between surgical \n and non - surgical vitreopathy as intermediate vitreopathy. \n the cases put in this class can shift to the surgical vitreopathy \n group if response to drug is adequate and to the non - surgical \n vitreopathy group if the drug treatment does not make the \n case operable .", "the term diabetic retinopathy may be replaced by \n diabetic retino - vitreopathy and may be classified as \n below . \n", "non - proliferative diabetic retinopathy ( mild / moderate/ \n severe / very severe)proliferative diabetic retinopathydiabetic maculopathy ( focal , diff use , ischemic , mixed)clinical diabetic vitreopathyi ) surgical vitreopathyii ) intermediate vitreopathyiii ) non - surgical vitreopathy \n non - proliferative diabetic retinopathy ( mild / moderate/ \n severe / very severe ) proliferative diabetic retinopathy diabetic maculopathy ( focal , diff use , ischemic , mixed ) clinical diabetic vitreopathy i ) surgical vitreopathy ii ) intermediate vitreopathy iii ) non - surgical vitreopathy \n posterior pole trd 5superior half trd 16tractional macular edema without macular \n ischemia 15premacular fibrosis 28recurrent vitreous hemorrhages in laser regressed \n pdr 4 , 17secondary rhegmatogenous retinal detachement 27 - 28optic disc traction 27 , 28macular heterotropia 41retinal wrinkling 41dense premacular hemorrhage 35 \n tractional macular edema without macular \n ischemia 15 premacular fibrosis 28 recurrent vitreous hemorrhages in laser regressed \n pdr 4 , 17 secondary rhegmatogenous retinal detachement 27 - 28 optic disc traction 27 , 28 macular heterotropia 41 dense premacular hemorrhage 35 \n florid neovascularization with /without any of the above indications 26anterior segment neovascularization 24neovascularization non - responsive to laser", "with/ \n without any of the above indications 24 - 26large non - resolving vitreous bleeding in laser - treated \n or untreated pdr 26 \n florid neovascularization with /without any of the above indications 26 anterior segment neovascularization 24 neovascularization non - responsive to laser with/ \n without any of the above indications 24 - 26 large non - resolving vitreous bleeding in laser - treated \n or untreated pdr 26 \n inferior peripheral trd 16recurrent vitreous hemorrhages in active pdr 28 , 36tractional macular edema with ischemia 15 , 27 \n inferior peripheral trd 16 recurrent vitreous hemorrhages in active pdr 28 , 36 tractional macular edema with ischemia 15 , 27 this classification represents all stages and manifestations \n of diabetic retinopathy including changes in the vitreous .", "the \n classification identifies surgical indications and places different \n manifestations in accordance with the pathology and indicated \n treatment .", "for example , tractional diabetic macular edema is \n classified as surgical vitreopathy as it is the vitreous traction \n which is the cause and surgery is the treatment .", "the classification incorporates the indications for using \n recent intravitreal anti - vegf drugs also .", "the study sets the direction for further research and \n investigations on vitreous changes using modern tools such as \n ultrasound and oct in diabetics before and after development \n of diabetic retinopathy and also long - term prospective \n observations on the retina and vitreous on a larger sample \n of pdr cases after prp .", "\n non - proliferative diabetic retinopathy ( mild / moderate/ \n severe / very severe)proliferative diabetic retinopathydiabetic maculopathy ( focal , diff use , ischemic , mixed)clinical diabetic vitreopathyi ) surgical vitreopathyii ) intermediate vitreopathyiii ) non - surgical vitreopathy \n non - proliferative diabetic retinopathy ( mild / moderate/ \n severe / very severe ) proliferative diabetic retinopathy diabetic maculopathy ( focal , diff use , ischemic , mixed ) clinical diabetic vitreopathy i ) surgical vitreopathy ii ) intermediate vitreopathy iii ) non - surgical vitreopathy", "\n posterior pole trd 5superior half trd 16tractional macular edema without macular \n ischemia 15premacular fibrosis 28recurrent vitreous hemorrhages in laser regressed \n pdr 4 , 17secondary rhegmatogenous retinal detachement 27 - 28optic disc traction 27 , 28macular heterotropia 41retinal wrinkling 41dense premacular hemorrhage 35 \n tractional macular edema without macular \n ischemia 15 premacular fibrosis 28 recurrent vitreous hemorrhages in laser regressed \n pdr 4 , 17 secondary rhegmatogenous retinal detachement 27 - 28 optic disc traction 27 , 28 macular heterotropia 41 dense premacular hemorrhage 35", "\n florid neovascularization with /without any of the above indications 26anterior segment neovascularization 24neovascularization non - responsive to laser with/ \n without any of the above indications 24 - 26large non - resolving vitreous bleeding in laser - treated \n or untreated pdr 26 \n florid neovascularization with /without any of the above indications 26 anterior segment neovascularization 24 neovascularization non - responsive to laser with/ \n without any of the above indications 24 - 26 large non - resolving vitreous bleeding in laser - treated \n or untreated pdr 26", "\n inferior peripheral trd 16recurrent vitreous hemorrhages in active pdr 28 , 36tractional macular edema with ischemia 15 , 27 \n inferior peripheral trd 16 recurrent vitreous hemorrhages in active pdr 28 , 36 tractional macular edema with ischemia 15 , 27 this classification represents all stages and manifestations \n of diabetic retinopathy including changes in the vitreous .", "the \n classification identifies surgical indications and places different \n manifestations in accordance with the pathology and indicated \n treatment .", "for example , tractional diabetic macular edema is \n classified as surgical vitreopathy as it is the vitreous traction \n which is the cause and surgery is the treatment .", "the classification incorporates the indications for using \n recent intravitreal anti - vegf drugs also .", "the study sets the direction for further research and \n investigations on vitreous changes using modern tools such as \n ultrasound and oct in diabetics before and after development \n of diabetic retinopathy and also long - term prospective \n observations on the retina and vitreous on a larger sample \n of pdr cases after prp .", "changes in the vitreous induced \n by diabetes mellitus are identified as primary diabetic \n vitreopathy / subclinical diabetic vitreopathy and changes \n induced by proliferative retinopathy as secondary diabetic \n vitreopathy / clinical diabetic vitreopathy .", "any indications for \n vitreous surgery in pdr are produced by vitreopathy and not \n retinopathy per se . in about", "two - thirds of pdr cases vitreopathy can be kept \n under control with adequate prp , the remaining one - third cases \n may require surgery due to vitreopathy .", "a new classification \n is proposed taking into consideration the element of diabetic \n vitreopathy as well as the clinical use of intravitreal anti - vegf \n drugs ." ]

[ "<S> background : many eyes with proliferative diabetic retinopathy ( pdr ) require vitreous surgery despite \n complete regression of new vessels with pan retinal laser photocoagulation ( prp ) . </S>", "<S> changes in the vitreous \n caused by diabetes mellitus and diabetic retinopathy may continue to progress independent of laser regressed \n status of retinopathy . </S>", "<S> diabetic vitreopathy can be an independent manifestation of the disease process.aim:to examine this concept by studying the long - term behavior of the vitreous in cases of pdr regressed \n with prp.materials and methods : seventy - four eyes with pure pdr ( without clinically evident vitreous traction ) \n showing fundus fluorescein angiography ( ffa ) proven regression of new vessels following prp were \n retrospectively studied out of a total of 1380 eyes photocoagulated between march 2001 and september 2006 \n for pdr of varying severity . </S>", "<S> follow - up was available from one to four years.results:twenty-three percent of eyes showing ffa - proven regression of new vessels with laser required \n to undergo surgery for indications produced by vitreous traction such as recurrent vitreous hemorrhage , \n tractional retinal detachment , secondary rhegmatogenous retinal detachment and tractional macular edema \n within one to four years.conclusion:vitreous changes continued to progress despite regression of pdr in many diabetics . </S>", "<S> we \n identifies this as clinical diabetic vitreopathy and propose an expanded classification for diabetic \n retinopathy to signify these changes and to redefine the indications for surgery . </S>" ]

[ "Materials and Methods", "Results", "Discussion", "Dubeys classification of diabetic retino-vitreopathy", "Surgical vitreopathy", "Intermediate vitreopathy", "Non-surgical vitreopathy", "Conclusion" ]

[ [ "a total of 1380 eyes were treated with prp for the indication \n of pdr from march 2001 to september 2006.the modified air \n lie house classification was followed .", "cases included pdr of \n varying severity such as flat new vessels , with raised new \n vessels , subhyaloid hemorrhage , vitreous hemorrhage , trd \n and macular edema with and without vitreous traction .", "thus in \n some of these cases the clinical evidence of vitreous contraction27 \n was already present before laser treatment was applied . in \n order to study the course of changes in the vitreous tissue over \n long term , we selected 100 eyes photocoagulated for pure pdr \n without clinically evident vitreous traction ( as defined below ) . out of these 100 eyes", "74 eyes showing complete regression of \n new blood vessels following prp were studied over a follow - up \n period of one to four years .", "criteria for pure pdr ( without clinically evident vitreous \n traction ) : selection ( 1 to 5 ) and follow - up ( 6 to 7 ) . \n", "proliferative diabetic retinopathy with flat new vessels \n on the disc ( nvd ) and /or flat new vessels elsewhere \n ( nve ) .", "27,28neither hemorrhage in the vitreous nor subhyaloid \n hemorrhageno tractional edema of maculano previous ocular surgeryabsence of systemic hypertension and renal diseasefundus fluorescein angiography ( ffa ) proven regression \n of new vessels aft er prp and no recurrence of new vessels \n during a follow - up of at least one year.presence of other signs of involution in addition to regression \n of new vessels such as decrease in venous dilatation , disc \n pallor , disappearance of retinal hemorrhages .", "29 \n proliferative diabetic retinopathy with flat new vessels \n on the disc ( nvd ) and /or flat new vessels elsewhere \n ( nve ) .", "27,28 neither hemorrhage in the vitreous nor subhyaloid \n hemorrhage no tractional edema of macula no previous ocular surgery absence of systemic hypertension and renal disease fundus fluorescein angiography ( ffa ) proven regression \n of new vessels aft er prp and no recurrence of new vessels \n during a follow - up of at least one year .", "presence of other signs of involution in addition to regression \n of new vessels such as decrease in venous dilatation , disc \n pallor , disappearance of retinal hemorrhages .", "29 careful history was obtained regarding duration of diabetes , \n type of diabetes and any other systemic illness ( hypertension , renal \n failure ) contributing to retinopathy , concurrent ocular disease \n and any previous ocular surgery .", "cases with such concomitant \n pathology were not included in the study [ table 1 ] .", "detailed ocular examination including indirect \n ophthalmoscopy , slit - lamp biomicroscopy , macular examination \n with three mirror contact lens / 90 d lens and ffa was done . \n", "optical coherence tomography ( oct ) was done in 32 eyes \n with csme out of 57 eyes ( 2004 onwards ) to rule out vitreous \n traction .", "eyes with any evidence of vitreous traction before prp \n were not included in the study .", "pan retinal laser photocoagulation with or without focal \n /grid macular photocoagulation was done in all the 100 eyes . \n", "green 532 and red 810 diode laser delivered through slit - lamp , \n were used .", "diode laser was preferentially used for macular \n photocoagulation in the presence of lenticular opacities.30 a \n mild grey whitening of retina was the end point of treatment \n with spot size 200 to 300 and exposure duration of 0.1 to 0.25 \n secs , with power level adjusted to produce the desired reaction . \n", "the prp was \n completed in three to four sittings at intervals of four to seven \n days each ; mainster 165 panfundoscopic lens was used .", "follow - up was available in terms of visual acuity and ocular \n examination at the first week in all cases .", "the ffa was done \n at six weeks ( 18 cases ) , eight weeks ( 64 cases ) and 11 weeks \n ( 18 cases ) ." ], [ "seventy - four out of 100 eyes responded by way of complete \n regression of new vessels with laser photocoagulation [ table \n 2 ] .", "twenty - six eyes required further prp or other adjunctive \n treatment . out of 57 eyes with macular edema visual \n improvement of two lines or more was observed in 34 eyes , \n stabilization of visual acuity in 11 eyes and drop of visual acuity \n by one line or more in 12 eyes . sixteen out of these 74 eyes \n required to undergo vitreo retinal surgery within a period of one \n to four years after complete regression of new vessels following \n laser photocoagulation [ table 3 ] .", "other signs of regression of \n pdr such as disc pallor , ghost vessels and reduced venous \n dilatation were present in only 39 out of 74 cases .", "incidence of pdr was significantly high in type i dm ( p \n 0.001 ) and was observed to increase with duration of dm ( p \n 0.001 ) [ table 1 ] .", "incidence of pdr with and without csme was \n significantly high in the age group 41 to 55 years ( p 0.05 ) and \n also regression of pdr with prp ( p 0.001 ) [ table 2 ] .", "indications \n for vitreo retinal surgery were significantly higher in type i dm \n ( p 0.01 ) , irrespective of age and duration of dm [ table 3 ] ." ], [ "diabetes induces pathology throughout the body and also \n in the vitreous via non - enzymatic glycation of proteins.7,10 \n advanced glycosylation end products ( ages ) have been \n found to be elevated in the vitreous of diabetics along with \n aggregation of collagen fibers and alterations in the cortex and \n hyalocytes.8,31 the vitreous in diabetics shows glycated collagen \n and increased amount of other proteins .", "7 - 9,32 degenerative \n vitreous changes occurring in diabetics at a much younger age \n produce anomalous pvd , which has been said to help formation \n of new retinal vessels.9,33 structural changes at the vitreoretinal \n interface promote migration and proliferation of vasogenic cells \n in the vitreous , consequent contraction can produce vitreous \n hemorrhage and macular edema.12,34 advanced glycation end products correlate with glycemic \n control and these reactive compounds form on dna , lipids and \n proteins where they represent pathophysiological modifications \n that precipitate dysfunction at a cellular and molecular level \n in diabetics.10,34 though the term diabetic vitreopathy exists in the \n literature it has not been used to address and identify the \n pathological complex of diabetic retinopathy in any of the \n existing classifications of diabetic retinopathy .", "19 - 22 we suggest \n that changes in the vitreous primarily due to diabetes mellitus \n and occurring independent of diabetic retinopathy can be \n called primary diabetic vitreopathy. the methods currently \n available for examination of the vitreous in vivo including oct \n give good information about the vitreo retinal interface but \n not the vitreous body , therefore such changes in the vitreous \n may not be easily detected clinically", ". we may therefore also \n call primary diabetic vitreopathy as subclinical diabetic \n vitreopathy.33 the hallmark of pdr is development of new vessels .", "the \n growing vascular endothelium combines with the collagen of the \n vitreous and gives it a contractile property.2,13,14 simultaneously \n fibrous tissue developing along the new vessels lines up on the \n posterior hyaloid , this also imparts contractile property to the \n vitreous.11 contraction of the vitreous can pull new retinal vessels \n to produce a bleeding in the subhyloid space to begin with [ fig . \n 1 ] .", "if the bleeding is forceful or the vitreous largely liquefied this \n blood can break into the vitreous tissue concurrently or later .", "it \n should be noted here that bleeding outside the tissue confines \n of retina ( internal limiting membrane ) is produced by vitreous \n contraction and not proliferative retinopathy per se and thus \n any hemorrhage outside the retinal tissue whether subhyloid \n or in the vitreous should be considered a sign of vitreopathy \n and not retinopathy .", "bleeding further augments the contractile \n property of the vitreous by inclusion of vasogenic and fibrogenic \n elements and such recurrent bleeding may result in the \n formation of fibrovascular tissue in the vitreous cavity .", "2 ] , secondary \n rhegmatogenous retinal detachment , persistent macular edema , \n premacular hemorrhage in addition to recurrent vitreous \n hemorrhage.35,36 we propose to call the changes induced in the \n vitreous tissue by proliferative retinopathy secondary diabetic \n vitreopathy or clinical diabetic vitreopathy. these are mainly \n in the form of an increase in the contractility and detachment \n of the vitreous .", "these changes are different from the changes \n of primary diabetic vitreopathy. if total vitreous detachment was present before development \n of pdr no bleeding or tractional retinal detachment may occur . \n", "similarly , if there was no separation of vitreous subsequent \n to development of retinal new vessels the above pathological \n events may not occur .", "there have been several attempts in \n the past for pharmacological vitreolysis so as to abort any \n complications of retinal neovascularization by pull of vitreous \n and there is a continuous suggestion in the literature for early \n vitrectomy in pdr cases with good vision so as to forestall \n the complications produced by vitreopathy .", "37 - 39 however , the \n changes in the vitreous have never been included in any of the \n classifications of diabetic retinopathy .", "laser ablation of the retina induces regression of new vessels \n in about two - thirds of cases .", "1 - 3 this results in the disappearance \n of a ready source of bleeding i.e. new vessels . with laser \n treatment the incidence of non - resolving massive vitreous \n hemorrhage has drastically reduced .", "but the other indications \n for vitreo retinal surgery such as trd , rhegmatogenous retinal \n detachment [ fig .", "3 ] , tractional macular edema , premacular \n fibrosis , small recurrent vitreous hemorrhages , retinal wrinkling , \n macular heterotropia and dense premacular hemorrhage have \n persisted .", "4 - 6 , 15 - 18 these are all produced by vitreopathy , vitreous \n traction and not by pdr alone . in our series , out of 74 eyes showing complete regression \n of new vessels with prp , 16 eyes ( 23% ) required to undergo \n vitreous surgery for the indications of recurrent vitreous \n hemorrhage ( eight ) , tractional retinal detachment ( four ) \n secondary rhegmatogenous retinal detachment(one ) and \n tractional macular edema ( three cases ) [ figs.4 a , b , c ] .", "in other \n words 23% of cases showed a continued contraction of vitreous \n strong enough to produce the indications for surgery despite \n successful regression of new vessels .", "we invited data from \n leading retina centers in the country on incidence of vitreous \n surgery in cases of pdr fully regressed with prp , over a period \n of one to four years .", "it is logical to say that in these cases the changes of primary \n /secondary diabetic vitreopathy were relatively independent of \n the effect of laser treatment and the regressed status of pdr . in \n the remaining 77% cases", "there are several factors which explain \n the absence of complications produced by diabetic vitreopathy . \n", "regression of new vessels is withdrawal of a ready source of \n bleeding , multiple chorioretinal adhesions produced by laser \n protect against trd and rhegmatogenous retinal detachment . \n", "laser treatment in cases with csme helps resolution of edema , \n it is effective in tractional macular edema also if the vitreous \n traction is not very strong .", "in addition to the above , prp induces \n posterior vitreous detachment in 50% cases and in the large \n majority this is eventless .", "a small percentage of these cases \n may show vitreous bleeding or even trd during completion \n of prp if it is too aggressive and spaced closely.40 we therefore \n understand that in the large majority of cases ( 77% in our \n series ) a balance is established between the beneficial effect of \n laser on retina and the damaging effect of diabetic vitreopathy . \n in the remaining cases ( 23% in our series )", "this balance might \n not be established and ongoing vitreous contraction might \n have produced indications for vitreo retinal surgery .", "another \n aspect of the effect of laser photocoagulation is that though the \n new vessels disappear the fibrous tissue does not and a slow , \n late cicatrization of fibrous tissue can produce indications like \n retinal wrinkling , macular heterotropia41 and shallow trd ; \n we had one such case in our series with retinal wrinkling and \n trd occurring in regressed retinopathy after a quiet period of \n three years [ fig .", "we suggest diabetic vitreopathy as a separate subdivision \n in the existing classification of diabetic retinopathy , identifying \n the changes induced by diabetes mellitus as primary diabetic \n vitreopathy and the changes induced by pdr as secondary \n diabetic vitreopathy. it is the changes of secondary diabetic \n vitreopathy which can be clinically observed and which \n produce the indications for vitreo retinal surgery , these can \n be called diabetic vitreopathy as such or clinical diabetic \n vitreopathy. we further observe that certain cases of clinical \n diabetic vitreopathy might not be operable because of extensive \n neovascularization both in the anterior and posterior segment \n or many complex fibrovascular membranes in the vitreous . in \n other words", "we can have cases which are operable and may \n benefit by vitreous surgery i.e. surgical vitreopathy and cases \n which are inoperable or may not benefit by vitreous surgery \n i.e. non surgical vitreopathy. recently , intravitreal drugs have been used to alter the course \n of pdr .", "first is \n purified ovine hyaluronidase ( vitrase , ista pharmaceuticals ) \n which produces vitreous liquefaction .", "the second group are \n anti - vegf drugs , which reduce neovascularization , contraction \n of fibrovascular proliferations when prp is applied and \n bleeding during surgery .", "avastin ( genetech pharmaceuticals ) , \n the commonest drug , has been used as a preoperative adjunct \n for pdr , trd with severe pdr , for iris rubeosis , florid disc \n neovascularization and for treatment of pdr complicated by \n vitreous hemorrhage .", "23 - 26 this would mean that anti - vegf drugs can be used \n in cases which are either inoperable because of extensive \n neovascularization , massive vitreous hemorrhage or are risky \n to operate due to the possibility of intraoperative bleeding \n and other complications .", "use of these drugs may render such \n cases suitable for surgery . in order to identify such cases in a \n logical manner", "we suggest a separate class in between surgical \n and non - surgical vitreopathy as intermediate vitreopathy. \n the cases put in this class can shift to the surgical vitreopathy \n group if response to drug is adequate and to the non - surgical \n vitreopathy group if the drug treatment does not make the \n case operable .", "the term diabetic retinopathy may be replaced by \n diabetic retino - vitreopathy and may be classified as \n below . \n", "non - proliferative diabetic retinopathy ( mild / moderate/ \n severe / very severe)proliferative diabetic retinopathydiabetic maculopathy ( focal , diff use , ischemic , mixed)clinical diabetic vitreopathyi ) surgical vitreopathyii ) intermediate vitreopathyiii ) non - surgical vitreopathy \n non - proliferative diabetic retinopathy ( mild / moderate/ \n severe / very severe ) proliferative diabetic retinopathy diabetic maculopathy ( focal , diff use , ischemic , mixed ) clinical diabetic vitreopathy i ) surgical vitreopathy ii ) intermediate vitreopathy iii ) non - surgical vitreopathy \n posterior pole trd 5superior half trd 16tractional macular edema without macular \n ischemia 15premacular fibrosis 28recurrent vitreous hemorrhages in laser regressed \n pdr 4 , 17secondary rhegmatogenous retinal detachement 27 - 28optic disc traction 27 , 28macular heterotropia 41retinal wrinkling 41dense premacular hemorrhage 35 \n tractional macular edema without macular \n ischemia 15 premacular fibrosis 28 recurrent vitreous hemorrhages in laser regressed \n pdr 4 , 17 secondary rhegmatogenous retinal detachement 27 - 28 optic disc traction 27 , 28 macular heterotropia 41 dense premacular hemorrhage 35 \n florid neovascularization with /without any of the above indications 26anterior segment neovascularization 24neovascularization non - responsive to laser", "with/ \n without any of the above indications 24 - 26large non - resolving vitreous bleeding in laser - treated \n or untreated pdr 26 \n florid neovascularization with /without any of the above indications 26 anterior segment neovascularization 24 neovascularization non - responsive to laser with/ \n without any of the above indications 24 - 26 large non - resolving vitreous bleeding in laser - treated \n or untreated pdr 26 \n inferior peripheral trd 16recurrent vitreous hemorrhages in active pdr 28 , 36tractional macular edema with ischemia 15 , 27 \n inferior peripheral trd 16 recurrent vitreous hemorrhages in active pdr 28 , 36 tractional macular edema with ischemia 15 , 27 this classification represents all stages and manifestations \n of diabetic retinopathy including changes in the vitreous .", "the \n classification identifies surgical indications and places different \n manifestations in accordance with the pathology and indicated \n treatment .", "for example , tractional diabetic macular edema is \n classified as surgical vitreopathy as it is the vitreous traction \n which is the cause and surgery is the treatment .", "the classification incorporates the indications for using \n recent intravitreal anti - vegf drugs also .", "the study sets the direction for further research and \n investigations on vitreous changes using modern tools such as \n ultrasound and oct in diabetics before and after development \n of diabetic retinopathy and also long - term prospective \n observations on the retina and vitreous on a larger sample \n of pdr cases after prp ." ], [ "\n non - proliferative diabetic retinopathy ( mild / moderate/ \n severe / very severe)proliferative diabetic retinopathydiabetic maculopathy ( focal , diff use , ischemic , mixed)clinical diabetic vitreopathyi ) surgical vitreopathyii ) intermediate vitreopathyiii ) non - surgical vitreopathy \n non - proliferative diabetic retinopathy ( mild / moderate/ \n severe / very severe ) proliferative diabetic retinopathy diabetic maculopathy ( focal , diff use , ischemic , mixed ) clinical diabetic vitreopathy i ) surgical vitreopathy ii ) intermediate vitreopathy iii ) non - surgical vitreopathy" ], [ "\n posterior pole trd 5superior half trd 16tractional macular edema without macular \n ischemia 15premacular fibrosis 28recurrent vitreous hemorrhages in laser regressed \n pdr 4 , 17secondary rhegmatogenous retinal detachement 27 - 28optic disc traction 27 , 28macular heterotropia 41retinal wrinkling 41dense premacular hemorrhage 35 \n tractional macular edema without macular \n ischemia 15 premacular fibrosis 28 recurrent vitreous hemorrhages in laser regressed \n pdr 4 , 17 secondary rhegmatogenous retinal detachement 27 - 28 optic disc traction 27 , 28 macular heterotropia 41 dense premacular hemorrhage 35" ], [ "\n florid neovascularization with /without any of the above indications 26anterior segment neovascularization 24neovascularization non - responsive to laser with/ \n without any of the above indications 24 - 26large non - resolving vitreous bleeding in laser - treated \n or untreated pdr 26 \n florid neovascularization with /without any of the above indications 26 anterior segment neovascularization 24 neovascularization non - responsive to laser with/ \n without any of the above indications 24 - 26 large non - resolving vitreous bleeding in laser - treated \n or untreated pdr 26" ], [ "\n inferior peripheral trd 16recurrent vitreous hemorrhages in active pdr 28 , 36tractional macular edema with ischemia 15 , 27 \n inferior peripheral trd 16 recurrent vitreous hemorrhages in active pdr 28 , 36 tractional macular edema with ischemia 15 , 27 this classification represents all stages and manifestations \n of diabetic retinopathy including changes in the vitreous .", "the \n classification identifies surgical indications and places different \n manifestations in accordance with the pathology and indicated \n treatment .", "for example , tractional diabetic macular edema is \n classified as surgical vitreopathy as it is the vitreous traction \n which is the cause and surgery is the treatment .", "the classification incorporates the indications for using \n recent intravitreal anti - vegf drugs also .", "the study sets the direction for further research and \n investigations on vitreous changes using modern tools such as \n ultrasound and oct in diabetics before and after development \n of diabetic retinopathy and also long - term prospective \n observations on the retina and vitreous on a larger sample \n of pdr cases after prp ." ], [ "changes in the vitreous induced \n by diabetes mellitus are identified as primary diabetic \n vitreopathy / subclinical diabetic vitreopathy and changes \n induced by proliferative retinopathy as secondary diabetic \n vitreopathy / clinical diabetic vitreopathy .", "any indications for \n vitreous surgery in pdr are produced by vitreopathy and not \n retinopathy per se . in about", "two - thirds of pdr cases vitreopathy can be kept \n under control with adequate prp , the remaining one - third cases \n may require surgery due to vitreopathy .", "a new classification \n is proposed taking into consideration the element of diabetic \n vitreopathy as well as the clinical use of intravitreal anti - vegf \n drugs ." ] ]

[ "we identified isolates for sequencing from 29 invasive gas cases diagnosed in patients in a northern arizona hospital during january ", "july 2015 and randomly selected an additional 99 gas isolates from a repository of > 2,000 arizona gas isolates collected during 20022006 ( no isolates from patients in arizona were available for 20072014 ) .", "four additional isolates from central arizona identified in 2015 were included in the analysis ( technical appendix table ) .", "all isolates were grown on 5% sheep blood tryptic soy agar plates ( hardy diagnostics , santa maria , ca ) , and incubated at 37c with 5% co2 .", "dna was extracted by using a dneasy blood and tissue kit ( qiagen , valencia , ca , usa ) following manufacturer s protocol .", "genomic dna libraries were prepared by using the nextera xt library prep kit ( illumina , san diego , ca ) and sequenced with paired - end reads ( 250 bp ) on an illumina miseq instrument , as previously described ( 9 ) . the finished genome of the emm59 canadian clone mgas15252 ( genbank accession no .", "cp003116 ) and high - quality publicly available sequence - read data from 44 us isolates , from ncbi short read archive ( bioproject # prjna194066 ) , were included in the subsequent phylogenetic analyses .", "the final core genome ( all nucleotide loci found in all genomes ) for single - nucleotide polymorphism ( snp ) detection was 1,636,024 bp ( 98.6% of reference ) .", "we used nasp snp analysis pipeline ( http://tgennorth.github.io/nasp/ ) for whole - genome snp typing as previously described ( 10 ) .", "we used mega version 5.2.2 software ( 11 ) to generate maximum parsimony phylogenetic trees .", "regions of high snp density were identified as possible regions of recombination and were further analyzed for impact on the consistency index .", "gas emm subtypes were assigned by using blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) , querying the study genome assemblies against the centers for disease control and prevention s ( cdc ) emm type - specific sequence database ( http://www.cdc.gov/streplab/m-proteingene-typing.html ) .", "we resolved dual emm - type hits using cdc s emm typing sanger sequencing primers ( http://www.cdc.gov/streplab/protocol-emm-type.html ) as a blast query and noting hit locations .", "we identified 18 of the 29 contemporary northern arizona isolates as subtype emm59 ; the remaining isolates were composed of 6 additional emm types : emm1 ( n = 2 ) , emm5 ( n = 2 ) , emm58 ( n = 1 ) , emm81 ( n = 2 ) , emm83 ( n = 1 ) , emm89 ( n = 2 ) , and emm94 ( n = 1 ) .", "the 99 historical and 4 contemporary background arizona isolates included 25 distinct emm types ( technical appendix table ) .", "no emm59 isolates were identified in this background set , and none had been previously reported in arizona .", "an emm59-only phylogenetic analysis demonstrated the apparent presence of multiple lineages of emm59 in the 2015 arizona isolates ( figure 1 ) .", "a distinct clone consisting of 14 of the 18 emm59 isolates were separated from each other by only 04 snps , genomically supporting the presence of an ongoing outbreak ; > 8 of these patients were epidemiologically linked to physical contact , cohabitation , or both with 1 other person ( data not shown ) .", "the additional emm59 isolates make up additional lineages separated from one other by 828 snps .", "a relatively large number of snps and indels were seen within an approximate 23-kilobase region ( figure 1 ) .", "this region has been previously reported to contain mutational hotspots associated with virulence ( 12,13 ) . considering the presumptive positive selective force on this region , snps within the region", "phylogenetic single - nucleotide polymorphism ( snp ) tree of emm59 isolates from a northern arizona hospital displaying distribution of mutations in a 23 kb positively selected region during invasive group a streptococcus outbreak , southwestern united states .", "maximum parsimony tree of all snp loci ( n = 58 ) in emm59 isolates ( n = 18 ) from arizona , 2 recent new mexico isolate genomes , and the canadian clone reference isolate mgas15252 .", "branch lengths represent numbers of snps between isolates ; unit bar is in the figure .", "numbered circles distinguish lineages of selected mutations in scpa , enn , sfbl , mga , sfbx , and sof genes in a 23-kb hotspot mutational region .", "when compared with all other publicly available us emm59 isolate genomes , nearly all the genomes identified in the united states were closely related to each other and to the canadian clone mgas15252 ; individual isolate snp branch lengths ranged from 0 to 10 ( figure 2 ) .", "the arizona outbreak isolates were separated from 2 new mexico isolates by 4 and 5 snps each ; these isolates fell within the overall arizona clade and were subsequently included in the arizona - only phylogenetic analysis ( figure 1 ) .", "conversley , the isolate from patient m appears more distant from the larger arizona population .", "the arizona clades , with the exception of that of the isolate from patient m , all appear to arise from the large minnesota polytomy .", "the previously estimated 1.32.1 snps / year mutation rates for gas ( 14,15 ) further support the arizona outbreak as being caused by a single clone , likely originating from new mexico and being spread over 612 months .", "phylogenetic single - nucleotide polymorphism ( snp ) tree of emm59 isolates from arizona during invasive group a streptococcus outbreak in the southwestern united states , previously analyzed us emm59 isolates , and the canadian clone .", "maximum parsimony tree of all 177 snp loci ( 44 parsimony informative snps ) in emm59 isolates from arizona ( n = 18 ) , minnesota ( n = 29 ) , oregon ( n = 8) , new mexico ( n = 3 ) , colorado ( n = 2 ) , and california ( n = 1 ) and the canadian clone reference isolate mgas15252 . tree has regions of recombination removed and is rooted with minnesota isolate srr11574570 .", "the emm59 subtype of gas , the etiologic agent of a substantial nationwide outbreak of invasive gas in canada during 20062009 ( 4 ) , is now present in arizona , causing at least 1 outbreak of epidemiologically and genomically linked cases and several additional epidemiologically unrelated cases .", "the lack of emm59 in background isolates in arizona from the previous decade , along with its low genetic diversity , suggests that emm59 emerged recently in arizona .", "following the emm59 epidemic in canada , this subtype was subsequently seen in a few us states ; a retrospective analyses of the centers for disease control and prevention active bacterial core surveillance ( abcs ) system ( http://www.cdc.gov/abcs/reports-findings/survreports.pdf ) identified 40 us emm59 isolates during 20002009 ( 6 ) and an additional 67 isolates during 20102012 ( 7 ) . of note , only 5 ( of the 40 emm59 isolates from 20002009 ( 2 from minnesota , 2 from california , and 1 from oregon ) appeared to be closely related to the canadian clone ( defined by the authors as being separated by < 16 snps ) ( 6 ) ; in contrast , all of the strains from the 20102012 survey appeared to be more closely related to the canadian clone .", "the more recent abcs analysis identified an increasing number of southwestern isolates , including 4 from colorado and 6 from new mexico ( 7 ) , although no outbreaks were specifically described in these states ( arizona is not included in the abcs system ) .", "( 7 ) , in an analysis of 60 mn emm59 isolates from case - patients with identified race , determined that 25 ( 42% ) were from native americans ; of 5 isolates from new mexico in that study , 3 were from native americans . ", "given the apparent distal nature of the arizona / new mexico isolates to the minnesota population in our study , it is reasonable to propose an unidentified epidemiologic relationship between these case populations .", "however , caution must be used in drawing conclusions regarding the relationships of isolates from disparate geographic regions because only limited comparable sequence data from previous emm59 studies in the united states ( 7 ) were publicly available to compare to the arizona isolates .", "epidemiologic investigations , along with healthcare provider and patient education activities , are ongoing in arizona to further determine the extent of the current outbreak and the associated risk factors and to help mitigate effects and limit or prevent further spread to at - risk populations ." ]

[ "<S> the hyper - virulent emm59 genotype of invasive group a streptococcus was identified in northern arizona in 2015 . </S>", "<S> eighteen isolates belonging to a genomic cluster grouped most closely with recently identified isolates in new mexico . </S>", "<S> the continued transmission of emm59 in the southwestern united states poses a public health concern . </S>" ]

[ "The Study", "Conclusions", "None" ]

[ [ "we identified isolates for sequencing from 29 invasive gas cases diagnosed in patients in a northern arizona hospital during january ", "july 2015 and randomly selected an additional 99 gas isolates from a repository of > 2,000 arizona gas isolates collected during 20022006 ( no isolates from patients in arizona were available for 20072014 ) .", "four additional isolates from central arizona identified in 2015 were included in the analysis ( technical appendix table ) .", "all isolates were grown on 5% sheep blood tryptic soy agar plates ( hardy diagnostics , santa maria , ca ) , and incubated at 37c with 5% co2 .", "dna was extracted by using a dneasy blood and tissue kit ( qiagen , valencia , ca , usa ) following manufacturer s protocol .", "genomic dna libraries were prepared by using the nextera xt library prep kit ( illumina , san diego , ca ) and sequenced with paired - end reads ( 250 bp ) on an illumina miseq instrument , as previously described ( 9 ) . the finished genome of the emm59 canadian clone mgas15252 ( genbank accession no .", "cp003116 ) and high - quality publicly available sequence - read data from 44 us isolates , from ncbi short read archive ( bioproject # prjna194066 ) , were included in the subsequent phylogenetic analyses .", "the final core genome ( all nucleotide loci found in all genomes ) for single - nucleotide polymorphism ( snp ) detection was 1,636,024 bp ( 98.6% of reference ) .", "we used nasp snp analysis pipeline ( http://tgennorth.github.io/nasp/ ) for whole - genome snp typing as previously described ( 10 ) .", "we used mega version 5.2.2 software ( 11 ) to generate maximum parsimony phylogenetic trees .", "regions of high snp density were identified as possible regions of recombination and were further analyzed for impact on the consistency index .", "gas emm subtypes were assigned by using blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) , querying the study genome assemblies against the centers for disease control and prevention s ( cdc ) emm type - specific sequence database ( http://www.cdc.gov/streplab/m-proteingene-typing.html ) .", "we resolved dual emm - type hits using cdc s emm typing sanger sequencing primers ( http://www.cdc.gov/streplab/protocol-emm-type.html ) as a blast query and noting hit locations .", "we identified 18 of the 29 contemporary northern arizona isolates as subtype emm59 ; the remaining isolates were composed of 6 additional emm types : emm1 ( n = 2 ) , emm5 ( n = 2 ) , emm58 ( n = 1 ) , emm81 ( n = 2 ) , emm83 ( n = 1 ) , emm89 ( n = 2 ) , and emm94 ( n = 1 ) .", "the 99 historical and 4 contemporary background arizona isolates included 25 distinct emm types ( technical appendix table ) .", "no emm59 isolates were identified in this background set , and none had been previously reported in arizona .", "an emm59-only phylogenetic analysis demonstrated the apparent presence of multiple lineages of emm59 in the 2015 arizona isolates ( figure 1 ) .", "a distinct clone consisting of 14 of the 18 emm59 isolates were separated from each other by only 04 snps , genomically supporting the presence of an ongoing outbreak ; > 8 of these patients were epidemiologically linked to physical contact , cohabitation , or both with 1 other person ( data not shown ) .", "the additional emm59 isolates make up additional lineages separated from one other by 828 snps .", "a relatively large number of snps and indels were seen within an approximate 23-kilobase region ( figure 1 ) .", "this region has been previously reported to contain mutational hotspots associated with virulence ( 12,13 ) . considering the presumptive positive selective force on this region , snps within the region", "phylogenetic single - nucleotide polymorphism ( snp ) tree of emm59 isolates from a northern arizona hospital displaying distribution of mutations in a 23 kb positively selected region during invasive group a streptococcus outbreak , southwestern united states .", "maximum parsimony tree of all snp loci ( n = 58 ) in emm59 isolates ( n = 18 ) from arizona , 2 recent new mexico isolate genomes , and the canadian clone reference isolate mgas15252 .", "branch lengths represent numbers of snps between isolates ; unit bar is in the figure .", "numbered circles distinguish lineages of selected mutations in scpa , enn , sfbl , mga , sfbx , and sof genes in a 23-kb hotspot mutational region .", "when compared with all other publicly available us emm59 isolate genomes , nearly all the genomes identified in the united states were closely related to each other and to the canadian clone mgas15252 ; individual isolate snp branch lengths ranged from 0 to 10 ( figure 2 ) .", "the arizona outbreak isolates were separated from 2 new mexico isolates by 4 and 5 snps each ; these isolates fell within the overall arizona clade and were subsequently included in the arizona - only phylogenetic analysis ( figure 1 ) .", "conversley , the isolate from patient m appears more distant from the larger arizona population .", "the arizona clades , with the exception of that of the isolate from patient m , all appear to arise from the large minnesota polytomy .", "the previously estimated 1.32.1 snps / year mutation rates for gas ( 14,15 ) further support the arizona outbreak as being caused by a single clone , likely originating from new mexico and being spread over 612 months .", "phylogenetic single - nucleotide polymorphism ( snp ) tree of emm59 isolates from arizona during invasive group a streptococcus outbreak in the southwestern united states , previously analyzed us emm59 isolates , and the canadian clone .", "maximum parsimony tree of all 177 snp loci ( 44 parsimony informative snps ) in emm59 isolates from arizona ( n = 18 ) , minnesota ( n = 29 ) , oregon ( n = 8) , new mexico ( n = 3 ) , colorado ( n = 2 ) , and california ( n = 1 ) and the canadian clone reference isolate mgas15252 . tree has regions of recombination removed and is rooted with minnesota isolate srr11574570 ." ], [ "the emm59 subtype of gas , the etiologic agent of a substantial nationwide outbreak of invasive gas in canada during 20062009 ( 4 ) , is now present in arizona , causing at least 1 outbreak of epidemiologically and genomically linked cases and several additional epidemiologically unrelated cases .", "the lack of emm59 in background isolates in arizona from the previous decade , along with its low genetic diversity , suggests that emm59 emerged recently in arizona .", "following the emm59 epidemic in canada , this subtype was subsequently seen in a few us states ; a retrospective analyses of the centers for disease control and prevention active bacterial core surveillance ( abcs ) system ( http://www.cdc.gov/abcs/reports-findings/survreports.pdf ) identified 40 us emm59 isolates during 20002009 ( 6 ) and an additional 67 isolates during 20102012 ( 7 ) . of note , only 5 ( of the 40 emm59 isolates from 20002009 ( 2 from minnesota , 2 from california , and 1 from oregon ) appeared to be closely related to the canadian clone ( defined by the authors as being separated by < 16 snps ) ( 6 ) ; in contrast , all of the strains from the 20102012 survey appeared to be more closely related to the canadian clone .", "the more recent abcs analysis identified an increasing number of southwestern isolates , including 4 from colorado and 6 from new mexico ( 7 ) , although no outbreaks were specifically described in these states ( arizona is not included in the abcs system ) .", "( 7 ) , in an analysis of 60 mn emm59 isolates from case - patients with identified race , determined that 25 ( 42% ) were from native americans ; of 5 isolates from new mexico in that study , 3 were from native americans . ", "given the apparent distal nature of the arizona / new mexico isolates to the minnesota population in our study , it is reasonable to propose an unidentified epidemiologic relationship between these case populations .", "however , caution must be used in drawing conclusions regarding the relationships of isolates from disparate geographic regions because only limited comparable sequence data from previous emm59 studies in the united states ( 7 ) were publicly available to compare to the arizona isolates .", "epidemiologic investigations , along with healthcare provider and patient education activities , are ongoing in arizona to further determine the extent of the current outbreak and the associated risk factors and to help mitigate effects and limit or prevent further spread to at - risk populations ." ], [ "" ] ]

[ "primary tumors of the trachea are rare and account for 0.1% of all malignancies . in adults ,", "approximately 90% of the primary tracheal tumors are malignant , whereas in children , the majority of these tumors are benign [ 2 , 3 ] . in a retrospective study by webb et al . , 55.4% of the patients were male and 77.3% of them were smokers .", "the frequency of this rare disease has been estimated to be 0.10.4% of all malignancies , with an annual incidence of 2.6 new cases per million per year .", "primary tracheal tumors can arise from the respiratory epithelium , salivary glands and mesenchymal structure of the trachea .", "squamous cell carcinoma ( scc ) and adenoid cystic carcinoma ( acc ) make up about 71.6% of all adult primary tracheal tumors . the remaining portion ( 28.4% )", "scc occurs predominantly in men in the sixth and seventh decades , whereas acc is equally distributed between the sexes and peaks in incidence in the fourth and fifth decades .", "in contrast to acc , scc ( the most common type of tracheal tumors ) has a more aggressive course .", "median survival time ranges from 6 to 14 months , and tumor disappearance is dependent on whether the primary lesion is resected .", "sccs spread to the lymph nodes and 1020% of the patients will have distant metastasis at presentation .", "carcinoids , lymphomas , granular cell tumors and small cell carcinomas have variable prognoses ; yet they seem to behave better than sccs , adenocarcinomas or sarcomas .", "the majority of the tracheal neoplasms are primary in nature , and other primary sites that can metastasize to the trachea are the lung and esophagus , the latter being the most common .", "patients with surgically resectable primary tracheal tumors have a better prognosis than those with tumors that can not be resected .", "preoperative radiation therapy has been attempted in some patients , yet the most compelling evidence for adjuvant radiation therapy comes from postoperative cases .", "the present study reports a rare case of primary tracheal malignancy treated with surgery and adjuvant radiation therapy .", "our 60-year - old diabetic and hypertensive male patient had a 1-month history of cough associated with marked respiratory distress and occasional dyspnea in the right lateral lying position .", "the patient consulted a chest physician and underwent bronchoscopy , which showed a polypoid growth in the trachea extending to the larynx ( fig .", "a computed tomography ( ct ) scan revealed irregular soft tissue thickening in the region of the trachea extending up to the adjacent larynx with no soft tissue calcification , and there was no definite lymphadenopathy in the cervical region and no involvement of the underlying bone . a repeat ct scan of the neck ( fig .", "2 ) showed evidence of an ill - defined , irregular , nodular , infiltrating soft tissue growth arising from the posterior tracheal wall , bridging the lumen and reaching up to the anterior wall , with an infiltration of about 2.2 1.8 cm .", "the location was opposite the c7-d1 level involving the 5th or 6th tracheal ring , approximately 7 cm proximal to the carina with no significant evidence of cervical and mediastinal lymphadenopathy .", "excision of the tracheal growth through a cervical transverse incision was performed , and a tracheostomy tube was inserted .", "soon after surgery , the cough subsided and the patient attended square hospital , dhaka , bangladesh , for postoperative irradiation .", "ninety - five percent of the planning target volume ( ptv ) covered 97.3% of the prescribed dose .", "the minimum and maximum dose received by the ptv was 50.05 and 63.68 gy , respectively .", "3 ) . three multileaf collimator fields ( left anterior oblique , right anterior oblique and posterior ) were used for three - dimensional conformal radiation therapy ( 3dcrt ) .", "complete response was documented , and the patient is still alive without any evidence of disease during 30 months of follow - up .", "primary malignant tumors of the trachea are uncommon , and therefore limited data supporting a standard management are available .", "the vast majority of the primary tracheal tumors in adults are malignant and most of them usually diagnosed at a later stage , which is due to a delayed presentation of specific symptoms like cough , dyspnea and hemoptysis .", "it is clear that patients who can be resected have a better prognosis than those who can not , prompting the recommendation of surgical resection for most primary tracheal tumors .", "all resected patients need postsurgical irradiation , regardless of tumor burden , margin status , histology or nodal status .", "grillo and mathisen experienced a median survival of 34 months in scc and 118 months in acc patients who underwent a combined modality like surgery and radiation therapy .", "attained a 61-month median survival using a combined surgery and radiation therapy in their study .", ". showed that 73% of the patients with a 5-year survival underwent radiation therapy postoperatively .", "the role of external beam radiation therapy as an adjuvant to surgical resection is better established .", "grillo and mathisen advocated maximal ( 4,5006,500 cgy ) radiation therapy after scc and acc resection because of the close margins necessary for resection and the high likelihood of local recurrence .", "modern ct - based 3dcrt or intensity - modulated radiation therapy theoretically allows higher and safer doses delivered to the trachea . for postoperative cases ,", "all patients with this tumor in the postoperative or definitive setting should be treated with the 3dcrt technique .", "an intraluminal boost technique after external beam radiation therapy may decrease the risk of late side effects .", "there are many studies showing the benefit of postoperative irradiation therapy in primary tracheal malignancies such as sccs and accs .", "however , little experience has been shared in the case of primary adenocarcinomas of the trachea , the tumor type described in our report .", "the prognosis of patients with malignant tumors of the trachea remains gloomy , and long - term median survival of tracheal adenocarcinoma patients undergoing combined modalities is unknown to us .", "primary adenocarcinoma of the trachea needs to be diagnosed at an early stage , and combined multimodal approaches may be explored to attain an extended median survival .", "none of the authors has any conflicts of interest regarding the content of this article ." ]

[ "<S> primary adenocarcinoma of the trachea is extremely rare and a standard treatment does not exist due to nonavailability of evidence - based randomized control studies . this paper reports the case of a 60-year - old male , who presented with cough and occasional respiratory distress . </S>", "<S> bronchoscopic examination and a computed tomography scan revealed a soft tissue mass in the trachea arising from the posterior tracheal wall . </S>", "<S> cytological examination and immunochemistry confirmed primary adenocarcinoma of the trachea . </S>", "<S> excision of the tumor followed by three - dimensional conformal radiation therapy was performed , and a dose of 56 gy was delivered to the primary site . </S>", "<S> two and a half years after treatment , the patient has no clinical or radiological evidence of the disease , and no late complication has occurred . </S>" ]

[ "Introduction", "Case Presentation", "Discussion", "Disclosure Statement" ]

[ [ "primary tumors of the trachea are rare and account for 0.1% of all malignancies . in adults ,", "approximately 90% of the primary tracheal tumors are malignant , whereas in children , the majority of these tumors are benign [ 2 , 3 ] . in a retrospective study by webb et al . , 55.4% of the patients were male and 77.3% of them were smokers .", "the frequency of this rare disease has been estimated to be 0.10.4% of all malignancies , with an annual incidence of 2.6 new cases per million per year .", "primary tracheal tumors can arise from the respiratory epithelium , salivary glands and mesenchymal structure of the trachea .", "squamous cell carcinoma ( scc ) and adenoid cystic carcinoma ( acc ) make up about 71.6% of all adult primary tracheal tumors . the remaining portion ( 28.4% )", "scc occurs predominantly in men in the sixth and seventh decades , whereas acc is equally distributed between the sexes and peaks in incidence in the fourth and fifth decades .", "in contrast to acc , scc ( the most common type of tracheal tumors ) has a more aggressive course .", "median survival time ranges from 6 to 14 months , and tumor disappearance is dependent on whether the primary lesion is resected .", "sccs spread to the lymph nodes and 1020% of the patients will have distant metastasis at presentation .", "carcinoids , lymphomas , granular cell tumors and small cell carcinomas have variable prognoses ; yet they seem to behave better than sccs , adenocarcinomas or sarcomas .", "the majority of the tracheal neoplasms are primary in nature , and other primary sites that can metastasize to the trachea are the lung and esophagus , the latter being the most common .", "patients with surgically resectable primary tracheal tumors have a better prognosis than those with tumors that can not be resected .", "preoperative radiation therapy has been attempted in some patients , yet the most compelling evidence for adjuvant radiation therapy comes from postoperative cases .", "the present study reports a rare case of primary tracheal malignancy treated with surgery and adjuvant radiation therapy ." ], [ "our 60-year - old diabetic and hypertensive male patient had a 1-month history of cough associated with marked respiratory distress and occasional dyspnea in the right lateral lying position .", "the patient consulted a chest physician and underwent bronchoscopy , which showed a polypoid growth in the trachea extending to the larynx ( fig .", "a computed tomography ( ct ) scan revealed irregular soft tissue thickening in the region of the trachea extending up to the adjacent larynx with no soft tissue calcification , and there was no definite lymphadenopathy in the cervical region and no involvement of the underlying bone . a repeat ct scan of the neck ( fig .", "2 ) showed evidence of an ill - defined , irregular , nodular , infiltrating soft tissue growth arising from the posterior tracheal wall , bridging the lumen and reaching up to the anterior wall , with an infiltration of about 2.2 1.8 cm .", "the location was opposite the c7-d1 level involving the 5th or 6th tracheal ring , approximately 7 cm proximal to the carina with no significant evidence of cervical and mediastinal lymphadenopathy .", "excision of the tracheal growth through a cervical transverse incision was performed , and a tracheostomy tube was inserted .", "soon after surgery , the cough subsided and the patient attended square hospital , dhaka , bangladesh , for postoperative irradiation .", "ninety - five percent of the planning target volume ( ptv ) covered 97.3% of the prescribed dose .", "the minimum and maximum dose received by the ptv was 50.05 and 63.68 gy , respectively .", "3 ) . three multileaf collimator fields ( left anterior oblique , right anterior oblique and posterior ) were used for three - dimensional conformal radiation therapy ( 3dcrt ) .", "complete response was documented , and the patient is still alive without any evidence of disease during 30 months of follow - up ." ], [ "primary malignant tumors of the trachea are uncommon , and therefore limited data supporting a standard management are available .", "the vast majority of the primary tracheal tumors in adults are malignant and most of them usually diagnosed at a later stage , which is due to a delayed presentation of specific symptoms like cough , dyspnea and hemoptysis .", "it is clear that patients who can be resected have a better prognosis than those who can not , prompting the recommendation of surgical resection for most primary tracheal tumors .", "all resected patients need postsurgical irradiation , regardless of tumor burden , margin status , histology or nodal status .", "grillo and mathisen experienced a median survival of 34 months in scc and 118 months in acc patients who underwent a combined modality like surgery and radiation therapy .", "attained a 61-month median survival using a combined surgery and radiation therapy in their study .", ". showed that 73% of the patients with a 5-year survival underwent radiation therapy postoperatively .", "the role of external beam radiation therapy as an adjuvant to surgical resection is better established .", "grillo and mathisen advocated maximal ( 4,5006,500 cgy ) radiation therapy after scc and acc resection because of the close margins necessary for resection and the high likelihood of local recurrence .", "modern ct - based 3dcrt or intensity - modulated radiation therapy theoretically allows higher and safer doses delivered to the trachea . for postoperative cases ,", "all patients with this tumor in the postoperative or definitive setting should be treated with the 3dcrt technique .", "an intraluminal boost technique after external beam radiation therapy may decrease the risk of late side effects .", "there are many studies showing the benefit of postoperative irradiation therapy in primary tracheal malignancies such as sccs and accs .", "however , little experience has been shared in the case of primary adenocarcinomas of the trachea , the tumor type described in our report .", "the prognosis of patients with malignant tumors of the trachea remains gloomy , and long - term median survival of tracheal adenocarcinoma patients undergoing combined modalities is unknown to us .", "primary adenocarcinoma of the trachea needs to be diagnosed at an early stage , and combined multimodal approaches may be explored to attain an extended median survival ." ], [ "none of the authors has any conflicts of interest regarding the content of this article ." ] ]

[ "intractable nausea and vomiting are very rare symptoms of medullary compression and there are approximately less than 10 cases reported in the literature of the aforementioned symptomatology mostly due to tumors .", "there is one reported case where vertebral artery compression of the medulla led to intractable nausea and vomiting .", "a 69-year - old woman presented with a 10-month history of intractable nausea and vomiting resulting in a 50 pound weight loss .", "she had an extensive medical workup at multiple outside hospitals including a comprehensive gastrointestinal workup which was significant for celiac disease .", "her pertinent past medical history includes a history of breast cancer , mastectomy , and cholecystectomy . during her evaluation by the gastroenterologists at our hospital ,", "the diagnosis of celiac disease was confirmed , and she was also noted to have a duodenal ulcer .", "hematologic and biochemical workup was only significant for anemia attributable to her ulcer and celiac disease .", "magnetic resonance imaging / angiography ( mri / mra ) of the brain and neck , and diagnostic four vessels cerebral angiogram were obtained to further evaluate her dizziness and double vision .", "the left vertebral artery angiogram revealed a tortuous left vertebral artery with a 9.6 5.6 mm dissecting aneurysm in the v3 segment .", "this , along with dolichoectasia of the vertebro - basilar arteries , resulted in compression of the medulla oblongata , which was also confirmed in the contrasted mri / mra of the brain and neck [ figure 1 ] . computed tomography angiography ( cta ) also confirmed that the vertebral artery was pushing the medulla medially [ figure 2 ] .", "the patient was offered a microvascular decompression ( mvd ) of the vertebral artery to attempt to decompress the brainstem and alleviate her intractable nausea and vomiting .", "preoperative magnetic resonance imaging ( mri ) t2 sequenceshowing compression of the medulla by the vertebral artery preoperative computed tomography angiography ( cta ) showing a tortuous left vertebral artery crossing the midline a left retrosigmoid craniotomy was completed to approach the vertebral artery and lower brainstem . the elongated , ectatic vertebral artery", "additionally , indentation of the lower part of the medulla by the vertebral artery was also recognized .", "apericranial graft was used as a sling and tacked to the dura , to decompress the brainstem .", "the postoperative course was uneventful , and the patient was kept in the hospital to transition her from gastrostomy tube feedings to regular oral feeding .", "postoperative cta demonstrated that the vertebral artery had been mobilized [ figure 3 ] away from the medulla .", "postoperative cta showing left vertebral artery away from the medulla the patient was discharged from the hospital without any antiemetic medication and was documented to have started gaining weight . at her 2-year postoperative visit , the patient had a nonfocal neurologic exam without recurrence of her prior nausea or vomiting .", "patients with central lesions often wait months or years and undergo countless tests before a central etiology is added to the differential diagnosis because most cases do not present with localizing neurological deficits", ". sustained hiccups with vomiting or isolated spontaneous vomiting with negative gastrointestinal symptoms should prompt further workup , including neuroimaging , to search for a central cause .", "the patient in our case was incidentally found to have dizziness and diplopia during the initial workup , but was not demonstrating positional vomiting or headaches , which would have prompted a neurological workup sooner . the tortuous vertebral artery in this case", "was found on the diagnostic mri / mra of the head and neck obtained secondary to her history of breast cancer and new onset symptoms of diplopia and dizziness . without a proper neurological examination", "this patient 's celiac disease and gastrointestinal reflux would have continued to mask the true cause of her symptoms . while compression of the medulla by a dilated vertebral artery is much less common than other brainstem compression syndromes such as trigeminal and glossopharyngeal neuralgias ; it is still significant .", "vascular compression of the medulla can cause disabling positional vertigo , hypertension , and hemifacial spasm ; but there are only a few reports of intractable nausea and vomiting as a symptom of an ectatic vertebral artery .", "less than 10 cases of nausea and vomiting due to vertebral artery compression have been reported in the literature .", "of the 20 cases of vertebral artery compression of the medulla reported in a meta - analysis by savitz et al .", ", only one patient experienced nausea and vomiting as a symptom . the majority of patients presented with hemiparesis and cranial nerve dysfunction .", "mvd was the choice of treatment for 17 of the patients , including the patient with symptoms of nausea , and it was shown to relieve symptoms in 16 patients .", "medullary compression can produce a wide spectrum of signs and symptoms from very few to several .", "regardless of the etiology , compressive forces on the neuronal tracts and nuclei that lie within the medulla can produce a number of clinical scenarios .", "when the etiology is vascular in origin , ischemic injury can produce symptoms such as headache , transient ischemic attacks ( tias ) , or infracts depending on the location of the compression . at the floor of the rhomboid fossa", "it contains specialized cells consisting of ependymal cells and tanycytes that allow for direct communication of cerebro spinal fluid and blood because it lacks an intact blood brain barrier . as a circumventricular organ ,", "the area postrema is able to detect toxins and drugs in the blood as well as hormones and other humoral signals to help maintain autonomic homeostasis .", "stimulation of the area postrema by vagal afferents from the nucleus ambiguous and gastrointestinal system or from emetogenic drugs and cytokines in the blood may trigger vomiting .", "the nucleus tractus solitarius ( nts ) lies adjacent to the area postrema , serves as a relay center to collect all afferent signals and activate the appropriate visceral nuclei to coordinate the action of vomiting .", "efferent pathways from the area postrema and nts project to the central pattern generator , ventral medulla , and hypothalamus .", "the central pattern generator is proposed to coordinate the activation of these nuclei within the medulla .", "the efferent signals stimulate the appropriate parasympathetic and sympathetic neurons , which produce the different phases of vomiting .", "the signal also diffuses through the brain via microglial messengers and cytokines such as substance p to allow for the cognitive recognition of emesis . a lesion to any of the afferent relay tracts or emetic reflex center proper may produce an abnormal emetic reflex and could produce intractable nausea and vomiting , as was observed in our case . on the preoperative mri ,", "the vertebral artery was seen to be compressing the medulla , where the area postrema is located , and nts at the inferior and posterior limit of the fourth ventricle .", "the potential disruption of the area postrema in this case may have caused an overstimulation of the emetic reflex .", "the first mvd was performed in 1966 by dr peter jannetta to relieve the facial pain in a patient with a compressed trigeminal nerve .", "it is now used to treat compression of the facial and glossopharyngeal nerves , as well as vascular abnormalities .", "the indications of mvd could potentially be expanded to treat compressive symptomology ; including , for example , intractable vomiting due to vascular compression of the medulla as demonstrated in this case report .", "this case demonstrated the importance of neurological investigations in the isolated intractable vomiting patient without a clear evidence of peripheral cause and including a central nervous system etiology on the differential , and the role of mvd in achieving cure ." ]

[ "<S> vertebral artery compressing the medulla and causing intractable vomiting has only been reported once previously . </S>", "<S> we report a case of a 69-year - old woman with intractable nausea and vomiting causing a 50 pound weight loss and who failed medical management and whose symptoms were completely reversed following microvascular decompression ( mvd ) . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSIONS" ]

[ [ "intractable nausea and vomiting are very rare symptoms of medullary compression and there are approximately less than 10 cases reported in the literature of the aforementioned symptomatology mostly due to tumors .", "there is one reported case where vertebral artery compression of the medulla led to intractable nausea and vomiting ." ], [ "a 69-year - old woman presented with a 10-month history of intractable nausea and vomiting resulting in a 50 pound weight loss .", "she had an extensive medical workup at multiple outside hospitals including a comprehensive gastrointestinal workup which was significant for celiac disease .", "her pertinent past medical history includes a history of breast cancer , mastectomy , and cholecystectomy . during her evaluation by the gastroenterologists at our hospital ,", "the diagnosis of celiac disease was confirmed , and she was also noted to have a duodenal ulcer .", "hematologic and biochemical workup was only significant for anemia attributable to her ulcer and celiac disease .", "magnetic resonance imaging / angiography ( mri / mra ) of the brain and neck , and diagnostic four vessels cerebral angiogram were obtained to further evaluate her dizziness and double vision .", "the left vertebral artery angiogram revealed a tortuous left vertebral artery with a 9.6 5.6 mm dissecting aneurysm in the v3 segment .", "this , along with dolichoectasia of the vertebro - basilar arteries , resulted in compression of the medulla oblongata , which was also confirmed in the contrasted mri / mra of the brain and neck [ figure 1 ] . computed tomography angiography ( cta ) also confirmed that the vertebral artery was pushing the medulla medially [ figure 2 ] .", "the patient was offered a microvascular decompression ( mvd ) of the vertebral artery to attempt to decompress the brainstem and alleviate her intractable nausea and vomiting .", "preoperative magnetic resonance imaging ( mri ) t2 sequenceshowing compression of the medulla by the vertebral artery preoperative computed tomography angiography ( cta ) showing a tortuous left vertebral artery crossing the midline a left retrosigmoid craniotomy was completed to approach the vertebral artery and lower brainstem . the elongated , ectatic vertebral artery", "additionally , indentation of the lower part of the medulla by the vertebral artery was also recognized .", "apericranial graft was used as a sling and tacked to the dura , to decompress the brainstem .", "the postoperative course was uneventful , and the patient was kept in the hospital to transition her from gastrostomy tube feedings to regular oral feeding .", "postoperative cta demonstrated that the vertebral artery had been mobilized [ figure 3 ] away from the medulla .", "postoperative cta showing left vertebral artery away from the medulla the patient was discharged from the hospital without any antiemetic medication and was documented to have started gaining weight . at her 2-year postoperative visit , the patient had a nonfocal neurologic exam without recurrence of her prior nausea or vomiting ." ], [ "patients with central lesions often wait months or years and undergo countless tests before a central etiology is added to the differential diagnosis because most cases do not present with localizing neurological deficits", ". sustained hiccups with vomiting or isolated spontaneous vomiting with negative gastrointestinal symptoms should prompt further workup , including neuroimaging , to search for a central cause .", "the patient in our case was incidentally found to have dizziness and diplopia during the initial workup , but was not demonstrating positional vomiting or headaches , which would have prompted a neurological workup sooner . the tortuous vertebral artery in this case", "was found on the diagnostic mri / mra of the head and neck obtained secondary to her history of breast cancer and new onset symptoms of diplopia and dizziness . without a proper neurological examination", "this patient 's celiac disease and gastrointestinal reflux would have continued to mask the true cause of her symptoms . while compression of the medulla by a dilated vertebral artery is much less common than other brainstem compression syndromes such as trigeminal and glossopharyngeal neuralgias ; it is still significant .", "vascular compression of the medulla can cause disabling positional vertigo , hypertension , and hemifacial spasm ; but there are only a few reports of intractable nausea and vomiting as a symptom of an ectatic vertebral artery .", "less than 10 cases of nausea and vomiting due to vertebral artery compression have been reported in the literature .", "of the 20 cases of vertebral artery compression of the medulla reported in a meta - analysis by savitz et al .", ", only one patient experienced nausea and vomiting as a symptom . the majority of patients presented with hemiparesis and cranial nerve dysfunction .", "mvd was the choice of treatment for 17 of the patients , including the patient with symptoms of nausea , and it was shown to relieve symptoms in 16 patients .", "medullary compression can produce a wide spectrum of signs and symptoms from very few to several .", "regardless of the etiology , compressive forces on the neuronal tracts and nuclei that lie within the medulla can produce a number of clinical scenarios .", "when the etiology is vascular in origin , ischemic injury can produce symptoms such as headache , transient ischemic attacks ( tias ) , or infracts depending on the location of the compression . at the floor of the rhomboid fossa", "it contains specialized cells consisting of ependymal cells and tanycytes that allow for direct communication of cerebro spinal fluid and blood because it lacks an intact blood brain barrier . as a circumventricular organ ,", "the area postrema is able to detect toxins and drugs in the blood as well as hormones and other humoral signals to help maintain autonomic homeostasis .", "stimulation of the area postrema by vagal afferents from the nucleus ambiguous and gastrointestinal system or from emetogenic drugs and cytokines in the blood may trigger vomiting .", "the nucleus tractus solitarius ( nts ) lies adjacent to the area postrema , serves as a relay center to collect all afferent signals and activate the appropriate visceral nuclei to coordinate the action of vomiting .", "efferent pathways from the area postrema and nts project to the central pattern generator , ventral medulla , and hypothalamus .", "the central pattern generator is proposed to coordinate the activation of these nuclei within the medulla .", "the efferent signals stimulate the appropriate parasympathetic and sympathetic neurons , which produce the different phases of vomiting .", "the signal also diffuses through the brain via microglial messengers and cytokines such as substance p to allow for the cognitive recognition of emesis . a lesion to any of the afferent relay tracts or emetic reflex center proper may produce an abnormal emetic reflex and could produce intractable nausea and vomiting , as was observed in our case . on the preoperative mri ,", "the vertebral artery was seen to be compressing the medulla , where the area postrema is located , and nts at the inferior and posterior limit of the fourth ventricle .", "the potential disruption of the area postrema in this case may have caused an overstimulation of the emetic reflex .", "the first mvd was performed in 1966 by dr peter jannetta to relieve the facial pain in a patient with a compressed trigeminal nerve .", "it is now used to treat compression of the facial and glossopharyngeal nerves , as well as vascular abnormalities .", "the indications of mvd could potentially be expanded to treat compressive symptomology ; including , for example , intractable vomiting due to vascular compression of the medulla as demonstrated in this case report ." ], [ "this case demonstrated the importance of neurological investigations in the isolated intractable vomiting patient without a clear evidence of peripheral cause and including a central nervous system etiology on the differential , and the role of mvd in achieving cure ." ] ]

[ "osteoarthritis ( oa ) is a chronic synovial joint disease , characterized by two main features : ( 1 ) progressive damage of articular cartilage , bone remodeling , and new bone formation ( osteophytes and subchondral bone sclerosis ) and ( 2 ) synovial inflammation and fibrosis of ligaments , tendons , menisci , and capsules .", "all joints may be affected , but the most commonly involved are knees , hands , and hips ( fig .", "1 ) . while chronic oa used to be regarded as a wear and tear disease , ", "researchers now believe that low - grade inflammation and growth of blood vessels and nerves from the subchondral bone into articular cartilage , as well as metabolic disorders , play a major role in disease pathology .", "pharmacological interventions are mostly palliative , focusing on alleviation of symptoms or slowing disease progression until damaged hip or knee joints are eventually replaced .", "differences in knee anatomy ( narrower femurs , thinner patellae , larger quadriceps angles , and differences in tibial condylar size ) , previous knee trauma , and genetic and hormonal influences may play a role .", "women present for treatment in more advanced stages of oa and have more debilitating pain than men .", "women also have less cartilage volume , greater cartilage wear , and overall differences in mechanical alignment .", "symptomatic knee oa typically presents with narrowing of the joint space and bone spurs ( arrows ) .", "( a and b ) during the development of oa , articular cartilage breaks down over time and becomes thin . as a result ,", "the bone surfaces rub against each other , further damaging the cartilage and bone and causing pain .", "( c and d ) joints with late - stage oa are often painful , warm to the touch , possibly red , swollen , have subchondral cysts , and notable loss of function .", "pain medications currently used to treat the symptoms of oa include acetaminophen , topical capsaicin , topical and oral nonsteroidal anti - inflammatory drugs ( nsaids ; i.e. , naproxen and ibuprofen ) , and the synthetic opioids tramadol and codeine . however , each of these therapies has potential drawbacks that may limit their widespread use .", "analgesics can be addictive , whereas acetaminophen can have serious side effects , such as kidney and liver damage .", "treatment with nsaids , which inhibit cyclooxygenases ( cox1 and cox2 ) , thereby blocking prostaglandin synthesis , improves quality of life and decreases pro - inflammatory cytokines including interleukin-6 ( il-6 ) , vascular endothelial growth factor ( vegf ) , and tumor necrosis factor- ( tnf- ) in synovial fluid and mitogen - activated protein kinases ( mapks ) in knee oa .", "however , nsaids can also cause serious side effects , including upper gastrointestinal ( gi ) toxicity ( dyspepsia , ulcers , perforation , obstructions , and bleeding ) and liver dysfunction .", "as such , they are typically prescribed for the shortest possible duration at the lowest effective dose . to reduce the risk of these upper gi complications , the us", "fda has approved the use of the nsaid hzt501 ( duexis ) , a drug containing 800 mg ibuprofen , in combination with 26.6 mg famotidine , a histamine h2-receptor antagonist .", "alternatively the nsaid celecoxib has less risk of upper gi complications by selectively inhibiting the isoenzyme cox-2 , which is specific to inflamed tissue , versus cox-1 , which is constitutive in many tissues including the gi tract .", "it should be noted that daily treatment with celecoxib is more effective in patients with normal body mass index ( bmi ) than obese patients .", "intra - articular injection of corticosteroids ( gc ) is recommended to relieve inflammation and pain in oa joints .", "however , gc injections are short acting , prone to adverse side effects , and have limited disease - modifying effects . for patients with knee oa , viscosupplementation with hyularonin may be used to replaces shock absorbing and lubricant material in the joint fluid , but the effects are similarly short - lived .", "currently , guidelines for oa management are available from numerous organizations , including the american academy of orthopedic surgeons ( aaos ) , the american college of rheumatology ( acr ) , the american geriatrics society ( ags ) , the american pain society ( aps ) , and the osteoarthritis research society international ( oarsi ) in the united states and the european league against rheumatism ( eular ) and the united kingdom s national institute for health and clinical excellence ( nice ) in europe .", "collectively , these guidelines reflect the experience of physicians across a variety of medical disciplines . whereas all generally use the same data sources ( i.e. , evidence - based research , expert opinion , patient experience , and cost - effectiveness analysis ) , they differ in focus .", "for instance , the aaos and ags guidelines reflect the perspective of specialists in orthopedic surgery , geriatrics , and pain management , whereas the eular and oarsi guidelines primarily emphasize the findings of experts in rheumatology .", "the nice guidelines are developed jointly by physicians and other health care professionals working in conjunction with a range of clinical researchers .", "in addition , the scope varies , with some guidelines ( e.g. , aaos , acr , eular , and oarsi ) addressing specific types of oa ( i.e. , knee , hip , or hand ) and others ( e.g. , ags , aps , and nice ) addressing oa more generally . as such", ", recommendations can vary widely , for instance , guidelines for use of nsaids . recommended nonpharmacologic interventions range between therapeutic exercises , patient education , transcutaneous electrical nerve stimulation , acupuncture , orthotics and insoles , heat and cryotherapy , patellar tapping , and weight control . in an effort to evaluate these varying guidelines ,", "the appraisal of guidelines research and evaluation ( agree ii ) scored 17 clinical practice guidelines ( cpgs ) including eular , nice , oarsi , aaos , and acr , on six different measures : d1 , scope and purpose ; d2 , stakeholder involvement ; d3 , rigor of development ; d4 , clarity and presentation ; d5 , applicability ; and d6 , editorial independence .", "the general clinical management recommendations tended to be similar among high - quality cpgs , although interventions addressed varied .", "new noninvasive , disease - modifying therapies for oa are lacking and needed by millions of patients .", "a number of prospective new treatments targeting pro - inflammatory mediators , cytokines , bone turnover , and angiogenic and neurogenic factors are being investigated , with varying success in clinical trials and clinical use .", "interleukin-1 ( il-1 ) may prove an effective target , as il-1 induces matrix metalloproteinase ( mmp ) production , resulting in the degradation of aggrecan and other matrix constituents .", "il-1 also induces high levels of cox2 and prostaglandin e2 ( pge2 ) , which may explain the pain associated with oa degeneration .", "the drug diacerein , an inhibitor of il-1 , may modify both disease symptoms and disease structure in oa .", "oral diacerein has proven effective in reducing pain , although evidence from clinical trials and scientific literature suggest that the effectiveness in oa is weak .", "it can be used in conjunction with nsaids or viscosupplementation therapies for additive effects due to its alternative mechanism of action .", "the most common side effects of diacerein are gastrointestinal , such as diarrhea , and changes in the color of urine .", "meanwhile , the il-1-receptor antagonists anakinra and orthokin are reported to improve western ontario and mcmaster universities arthritis index ( womac ) scores .", "gevokizumab is in phase ii clinical trials for safety and biological activity in the treatment of hand oa .", "nerve growth factor ( ngf ) has also been recognized as an important mediator of chronic pain in oa .", "tanezumab , a monoclonal antibody against -ngf receptor tyrosine kinase ( trka ) , inhibits ngf action and reduces pain in patients .", "two randomized phase iii clinical trials indicate that tanezumab provides superior pain relief while improving physical function and global disease assessment scores in patients with painful hip oa .", "although in most cases tanezumab is well tolerated , the unexpected occurrence of rapid destructive arthropathies suggests there may be safety issues .", "alternatively , using the drug adalimumab to inhibit tnf- , which upregulates -ngf , does not improve global disease assessment scores in oa of the hand .", "strontium ranelate ( srra ) , an element similar to calcium , is easily taken up by the body and incorporated into bones in place of calcium .", "the sekoia ( srra efficacy in knee osteoarthritis trial ) trial , a 3-year randomized , double - blind , placebo - controlled trial , evaluated the efficacy , safety , and disease - modifying effects of srra given at 1 to 2 g / day in patients with knee", "magnetic resonance imaging ( mri ) data indicate that srra significantly reduced cartilage volume loss and bone marrow lesion progression .", "symptoms also improved in terms of pain and physical function after 6 and 12 months , respectively , although treatment was deemed safe and well tolerated .", "these data indicate that srra could be a promising new symptom and disease - modifying treatment for oa .", "additionally , there is a need for further investigations to establish the optimal dosage and to better clarify the mechanism of action of srra in oa .", "several clinical studies have investigated the effects of anti - resorptive therapies such as bisphosphonates on oa symptoms . a study by carbone et al .", "found that alendronate ( aln ) use in oa patients decreased bone abnormalities and attenuated knee pain , yet cartilage degeneration was still present in the mri scans of treated patients .", "determined that risedronate use led to significant improvements in womac scores and preservation of knee joint space compared with placebo in a 1-year randomized control trial involving patients with moderate oa .", "however , a 2-year randomized control trial of risedronate treatment revealed contradictory results , with no significant improvement of womac score or joint space retention in the knee . similarly , nishii et al . observed no inhibition of oa progression in treated hip oa patients after 2 years of aln treatment", "therefore , in spite of the growing body of clinical work investigating the subject , no definitive conclusion can be reached on the practicality of using bisphosphonates to treat patients with oa .", "antidepressants have shown promising preliminary results for treatment of pain associated with oa by increasing serotonin levels in the brain .", "serotonin - norepinephrine reuptake inhibitors duloxetine ( cymbalta ) and milnacipran significantly improve pain in oa .", "an open - label trial also suggested analgesic effectiveness of methotrexate , an anti - inflammatory drug that acts by inhibiting the metabolism of folic acid , demonstrating that up to 20 mg / week for 6 months achieved oarsi responder criteria in knee oa and warranted a randomized controlled trial .", "the small molecule kartogenin was identified in an image - based high - throughput screen to promote chondrocyte differentiation .", "it shows chondroprotective effects in vitro and is efficacious in two animal models of oa .", "kartogenin induces chondrogenesis by disrupting the interaction between filamin a and the transcription factor core - binding factor b subunit ( cbf ) , thereby altering cbf-runx1 and possibly runx2 transcriptional programs .", "autologous injection of platelet - rich plasma ( prp ) has been used to stimulate cartilage repair and healing in oa patients , but the presence of other growth factors in prp may be problematic .", "furthermore , bone morphogenic protein 7 ( bmp7 ) , fgf-8 , and botulium toxin a ( bont - a ) are used in the treatment of knee oa .", "bont - a has an analgesic effect by temporarily suppressing acetylcholine secretion at presynaptic nueuromuscular junctions and appears to be effective and safe for the management of advanced knee oa .", "however , these results can not be generalized to patients with mild knee joint pain or nonspecific soft tissue pain in the knee joint region .", "further research is necessary to investigate possible complications such as aggravation of infection , effect on muscle strength , and neuropathic joint degeneration .", "recently , a phase i trial was reported in which chondrocytes were modified via intra - articular dna injection to produce tgf-1 in patients with advanced knee", "intra - articular injection of adipose - derived stem cell ( adsc ) therapy in a new european program is also under investigation .", "adsc induced the release of trophic factors that exerted anti - inflammatory effects on both synoviocytes and chondrocytes , with no mmp1 , mmp3 , or mmp13 production , suggesting safe and effective use of adscs for clinical applications . however , both treatments need proof - of - concept studies in larger patient populations .", "alternatively , intra - articular injection of human mesenchymal stem cells can lead to articular cartilage protection through the sdf-1/cxcr4 axis .", "natural products can be safer than prescription medications with less undesirable side effects . dietary supplements including avocado soybean unsaponifiables ( asu ) , chondroitin sulfate , hyaluronan , and glucosamine sulfate", "they are used to treat mild to moderate pain and alleviate symptoms to reduce the consumption of nsaids .", "several trials for chondroitin sulfate , glucosamine sulfate , and hyaluronan ( c14h21no11 ) are in process .", "chondroitin sulfate , glucosamine sulfate , and hyaluronan are building blocks for proteoglycan synthesis , and major constituents of the extracellular matrix in cartilage and synovial fluid .", "hyaluronan and hyaluronic acid ( hyalgan hylan - gf20/synvisc ) can be injected into the knee joint of patients with oa who can not tolerate nsaids or are awaiting joint surgery .", "a recent report indicates that viscosupplementation with hylan - gf20 slows type ii collagen degradation and joint inflammation in patients with oa .", "however , hylan - gf20 was not present in granulomas , an indicator of inflammation , raising the question of clinical significance in pain reduction .", "also , viscosupplementation with hyaluronic acid itself does not significantly improve disease outcome , and little is known about long - term effects .", "the efficacy of glucosamine and/or chondroitin in treating knee oa pain was evaluated in the glucosamine / chondroitin arthritis intervention trial , funded by the national center for complementary and alternative medicine and the national institute of arthritis and musculoskeletal and skin diseases .", "patients were treated daily for 24 weeks with glucosamine alone ( 1,500 mg ) , chondroitin sulfate alone ( 1,200 mg ) , glucosamine and chondroitin sulfate combined ( same doses ) , a placebo , or celecoxib ( 200 mg ) , which served as a positive control .", "although there were no statistically significant differences between any of the experimental treatments and the placebo overall , patients with moderate - to - severe pain given both glucosamine and chondroitin sulfate did show improvement ( 79% experienced pain reduction vs. 54% for placebo ) .", "because of the small size of this subgroup , these findings should be considered preliminary and need to be confirmed in further studies .", "glucosamine does not appear to slow arthritis progression over the long term and has many potential complications .", "the most common adverse effects are epigastric pain or tenderness , heartburn , diarrhea , and nausea .", "glucosamine also may cause allergic reactions in patients with seafood allergies , as a product of lobster , crab , and shrimp shells .", "glucosamine may interact with various pharmaceuticals , such as warfarin ( coumadin ) and diabetes medications , dangerously modifying their efficacy .", "similarly , chondroitin sulfate appears not to provide meaningful benefit for patients with oa , and their combination has not proven effective for either pain management or functional improvement .", "oarsi and nice no longer recommend the use of glucosamine or chondroitin sulfate alone , or in combination , if no effects are observed after 6 months or radiographic changes are marginal . a network meta - analysis of 10 trials in 3,803 patients by juni and collaborators in 2012 found no clinically significant improvements in oa pain alleviation or jsn parameters with glucosamine , chondroitin , or combined treatment compared with placebo . despite these results ,", "many patients believe otherwise , potentially due to the natural course of disease , regression to the mean , or the placebo effect .", "the authors conclude that such patients should be permitted to use these supplements if they cover the cost themselves , since neither of these preparations was found to be dangerous .", "sierrasil is a dietary supplement marketed for joint pain relief that is derived from the mineral - rich clay found in the high sierra mountains in the united states .", "clinical trial testing short - term efficacy of sierrasil at doses of 2 and 3 g per day failed to show sustained benefits over placebo , and iron toxicity has been reported .", "some oa patients experience pain relief from topical creams containing capsaicin , the active component of chili peppers .", "however , use of these creams may introduce side effects such as burning , stinging , and redness of the skin and eyes .", "avocado / soybean unsaponifiables are natural vegetable extracts made from avocado and soybean oils , consisting of the leftover fraction ( approximately 1% ) that can not be made into soap after saponification .", "asu is composed of one third avocado and two thirds soybean unsaponifiables ( a1s2u ) .", "the major components of asu are phytosterols -sitosterol , campesterol , and stigmasterol , which are rapidly incorporated into cells .", "asu is a complex mixture of many compounds including fat - soluble vitamins , sterols , triterpene alcohols , and possibly furan fatty acids .", "the sterol contents of asu preparations are the primary contributors to biological activity in articular chondrocytes .", "preclinical in vitro and in vivo studies have demonstrated that asus have beneficial effects on oa .", "it inhibits the breakdown of cartilage and promotes cartilage repair by inhibiting a number of molecules and pathways implicated in oa ( tables 1 and 2 ) .", "asu stimulates the synthesis of collagen and aggrecan by inhibiting inflammatory cytokines such as il-1 , il-6 , il-8 , tnf , and pge2 through modulation of nf - kappab .", "the combination of asu and epigallocatechin gallate ( egcg ; a major component of green tea catechins ) affects an array of inflammatory molecules including expression of cox-2 and production of pge2 in chondrocytes .", "cox-2 regulates the production of pge2 ; both are mediators involved in the process of cartilage breakdown .", "asu also inhibits the release and activity of collagenase ( mmp2 ) and stromelysin 1 ( mmp3 ) in cultured chondrocytes , increases tissue inhibitors of metalloproteinases ( timp-1 ) , and inhibits il1-induced erk but not p38 or jnk in chondrocytes in vitro .", "stimulatory effects of avocado soybean unsaponifiable on anti - inflammatory , anabolic mediators that protect against osteoarthritis .", "inhibitory effects of avocado soybean unsaponifiable on inflammatory and catabolic mediators of osteoarthritis . in vitro studies show that asu inhibits fibrinolysis by stimulating the expression of plasminogen activator inhibitor ( pai-1 ) .", "pai-1 inhibits tissue plasminogen activator and urokinase ( upa ) , thereby blocking plasminogen activation and inhibiting fibrinolysis ( the physiological breakdown of blood clots )", ". this fibrinolytic and tissue destructive proteinase cascade may play a role in oa joint inflammation via altered expression of upa receptors .", "asus alter growth factor levels implicated in oa pathogenesis , increasing tgf-1 and tgf-2 in the canine knee joint fluid , to repair cartilage and decreasing vegf , which is markedly elevated in synovial fluid of patients . in a study of implant osseointegration in rat tibiae , asu administration improved markers of bone growth , including bone morphogenic protein 2 ( bmp-2 ) and transforming growth factor beta 1 ( tgf-1 ) , though histomorphometric analysis of implant osseointegration was only slightly improved .", "sixty percent of patients with oa exhibit high levels of oxidized low - density lipoproteins ( oldl ) in serum , which mediates reactive oxygen species ( ros ) activity in chondrocytes and oa pathology . treating patients with a daily dose of 300 mg asu for 3 months decreased oldl levels . at the clinical level", ", asu reduces pain and stiffness while improving function in joints , resulting in decreased dependence on analgesics . asu efficacy and safety during and after treatment", "have been assessed in various randomized , double - blind , multicenter trials in patients with symptomatic knee or hip oa .", "two studies conducted over a 3-month period report that standard treatment with 300 mg / day of asu improved indices of pain , stiffness , and physical function , as measured by womac , and decreased analgesic drug demanded in patients with oa .", "a third trial conducted over 6 months reports similarly improved function compared with placebo , measured by the lequense functional index , with persistent effects after termination of treatment . in a 6-month trial on patients with femorotibial gonarthrosis , asu was as effective as 400 mg of chondroitin sulfate three times per day , as measured by womac .", "most recently , a 3-year randomized trial on patients with hip oa , performed under the acr criteria ( minimum of 1 - 4 mm hip jsw on the pelvic radiographs ) , reports excellent safety , but no significant reduction in the mean rate of jsn after 1 year .", "however , analyzing the results under different parameters reveals a significant 20% reduction in the rate of progression in patients with severe hip oa ( p = 0.04 ) , indicating a potential structure modifying effect of asu , as confirmed in the eradias study . in a clinical trial of patients with hip oa", ", the effects of asu treatment over 3 years were evaluated by radiography to identify joint pathology and disease progression on the structural level . although jsn was not statistically significant between asu and placebo treatment , secondary analysis of disease progression , measured by jsn ( 0.5 mm ) or total hip replacements , indicated 20% improvement with asu ( 42.2% vs. 51.4% of placebo group , p = 0.054 ) .", "computerized image analysis also showed significant histological differences not detectable by traditional scoring methods . in sheep ,", "asu treatment following cartilage insult improved articular integrity , as measured by toluidine blue staining , after 6 months compared with untreated animals .", "these improvements were the result of decreased catabolism and increased anabolism of cartilage by asu .", "indeed , asu reduces inflammation - mediated cartilage degradation by reducing il-1 , pge2 , and mmp-3 production , while also inducing proteoglycan , noncollagenous protein ( ncp ) , and collagen synthesis within 72 hours of administration to bovine cells in culture . a recent study in patients with nonspecific dorsalgia demonstrated analgesic effect of piascledine with positive outcome after 1 month", "however , a randomized , double - blind , placebo - controlled clinical trial carried out in 14 obese adult volunteers over 3 months reports no significant effect on these parameters , as measured by hyperglycemic ", "four double - blind placebo - controlled randomized human clinical trials ( rcts ) evaluate asu s impact on knee and hip oa .", "another found that asu improves lfi compared with placebo over the course of 6 months , and also that improvements took 2 months to take effect , and subside after treatment ended .", "alternatively , a long - term study indicated no significant difference in jsn , or other parameters of disease , after 2 years of asu treatment , indicating that the beneficial impact of asu on oa may be limited to short - term effects .", "however , this study also focused on identifying structure - modifying effects , versus symptom - modifying effects ; although these two different measures of oa severity often correlate , asu may affect each uniquely .", "evidence for symptom - modifying effects of asu is much stronger , and thus an alternative explanation for these contradictory findings is that while asu does not improve structural damage of oa , as measured in this study , it does improve symptoms such as pain and mobility , as measured in previous studies . in a study of chronic nonspecific back", "pain , treatment with asu ( piascledine ) combined with the nsaid artrosiline ( 320 mg / day ) showed significant analgesic effect over nsaid treatment alone .", ", the eradias trial determined whether asu expanscience treatment slowed the radiological progression of hip oa .", "as for safety , none of the four rcts reported significant differences in adverse effects between asu and placebo .", "factors like bmi , severity of disease , and activity level may influence the effect of asu , as these conditions exacerbate inflammatory conditions and mechanical stresses that contribute to oa .", "adipose tissue plays an important role by producing metabolic factors with catabolic and pro - inflammatory properties , including cytokines , chemokines , and adipokines ( il-6 and tnf- , il-8 , ifn- ) , which orchestrate pathophysiological processes in oa .", "soluble mediators produced by adipocytes may also modulate chondrocyte metabolism and contribute to cartilage degradation .", "asu may counteract these inflammatory processes by inhibiting the translocation of the transcription factor nf-b from the cytoplasm to the nucleus , which controls transcription of many pro - inflammatory factors ( table 2 ) .", "as such , asu acts as an anabolic agent in vitro , reducing the production of pro - inflammatory mediators , including il-1 , il-6 , il-8 , macrophage inflammatory protein-1 , no , mmp-13 , tnf- , and cox2/pge2 from various cell types ( table 1 ) . in mice , asu decreases pro - inflammatory interferon- ( ifn- ) and il-4 production , in the context of parasitic diseases .", "although more studies need to be conducted to show the effects of asu in patients with varying bmi , the anti - inflammatory effects of asu are likely to protect cartilage from obesity - associated inflammatory degradation and improve oa symptoms . indeed , asu significantly decreased the rate of oa progression to 40% compared with 50% in the placebo group in one study .", "however , this study showed that asu did not influence the rate of oa progression in the obese subset of patients with mean symptom duration 4 and bmi of 27 kg / m .", "however , excessive inflammation associated with obesity may also impede efficacy , as it does with celecoxib ( nsaid ) treatment , which is not as effective in obese patients ( bmi in excess of 30 kg / m ) .", "the influence of obesity , and how it influences asu efficacy , may also depend on the parameters used to measure and define disease . in a study examining the relationship between bmi and oa in patients scheduled to undergo hip replacement , increasing bmi", "was associated with increasing levels of pain and functional disability , but not radiographic joint damage .", "this should be taken into account when designing and assessing studies intended to examine the impact of obesity on treatment efficacy .", "asu has anti - inflammatory effects in mice when administered in conjunction with the anti - parasitic drug praziquantel , reducing inflammatory cytokines ifn- and il-4 , as well as granuloma size , while increasing cidal activity .", "asu also protects gingival elastic fibers from degradation by human leukocyte elastase , hypodermatitis , and ischemic damage .", "a recent electronic database analysis demonstrated the benefits and harms of oral medicinal plant products in treating oa .", "the authors used standard methods for trial selection and data extraction , and they assessed the quality of the body of evidence using the grade approach for major outcomes such as pain , function , radiographic joint changes , quality of life , withdrawals due to adverse events , total adverse events , and serious adverse events .", "the asu product piasclidine formed a small and clinically questionable improvement in symptoms , compared with placebo after 3 to 12 months treatment .", "radiographic joint changes , as change in joint space width ( jsw ) , did not differ between asu 300 mg treatment and placebo .", "moderate - quality evidence from a single study confirmed possible benefits of asu 600 mg over placebo .", "there is no evidence that piasclidine significantly improves joint structure , and limited evidence that it prevents joint space narrowing .", "the authors suggest further investigations are required to determine optimum daily doses producing clinical benefits without adverse events .", "asu is considered as drug in most countries and is therefore prescribed by physicians . however , in the united states it is classified as dietary supplement and can be purchased as over - the - counter supplements , avoca asu ( asu - nmx1000 , nutramax laboratories inc . ,", "avoca asu , a combination of asu and glucosamine sulfate , has been shown to suppress tnf- , il-1 , cox2 , inos , pge2 , nf-b activation and nitrite production in articular chondrocytes and monocytes / macrophages , reducing pain and inflammation in oa patients . however ,", "conflicting reports indicate the complete absence of specific asu molecules in avoca asu when compared with piascledine .", "questions remain about the efficacy and safety of asus for treatment of oa ( table 4 ) .", "macaigne and colleagues published a case report in 2004 describing a female with lymphatic colitis associated with piascledine treatment .", "further prospective multicenter studies are warranted to investigate whether other microscopic colitis cases are observed in patients treated with piascledine .", "avoca asu that contains glucosamine can induce allergic reaction in people with shellfish allergy . even in very small quantities", ", these people may experience mild symptoms , such as hives or nasal congestion , or more severe , even life - threatening , symptoms . an alternative asu formulation is arthrocen ( pharmin , usa , llc , san jose , ca ) .", "arthrocen is an extract from avocado and soybean oils that does not contain any ingredients of animal origin , artificial flavor , sweetener , preservative , or color .", "each capsule contains 100 mg unsaponifiable persea gratissima unsaponifiable ( avocado ) and 200 mg unsaponifiable glycine max ( soybean ) extracts , silica , magnesium stearate ( e470b manufactured from vegetable oil ) , and gelatin fines . in general", ", the fda does not hold dietary supplements to the stringent standards of pharmaceutical manufacture .", "if asu is to be widely used for the treatment of oa , serious consideration should be given to their current regulatory status in order to ensure potency , purity , and as well as the excipients .", "many studies have demonstrated substantial variation between the content listed on the labels of these products and the actual content .", "the sterols content of asu have been demonstrated to have biological activities in culture and in animal models .", "2 ) . we found multiple peaks were present in the piascledine-300 ( expanscience ) mass spectrometry analysis ( agilent 7890 gc system , 7693 auto sampler , agilent vl msd with triple axis detector , brea , ca ) , compared to the arthrocen 300 mg ( pharminusa ) or asu300-avocado soy unsaponifiable with sierra 600 mg ( maximize , maximum int . ) preparations ( fig .", "similar results were found for piascledine-300 , with mass spectra sterol content of c20h30o2 , c20h28o2 , sitosterol , stigmasterol , campesterol , squalene , -tocopherol , desmethyl tocopherol , oleic acid docosane , -amyrin , and cholesterol . in two letters to the editor , msika et al .", "and henroitin claimed that the exact ingredients and preparation of asu - expanscience was an intellectual proprietary , protected by patent .", "msika further emphasized that the tocopherols , sterols , and patented specific molecules from avocado contribute to the originality of the product , different from natural avocado unsaponifiables .", "in contrast , they analyzed dasuquin with msm , dasuquin , and avoca asu ( nutramax ) , and compared them with asu expanscience .", "the analyses revealed content of products were significantly different from those indicated on the nutramax labels with no citrostadienol , and brassicasterol present in asu expanscience .", "contrary to that they found contents included high level of rapeseed oil and unsaponifiables products with very low tocopherol , and without respected ratio of 1:2 for avocado to soybean unsaponifiables .", "the original asu piascledine 300 pills contains 100 mg of avocado unsaponifiables and 200 mg of soybean unsaponifiables .", "the difference in sterol content is based on the a / s ratio and avocado - specific modified unsaponifiables obtained by a patented process .", "have shown asu effects are best when the asu ratio is 2:1 . in support ,", "these issues and reported adverse effects of asu ( table 4 ) raises concerns about the content and purity of asu supplements on the market , with implications for patient safety .", " mass spectrometry analysis of major sterol components of piascledine 300 , avocado 300 soy unsaponifiable with sierra 600 mg , and arthrocen 300 .", "control sample exhibited characteristic peaks corresponding to 1 = dihydrocholesterol ( 5-cholestan-3-ol ; internal control ; sigma aldrich ) , 2 = brass ( brassicasterol ) , 3 = camp ( campesterol ) , 4 = stigmn ( stigmastanol ) , 5 = -sito ( -sitosterol ) , 6 = stigma ( stigmasterol ) .", "improving joint function and patient activity is a central public health concern to improve quality and length of life .", "the aim is not only to treat pain but also to prevent the onset of disease .", "there is no cure for oa , and even symptomatic treatment options are scarce , dominated by pain management and surgical intervention .", "asus may prove to be an effective treatment option for symptomatic oa , as they have been shown to possess chondroprotective , anabolic , and anticatabolic properties , as well as anti - inflammatory properties . at the clinical level , asus reduce pain and stiffness while improving joint function .", "importantly , asus are a natural , slow - acting agent that do not merely address acute pain but actively prevent progression of oa symptoms .", "further studies are required to determine the specific mechanisms and target molecules of asu function on oa at the cellular and metabolic level ." ]

[ "<S> osteoarthritis ( oa ) is a painful and life - altering disease that severely limits the daily activities of millions of americans , and it is one of the most common causes of disability in the world . with obesity on the rise and the world </S>", "<S> s population living longer , the prevalence of oa is expected to increase dramatically in the coming decades , generating burdensome socioeconomic costs . </S>", "<S> this review summarizes current pharmaceutical , nonpharmaceutical , and prospective new treatments for oa , with primary focus on the dietary supplement avocado / soybean unsaponifiables ( asu ) . </S>", "<S> asu modulates oa pathogenesis by inhibiting a number of molecules and pathways implicated in oa . </S>", "<S> anticatabolic properties prevent cartilage degradation by inhibiting the release and activity of matrix metalloproteinases and increasing tissue inhibitors of these catabolic enzymes . </S>", "<S> asu also inhibits fibrinolysis by stimulating the expression of plasminogen activator inhibitor . </S>", "<S> anabolic properties promote cartilage repair by stimulating collagen and aggrecan synthesis via inhibition of inflammatory cytokines such as interleukin ( il)-1 , il-6 , il-8 , tumor necrosis factor , erk , and prostaglandin e2 . </S>", "<S> chondroprotective effects are mediated by correcting growth factor abnormalities , increasing tgf- , and decreasing vascular endothelial growth factor ( vegf ) in synovial fluid . </S>", "<S> asu also inhibits cholesterol absorption and endogenous cholesterol biosynthesis , which mediate reactive oxygen species pathology in chondrocytes . at the clinical level </S>", "<S> , asu reduces pain and stiffness while improving joint function , resulting in decreased dependence on analgesics . </S>" ]

[ "Introduction", "Current Pharmacologic Therapies", "Current Nonpharmacologic Therapies", "Prospective New Treatments", "Dietary Supplements", "Avocado and Soybean Unsaponifiables", "Conclusion" ]

[ [ "osteoarthritis ( oa ) is a chronic synovial joint disease , characterized by two main features : ( 1 ) progressive damage of articular cartilage , bone remodeling , and new bone formation ( osteophytes and subchondral bone sclerosis ) and ( 2 ) synovial inflammation and fibrosis of ligaments , tendons , menisci , and capsules .", "all joints may be affected , but the most commonly involved are knees , hands , and hips ( fig .", "1 ) . while chronic oa used to be regarded as a wear and tear disease , ", "researchers now believe that low - grade inflammation and growth of blood vessels and nerves from the subchondral bone into articular cartilage , as well as metabolic disorders , play a major role in disease pathology .", "pharmacological interventions are mostly palliative , focusing on alleviation of symptoms or slowing disease progression until damaged hip or knee joints are eventually replaced .", "differences in knee anatomy ( narrower femurs , thinner patellae , larger quadriceps angles , and differences in tibial condylar size ) , previous knee trauma , and genetic and hormonal influences may play a role .", "women present for treatment in more advanced stages of oa and have more debilitating pain than men .", "women also have less cartilage volume , greater cartilage wear , and overall differences in mechanical alignment .", "symptomatic knee oa typically presents with narrowing of the joint space and bone spurs ( arrows ) .", "( a and b ) during the development of oa , articular cartilage breaks down over time and becomes thin . as a result ,", "the bone surfaces rub against each other , further damaging the cartilage and bone and causing pain .", "( c and d ) joints with late - stage oa are often painful , warm to the touch , possibly red , swollen , have subchondral cysts , and notable loss of function ." ], [ "pain medications currently used to treat the symptoms of oa include acetaminophen , topical capsaicin , topical and oral nonsteroidal anti - inflammatory drugs ( nsaids ; i.e. , naproxen and ibuprofen ) , and the synthetic opioids tramadol and codeine . however , each of these therapies has potential drawbacks that may limit their widespread use .", "analgesics can be addictive , whereas acetaminophen can have serious side effects , such as kidney and liver damage .", "treatment with nsaids , which inhibit cyclooxygenases ( cox1 and cox2 ) , thereby blocking prostaglandin synthesis , improves quality of life and decreases pro - inflammatory cytokines including interleukin-6 ( il-6 ) , vascular endothelial growth factor ( vegf ) , and tumor necrosis factor- ( tnf- ) in synovial fluid and mitogen - activated protein kinases ( mapks ) in knee oa .", "however , nsaids can also cause serious side effects , including upper gastrointestinal ( gi ) toxicity ( dyspepsia , ulcers , perforation , obstructions , and bleeding ) and liver dysfunction .", "as such , they are typically prescribed for the shortest possible duration at the lowest effective dose . to reduce the risk of these upper gi complications , the us", "fda has approved the use of the nsaid hzt501 ( duexis ) , a drug containing 800 mg ibuprofen , in combination with 26.6 mg famotidine , a histamine h2-receptor antagonist .", "alternatively the nsaid celecoxib has less risk of upper gi complications by selectively inhibiting the isoenzyme cox-2 , which is specific to inflamed tissue , versus cox-1 , which is constitutive in many tissues including the gi tract .", "it should be noted that daily treatment with celecoxib is more effective in patients with normal body mass index ( bmi ) than obese patients .", "intra - articular injection of corticosteroids ( gc ) is recommended to relieve inflammation and pain in oa joints .", "however , gc injections are short acting , prone to adverse side effects , and have limited disease - modifying effects . for patients with knee oa , viscosupplementation with hyularonin may be used to replaces shock absorbing and lubricant material in the joint fluid , but the effects are similarly short - lived ." ], [ "currently , guidelines for oa management are available from numerous organizations , including the american academy of orthopedic surgeons ( aaos ) , the american college of rheumatology ( acr ) , the american geriatrics society ( ags ) , the american pain society ( aps ) , and the osteoarthritis research society international ( oarsi ) in the united states and the european league against rheumatism ( eular ) and the united kingdom s national institute for health and clinical excellence ( nice ) in europe .", "collectively , these guidelines reflect the experience of physicians across a variety of medical disciplines . whereas all generally use the same data sources ( i.e. , evidence - based research , expert opinion , patient experience , and cost - effectiveness analysis ) , they differ in focus .", "for instance , the aaos and ags guidelines reflect the perspective of specialists in orthopedic surgery , geriatrics , and pain management , whereas the eular and oarsi guidelines primarily emphasize the findings of experts in rheumatology .", "the nice guidelines are developed jointly by physicians and other health care professionals working in conjunction with a range of clinical researchers .", "in addition , the scope varies , with some guidelines ( e.g. , aaos , acr , eular , and oarsi ) addressing specific types of oa ( i.e. , knee , hip , or hand ) and others ( e.g. , ags , aps , and nice ) addressing oa more generally . as such", ", recommendations can vary widely , for instance , guidelines for use of nsaids . recommended nonpharmacologic interventions range between therapeutic exercises , patient education , transcutaneous electrical nerve stimulation , acupuncture , orthotics and insoles , heat and cryotherapy , patellar tapping , and weight control . in an effort to evaluate these varying guidelines ,", "the appraisal of guidelines research and evaluation ( agree ii ) scored 17 clinical practice guidelines ( cpgs ) including eular , nice , oarsi , aaos , and acr , on six different measures : d1 , scope and purpose ; d2 , stakeholder involvement ; d3 , rigor of development ; d4 , clarity and presentation ; d5 , applicability ; and d6 , editorial independence .", "the general clinical management recommendations tended to be similar among high - quality cpgs , although interventions addressed varied ." ], [ "new noninvasive , disease - modifying therapies for oa are lacking and needed by millions of patients .", "a number of prospective new treatments targeting pro - inflammatory mediators , cytokines , bone turnover , and angiogenic and neurogenic factors are being investigated , with varying success in clinical trials and clinical use .", "interleukin-1 ( il-1 ) may prove an effective target , as il-1 induces matrix metalloproteinase ( mmp ) production , resulting in the degradation of aggrecan and other matrix constituents .", "il-1 also induces high levels of cox2 and prostaglandin e2 ( pge2 ) , which may explain the pain associated with oa degeneration .", "the drug diacerein , an inhibitor of il-1 , may modify both disease symptoms and disease structure in oa .", "oral diacerein has proven effective in reducing pain , although evidence from clinical trials and scientific literature suggest that the effectiveness in oa is weak .", "it can be used in conjunction with nsaids or viscosupplementation therapies for additive effects due to its alternative mechanism of action .", "the most common side effects of diacerein are gastrointestinal , such as diarrhea , and changes in the color of urine .", "meanwhile , the il-1-receptor antagonists anakinra and orthokin are reported to improve western ontario and mcmaster universities arthritis index ( womac ) scores .", "gevokizumab is in phase ii clinical trials for safety and biological activity in the treatment of hand oa .", "nerve growth factor ( ngf ) has also been recognized as an important mediator of chronic pain in oa .", "tanezumab , a monoclonal antibody against -ngf receptor tyrosine kinase ( trka ) , inhibits ngf action and reduces pain in patients .", "two randomized phase iii clinical trials indicate that tanezumab provides superior pain relief while improving physical function and global disease assessment scores in patients with painful hip oa .", "although in most cases tanezumab is well tolerated , the unexpected occurrence of rapid destructive arthropathies suggests there may be safety issues .", "alternatively , using the drug adalimumab to inhibit tnf- , which upregulates -ngf , does not improve global disease assessment scores in oa of the hand .", "strontium ranelate ( srra ) , an element similar to calcium , is easily taken up by the body and incorporated into bones in place of calcium .", "the sekoia ( srra efficacy in knee osteoarthritis trial ) trial , a 3-year randomized , double - blind , placebo - controlled trial , evaluated the efficacy , safety , and disease - modifying effects of srra given at 1 to 2 g / day in patients with knee", "magnetic resonance imaging ( mri ) data indicate that srra significantly reduced cartilage volume loss and bone marrow lesion progression .", "symptoms also improved in terms of pain and physical function after 6 and 12 months , respectively , although treatment was deemed safe and well tolerated .", "these data indicate that srra could be a promising new symptom and disease - modifying treatment for oa .", "additionally , there is a need for further investigations to establish the optimal dosage and to better clarify the mechanism of action of srra in oa .", "several clinical studies have investigated the effects of anti - resorptive therapies such as bisphosphonates on oa symptoms . a study by carbone et al .", "found that alendronate ( aln ) use in oa patients decreased bone abnormalities and attenuated knee pain , yet cartilage degeneration was still present in the mri scans of treated patients .", "determined that risedronate use led to significant improvements in womac scores and preservation of knee joint space compared with placebo in a 1-year randomized control trial involving patients with moderate oa .", "however , a 2-year randomized control trial of risedronate treatment revealed contradictory results , with no significant improvement of womac score or joint space retention in the knee . similarly , nishii et al . observed no inhibition of oa progression in treated hip oa patients after 2 years of aln treatment", "therefore , in spite of the growing body of clinical work investigating the subject , no definitive conclusion can be reached on the practicality of using bisphosphonates to treat patients with oa .", "antidepressants have shown promising preliminary results for treatment of pain associated with oa by increasing serotonin levels in the brain .", "serotonin - norepinephrine reuptake inhibitors duloxetine ( cymbalta ) and milnacipran significantly improve pain in oa .", "an open - label trial also suggested analgesic effectiveness of methotrexate , an anti - inflammatory drug that acts by inhibiting the metabolism of folic acid , demonstrating that up to 20 mg / week for 6 months achieved oarsi responder criteria in knee oa and warranted a randomized controlled trial .", "the small molecule kartogenin was identified in an image - based high - throughput screen to promote chondrocyte differentiation .", "it shows chondroprotective effects in vitro and is efficacious in two animal models of oa .", "kartogenin induces chondrogenesis by disrupting the interaction between filamin a and the transcription factor core - binding factor b subunit ( cbf ) , thereby altering cbf-runx1 and possibly runx2 transcriptional programs .", "autologous injection of platelet - rich plasma ( prp ) has been used to stimulate cartilage repair and healing in oa patients , but the presence of other growth factors in prp may be problematic .", "furthermore , bone morphogenic protein 7 ( bmp7 ) , fgf-8 , and botulium toxin a ( bont - a ) are used in the treatment of knee oa .", "bont - a has an analgesic effect by temporarily suppressing acetylcholine secretion at presynaptic nueuromuscular junctions and appears to be effective and safe for the management of advanced knee oa .", "however , these results can not be generalized to patients with mild knee joint pain or nonspecific soft tissue pain in the knee joint region .", "further research is necessary to investigate possible complications such as aggravation of infection , effect on muscle strength , and neuropathic joint degeneration .", "recently , a phase i trial was reported in which chondrocytes were modified via intra - articular dna injection to produce tgf-1 in patients with advanced knee", "intra - articular injection of adipose - derived stem cell ( adsc ) therapy in a new european program is also under investigation .", "adsc induced the release of trophic factors that exerted anti - inflammatory effects on both synoviocytes and chondrocytes , with no mmp1 , mmp3 , or mmp13 production , suggesting safe and effective use of adscs for clinical applications . however , both treatments need proof - of - concept studies in larger patient populations .", "alternatively , intra - articular injection of human mesenchymal stem cells can lead to articular cartilage protection through the sdf-1/cxcr4 axis ." ], [ "natural products can be safer than prescription medications with less undesirable side effects . dietary supplements including avocado soybean unsaponifiables ( asu ) , chondroitin sulfate , hyaluronan , and glucosamine sulfate", "they are used to treat mild to moderate pain and alleviate symptoms to reduce the consumption of nsaids .", "several trials for chondroitin sulfate , glucosamine sulfate , and hyaluronan ( c14h21no11 ) are in process .", "chondroitin sulfate , glucosamine sulfate , and hyaluronan are building blocks for proteoglycan synthesis , and major constituents of the extracellular matrix in cartilage and synovial fluid .", "hyaluronan and hyaluronic acid ( hyalgan hylan - gf20/synvisc ) can be injected into the knee joint of patients with oa who can not tolerate nsaids or are awaiting joint surgery .", "a recent report indicates that viscosupplementation with hylan - gf20 slows type ii collagen degradation and joint inflammation in patients with oa .", "however , hylan - gf20 was not present in granulomas , an indicator of inflammation , raising the question of clinical significance in pain reduction .", "also , viscosupplementation with hyaluronic acid itself does not significantly improve disease outcome , and little is known about long - term effects .", "the efficacy of glucosamine and/or chondroitin in treating knee oa pain was evaluated in the glucosamine / chondroitin arthritis intervention trial , funded by the national center for complementary and alternative medicine and the national institute of arthritis and musculoskeletal and skin diseases .", "patients were treated daily for 24 weeks with glucosamine alone ( 1,500 mg ) , chondroitin sulfate alone ( 1,200 mg ) , glucosamine and chondroitin sulfate combined ( same doses ) , a placebo , or celecoxib ( 200 mg ) , which served as a positive control .", "although there were no statistically significant differences between any of the experimental treatments and the placebo overall , patients with moderate - to - severe pain given both glucosamine and chondroitin sulfate did show improvement ( 79% experienced pain reduction vs. 54% for placebo ) .", "because of the small size of this subgroup , these findings should be considered preliminary and need to be confirmed in further studies .", "glucosamine does not appear to slow arthritis progression over the long term and has many potential complications .", "the most common adverse effects are epigastric pain or tenderness , heartburn , diarrhea , and nausea .", "glucosamine also may cause allergic reactions in patients with seafood allergies , as a product of lobster , crab , and shrimp shells .", "glucosamine may interact with various pharmaceuticals , such as warfarin ( coumadin ) and diabetes medications , dangerously modifying their efficacy .", "similarly , chondroitin sulfate appears not to provide meaningful benefit for patients with oa , and their combination has not proven effective for either pain management or functional improvement .", "oarsi and nice no longer recommend the use of glucosamine or chondroitin sulfate alone , or in combination , if no effects are observed after 6 months or radiographic changes are marginal . a network meta - analysis of 10 trials in 3,803 patients by juni and collaborators in 2012 found no clinically significant improvements in oa pain alleviation or jsn parameters with glucosamine , chondroitin , or combined treatment compared with placebo . despite these results ,", "many patients believe otherwise , potentially due to the natural course of disease , regression to the mean , or the placebo effect .", "the authors conclude that such patients should be permitted to use these supplements if they cover the cost themselves , since neither of these preparations was found to be dangerous .", "sierrasil is a dietary supplement marketed for joint pain relief that is derived from the mineral - rich clay found in the high sierra mountains in the united states .", "clinical trial testing short - term efficacy of sierrasil at doses of 2 and 3 g per day failed to show sustained benefits over placebo , and iron toxicity has been reported .", "some oa patients experience pain relief from topical creams containing capsaicin , the active component of chili peppers .", "however , use of these creams may introduce side effects such as burning , stinging , and redness of the skin and eyes ." ], [ "avocado / soybean unsaponifiables are natural vegetable extracts made from avocado and soybean oils , consisting of the leftover fraction ( approximately 1% ) that can not be made into soap after saponification .", "asu is composed of one third avocado and two thirds soybean unsaponifiables ( a1s2u ) .", "the major components of asu are phytosterols -sitosterol , campesterol , and stigmasterol , which are rapidly incorporated into cells .", "asu is a complex mixture of many compounds including fat - soluble vitamins , sterols , triterpene alcohols , and possibly furan fatty acids .", "the sterol contents of asu preparations are the primary contributors to biological activity in articular chondrocytes .", "preclinical in vitro and in vivo studies have demonstrated that asus have beneficial effects on oa .", "it inhibits the breakdown of cartilage and promotes cartilage repair by inhibiting a number of molecules and pathways implicated in oa ( tables 1 and 2 ) .", "asu stimulates the synthesis of collagen and aggrecan by inhibiting inflammatory cytokines such as il-1 , il-6 , il-8 , tnf , and pge2 through modulation of nf - kappab .", "the combination of asu and epigallocatechin gallate ( egcg ; a major component of green tea catechins ) affects an array of inflammatory molecules including expression of cox-2 and production of pge2 in chondrocytes .", "cox-2 regulates the production of pge2 ; both are mediators involved in the process of cartilage breakdown .", "asu also inhibits the release and activity of collagenase ( mmp2 ) and stromelysin 1 ( mmp3 ) in cultured chondrocytes , increases tissue inhibitors of metalloproteinases ( timp-1 ) , and inhibits il1-induced erk but not p38 or jnk in chondrocytes in vitro .", "stimulatory effects of avocado soybean unsaponifiable on anti - inflammatory , anabolic mediators that protect against osteoarthritis .", "inhibitory effects of avocado soybean unsaponifiable on inflammatory and catabolic mediators of osteoarthritis . in vitro studies show that asu inhibits fibrinolysis by stimulating the expression of plasminogen activator inhibitor ( pai-1 ) .", "pai-1 inhibits tissue plasminogen activator and urokinase ( upa ) , thereby blocking plasminogen activation and inhibiting fibrinolysis ( the physiological breakdown of blood clots )", ". this fibrinolytic and tissue destructive proteinase cascade may play a role in oa joint inflammation via altered expression of upa receptors .", "asus alter growth factor levels implicated in oa pathogenesis , increasing tgf-1 and tgf-2 in the canine knee joint fluid , to repair cartilage and decreasing vegf , which is markedly elevated in synovial fluid of patients . in a study of implant osseointegration in rat tibiae , asu administration improved markers of bone growth , including bone morphogenic protein 2 ( bmp-2 ) and transforming growth factor beta 1 ( tgf-1 ) , though histomorphometric analysis of implant osseointegration was only slightly improved .", "sixty percent of patients with oa exhibit high levels of oxidized low - density lipoproteins ( oldl ) in serum , which mediates reactive oxygen species ( ros ) activity in chondrocytes and oa pathology . treating patients with a daily dose of 300 mg asu for 3 months decreased oldl levels . at the clinical level", ", asu reduces pain and stiffness while improving function in joints , resulting in decreased dependence on analgesics . asu efficacy and safety during and after treatment", "have been assessed in various randomized , double - blind , multicenter trials in patients with symptomatic knee or hip oa .", "two studies conducted over a 3-month period report that standard treatment with 300 mg / day of asu improved indices of pain , stiffness , and physical function , as measured by womac , and decreased analgesic drug demanded in patients with oa .", "a third trial conducted over 6 months reports similarly improved function compared with placebo , measured by the lequense functional index , with persistent effects after termination of treatment . in a 6-month trial on patients with femorotibial gonarthrosis , asu was as effective as 400 mg of chondroitin sulfate three times per day , as measured by womac .", "most recently , a 3-year randomized trial on patients with hip oa , performed under the acr criteria ( minimum of 1 - 4 mm hip jsw on the pelvic radiographs ) , reports excellent safety , but no significant reduction in the mean rate of jsn after 1 year .", "however , analyzing the results under different parameters reveals a significant 20% reduction in the rate of progression in patients with severe hip oa ( p = 0.04 ) , indicating a potential structure modifying effect of asu , as confirmed in the eradias study . in a clinical trial of patients with hip oa", ", the effects of asu treatment over 3 years were evaluated by radiography to identify joint pathology and disease progression on the structural level . although jsn was not statistically significant between asu and placebo treatment , secondary analysis of disease progression , measured by jsn ( 0.5 mm ) or total hip replacements , indicated 20% improvement with asu ( 42.2% vs. 51.4% of placebo group , p = 0.054 ) .", "computerized image analysis also showed significant histological differences not detectable by traditional scoring methods . in sheep ,", "asu treatment following cartilage insult improved articular integrity , as measured by toluidine blue staining , after 6 months compared with untreated animals .", "these improvements were the result of decreased catabolism and increased anabolism of cartilage by asu .", "indeed , asu reduces inflammation - mediated cartilage degradation by reducing il-1 , pge2 , and mmp-3 production , while also inducing proteoglycan , noncollagenous protein ( ncp ) , and collagen synthesis within 72 hours of administration to bovine cells in culture . a recent study in patients with nonspecific dorsalgia demonstrated analgesic effect of piascledine with positive outcome after 1 month", "however , a randomized , double - blind , placebo - controlled clinical trial carried out in 14 obese adult volunteers over 3 months reports no significant effect on these parameters , as measured by hyperglycemic ", "four double - blind placebo - controlled randomized human clinical trials ( rcts ) evaluate asu s impact on knee and hip oa .", "another found that asu improves lfi compared with placebo over the course of 6 months , and also that improvements took 2 months to take effect , and subside after treatment ended .", "alternatively , a long - term study indicated no significant difference in jsn , or other parameters of disease , after 2 years of asu treatment , indicating that the beneficial impact of asu on oa may be limited to short - term effects .", "however , this study also focused on identifying structure - modifying effects , versus symptom - modifying effects ; although these two different measures of oa severity often correlate , asu may affect each uniquely .", "evidence for symptom - modifying effects of asu is much stronger , and thus an alternative explanation for these contradictory findings is that while asu does not improve structural damage of oa , as measured in this study , it does improve symptoms such as pain and mobility , as measured in previous studies . in a study of chronic nonspecific back", "pain , treatment with asu ( piascledine ) combined with the nsaid artrosiline ( 320 mg / day ) showed significant analgesic effect over nsaid treatment alone .", ", the eradias trial determined whether asu expanscience treatment slowed the radiological progression of hip oa .", "as for safety , none of the four rcts reported significant differences in adverse effects between asu and placebo .", "factors like bmi , severity of disease , and activity level may influence the effect of asu , as these conditions exacerbate inflammatory conditions and mechanical stresses that contribute to oa .", "adipose tissue plays an important role by producing metabolic factors with catabolic and pro - inflammatory properties , including cytokines , chemokines , and adipokines ( il-6 and tnf- , il-8 , ifn- ) , which orchestrate pathophysiological processes in oa .", "soluble mediators produced by adipocytes may also modulate chondrocyte metabolism and contribute to cartilage degradation .", "asu may counteract these inflammatory processes by inhibiting the translocation of the transcription factor nf-b from the cytoplasm to the nucleus , which controls transcription of many pro - inflammatory factors ( table 2 ) .", "as such , asu acts as an anabolic agent in vitro , reducing the production of pro - inflammatory mediators , including il-1 , il-6 , il-8 , macrophage inflammatory protein-1 , no , mmp-13 , tnf- , and cox2/pge2 from various cell types ( table 1 ) . in mice , asu decreases pro - inflammatory interferon- ( ifn- ) and il-4 production , in the context of parasitic diseases .", "although more studies need to be conducted to show the effects of asu in patients with varying bmi , the anti - inflammatory effects of asu are likely to protect cartilage from obesity - associated inflammatory degradation and improve oa symptoms . indeed , asu significantly decreased the rate of oa progression to 40% compared with 50% in the placebo group in one study .", "however , this study showed that asu did not influence the rate of oa progression in the obese subset of patients with mean symptom duration 4 and bmi of 27 kg / m .", "however , excessive inflammation associated with obesity may also impede efficacy , as it does with celecoxib ( nsaid ) treatment , which is not as effective in obese patients ( bmi in excess of 30 kg / m ) .", "the influence of obesity , and how it influences asu efficacy , may also depend on the parameters used to measure and define disease . in a study examining the relationship between bmi and oa in patients scheduled to undergo hip replacement , increasing bmi", "was associated with increasing levels of pain and functional disability , but not radiographic joint damage .", "this should be taken into account when designing and assessing studies intended to examine the impact of obesity on treatment efficacy .", "asu has anti - inflammatory effects in mice when administered in conjunction with the anti - parasitic drug praziquantel , reducing inflammatory cytokines ifn- and il-4 , as well as granuloma size , while increasing cidal activity .", "asu also protects gingival elastic fibers from degradation by human leukocyte elastase , hypodermatitis , and ischemic damage .", "a recent electronic database analysis demonstrated the benefits and harms of oral medicinal plant products in treating oa .", "the authors used standard methods for trial selection and data extraction , and they assessed the quality of the body of evidence using the grade approach for major outcomes such as pain , function , radiographic joint changes , quality of life , withdrawals due to adverse events , total adverse events , and serious adverse events .", "the asu product piasclidine formed a small and clinically questionable improvement in symptoms , compared with placebo after 3 to 12 months treatment .", "radiographic joint changes , as change in joint space width ( jsw ) , did not differ between asu 300 mg treatment and placebo .", "moderate - quality evidence from a single study confirmed possible benefits of asu 600 mg over placebo .", "there is no evidence that piasclidine significantly improves joint structure , and limited evidence that it prevents joint space narrowing .", "the authors suggest further investigations are required to determine optimum daily doses producing clinical benefits without adverse events .", "asu is considered as drug in most countries and is therefore prescribed by physicians . however , in the united states it is classified as dietary supplement and can be purchased as over - the - counter supplements , avoca asu ( asu - nmx1000 , nutramax laboratories inc . ,", "avoca asu , a combination of asu and glucosamine sulfate , has been shown to suppress tnf- , il-1 , cox2 , inos , pge2 , nf-b activation and nitrite production in articular chondrocytes and monocytes / macrophages , reducing pain and inflammation in oa patients . however ,", "conflicting reports indicate the complete absence of specific asu molecules in avoca asu when compared with piascledine .", "questions remain about the efficacy and safety of asus for treatment of oa ( table 4 ) .", "macaigne and colleagues published a case report in 2004 describing a female with lymphatic colitis associated with piascledine treatment .", "further prospective multicenter studies are warranted to investigate whether other microscopic colitis cases are observed in patients treated with piascledine .", "avoca asu that contains glucosamine can induce allergic reaction in people with shellfish allergy . even in very small quantities", ", these people may experience mild symptoms , such as hives or nasal congestion , or more severe , even life - threatening , symptoms . an alternative asu formulation is arthrocen ( pharmin , usa , llc , san jose , ca ) .", "arthrocen is an extract from avocado and soybean oils that does not contain any ingredients of animal origin , artificial flavor , sweetener , preservative , or color .", "each capsule contains 100 mg unsaponifiable persea gratissima unsaponifiable ( avocado ) and 200 mg unsaponifiable glycine max ( soybean ) extracts , silica , magnesium stearate ( e470b manufactured from vegetable oil ) , and gelatin fines . in general", ", the fda does not hold dietary supplements to the stringent standards of pharmaceutical manufacture .", "if asu is to be widely used for the treatment of oa , serious consideration should be given to their current regulatory status in order to ensure potency , purity , and as well as the excipients .", "many studies have demonstrated substantial variation between the content listed on the labels of these products and the actual content .", "the sterols content of asu have been demonstrated to have biological activities in culture and in animal models .", "2 ) . we found multiple peaks were present in the piascledine-300 ( expanscience ) mass spectrometry analysis ( agilent 7890 gc system , 7693 auto sampler , agilent vl msd with triple axis detector , brea , ca ) , compared to the arthrocen 300 mg ( pharminusa ) or asu300-avocado soy unsaponifiable with sierra 600 mg ( maximize , maximum int . ) preparations ( fig .", "similar results were found for piascledine-300 , with mass spectra sterol content of c20h30o2 , c20h28o2 , sitosterol , stigmasterol , campesterol , squalene , -tocopherol , desmethyl tocopherol , oleic acid docosane , -amyrin , and cholesterol . in two letters to the editor , msika et al .", "and henroitin claimed that the exact ingredients and preparation of asu - expanscience was an intellectual proprietary , protected by patent .", "msika further emphasized that the tocopherols , sterols , and patented specific molecules from avocado contribute to the originality of the product , different from natural avocado unsaponifiables .", "in contrast , they analyzed dasuquin with msm , dasuquin , and avoca asu ( nutramax ) , and compared them with asu expanscience .", "the analyses revealed content of products were significantly different from those indicated on the nutramax labels with no citrostadienol , and brassicasterol present in asu expanscience .", "contrary to that they found contents included high level of rapeseed oil and unsaponifiables products with very low tocopherol , and without respected ratio of 1:2 for avocado to soybean unsaponifiables .", "the original asu piascledine 300 pills contains 100 mg of avocado unsaponifiables and 200 mg of soybean unsaponifiables .", "the difference in sterol content is based on the a / s ratio and avocado - specific modified unsaponifiables obtained by a patented process .", "have shown asu effects are best when the asu ratio is 2:1 . in support ,", "these issues and reported adverse effects of asu ( table 4 ) raises concerns about the content and purity of asu supplements on the market , with implications for patient safety .", " mass spectrometry analysis of major sterol components of piascledine 300 , avocado 300 soy unsaponifiable with sierra 600 mg , and arthrocen 300 .", "control sample exhibited characteristic peaks corresponding to 1 = dihydrocholesterol ( 5-cholestan-3-ol ; internal control ; sigma aldrich ) , 2 = brass ( brassicasterol ) , 3 = camp ( campesterol ) , 4 = stigmn ( stigmastanol ) , 5 = -sito ( -sitosterol ) , 6 = stigma ( stigmasterol ) ." ], [ "improving joint function and patient activity is a central public health concern to improve quality and length of life .", "the aim is not only to treat pain but also to prevent the onset of disease .", "there is no cure for oa , and even symptomatic treatment options are scarce , dominated by pain management and surgical intervention .", "asus may prove to be an effective treatment option for symptomatic oa , as they have been shown to possess chondroprotective , anabolic , and anticatabolic properties , as well as anti - inflammatory properties . at the clinical level , asus reduce pain and stiffness while improving joint function .", "importantly , asus are a natural , slow - acting agent that do not merely address acute pain but actively prevent progression of oa symptoms .", "further studies are required to determine the specific mechanisms and target molecules of asu function on oa at the cellular and metabolic level ." ] ]

[ "in modern society , the fashion of wearing tight clothes for stylish dressed states or \n comeliness is getting very popular1 . \n", "however , specialists reported that excessively pressing certain areas of the human body \n could cause many problems in the cardiovascular systems and visceral organs1 .", "in addition to this pressure inflicted on \n the human body may deform muscles , the skeletal system , and even the overall body type2 , 3 . \n", "jeans that are too tight compress a nerve that cuts off sensation to the thighs , and this is \n consistent with human anatomy and physiology4 .", "trousers that are too tight can squeeze a sensory nerve under the \n hip bone , causing a tingling , burning sensation called paresthesia1 , 4 , 5 . with this as the background , the present study aimed to understand \n the hazards of the habit of wearing tightly fitting clothes in relation to deformations of \n the musculoskeletal system and the movement of the lumbar spine and pelvis for the purpose \n of providing basic data regarding proper habits in wearing clothes for the prevention of \n pain in the musculoskeletal systems2 , 3 .", "so the present study evaluated the effect \n of wearing tight pants on the trunk flexion and pelvic tilting angles in stand - to - sit \n movement and in a seated posture .", "this study was performed on nine males aged 2027 years ( 23.22.0 years , meansd ) whose \n height and weight were 175.13.2 cm and 62.13.4 kg , respectively .", "subjects with conditions \n that might affect trunk mobility , such as injury or neurologic deficits of the hip and lower \n extremities during the past year , were excluded from study . the study purpose and methods \n were explained to all the subjects , who provided informed consent according to the \n principles of the declaration of helsinki before participating .", "data were collected at a sampling rate of 100 hz with a motion capture system \n ( vicon mx , oxford metrics , oxford , uk ) that consisted of eight infrared cameras .", "sixteen reflective markers were \n attached to the lower body according to the plug - in - gait marker set ( oxford metrics ) using \n double - sided tape .", "the software used for kinematic data collection was nexus 1.4.1 ( oxford \n metrics ) , and the data were analyzed with the polygon 3.1 software ( oxford metrics ) .", "the \n experimental protocol required the completion of two stand - to - sit trials for each of the two \n pants conditions .", "the worn pants were made from the same material , cotton , woven into a \n rugged cotton textile .", "we used general and tight pants from the same company ( g company ) . \n", "the pants were worn under two conditions in this study : ( 1 ) general pants , worn with sizes \n equivalent to 105110% of the subjects hip , thigh , and calf circumferences , and ( 2 ) tight \n pants , worn with sizes equivalent to 9095% of the subjects hip , thigh , and calf \n circumferences .", "each subject was asked to stand up at a \n self - selected speed from a seated posture and to stand in an erect spine posture . for time \n normalization , the time required for a complete stand - to - sit movement cycle ,", "that is , from \n movement onset to completion , was considered to be 100% ; values were determined for each 2% \n of the movement , beginning at 0% .", "the changes in trunk flexion and pelvic posterior tilting \n angles were calculated based on the difference between the maximal and initial angles . as \n the analysis was performed with a withwn - subject design ,", "the paired t - test was conducted to \n test for differences in pelvic and trunk kinematics values during the maneuver .", "all \n significance levels were set at p<0.05 , and spss version 12.0 ( spss , chicago , il , usa ) \n was used for statistical analyses .", "the change in the posterior pelvic tilting angle ( 9.3 4.1 ) during the stand - to - sit \n movement when wearing the tight pants increased significantly compared with that when \n wearing the general pants ( 7.0 3.3 ) ( p<0.05 ) .", "the change of the trunk flexion angle \n ( 19.3 5.4 ) during the stand - to - sit movement when wearing the tight pants increased \n significantly compared with that when wearing the general pants ( 12.0 3.8 ) ( p<0.05 ) . \n the change in the posterior pelvic tilting angle ( 13.3 3.2 ) in the seated posture when \n wearing the tight pants increased significantly compared with that when wearing of the \n general pants ( 10.3 2.9 ) ( p<0.05 ) .", "the change in the trunk flexion angle ( 25.3 6.1 ) \n in the seated posture when wearing the tight pants increased significantly compared with \n that when wearing of the general pants ( 20.1 5.4 ) ( p<0.05 ) .", "park and yoo reported that the tightness of a waist belt might restricts forward movement \n of the center of mass and that pelvic inclination might be increased as a compensatory \n mechanism2 .", "they showed that the wearing \n a tight belt could interrupt normal lumbo - pelvic coordination , which might contribute to \n muscle imbalance2 .", "elevated abdominal \n pressure has been shown to cause multidirectional stiffness of the spine5 , 6 .", "a \n previous study that used a wide belt reported that the intramuscular pressure on the erector \n spinae could influence spinal stiffness separately from muscle7 .", "the results of the present study showed that the trunk flexion and \n posterior pelvic tilting angles during the stand - to - sit movement when wearing tight pants \n significantly increased when compared with wearing general pants .", "also , the trunk flexion \n and posterior pelvic tilting angles in the seated posture when wearing tight pants \n significantly increased when compared with those when wearing of general pants .", "the lumbar \n and hip rhythm and interaction between the lumbar spine and hip are important kinematic \n factors that are used not only in experimental research but also in clinical \n examination8 , 9 . wearing tight jeans induced excessive lumbar flexion during stand \n to sit movement and in the seated posture .", "the exaggerated lumbar flexion may overstretch \n posterior connective tissues , such as the interspinous ligament , apophyseal joint capsule , \n and thoracolumbar fascia , or increase stress on discs and apophyseal joints5 .", "posterior pelvic tilt decreases lordosis \n via flexion of the lumbar spine , causes posterior movement of the nucleus pulposus , and \n increases the diameter of the intervertebral foramina5 , 8 .", "slump sitting reduces the \n activation of the spinal stabilizing muscles and is associated with increases in loading on \n the intervertebral disc and connective tissue8 , 9 .", "therefore , wearing tight pants could \n produce musculoskeletal disorders via abnormal movement and posture in the lumbar spine and \n pelvis .", "so the effects of wearing tight pants need to be investigated in further studies to \n reveal their direct relationship to musculoskeletal problems .", "the effects of tight clothes \n and accessory items on the human body should also be investigated in order to provide basic \n data for proper use .", "further studies will become the starting point for studies regarding \n the effects of wearing tight clothes and tight fashion accessories on the musculoskeletal \n system ." ]

[ "<S> [ purpose ] the purpose of this study was to evaluate the effect of wearing the tight pants \n on the trunk flexion and pelvic tilting angles in the stand - to - sit movement and a seated \n posture . </S>", "<S> [ subjects ] nine male subjects were recruited . </S>", "<S> [ methods ] the trunk flexion angle \n and pelvic posterior tilting angle were measured using a motion - capture system during the \n stand - to - sit movement and in a seated posture . [ results ] the trunk flexion and the \n posterior pelvic tilting angles during the stand - to - sit movement and in the seated posture \n when wearing tight pants significantly increased compared with those when wearing of \n general pants . </S>", "<S> [ conclusion ] therefore , wearing tight pants could produce musculoskeletal \n disorders via abnormal movement and posture in the lumbar spine and pelvis . </S>", "<S> so the effects \n of wearing tight pants need to be investigated in further studies to reveal their direct \n relationship to musculoskeletal problems . </S>" ]

[ "INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "in modern society , the fashion of wearing tight clothes for stylish dressed states or \n comeliness is getting very popular1 . \n", "however , specialists reported that excessively pressing certain areas of the human body \n could cause many problems in the cardiovascular systems and visceral organs1 .", "in addition to this pressure inflicted on \n the human body may deform muscles , the skeletal system , and even the overall body type2 , 3 . \n", "jeans that are too tight compress a nerve that cuts off sensation to the thighs , and this is \n consistent with human anatomy and physiology4 .", "trousers that are too tight can squeeze a sensory nerve under the \n hip bone , causing a tingling , burning sensation called paresthesia1 , 4 , 5 . with this as the background , the present study aimed to understand \n the hazards of the habit of wearing tightly fitting clothes in relation to deformations of \n the musculoskeletal system and the movement of the lumbar spine and pelvis for the purpose \n of providing basic data regarding proper habits in wearing clothes for the prevention of \n pain in the musculoskeletal systems2 , 3 .", "so the present study evaluated the effect \n of wearing tight pants on the trunk flexion and pelvic tilting angles in stand - to - sit \n movement and in a seated posture ." ], [ "this study was performed on nine males aged 2027 years ( 23.22.0 years , meansd ) whose \n height and weight were 175.13.2 cm and 62.13.4 kg , respectively .", "subjects with conditions \n that might affect trunk mobility , such as injury or neurologic deficits of the hip and lower \n extremities during the past year , were excluded from study . the study purpose and methods \n were explained to all the subjects , who provided informed consent according to the \n principles of the declaration of helsinki before participating .", "data were collected at a sampling rate of 100 hz with a motion capture system \n ( vicon mx , oxford metrics , oxford , uk ) that consisted of eight infrared cameras .", "sixteen reflective markers were \n attached to the lower body according to the plug - in - gait marker set ( oxford metrics ) using \n double - sided tape .", "the software used for kinematic data collection was nexus 1.4.1 ( oxford \n metrics ) , and the data were analyzed with the polygon 3.1 software ( oxford metrics ) .", "the \n experimental protocol required the completion of two stand - to - sit trials for each of the two \n pants conditions .", "the worn pants were made from the same material , cotton , woven into a \n rugged cotton textile .", "we used general and tight pants from the same company ( g company ) . \n", "the pants were worn under two conditions in this study : ( 1 ) general pants , worn with sizes \n equivalent to 105110% of the subjects hip , thigh , and calf circumferences , and ( 2 ) tight \n pants , worn with sizes equivalent to 9095% of the subjects hip , thigh , and calf \n circumferences .", "each subject was asked to stand up at a \n self - selected speed from a seated posture and to stand in an erect spine posture . for time \n normalization , the time required for a complete stand - to - sit movement cycle ,", "that is , from \n movement onset to completion , was considered to be 100% ; values were determined for each 2% \n of the movement , beginning at 0% .", "the changes in trunk flexion and pelvic posterior tilting \n angles were calculated based on the difference between the maximal and initial angles . as \n the analysis was performed with a withwn - subject design ,", "the paired t - test was conducted to \n test for differences in pelvic and trunk kinematics values during the maneuver .", "all \n significance levels were set at p<0.05 , and spss version 12.0 ( spss , chicago , il , usa ) \n was used for statistical analyses ." ], [ "the change in the posterior pelvic tilting angle ( 9.3 4.1 ) during the stand - to - sit \n movement when wearing the tight pants increased significantly compared with that when \n wearing the general pants ( 7.0 3.3 ) ( p<0.05 ) .", "the change of the trunk flexion angle \n ( 19.3 5.4 ) during the stand - to - sit movement when wearing the tight pants increased \n significantly compared with that when wearing the general pants ( 12.0 3.8 ) ( p<0.05 ) . \n the change in the posterior pelvic tilting angle ( 13.3 3.2 ) in the seated posture when \n wearing the tight pants increased significantly compared with that when wearing of the \n general pants ( 10.3 2.9 ) ( p<0.05 ) .", "the change in the trunk flexion angle ( 25.3 6.1 ) \n in the seated posture when wearing the tight pants increased significantly compared with \n that when wearing of the general pants ( 20.1 5.4 ) ( p<0.05 ) ." ], [ "park and yoo reported that the tightness of a waist belt might restricts forward movement \n of the center of mass and that pelvic inclination might be increased as a compensatory \n mechanism2 .", "they showed that the wearing \n a tight belt could interrupt normal lumbo - pelvic coordination , which might contribute to \n muscle imbalance2 .", "elevated abdominal \n pressure has been shown to cause multidirectional stiffness of the spine5 , 6 .", "a \n previous study that used a wide belt reported that the intramuscular pressure on the erector \n spinae could influence spinal stiffness separately from muscle7 .", "the results of the present study showed that the trunk flexion and \n posterior pelvic tilting angles during the stand - to - sit movement when wearing tight pants \n significantly increased when compared with wearing general pants .", "also , the trunk flexion \n and posterior pelvic tilting angles in the seated posture when wearing tight pants \n significantly increased when compared with those when wearing of general pants .", "the lumbar \n and hip rhythm and interaction between the lumbar spine and hip are important kinematic \n factors that are used not only in experimental research but also in clinical \n examination8 , 9 . wearing tight jeans induced excessive lumbar flexion during stand \n to sit movement and in the seated posture .", "the exaggerated lumbar flexion may overstretch \n posterior connective tissues , such as the interspinous ligament , apophyseal joint capsule , \n and thoracolumbar fascia , or increase stress on discs and apophyseal joints5 .", "posterior pelvic tilt decreases lordosis \n via flexion of the lumbar spine , causes posterior movement of the nucleus pulposus , and \n increases the diameter of the intervertebral foramina5 , 8 .", "slump sitting reduces the \n activation of the spinal stabilizing muscles and is associated with increases in loading on \n the intervertebral disc and connective tissue8 , 9 .", "therefore , wearing tight pants could \n produce musculoskeletal disorders via abnormal movement and posture in the lumbar spine and \n pelvis .", "so the effects of wearing tight pants need to be investigated in further studies to \n reveal their direct relationship to musculoskeletal problems .", "the effects of tight clothes \n and accessory items on the human body should also be investigated in order to provide basic \n data for proper use .", "further studies will become the starting point for studies regarding \n the effects of wearing tight clothes and tight fashion accessories on the musculoskeletal \n system ." ] ]

[ "in commonly accepted opinion every searching for new , more effective drugs should be rationalized i.e. , determined by the low cost and non time - consuming procedures .", "these procedures are especially useful on the preliminary stage of searching for new chemical structures of potential biological activity ( jorgensen , 2004 ; leeson and springthorpe , 2007 ; ou - yang et al . , 2012 ) .", "in general , on this purpose there are employed various correlation qsar methods ( dudek et al . , 2006 ; yang and huang , 2006 ; shailesh et al . , 2012 ) . however , in particular cases it is more convenient to develop the procedure of selection of the appropriate structures based on more direct and easier interpretatively criteria .", "it seems that just such a case is a search for effective ligands of 5ht1a , 5ht2a , and d2 receptors since many structural data on their agonist and antagonist as well as the models of these receptors are well - known ( klabunde and hessler , 2002 ; bissantz et al .", ", 2003 ; teeter et al . , 1994 ; chambers and nichols , 2002 ; homan et al . , 1999 ) .", "in addition , wide availability of various bases containing a lot of structural data on very active ligands allows to generate pretty accurate pharmacophore patterns ( nelson , 1991 ; bojarski , 2006 ) .", "thanks to these all literature data it is possible to estimate the affinity of potential ligand for receptor of interest .", "the chemical structure of pharmacophore of being selected potential ligand and its affinity to the receptor seem to be sufficiently unambiguous discriminators , on a preliminary stage , in the search for new effective antipsychotics . to verify this hypothesis ,", "the first step includes determination of pharmacophores for two tested compounds of well - known affinity ( previously in vitro determined ) to the same receptors as well as pharmacophore pertinent to well - known d2 receptor agonists or antagonists and finally comparison of their properties to in vitro binding data .", "the pharmacophore model of d2 receptor ligands was found on the basis of 15 compounds of high affinity to d2 receptor reported in literature ( sowiski et al . , 2011 ) .", "these two tested compounds were 3-acylamine derivatives of tropane : n-(8-furan-2-ylmethyl-8-azabicyclo[3.2.1]oct-3-yl)-2-methoxybenzamide ( compounds i ) and n-(8-furan-2-ylmethyl-8-azabicyclo[3.2.1]oct-3-yl)-2.3-dimethoxybenzamide ( compound ii ) ( fig . 1 ) .", "their synthesis have been developed and described in the previously published paper on tropane derivatives ( sowiski et al .", ". 1the chemical formulas of compound i and compound ii the chemical formulas of compound i and compound ii the pharmacophores of compounds i and ii were found on the basis of their structures determined by x - ray diffraction method .", "the ccdc ( cambridge crystallographic data centre ) numbers of compounds i and ii are : 905689 and 905690 , respectively ( figs . 2 , 3).fig . ", "3the x - ray diffraction structure of compound ii the x - ray diffraction structure of compound i the x - ray diffraction structure of compound ii the molecular structure of compound i shows an intramolecular hydrogen bond between the o atom of the methoxy group and the nh of the amide function leads to a six - membered ring .", "the dihedral angle between the least - squares planes of the phenyl and this virtual ring is only 2.50(7). the piperidine moiety adopts a chair conformation .", "the best plane of the furan ring and the c1/c2/c4/c5 plane make an angle 69.42(9) and the dihedral angle between the planes of the furan and benzene rings is 72.50(8). the compound ii molecule adopts a folded conformation with an angle between the furan and benzene rings of 63.29(8) and between the best plane of the furan ring and the c1/c2/c4/c5 plane of 87.56(9). this conformation is stabilized by an intramolecular n15h15ao25 and c26h26co27 hydrogen bonds . as a result of n15h15ao25 interaction a six - membered ring", "the piperidine moiety assumes a chair conformation and the substituent at n8 is in an equatorial position .", "conformations of both methoxy groups are different . the disposition of these groups with respect to the phenyl ring can be described by the torsion angles c18c19o25c26 of 107.8(2) and c21c20o27c28 of 11.1(3). in consequence , the methyl carbon atom c26 is found to be 1.107(4 ) out of the phenyl plane , and c28 atom is almost coplanar with this ring .", "the pharmacophore structure is a reflection template of the geometrical distribution of property centers localized in molecule and determines to large extent its biological activity .", "it means that even subtle differences in the geometry of structurally similar molecules can significantly impact on their affinity to receptor binding site .", "the comparative analysis of the studied pharmacophores was intended to find the specific properties and geometrical parameters which are crucial for the strength of binding of potential ligands to the receptors of interest .", "the second step of the applied procedure devoted to the selection of the potential agonists or antagonists of the studied receptors relies on docking of the reference compounds i and ii to the models of the d2 receptor ( sakhteman et al . , 2011 ) .", "from analysis of in vitro results ( table 1 ) follows that the both studied compounds ( i , ii ) are very poorly being bounded to 5-ht1a and 5-ht2a receptors . indeed , the model docking of compounds i and ii to these receptors also showed that such binding can not take place .", "the both molecules of compounds i and ii were placed outside the receptor binding pockets .", "thus , only docking of compounds i and ii to d2 receptor is detailed analyzed .", "the most discriminative parameters which distinctly classify the quality of docking are number and strength ( equivalently length and geometry ) of the hydrogen bonds formed between ligand and specific amino acids not only inside the receptor binding pocket but also , although to a less degree , intermolecular interactions of other types e.g. , hydrophobic and edge-to-face.table 15ht1a , 5ht2a , and d2 receptor affinitiesligandreceptor [ k(nm)]5ht1a5ht2ad2compound", "i6,1006,0001,000compound ii3,000744.526.3 5ht1a , 5ht2a , and d2 receptor affinities the used 3d homology model of d2 receptor has been revealed by comparative modeling using the crystal structure of the human 2-adrenergic receptor and the bovine rhodopsin as the templates ( sakhteman et al . , 2011 ; strzelczyk et al . , 2004 ;", "the quite recently reported x - ray structure of the human 2-adrenergic receptor opens new possibilities for modeling of the correct structures of the dopamine ones .", "currently , the human 2-adrenergic receptor is considered to be more homologous to the dopamine receptors than bovine rhodopsin ( cherezov et al . , 2007 ) .", "all modeling of the pharmacophores as well as docking of the compounds i and ii to the d2 receptor model were done by discovery studio software ( accelrys software inc . , discovery studio modeling environment , 2005 ) .", "crystals of compounds i and ii suitable for x - ray analysis were grown by slow evaporation from acetate / diisopropyl ether ( compound i ) and hexane / ethanol ( compound ii ) solutions .", "the data were collected on an oxford diffraction km4ccd diffractometer at 293 k , using graphite - monochromated mo k radiation .", "the unit cell parameters were determined by least - squares treatment of setting angles of highest - intensity reflections chosen from the whole experiment .", "the structure was solved by direct methods using the shelxs97 program ( sheldric , 1990 ) and refined by the full - matrix least - squares method with the shelxl97 program ( sheldric , 1997 ) . the function w(|fo| ", "|fc| ) was minimized with w= [ (fo ) + ( 0.0688p ) ] , where p = ( fo2 + 2fc2)/3 .", "= kfc[1 + ( 0.001fc2/sin2 ) ] ( sheldric , 1997 ) and the extinction coefficient was equal to 0.014(2 ) .", "the coordinates of the hydrogen atoms were calculated in idealized positions and refined as a riding model with their thermal parameters calculated as 1.2 ( 1.5 for methyl group ) times ueq of the respective carrier carbon atom .", "crystals of compounds i and ii suitable for x - ray analysis were grown by slow evaporation from acetate / diisopropyl ether ( compound i ) and hexane / ethanol ( compound ii ) solutions .", "the data were collected on an oxford diffraction km4ccd diffractometer at 293 k , using graphite - monochromated mo k radiation .", "the unit cell parameters were determined by least - squares treatment of setting angles of highest - intensity reflections chosen from the whole experiment .", "the structure was solved by direct methods using the shelxs97 program ( sheldric , 1990 ) and refined by the full - matrix least - squares method with the shelxl97 program ( sheldric , 1997 ) . the function w(|fo| ", "|fc| ) was minimized with w= [ (fo ) + ( 0.0688p ) ] , where p = ( fo2 + 2fc2)/3 .", "= kfc[1 + ( 0.001fc2/sin2 ) ] ( sheldric , 1997 ) and the extinction coefficient was equal to 0.014(2 ) .", "the coordinates of the hydrogen atoms were calculated in idealized positions and refined as a riding model with their thermal parameters calculated as 1.2 ( 1.5 for methyl group ) times ueq of the respective carrier carbon atom .", "the in vitro binding data for compounds i , ii as ligands of 5ht1a , 5ht2a , and d2 receptors are given in table 1 ( sowiski et al .", ", 2011 ) . these experimental binding data unambiguously points at very low affinity of compound i to 5ht1a and 5ht2a receptors and somewhat better to d2 one , yet", ", compound ii displayed very weak binding activity to 5ht1a , moderate to 5ht2a and very high to d2 receptors .", "the differences between parameters ( geometrical and property types ) of the reference pharmacophores and the pharmacophores pertinent to compounds i and ii are expected to reflect the differences in affinity of tested compounds to the receptors of interest . the found structures of pharmacophores described by their specific properties are given on figs . ", "the particular colors denote the following properties : red positive ionization ( nitrogen atom ) , green hydrogen bond acceptor , magenta hydrogen bond donor , pale blue hydrophobic , aromatic , ultramarine hydrophobic , aliphatic , orange aromatic ring ( table 2).fig . ", "4the spatial distribution of pharmacophore properties on a background of compound i x - ray diffraction structure .", "a green square depicts the plane of a phenyl ring ( color figure online)fig . ", "5the spatial distribution of pharmacophore properties on a background of compound ii x - ray diffraction structure .", "a green square depicts the plane of a phenyl ring ( color figure online)fig . ", "aliphatic property ( color figure online)table 2pharmacophore properties of compound i and iipharmacophore feature / propertycompound icompound iipositive ionization ( red)nitrogen atomnitrogen atomhydrogen bond acceptor ( hba , green)carbonyl group of amide bondcarbonyl group of amide bondaromatic ring ( orange)benzene ring substituted with methoxy groupbenzene ring substituted with two methoxy groupshydrophobic , aromatic ( pale blue)furane ringfurane ringhydrophobic , aliphatic ( ultramarine)one methyl group in methoxy moiety attached to the benzene ringtwo methyl groups in methoxy moieties attached to the benzene ring the spatial distribution of pharmacophore properties on a background of compound i x - ray diffraction structure .", "a green square depicts the plane of a phenyl ring ( color figure online ) the spatial distribution of pharmacophore properties on a background of compound ii x - ray diffraction structure .", "a green square depicts the plane of a phenyl ring ( color figure online ) the spatial distribution of pharmacophore properties of d2 receptor ligands .", "aliphatic property ( color figure online ) pharmacophore properties of compound i and ii the geometry of a spatial distribution of pharmacophore properties in obtained models is an exact reflection of the x - ray diffraction structure of compounds i and ii ( table 3 ) .", "it is worthy to note that in spite of the high similarity of chemical structures of these compounds , that their conformations significantly differ each from other .", "obviously , it should be taken into account some flexibility of the spatial pharmacophore geometry and possibility of its change during docking of studied compounds to particular receptors .", "however , such changes are often possible only to small degree or impossible at all on account of the high energetic rotation barriers . in this context , the presence of two separate aliphatic hydrophobic centers in pharmacophore of compound ii takes on a special importance for explanation of very high affinity of this compound , in contrast to compound i , for d2 receptor .", "it is likely that just second methoxy group in compound ii molecule underlies its high binding to d2 receptor while the same group do not affect the affinity of compound ii to 5-ht1a and 5-ht2a receptors .", "the comparative analysis of the d2 receptor ligand pharmacophore ( fig . 6 ) and pharmacophores of compounds i and ii also leads to the same conclusion ( figs . 4 and 5 ) .", "the pharmacophore of d2 ligand quite well matches the pharmacophore of compound ii but does not the pharmacophore of compound i ( c.f .", "in addition , specificity of the structural relation between these pharmacophores results from the identical spatial localization of the aliphatic property of d2 ligand pharmacophore and its analog present in pharmacophore of compound ii but absent in pharmacophore of compound i ( table 2 ) . \n fig . ", "7superposition of the d2 receptor ligand pharmacophore and pharmacophore of compound iitable 3pharmacophore geometry parameterspharmacophore geometry parameterscompound icompound iidistance between piperidine nitrogen atom and center of the benzene ring7.85 7.76 dihedral angle between benzene ring plane and furane ring plane72.5063.29dihedral angle between piperidine ring ( c1/c2/c4/c5 ) plane and benzene ring plane65.7950.97dihedral angle between piperidine ring ( c1/c2/c4/c5 ) plane and furane ring plane69.4287.56dihedral angle between carbonyl group plane and piperidine ring plane73.5086.72 superposition of the d2 receptor ligand pharmacophore and pharmacophore of compound ii pharmacophore geometry parameters docking of both tested compounds to d2 receptor model turned out to be non discriminative investigation not giving criteria for explanation of difference in ability to the binding of compounds i and ii with d2 receptor .", "both compounds docked to d2 receptor interact with its amino acids via the same hydrogen bonds . in case of compound", "i the hydrogen bonds are : ligand thyrosine 379 ( length 2.198 ) , ligand alanine 185 ( length 2.315 ) , and compound ii ligand ", "thyrosine 379 ( length 2.310 ) , ligand alanine 185 ( length 2.139 ) .", "in addition , both compounds interact similarly with d2 receptor with hydrophobic forces ( fig . ", "8the molecules of compounds i and ii ( green ) inside binding pocket of d2 receptor .", "yellow dashed lines denote hydrogen bonds ( color figure online ) the molecules of compounds i and ii ( green ) inside binding pocket of d2 receptor .", "yellow dashed lines denote hydrogen bonds ( color figure online ) the obtained docking results are not unexpected since , purposely , the structurally similar compounds were investigated to point out that even very subtle differences in the chemical structure of compounds , to which docking procedure is insensitive , may impact crucially on their therapeutic activity .", "thus , it should be stated that two stages pharmacophore and docking investigations are necessary to estimate properly an affinity of newly designed receptor ligands . on the whole , these studies were intended to prove that postulated two - stages procedure can be applied to verification of the properties of even very similar structurally potential and being designed antipsychotics ." ]

[ "<S> the two - stages studies of structure activity relationship for model ligands of 5ht1a , 5ht2a , and d2 receptors were performed . on the first stage , the pharmacophores of two potential ligands of known in vitro binding to 5ht1a , 5ht2a , d2 receptors and model pharmacophore of strongly interacting d2 receptor ligands were found and their parameters were related to affinity data . </S>", "<S> the analyzed parameters were hydrophobic , hydrophilic , aromatic , donor and acceptor of proton centers . </S>", "<S> the geometry of spatial distribution of these properties was also investigated in comparative analysis . </S>", "<S> the studied , model compounds were two 3-acylamine derivatives of tropane . </S>", "<S> the second stage includes docking of studied compounds to d2 receptor model and the comparison of its quality with in vivo binding data . </S>", "<S> the obtained results are consistent with in vitro binding data and applied procedure accurate estimates the affinity of potential ligands to d2 receptors . </S>" ]

[ "Introduction", "Materials and methods", "X-ray diffraction measurements", "Results and discussion" ]

[ [ "in commonly accepted opinion every searching for new , more effective drugs should be rationalized i.e. , determined by the low cost and non time - consuming procedures .", "these procedures are especially useful on the preliminary stage of searching for new chemical structures of potential biological activity ( jorgensen , 2004 ; leeson and springthorpe , 2007 ; ou - yang et al . , 2012 ) .", "in general , on this purpose there are employed various correlation qsar methods ( dudek et al . , 2006 ; yang and huang , 2006 ; shailesh et al . , 2012 ) . however , in particular cases it is more convenient to develop the procedure of selection of the appropriate structures based on more direct and easier interpretatively criteria .", "it seems that just such a case is a search for effective ligands of 5ht1a , 5ht2a , and d2 receptors since many structural data on their agonist and antagonist as well as the models of these receptors are well - known ( klabunde and hessler , 2002 ; bissantz et al .", ", 2003 ; teeter et al . , 1994 ; chambers and nichols , 2002 ; homan et al . , 1999 ) .", "in addition , wide availability of various bases containing a lot of structural data on very active ligands allows to generate pretty accurate pharmacophore patterns ( nelson , 1991 ; bojarski , 2006 ) .", "thanks to these all literature data it is possible to estimate the affinity of potential ligand for receptor of interest .", "the chemical structure of pharmacophore of being selected potential ligand and its affinity to the receptor seem to be sufficiently unambiguous discriminators , on a preliminary stage , in the search for new effective antipsychotics . to verify this hypothesis ,", "the first step includes determination of pharmacophores for two tested compounds of well - known affinity ( previously in vitro determined ) to the same receptors as well as pharmacophore pertinent to well - known d2 receptor agonists or antagonists and finally comparison of their properties to in vitro binding data .", "the pharmacophore model of d2 receptor ligands was found on the basis of 15 compounds of high affinity to d2 receptor reported in literature ( sowiski et al . , 2011 ) .", "these two tested compounds were 3-acylamine derivatives of tropane : n-(8-furan-2-ylmethyl-8-azabicyclo[3.2.1]oct-3-yl)-2-methoxybenzamide ( compounds i ) and n-(8-furan-2-ylmethyl-8-azabicyclo[3.2.1]oct-3-yl)-2.3-dimethoxybenzamide ( compound ii ) ( fig . 1 ) .", "their synthesis have been developed and described in the previously published paper on tropane derivatives ( sowiski et al .", ". 1the chemical formulas of compound i and compound ii the chemical formulas of compound i and compound ii the pharmacophores of compounds i and ii were found on the basis of their structures determined by x - ray diffraction method .", "the ccdc ( cambridge crystallographic data centre ) numbers of compounds i and ii are : 905689 and 905690 , respectively ( figs . 2 , 3).fig . ", "3the x - ray diffraction structure of compound ii the x - ray diffraction structure of compound i the x - ray diffraction structure of compound ii the molecular structure of compound i shows an intramolecular hydrogen bond between the o atom of the methoxy group and the nh of the amide function leads to a six - membered ring .", "the dihedral angle between the least - squares planes of the phenyl and this virtual ring is only 2.50(7). the piperidine moiety adopts a chair conformation .", "the best plane of the furan ring and the c1/c2/c4/c5 plane make an angle 69.42(9) and the dihedral angle between the planes of the furan and benzene rings is 72.50(8). the compound ii molecule adopts a folded conformation with an angle between the furan and benzene rings of 63.29(8) and between the best plane of the furan ring and the c1/c2/c4/c5 plane of 87.56(9). this conformation is stabilized by an intramolecular n15h15ao25 and c26h26co27 hydrogen bonds . as a result of n15h15ao25 interaction a six - membered ring", "the piperidine moiety assumes a chair conformation and the substituent at n8 is in an equatorial position .", "conformations of both methoxy groups are different . the disposition of these groups with respect to the phenyl ring can be described by the torsion angles c18c19o25c26 of 107.8(2) and c21c20o27c28 of 11.1(3). in consequence , the methyl carbon atom c26 is found to be 1.107(4 ) out of the phenyl plane , and c28 atom is almost coplanar with this ring .", "the pharmacophore structure is a reflection template of the geometrical distribution of property centers localized in molecule and determines to large extent its biological activity .", "it means that even subtle differences in the geometry of structurally similar molecules can significantly impact on their affinity to receptor binding site .", "the comparative analysis of the studied pharmacophores was intended to find the specific properties and geometrical parameters which are crucial for the strength of binding of potential ligands to the receptors of interest .", "the second step of the applied procedure devoted to the selection of the potential agonists or antagonists of the studied receptors relies on docking of the reference compounds i and ii to the models of the d2 receptor ( sakhteman et al . , 2011 ) .", "from analysis of in vitro results ( table 1 ) follows that the both studied compounds ( i , ii ) are very poorly being bounded to 5-ht1a and 5-ht2a receptors . indeed , the model docking of compounds i and ii to these receptors also showed that such binding can not take place .", "the both molecules of compounds i and ii were placed outside the receptor binding pockets .", "thus , only docking of compounds i and ii to d2 receptor is detailed analyzed .", "the most discriminative parameters which distinctly classify the quality of docking are number and strength ( equivalently length and geometry ) of the hydrogen bonds formed between ligand and specific amino acids not only inside the receptor binding pocket but also , although to a less degree , intermolecular interactions of other types e.g. , hydrophobic and edge-to-face.table 15ht1a , 5ht2a , and d2 receptor affinitiesligandreceptor [ k(nm)]5ht1a5ht2ad2compound", "i6,1006,0001,000compound ii3,000744.526.3 5ht1a , 5ht2a , and d2 receptor affinities the used 3d homology model of d2 receptor has been revealed by comparative modeling using the crystal structure of the human 2-adrenergic receptor and the bovine rhodopsin as the templates ( sakhteman et al . , 2011 ; strzelczyk et al . , 2004 ;", "the quite recently reported x - ray structure of the human 2-adrenergic receptor opens new possibilities for modeling of the correct structures of the dopamine ones .", "currently , the human 2-adrenergic receptor is considered to be more homologous to the dopamine receptors than bovine rhodopsin ( cherezov et al . , 2007 ) .", "all modeling of the pharmacophores as well as docking of the compounds i and ii to the d2 receptor model were done by discovery studio software ( accelrys software inc . , discovery studio modeling environment , 2005 ) ." ], [ "crystals of compounds i and ii suitable for x - ray analysis were grown by slow evaporation from acetate / diisopropyl ether ( compound i ) and hexane / ethanol ( compound ii ) solutions .", "the data were collected on an oxford diffraction km4ccd diffractometer at 293 k , using graphite - monochromated mo k radiation .", "the unit cell parameters were determined by least - squares treatment of setting angles of highest - intensity reflections chosen from the whole experiment .", "the structure was solved by direct methods using the shelxs97 program ( sheldric , 1990 ) and refined by the full - matrix least - squares method with the shelxl97 program ( sheldric , 1997 ) . the function w(|fo| ", "|fc| ) was minimized with w= [ (fo ) + ( 0.0688p ) ] , where p = ( fo2 + 2fc2)/3 .", "= kfc[1 + ( 0.001fc2/sin2 ) ] ( sheldric , 1997 ) and the extinction coefficient was equal to 0.014(2 ) .", "the coordinates of the hydrogen atoms were calculated in idealized positions and refined as a riding model with their thermal parameters calculated as 1.2 ( 1.5 for methyl group ) times ueq of the respective carrier carbon atom ." ], [ "crystals of compounds i and ii suitable for x - ray analysis were grown by slow evaporation from acetate / diisopropyl ether ( compound i ) and hexane / ethanol ( compound ii ) solutions .", "the data were collected on an oxford diffraction km4ccd diffractometer at 293 k , using graphite - monochromated mo k radiation .", "the unit cell parameters were determined by least - squares treatment of setting angles of highest - intensity reflections chosen from the whole experiment .", "the structure was solved by direct methods using the shelxs97 program ( sheldric , 1990 ) and refined by the full - matrix least - squares method with the shelxl97 program ( sheldric , 1997 ) . the function w(|fo| ", "|fc| ) was minimized with w= [ (fo ) + ( 0.0688p ) ] , where p = ( fo2 + 2fc2)/3 .", "= kfc[1 + ( 0.001fc2/sin2 ) ] ( sheldric , 1997 ) and the extinction coefficient was equal to 0.014(2 ) .", "the coordinates of the hydrogen atoms were calculated in idealized positions and refined as a riding model with their thermal parameters calculated as 1.2 ( 1.5 for methyl group ) times ueq of the respective carrier carbon atom ." ], [ "the in vitro binding data for compounds i , ii as ligands of 5ht1a , 5ht2a , and d2 receptors are given in table 1 ( sowiski et al .", ", 2011 ) . these experimental binding data unambiguously points at very low affinity of compound i to 5ht1a and 5ht2a receptors and somewhat better to d2 one , yet", ", compound ii displayed very weak binding activity to 5ht1a , moderate to 5ht2a and very high to d2 receptors .", "the differences between parameters ( geometrical and property types ) of the reference pharmacophores and the pharmacophores pertinent to compounds i and ii are expected to reflect the differences in affinity of tested compounds to the receptors of interest . the found structures of pharmacophores described by their specific properties are given on figs . ", "the particular colors denote the following properties : red positive ionization ( nitrogen atom ) , green hydrogen bond acceptor , magenta hydrogen bond donor , pale blue hydrophobic , aromatic , ultramarine hydrophobic , aliphatic , orange aromatic ring ( table 2).fig . ", "4the spatial distribution of pharmacophore properties on a background of compound i x - ray diffraction structure .", "a green square depicts the plane of a phenyl ring ( color figure online)fig . ", "5the spatial distribution of pharmacophore properties on a background of compound ii x - ray diffraction structure .", "a green square depicts the plane of a phenyl ring ( color figure online)fig . ", "aliphatic property ( color figure online)table 2pharmacophore properties of compound i and iipharmacophore feature / propertycompound icompound iipositive ionization ( red)nitrogen atomnitrogen atomhydrogen bond acceptor ( hba , green)carbonyl group of amide bondcarbonyl group of amide bondaromatic ring ( orange)benzene ring substituted with methoxy groupbenzene ring substituted with two methoxy groupshydrophobic , aromatic ( pale blue)furane ringfurane ringhydrophobic , aliphatic ( ultramarine)one methyl group in methoxy moiety attached to the benzene ringtwo methyl groups in methoxy moieties attached to the benzene ring the spatial distribution of pharmacophore properties on a background of compound i x - ray diffraction structure .", "a green square depicts the plane of a phenyl ring ( color figure online ) the spatial distribution of pharmacophore properties on a background of compound ii x - ray diffraction structure .", "a green square depicts the plane of a phenyl ring ( color figure online ) the spatial distribution of pharmacophore properties of d2 receptor ligands .", "aliphatic property ( color figure online ) pharmacophore properties of compound i and ii the geometry of a spatial distribution of pharmacophore properties in obtained models is an exact reflection of the x - ray diffraction structure of compounds i and ii ( table 3 ) .", "it is worthy to note that in spite of the high similarity of chemical structures of these compounds , that their conformations significantly differ each from other .", "obviously , it should be taken into account some flexibility of the spatial pharmacophore geometry and possibility of its change during docking of studied compounds to particular receptors .", "however , such changes are often possible only to small degree or impossible at all on account of the high energetic rotation barriers . in this context , the presence of two separate aliphatic hydrophobic centers in pharmacophore of compound ii takes on a special importance for explanation of very high affinity of this compound , in contrast to compound i , for d2 receptor .", "it is likely that just second methoxy group in compound ii molecule underlies its high binding to d2 receptor while the same group do not affect the affinity of compound ii to 5-ht1a and 5-ht2a receptors .", "the comparative analysis of the d2 receptor ligand pharmacophore ( fig . 6 ) and pharmacophores of compounds i and ii also leads to the same conclusion ( figs . 4 and 5 ) .", "the pharmacophore of d2 ligand quite well matches the pharmacophore of compound ii but does not the pharmacophore of compound i ( c.f .", "in addition , specificity of the structural relation between these pharmacophores results from the identical spatial localization of the aliphatic property of d2 ligand pharmacophore and its analog present in pharmacophore of compound ii but absent in pharmacophore of compound i ( table 2 ) . \n fig . ", "7superposition of the d2 receptor ligand pharmacophore and pharmacophore of compound iitable 3pharmacophore geometry parameterspharmacophore geometry parameterscompound icompound iidistance between piperidine nitrogen atom and center of the benzene ring7.85 7.76 dihedral angle between benzene ring plane and furane ring plane72.5063.29dihedral angle between piperidine ring ( c1/c2/c4/c5 ) plane and benzene ring plane65.7950.97dihedral angle between piperidine ring ( c1/c2/c4/c5 ) plane and furane ring plane69.4287.56dihedral angle between carbonyl group plane and piperidine ring plane73.5086.72 superposition of the d2 receptor ligand pharmacophore and pharmacophore of compound ii pharmacophore geometry parameters docking of both tested compounds to d2 receptor model turned out to be non discriminative investigation not giving criteria for explanation of difference in ability to the binding of compounds i and ii with d2 receptor .", "both compounds docked to d2 receptor interact with its amino acids via the same hydrogen bonds . in case of compound", "i the hydrogen bonds are : ligand thyrosine 379 ( length 2.198 ) , ligand alanine 185 ( length 2.315 ) , and compound ii ligand ", "thyrosine 379 ( length 2.310 ) , ligand alanine 185 ( length 2.139 ) .", "in addition , both compounds interact similarly with d2 receptor with hydrophobic forces ( fig . ", "8the molecules of compounds i and ii ( green ) inside binding pocket of d2 receptor .", "yellow dashed lines denote hydrogen bonds ( color figure online ) the molecules of compounds i and ii ( green ) inside binding pocket of d2 receptor .", "yellow dashed lines denote hydrogen bonds ( color figure online ) the obtained docking results are not unexpected since , purposely , the structurally similar compounds were investigated to point out that even very subtle differences in the chemical structure of compounds , to which docking procedure is insensitive , may impact crucially on their therapeutic activity .", "thus , it should be stated that two stages pharmacophore and docking investigations are necessary to estimate properly an affinity of newly designed receptor ligands . on the whole , these studies were intended to prove that postulated two - stages procedure can be applied to verification of the properties of even very similar structurally potential and being designed antipsychotics ." ] ]

[ "the treatment of lung cancer with radiation has undergone significant improvements over the past decade . ", "first , it is possible to better delineate the target volume through the advancement of imaging such as positron emission tomography ( pet ) fusion [ 1 , 2 ] .", "second , there has been an increase in the utilization of radiation planning and delivery systems such as intensity - modulated radiation therapy ( imrt ) , stereotactic body radiation therapy ( sbrt ) , and proton therapy that allow more conformal delivery of radiation to achieve dose escalation while minimizing toxicity to normal structures [ 6 , 7 ] . ", "finally , the advancement of imaging modalities available during the planning and delivery process has made it feasible to adapt the target volume , both within a given fraction of treatment and between fractions , for such factors as internal motion , tumor response , anatomical changes , and weight loss . with the availability of 4d ct(four - dimensional computerized tomography)based radiotherapy planning and on - board imaging ( obi ) , accurate positioning of the target using daily image guidance", "may result in many advantages , such as a decreased probability of target miss , smaller setup margins , and less normal tissue exposed to high radiation doses .", "in addition , novel treatment adaptation algorithms can provide more effective modification of treatment plans to adapt to the changes in a patient 's anatomy and organ motion during and/or between ( intra- and/or inter- ) treatment fractions .", "adaptive radiotherapy , and will be the focus of this paper . in this examination of adaptive radiation in the setting of lung cancer , we will assess the impact of this development in sbrt , conventionally fractionated imrt , and proton therapy . ", "we will first define key terms in adaptive radiation , and will then review prior studies assessing the magnitude of intrafractional changes , which we define as changes within a given fraction of radiation , from setup to delivery .", "this discussion will be followed by the effect of these changes on delivered dose and specific planning techniques that have been utilized to decrease this variation , specifically cone beam computed tomography ( cbct ) and respiratory gating . ", "we will then focus on interfraction changes that occur during the length of treatment ( over a period of several weeks ) , such as changes in tumor mobility , tumor volume , anatomy , and body weight , and examine specific studies that assess the impact of adaptive planning on improving dose distributions in these setting . ", "finally , we will appraise future directions of adaptive radiation , including selective dose escalation through the visualization of nonresponsive regions and internal tracking to monitor intrafraction motion in real - time , both of which may be able to allow for further sparing of critical structures while maintaining accurate delivery for treatment of this aggressive disease .", "50 : . tumor visible by any imaging modality , to include both the primary tumor and any involved lymph nodes . ", "lymph nodes greater than 1 cm in size in the shortest axis are generally considered positive for disease , but functional imaging such as pet scanning is critical for target delineation [ 10 , 11 ] .", "this region can not be visualized as a discrete structure with radiographic imaging , and the extent has been defined by surgical series with pathologic correlates , as well as autopsy series . as defined by giraud et al .", ", based on nsclc surgical specimens , an appropriate gtv to ctv margin for adenocarcinoma is 8 mm , and for squamous cell carcinoma , 6 mm . ", "another study found that in stage i adenocarcinoma , a margin of 9 mm is sufficient to cover 90% of disease . in terms of mediastinal disease , a gtv to ctv margin has not been rigorously analyzed , but based on an abstract presented at the 2006 meeting of the american society for therapeutic radiology and oncology that found a maximal microscopic extension of lymph nodes is between 0.5 and 8.9 mm , we routinely use margins of 8 mm for nodal disease . ctv with a margin to account for organ motion ; or , in other words , changes in the size , shape , and position of the target during treatment . delineating the itv from 4d ct images involves assessing the target volume ( ctv ) on expiratory - phase images and then registering the outline to the images from other phases to create a union of target contours enclosing all possible positions of the target .", "an alternative method is to create a maximum intensity projection ( mip ) image by combining data from the multiple ct data sets with data from the whole - breath cycle and modifying the itv by visually verifying the target volume throughout the breathing phases ( typically 10 ) . in this process", ", attention should be paid to irregular breathing and breathing pattern variations during each treatment session and over the entire treatment course , as well as to the effects of these irregularities on the itv margin . because it is", "often more straightforward to delineate the boundaries of the gross tumor volume with motion on 4d ct image data sets as opposed to the clinical target volume , we previously proposed the concept of the internal gross tumor volume ( igtv ) , which envelops the gtv motion throughout the whole - breath cycle . in this process , rather than delineating the gtv , expanding to the ctv , and then adding the itv , the gtv is contoured , motion is assessed as outlined above ( i.e. , through the mip images or through all breathing phases ) , and this target is expanded to the igtv . ", "then , the igtv is expanded to the itv ( itv = igtv + ctv ) . ", "this latter method is what is commonly utilized at our institution to account for organ motion in the treatment of nsclc . ", "ctv plus a margin to account for both organ motion and daily setup . or", ", the itv with a margin to account for daily setup . the application and revision of margins for the ptv and itv will be discussed in detail below .", "bissonnette et al . examined cbct images during each fraction of 18 patients receiving sbrt for medically inoperable stage i nonsmall cell lung cancer ( nsclc ) . ", "the cbct images were performed at the beginning , midpoint , and end of each fraction to determine differences in tumor motion amplitude . ", "the authors found that at a mean time of 35 minutes ( from the beginning of each treatment to the end ) , the mean change in tumor amplitude was 0.4 , 1.0 , and 0.4 mm in the medial - lateral ( ml ) , superior - inferior ( si ) , and anterior - posterior ( ap ) directions , respectively . ", "these values were not statistically significantly different when compared to the initial respiratory correlated ( 4d ) ct scan , other than in patients in which abdominal compression was used , in which longer times on the couch were hypothesized to increase these differences . in another study ,", "michalski et al . compared the amount of motion between the three aforementioned directional axes ( ml , si , and ap ) in 23 patients undergoing 4d ct scans . the authors found that the largest intrafractional extent of motion was in the si direction , with the largest range being 3.59 cm . ", "a similar study from our institution also assessed respiration - induced tumor motion during radiation for lung cancer . ", "this study by liu et al . assessed 166 tumors in 152 lung cancer patients , 57% of whom had stage iii or iv disease . ", "the authors also found that the largest axis of motion was in the si direction , and that 39% of tumors moved > 0.5 cm in this direction , compared to 1.8% and 5.4% in the ml and ap directions , respectively . ", "tumor motion was also found to be correlated with the amount of diaphragm motion , the si location of the tumor , and the size of the gtv , with smaller tumors exhibiting a greater degree of intrafractional motion . a study by thomas et al .", "demonstrated that mediastinal nodal regions also move substantially with the respiratory cycle , generally posteriorly and superiorly with exhalation , and that inferior nodal stations exhibit a greater degree of motion , such that the same issues with respiratory variation in parenchymal tumors can also be applied to mediastinal disease . ", "thus , it can be concluded that : ( 1 ) inferior tumors move more than superior tumors , ( 2 ) the largest axis of motion is in the si direction , and ( 3 ) mediastinal lymph nodes are also subject to a significant degree of tumor motion .", "there are several methods to detect setup error and intrafractional variation during radiation therapy for nsclc . in this technique ,", "two - dimensional images are composed in the treatment position , and the patient is set up on patient anatomical landmarks such as bony anatomy . ", "historically , megavoltage ( mv ) imaging had been used , but the images produced with this technique were generally of poor image quality and exposed the patient to high radiation doses . in the past decade , the utilization of kilovoltage ( kv ) imaging has increased , which has improved image quality and decreased radiation exposure . in this method ,", "a ct image is reconstructed on the treatment table from a set of projection images acquired at multiple angles around the patient . ", "image reconstruction with this modality differs from conventional ct scans in that rather than a linear array of detectors being back projected to construct a 2d slice , a detector array ", "( e.g. , a flat - panel portal imager ) is used to reconstruct a 3d data set . ", "( figure 1 ) it follows that the ct images that are a result of this process are theoretically superior in improving intrafractional error as compared to portal images , and multiple studies have exhibited this improvement , particularly in sbrt where longer treatment times can lead to greater instability and less reproducibility of the setup process . ", "intrafraction treatment variation has two components in lung cancer : uncertainties in patient positioning and variations in internal motion .", "nelson et al . examined the effect of lung tumor motion and setup uncertainties using implanted fiducial markers . ", "the authors found that systematic and random uncertainties ranged between 4 and 6 mm in all three directions . with daily portal imaging , the authors found in a subsequent study that alignment based on implanted fiducials reduced systematic errors in the left - right and superior - inferior direction each by 3 mm . as a result , daily portal imaging", "is often utilized to decrease setup error in conventionally fractionated regimens , with a daily setup error of up to 5 mm . ", "borst et al . compared cbct setup with portal imaging in daily setup for 62 patients in 524 scans with nsclc , and found that cbct reduced the setup error to less than 5 mm , from 51% of patients with setup errors more than 5 mm to 2% with cbct .", "bissonnette et al . assessed the accuracy of cbct in rt for lung cancer in patients receiving both sbrt for early - stage malignancies and in those patients undergoing conventionally fractionated treatment for locally advanced disease . ", "the couch position was adjusted for discrepancies greater than 3 mm between the initial setup and treatment images . ", "the accuracy of this adjustment was then verified with a second cbct . without cbct adjustments ,", "positioning errors were found to exceed 5 mm in approximately 55% of patients . however , cbct decreased this error , such that systematic and random setup margin was within 3 mm for 82% of fractions in the sbrt group , and between 76% and 84% of the patients in the conventionally fractionated group .", "similar findings were published by grills et al . , who found that cbct image guidance significantly decreased setup margins in sbrt , with calculated precorrection population margins of 913 mm and 1014 mm with a stereotactic body frame and alpha cradle , respectively , while these same margins were 1 - 2 mm and 2 - 3 mm postcorrection , and 24 mm and 25 mm postreatment , respectively .", "the conclusion of these studies is that consistent cbct imaging can decrease the amount of systematic and random error on a daily basis ( to within 3 mm ) , and thus decrease intrafraction variation .", "lung tumors differ from many other treatment sites in that internal motion can account for large variations in tumor position , and as a result , dosimetry . ", "mechalakos et al . examined treatment plans for 12 patients receiving radiation therapy for nsclc . ", "the authors found that the dose to 95% of the gross tumor volume ( gtv ) , also known as the d95 , changed on an average of only 1.4% when normal breathing effects were incorporated . ", "therefore , the authors concluded that while the chance of a 10% or greater decrease in d95 was less than 4% , patients with a large degree of respiratory motion could have significant effects , and thus these patients should be identified . a study from the university hospital rotterdam in the netherlands found that with a gtv to planning target volume ( ptv ) margin of 1.5 cm , approximately 11% of the tumor was not covered in mobile tumors . ", "engelsman et al . found that when combined with setup error , respiratory motion reduced the tumor control probability ( tcp ) by almost 9% ( from 50% to 42% ) in patients receiving conventionally fractionated radiation to 70 gy in lung tumors . ", "there are several methods in which tumor motion can be taken into account with the delivery of radiation therapy and which are utilized at our institution . ", "first , breathing can be monitored during simulation and the igtv / itv can then be added as a margin to ensure that the tumor is treated adequately throughout all phases of the respiratory cycles as outlined above ( e.g. , if the tumor is noted to move 1.5 cm during treatment , then a 1.5 cm margin , termed the itv / igtv , is added to account for this respiratory motion . ) . ", "if 4d ct imaging is not available at the institution , then an alternative method to account for respiratory motion is through the use of breath - hold spiral ct simulation . in this method ,", "the patient is instructed to hold his breath during simulation , both at end inspiration and expiration . ", "images are then taken at end - inspiration and end - expiration , such that an itv can be generated by combining the two ctvs from the inspiratory and expiratory scans . a disadvantage to the free - breathing method", "is that in tumors that have a large magnitude of motion , the amount of normal tissue that is treated can be relatively large , since radiation is being delivered throughout the breathing cycle . ", "therefore , an alternative method of radiation delivery is to instruct the patient to hold their breath during treatment and activate the radiation while the tumor is in this full inspiratory , fixed position . ", "the disadvantage to this technique ( often termed deep inspiratory breath hold , or disb ) is that it requires full cooperation of the patient , in that the patient will need to hold their breath for 15 seconds or longer . as an alternative to this method", ", radiation could be delivered in either a relaxed inspiratory position or in expiration , which is often more reproducible than disb . in patients that are not able to comply with any of these instructions , a third method of delivering radiation in tumors that have a great deal of motion", "is through a ventilatory - gated approach , during which the radiation beam is coordinated with the respiratory cycle through the placement of externally placed fiducial markers . or , in other words , the radiation is only delivered at certain phases , most typically full expiration .", "this type of therapy is generally most useful for tumors less than 5 cm in diameter and for a tumor motion of greater than 1 cm . ", "figure 2 demonstrates the motion of a tumor in the inferior portion of the lung . ", "it is evident that with timed radiation delivery , a great deal of normal tissue could be spared by reducing the treatment margins .", "vlachaki et al . assessed 10 patients with lung tumors to determine the effect of respiratory gating on dosimetry . ", "gated images were acquired at full inspiration , full expiration , and at each quartile of respiratory movement . the authors found that gating led to higher minimum target volume doses , and that the v20 was reduced from 35% to 26% for gated plans . ", "the mean lung , heart , and esophageal doses were also lower with gated plans . ", "the authors concluded that gating could improve the dose to normal structures while maintaining target tissue coverage .", "underberg et al . examined 31 patients simulated with 4d ct scans and compared the dose to normal tissue in three different techniques to account for breathing motion in target delineation : ( 1 ) standard , population - based margins to account for internal motion , ( 2 ) the generation of an itv based on tumor mobility in three consecutive phases , and ( 3 ) a ptv generated from respiratory gating . ", "margins led to unnecessary normal tissue irradiation , and that the risk was best minimized if gating was utilized .", "a recent study from fox chase cancer center arrived at a similar conclusion , in that 4d ct - based treatment planning maintains target coverage while reducing normal tissue dose . ", "it is important to note that the intrafractional adaptive radiotherapy techniques of cbct and breathing control are not mutually exclusive , but in contrast should be utilized together to optimize the therapeutic ratio , as demonstrated by several studies . ", "koch et al . found that the relationship between internal lung motion and skin fiducial motion is complex and unpredictable , and that the ap motion of tumors correlated poorly with skin surface markers . ", "they also found that there is significant intersubject variability . in a follow - up study , liu et al . found that this variability ( and in turn , the target volume margins ) could be substantially reduced with the use of respiratory gating techniques . and in a study by nelson et al . , the authors assessed the margins necessary to account for uncertainties in tumor position with respiratory gating , image - guided patient setup , neither , or both . ", "the authors found that utilizing both methods simultaneously allowed for the greatest reduction in margins that completely encompassed the tumor , and therefore concluded that when respiratory motion management is used , it should be used in conjunction with image - guided patient setup in order to reduce the overall treatment margin effectively .", "another delivery system of increasingly widespread use is cyberknife therapy , which utilizes a stereotactic guidance system designed primarily for radiosurgery in multiple organ systems . ", "the premise behind this system is that the linear accelerator is mounted on a robotic arm , which can then track the target during treatment . ", "cyberknife has been reported in single institution studies in the setting of sbrt for lung cancers . ", "for example , le et al . reported 32 patients with lung tumors treated in a dose escalation study using single fractions , and found a 1-year local control rate of 91% for doses greater than 20 gy and at doses less than 25 gy there was no significant toxicity .", "brown et al . assessed the efficacy of this technique in peripherally located stage i nonsmall cell lung cancers . in a cohort of 31 patients ,", "the authors reported no grade 3 or higher toxicities and 1-year local control rates of 93.2% .", "further studies on this technique will continue to define its role in adaptive radiotherapy and the definitive treatment of nsclc . ", "more recently , novalis tx provides stereo x - ray targeting and adaptive gating using exactrac x - ray 6d and snap verification system .", "reported 94.4% local control with minimal toxicities with 50 gy delivered in 5 fractions using the novalis / brain lab system .", "all of these novel systems may provide an optimal treatment for clinical challenging cases such as patients with poor lung function and/or lesions close to critical structures .", "several factors change during the course of radiation can affect target and normal tissue dose . ", "these factors include but are not limited to changes in tumor size , alterations in tissue anatomy , variations in respiratory patterns , and reductions in patient weight . in this setting , adaptive radiotherapy refers to repeat assessment of the target volume , either through repeat cbct , 4d imaging , or both . ", "multiple studies have been performed examining causative factors of interfractional changes and the effect of these variations on dosimetry throughout treatment . ", "examined 10 patients to determine whether tumor excursion due to respiratory motion was stable when comparing images acquired at the time of simulation with those during treatment . ", "the authors found that while there was interfraction consistency in tumor excursion during treatment , the relationship between the gtv and other anatomic structures between respiratory changes varied with rescanning . ", "the authors therefore expressed caution in relying on surrogate anatomic markers to assess tumor motion throughout treatment . ", "bosmans et al . assessed 23 patients with locally advanced nsclc who underwent ct - pet and respiration - correlated imaging prior to treatment , and repeated at the first and second weeks after the start of radiation . ", "the authors observed that while changes in tumor motion were relatively small , there was a great deal of variation in tumor size during therapy . ", "these changes ranged from an increase in greater than 30% to a decrease of the same magnitude . ", "the authors concluded that the changes in tumor size warranted assessment for replanning during the course of therapy .", "van zwienen et al . found that clinically evident regression occurred in approximately 40% of patients undergoing definitive treatment for nsclc , with approximately 10% of patients undergoing > 25% reduction in size by week three and in 24 out of 114 patients by week four . ", "a study from johns hopkins university supported this finding of large variations in the gtv , and thus also recommended an adaptive approach in conventionally fractionated patients . ", "britton et al . analyzed the effect of gross tumor volume regression and motion changes during the course of radiation therapy in locally advanced nsclc at md anderson cancer center . ", "the authors found that in 8 patients with weekly 4dct data sets , the tumor volume was reduced by a range of 1571% . ", "there was also noted to be increased tumor mobility in the si and ap directions throughout treatment , without any clear trends in tumor motion . in a follow - up study at the same institution , the authors found that the dose to 95% of the ptv and itv with weekly ct scans changed by approximately 12% and 2.5% , respectively . while the lung v20 and mean lung dose only increased by a mean of 3.1% and 2.2% , respectively , the spinal cord dose changed by an average of 34.3% .", "these studies together imply that continued assessment of the target volume is recommended and is essential in tumors that lie near the spinal cord . ", "figure 3 demonstrates the significant reduction in size of a tumor throughout treatment , and the large effect that this reduction has on the dose distribution . a study from denmark demonstrated that anatomical and motion changes persist even with respiratory gating . in a study by juher - nottrup et al .", ", ten patients receiving 60 gy in 2 gy fractions underwent serial 4d ct scans during treatment . ", "the authors found that the interfractional overlap of lung tumors was only 80%87% when bony landmarks were used with a gating technique , and that this overlap decreased to 70%76% when skin tattoos were present . with mediastinal tumors ,", "it can therefore be concluded that gating does not preclude interfractional adaptive planning in patients with lung or mediastinal tumors . ", "weight loss can be a factor in changing anatomy and in turn the dose to target volume , such as in the case of a patient that loses a great deal of weight that causes the effective beam path to change . ", "some investigators have questioned whether weight loss in itself can be a cause of setup error during the course of treatment due to factors such as changes in the position of skin marks . ", "johansen et al . attempted to answer this question by evaluating the relationship of interfractional setup errors with body mass index and weight loss . ", "the authors assessed 34 head and neck cancer patients and 20 lung cancer patients who received serial cbct images to evaluate whether there was a change in 3d position between the initial cbct scans and those from the 10th and 20th treatment session . ", "the study did not find a statistically significant correlation between setup error and either patient body mass index or weight loss .", "therefore , while it is still recommended that replanning is performed on the basis of weight changes during treatment , interfractional changes as a result of weight loss may be more a function of subsequent anatomical variations rather than true setup error .", "similar studies as above quantitating interfractional dosimetric error have been performed in patients being treated with sbrt . ", "matsugi et al . examined 4d ct scans for 8 patients being treated with sbrt , to measure interfraction variations in position and the size of target volumes with this technique . ", "in contrast to a conventionally fractionated treatment which is several weeks in duration , the authors found that the size of the gtv did not change significantly during treatment and that variations in motion range and position were also small .", ". however , the target volume is usually small in sbrt and a large fraction size is delivered in less than 5 fractions ; therefore , missing a small volume of target even just in one fraction can cause significant underdosing of the tumor and/or overdosing surrounding normal tissues .", "we therefore reiterate that we recommend daily volumetric verification for target coverage in sbrt . with the advent of proton therapy ,", "investigators have recently begun to examine the effect of interfractional motion and anatomic changes on dose distribution with this technique . ", "this issue is particularly important with protons because the impact of motion and anatomy changes is more significant in proton compared with photon ( figure 3 ) . ", "hui and chang et al . acquired weekly 4d - ct scans on 8 patients with locally advanced nsclc treated with imrt .", "a conformal passive scattering plan was generated for each patient and compared with imrt plan over 7 weeks of radiotherapy . ", "the authors found that normal tissue doses were increased and ctv coverage was significantly compromised in one patient ( with a decrease in approximately 8% ) with proton therapy but much less significantly in the imrt plan . ", "the authors concluded that proton therapy is more sensitive to motion and anatomy changes compared with photon and interfractional adaptive planning and is indicated in select patients . at our institution , all patients undergo a 4d ct simulation to assess for internal motion . ", "patients are immobilized with an upper body cradleand t - bar , which has a daily setup uncertainty of approximately 7 mm . at the time of simulation ,", "whose tumor motion is less than 1 cm , a free - breathing ", "technique is typically used , with the creation of an itv or igtv and radiation treatment delivery in all phases of the breathing cycle . ", "if the target volume moves more than 1 cm and the patient can breathe reproducibly , then radiation is either timed with certain phases of the breathing cycle while the patient breathes freely ( ventilatory gated technique ) , or the patient is instructed to hold their breath for at least 15 seconds while radiation is delivered at deep inspiration ( disb ) . ", "we utilize visual and/or audio feedback guidance for patients who can comply with these devices . ", "patients undergoing standard fractionated regimens are initially set up daily with kv imaging for verification , which reduces the setup margin to 5 mm . ", "all patients are assessed for concordance between bony / skin landmarks and tumor setup . if it is found that these setup parameters are discordant , or that the tumor is changing rapidly , cbct is employed , which reduces the itv to ptv margin to 3 mm . ", "all patients receiving sbrt undergo volumetric verification of set up and motion such as cbct prior to each treatment . ", "repeat 4d simulations are performed selectively at the physician 's discretion in patients with nsclc undergoing conventional fractionation for 6 - 7 weeks and are routinely performed in patients with small cell lung carcinoma undergoing hyperfractionated ( twice a day ) or dose escalated / accelerated radiotherapy for 3 to 7 weeks .", "adaptive 4d replanning is performed if motion / anatomy changes may change the target coverage and/or increase dose to surrounding critical structures .", "interfractional adaptive 4d planning is not routinely utilized on patients undergoing sbrt ( 4 - 10 fractions ) , as these patients receive daily volumetric imaging . ", "it is notable that it is still unclear if target volume reductions are warranted in the scenario of gtv shrinkage during the course of radiotherapy [ 5052 ] .", "some physicians advocate an approach in which the target volume remains constant due to concerns for residual microscopic disease . to address these concerns , one option is to deliver at least 50 gy , the standard dose for microscopic disease , to the original target volume , and then to boost the reduced volume to the full dose . ", "new technologies are evolving to improve adaptive radiation therapy , such that the high dose region is focused on the target while sparing critical normal tissues .", "treatment planning based on 4d ct images and on - board image - guided adaptive treatment delivery assists the radiation oncologist in tracking tumor motion and targeting the tumor precisely . as advancements in technology have allowed for both intra- and interfraction replanning , investigators have begun to assess whether or not the response of tumor during treatment can lead to changes in total dose to portions of the target volume based on the magnitude of tumor response to the initial phase of treatment ( image - guided adaptive treatment ) . ", "this strategy is particularly relevant because several studies have shown that dose escalation in lung cancer improves local control . furthermore , prior studies have demonstrated that regions with high suvs and hypoxic areas are more likely to exhibit local failure when treated with radiation [ 54 , 55 ] .", "the information thus suggests that the radioresistance and propensity for distant metastasis in gross disease varies based on factors other than histologic type , stage , and tumor grade . ", "it is in this realm that adaptive dose escalation may have a role in addition to the utility of imrt dose painting .", "performed a pilot study in which 14 patients with nsclc underwent repeat pet - ct scans prior to the start of radiation therapy and mid - way through treatment . ", "boost fields were then designed based on residual pet avidity . in this dosimetric study , the authors found that this method allowed for a mean dose escalation of 58 gy or a reduction in normal tissue complication probability ( ntcp ) of up to 3% in patients with a reduction in tumor size . in a similar study , gillham et al .", "performed an additional pet - ct scan in week 5 or 6 of radiotherapy for patients being treated with localized inoperable nsclc to 66 gy in 2 gy fractions . ", "the authors found that there was a median ptv reduction of 20% , and that in this dosimetric study , dose escalation to 78 gy was feasible in four out of ten patients without exceeding normal tissue constraints .", "future studies will determine whether this technique of adaptive dose escalation is feasible from a clinical standpoint without increasing toxicity and while maintaining tumor control .", "an additional technologic advancement in the context of adaptive radiotherapy is the development of real - time ", "tracking of tumor motion so that the treatment can be actively and variably adapted in concordance with intrafractional changes . ", "in addition to cyberknife , which has been used clinically as discussed above , another commercially available system is the calypso medical three- or four - dimensional electromagnetic tracking system . ", "the system has undergone initial assessment in a wide variety of tumor sites and several studies have been published describing the technique . ", "for example , smith et al . compared dosimetric results with and without real - time tracking . ", "the authors found that the dose profiles were comparable with an idealized gating algorithm while minimizing the uncertainties inherent in the use of anatomical surrogates for target location , with a high level of efficiency . in a follow - up study ,", "the same authors proposed a method for linear accelerator gating with wireless internal fiducial markers without the requirement for ionizing radiation for imaging , leading to improvements in the dose distribution . adaptive radiotherapy may improve tumor control in lung cancer by reducing target misses , escalating target dose , and minimizing side effects by avoiding critical structures over the course of radiotherapy ." ]

[ "<S> the challenges of lung cancer radiotherapy are intra / inter - fraction tumor / organ anatomy / motion changes and the \n need to spare surrounding critical structures . evolving radiotherapy technologies , such as four - dimensional ( 4d ) image - based motion management , daily on - board imaging and adaptive radiotherapy based on volumetric images over the course of radiotherapy , </S>", "<S> have enabled us to deliver higher dose to target while minimizing normal tissue toxicities . </S>", "<S> the image - guided radiotherapy adapted to changes of motion and anatomy has made the radiotherapy more precise and allowed ablative dose delivered to the target using novel treatment approaches such as intensity - modulated radiation therapy , stereotactic body radiation therapy , and proton therapy in lung cancer , techniques used to be considered very sensitive to motion change . </S>", "<S> future clinical trials using real time tracking and biological adaptive radiotherapy based on functional images are proposed . </S>" ]

[ "1. Introduction", "2. Important Definitions in Adaptive Radiation for Lung Cancer", "3. The Role of Adaptive Radiotherapy in Intrafraction Adaptive Planning", "4. The Role of Adaptive Radiotherapy in Interfractional Changes", "5. Future Directions in Adaptive Radiotherapy" ]

[ [ "the treatment of lung cancer with radiation has undergone significant improvements over the past decade . ", "first , it is possible to better delineate the target volume through the advancement of imaging such as positron emission tomography ( pet ) fusion [ 1 , 2 ] .", "second , there has been an increase in the utilization of radiation planning and delivery systems such as intensity - modulated radiation therapy ( imrt ) , stereotactic body radiation therapy ( sbrt ) , and proton therapy that allow more conformal delivery of radiation to achieve dose escalation while minimizing toxicity to normal structures [ 6 , 7 ] . ", "finally , the advancement of imaging modalities available during the planning and delivery process has made it feasible to adapt the target volume , both within a given fraction of treatment and between fractions , for such factors as internal motion , tumor response , anatomical changes , and weight loss . with the availability of 4d ct(four - dimensional computerized tomography)based radiotherapy planning and on - board imaging ( obi ) , accurate positioning of the target using daily image guidance", "may result in many advantages , such as a decreased probability of target miss , smaller setup margins , and less normal tissue exposed to high radiation doses .", "in addition , novel treatment adaptation algorithms can provide more effective modification of treatment plans to adapt to the changes in a patient 's anatomy and organ motion during and/or between ( intra- and/or inter- ) treatment fractions .", "adaptive radiotherapy , and will be the focus of this paper . in this examination of adaptive radiation in the setting of lung cancer , we will assess the impact of this development in sbrt , conventionally fractionated imrt , and proton therapy . ", "we will first define key terms in adaptive radiation , and will then review prior studies assessing the magnitude of intrafractional changes , which we define as changes within a given fraction of radiation , from setup to delivery .", "this discussion will be followed by the effect of these changes on delivered dose and specific planning techniques that have been utilized to decrease this variation , specifically cone beam computed tomography ( cbct ) and respiratory gating . ", "we will then focus on interfraction changes that occur during the length of treatment ( over a period of several weeks ) , such as changes in tumor mobility , tumor volume , anatomy , and body weight , and examine specific studies that assess the impact of adaptive planning on improving dose distributions in these setting . ", "finally , we will appraise future directions of adaptive radiation , including selective dose escalation through the visualization of nonresponsive regions and internal tracking to monitor intrafraction motion in real - time , both of which may be able to allow for further sparing of critical structures while maintaining accurate delivery for treatment of this aggressive disease ." ], [ "50 : . tumor visible by any imaging modality , to include both the primary tumor and any involved lymph nodes . ", "lymph nodes greater than 1 cm in size in the shortest axis are generally considered positive for disease , but functional imaging such as pet scanning is critical for target delineation [ 10 , 11 ] .", "this region can not be visualized as a discrete structure with radiographic imaging , and the extent has been defined by surgical series with pathologic correlates , as well as autopsy series . as defined by giraud et al .", ", based on nsclc surgical specimens , an appropriate gtv to ctv margin for adenocarcinoma is 8 mm , and for squamous cell carcinoma , 6 mm . ", "another study found that in stage i adenocarcinoma , a margin of 9 mm is sufficient to cover 90% of disease . in terms of mediastinal disease , a gtv to ctv margin has not been rigorously analyzed , but based on an abstract presented at the 2006 meeting of the american society for therapeutic radiology and oncology that found a maximal microscopic extension of lymph nodes is between 0.5 and 8.9 mm , we routinely use margins of 8 mm for nodal disease . ctv with a margin to account for organ motion ; or , in other words , changes in the size , shape , and position of the target during treatment . delineating the itv from 4d ct images involves assessing the target volume ( ctv ) on expiratory - phase images and then registering the outline to the images from other phases to create a union of target contours enclosing all possible positions of the target .", "an alternative method is to create a maximum intensity projection ( mip ) image by combining data from the multiple ct data sets with data from the whole - breath cycle and modifying the itv by visually verifying the target volume throughout the breathing phases ( typically 10 ) . in this process", ", attention should be paid to irregular breathing and breathing pattern variations during each treatment session and over the entire treatment course , as well as to the effects of these irregularities on the itv margin . because it is", "often more straightforward to delineate the boundaries of the gross tumor volume with motion on 4d ct image data sets as opposed to the clinical target volume , we previously proposed the concept of the internal gross tumor volume ( igtv ) , which envelops the gtv motion throughout the whole - breath cycle . in this process , rather than delineating the gtv , expanding to the ctv , and then adding the itv , the gtv is contoured , motion is assessed as outlined above ( i.e. , through the mip images or through all breathing phases ) , and this target is expanded to the igtv . ", "then , the igtv is expanded to the itv ( itv = igtv + ctv ) . ", "this latter method is what is commonly utilized at our institution to account for organ motion in the treatment of nsclc . ", "ctv plus a margin to account for both organ motion and daily setup . or", ", the itv with a margin to account for daily setup . the application and revision of margins for the ptv and itv will be discussed in detail below ." ], [ "bissonnette et al . examined cbct images during each fraction of 18 patients receiving sbrt for medically inoperable stage i nonsmall cell lung cancer ( nsclc ) . ", "the cbct images were performed at the beginning , midpoint , and end of each fraction to determine differences in tumor motion amplitude . ", "the authors found that at a mean time of 35 minutes ( from the beginning of each treatment to the end ) , the mean change in tumor amplitude was 0.4 , 1.0 , and 0.4 mm in the medial - lateral ( ml ) , superior - inferior ( si ) , and anterior - posterior ( ap ) directions , respectively . ", "these values were not statistically significantly different when compared to the initial respiratory correlated ( 4d ) ct scan , other than in patients in which abdominal compression was used , in which longer times on the couch were hypothesized to increase these differences . in another study ,", "michalski et al . compared the amount of motion between the three aforementioned directional axes ( ml , si , and ap ) in 23 patients undergoing 4d ct scans . the authors found that the largest intrafractional extent of motion was in the si direction , with the largest range being 3.59 cm . ", "a similar study from our institution also assessed respiration - induced tumor motion during radiation for lung cancer . ", "this study by liu et al . assessed 166 tumors in 152 lung cancer patients , 57% of whom had stage iii or iv disease . ", "the authors also found that the largest axis of motion was in the si direction , and that 39% of tumors moved > 0.5 cm in this direction , compared to 1.8% and 5.4% in the ml and ap directions , respectively . ", "tumor motion was also found to be correlated with the amount of diaphragm motion , the si location of the tumor , and the size of the gtv , with smaller tumors exhibiting a greater degree of intrafractional motion . a study by thomas et al .", "demonstrated that mediastinal nodal regions also move substantially with the respiratory cycle , generally posteriorly and superiorly with exhalation , and that inferior nodal stations exhibit a greater degree of motion , such that the same issues with respiratory variation in parenchymal tumors can also be applied to mediastinal disease . ", "thus , it can be concluded that : ( 1 ) inferior tumors move more than superior tumors , ( 2 ) the largest axis of motion is in the si direction , and ( 3 ) mediastinal lymph nodes are also subject to a significant degree of tumor motion .", "there are several methods to detect setup error and intrafractional variation during radiation therapy for nsclc . in this technique ,", "two - dimensional images are composed in the treatment position , and the patient is set up on patient anatomical landmarks such as bony anatomy . ", "historically , megavoltage ( mv ) imaging had been used , but the images produced with this technique were generally of poor image quality and exposed the patient to high radiation doses . in the past decade , the utilization of kilovoltage ( kv ) imaging has increased , which has improved image quality and decreased radiation exposure . in this method ,", "a ct image is reconstructed on the treatment table from a set of projection images acquired at multiple angles around the patient . ", "image reconstruction with this modality differs from conventional ct scans in that rather than a linear array of detectors being back projected to construct a 2d slice , a detector array ", "( e.g. , a flat - panel portal imager ) is used to reconstruct a 3d data set . ", "( figure 1 ) it follows that the ct images that are a result of this process are theoretically superior in improving intrafractional error as compared to portal images , and multiple studies have exhibited this improvement , particularly in sbrt where longer treatment times can lead to greater instability and less reproducibility of the setup process . ", "intrafraction treatment variation has two components in lung cancer : uncertainties in patient positioning and variations in internal motion .", "nelson et al . examined the effect of lung tumor motion and setup uncertainties using implanted fiducial markers . ", "the authors found that systematic and random uncertainties ranged between 4 and 6 mm in all three directions . with daily portal imaging , the authors found in a subsequent study that alignment based on implanted fiducials reduced systematic errors in the left - right and superior - inferior direction each by 3 mm . as a result , daily portal imaging", "is often utilized to decrease setup error in conventionally fractionated regimens , with a daily setup error of up to 5 mm . ", "borst et al . compared cbct setup with portal imaging in daily setup for 62 patients in 524 scans with nsclc , and found that cbct reduced the setup error to less than 5 mm , from 51% of patients with setup errors more than 5 mm to 2% with cbct .", "bissonnette et al . assessed the accuracy of cbct in rt for lung cancer in patients receiving both sbrt for early - stage malignancies and in those patients undergoing conventionally fractionated treatment for locally advanced disease . ", "the couch position was adjusted for discrepancies greater than 3 mm between the initial setup and treatment images . ", "the accuracy of this adjustment was then verified with a second cbct . without cbct adjustments ,", "positioning errors were found to exceed 5 mm in approximately 55% of patients . however , cbct decreased this error , such that systematic and random setup margin was within 3 mm for 82% of fractions in the sbrt group , and between 76% and 84% of the patients in the conventionally fractionated group .", "similar findings were published by grills et al . , who found that cbct image guidance significantly decreased setup margins in sbrt , with calculated precorrection population margins of 913 mm and 1014 mm with a stereotactic body frame and alpha cradle , respectively , while these same margins were 1 - 2 mm and 2 - 3 mm postcorrection , and 24 mm and 25 mm postreatment , respectively .", "the conclusion of these studies is that consistent cbct imaging can decrease the amount of systematic and random error on a daily basis ( to within 3 mm ) , and thus decrease intrafraction variation .", "lung tumors differ from many other treatment sites in that internal motion can account for large variations in tumor position , and as a result , dosimetry . ", "mechalakos et al . examined treatment plans for 12 patients receiving radiation therapy for nsclc . ", "the authors found that the dose to 95% of the gross tumor volume ( gtv ) , also known as the d95 , changed on an average of only 1.4% when normal breathing effects were incorporated . ", "therefore , the authors concluded that while the chance of a 10% or greater decrease in d95 was less than 4% , patients with a large degree of respiratory motion could have significant effects , and thus these patients should be identified . a study from the university hospital rotterdam in the netherlands found that with a gtv to planning target volume ( ptv ) margin of 1.5 cm , approximately 11% of the tumor was not covered in mobile tumors . ", "engelsman et al . found that when combined with setup error , respiratory motion reduced the tumor control probability ( tcp ) by almost 9% ( from 50% to 42% ) in patients receiving conventionally fractionated radiation to 70 gy in lung tumors . ", "there are several methods in which tumor motion can be taken into account with the delivery of radiation therapy and which are utilized at our institution . ", "first , breathing can be monitored during simulation and the igtv / itv can then be added as a margin to ensure that the tumor is treated adequately throughout all phases of the respiratory cycles as outlined above ( e.g. , if the tumor is noted to move 1.5 cm during treatment , then a 1.5 cm margin , termed the itv / igtv , is added to account for this respiratory motion . ) . ", "if 4d ct imaging is not available at the institution , then an alternative method to account for respiratory motion is through the use of breath - hold spiral ct simulation . in this method ,", "the patient is instructed to hold his breath during simulation , both at end inspiration and expiration . ", "images are then taken at end - inspiration and end - expiration , such that an itv can be generated by combining the two ctvs from the inspiratory and expiratory scans . a disadvantage to the free - breathing method", "is that in tumors that have a large magnitude of motion , the amount of normal tissue that is treated can be relatively large , since radiation is being delivered throughout the breathing cycle . ", "therefore , an alternative method of radiation delivery is to instruct the patient to hold their breath during treatment and activate the radiation while the tumor is in this full inspiratory , fixed position . ", "the disadvantage to this technique ( often termed deep inspiratory breath hold , or disb ) is that it requires full cooperation of the patient , in that the patient will need to hold their breath for 15 seconds or longer . as an alternative to this method", ", radiation could be delivered in either a relaxed inspiratory position or in expiration , which is often more reproducible than disb . in patients that are not able to comply with any of these instructions , a third method of delivering radiation in tumors that have a great deal of motion", "is through a ventilatory - gated approach , during which the radiation beam is coordinated with the respiratory cycle through the placement of externally placed fiducial markers . or , in other words , the radiation is only delivered at certain phases , most typically full expiration .", "this type of therapy is generally most useful for tumors less than 5 cm in diameter and for a tumor motion of greater than 1 cm . ", "figure 2 demonstrates the motion of a tumor in the inferior portion of the lung . ", "it is evident that with timed radiation delivery , a great deal of normal tissue could be spared by reducing the treatment margins .", "vlachaki et al . assessed 10 patients with lung tumors to determine the effect of respiratory gating on dosimetry . ", "gated images were acquired at full inspiration , full expiration , and at each quartile of respiratory movement . the authors found that gating led to higher minimum target volume doses , and that the v20 was reduced from 35% to 26% for gated plans . ", "the mean lung , heart , and esophageal doses were also lower with gated plans . ", "the authors concluded that gating could improve the dose to normal structures while maintaining target tissue coverage .", "underberg et al . examined 31 patients simulated with 4d ct scans and compared the dose to normal tissue in three different techniques to account for breathing motion in target delineation : ( 1 ) standard , population - based margins to account for internal motion , ( 2 ) the generation of an itv based on tumor mobility in three consecutive phases , and ( 3 ) a ptv generated from respiratory gating . ", "margins led to unnecessary normal tissue irradiation , and that the risk was best minimized if gating was utilized .", "a recent study from fox chase cancer center arrived at a similar conclusion , in that 4d ct - based treatment planning maintains target coverage while reducing normal tissue dose . ", "it is important to note that the intrafractional adaptive radiotherapy techniques of cbct and breathing control are not mutually exclusive , but in contrast should be utilized together to optimize the therapeutic ratio , as demonstrated by several studies . ", "koch et al . found that the relationship between internal lung motion and skin fiducial motion is complex and unpredictable , and that the ap motion of tumors correlated poorly with skin surface markers . ", "they also found that there is significant intersubject variability . in a follow - up study , liu et al . found that this variability ( and in turn , the target volume margins ) could be substantially reduced with the use of respiratory gating techniques . and in a study by nelson et al . , the authors assessed the margins necessary to account for uncertainties in tumor position with respiratory gating , image - guided patient setup , neither , or both . ", "the authors found that utilizing both methods simultaneously allowed for the greatest reduction in margins that completely encompassed the tumor , and therefore concluded that when respiratory motion management is used , it should be used in conjunction with image - guided patient setup in order to reduce the overall treatment margin effectively .", "another delivery system of increasingly widespread use is cyberknife therapy , which utilizes a stereotactic guidance system designed primarily for radiosurgery in multiple organ systems . ", "the premise behind this system is that the linear accelerator is mounted on a robotic arm , which can then track the target during treatment . ", "cyberknife has been reported in single institution studies in the setting of sbrt for lung cancers . ", "for example , le et al . reported 32 patients with lung tumors treated in a dose escalation study using single fractions , and found a 1-year local control rate of 91% for doses greater than 20 gy and at doses less than 25 gy there was no significant toxicity .", "brown et al . assessed the efficacy of this technique in peripherally located stage i nonsmall cell lung cancers . in a cohort of 31 patients ,", "the authors reported no grade 3 or higher toxicities and 1-year local control rates of 93.2% .", "further studies on this technique will continue to define its role in adaptive radiotherapy and the definitive treatment of nsclc . ", "more recently , novalis tx provides stereo x - ray targeting and adaptive gating using exactrac x - ray 6d and snap verification system .", "reported 94.4% local control with minimal toxicities with 50 gy delivered in 5 fractions using the novalis / brain lab system .", "all of these novel systems may provide an optimal treatment for clinical challenging cases such as patients with poor lung function and/or lesions close to critical structures ." ], [ "several factors change during the course of radiation can affect target and normal tissue dose . ", "these factors include but are not limited to changes in tumor size , alterations in tissue anatomy , variations in respiratory patterns , and reductions in patient weight . in this setting , adaptive radiotherapy refers to repeat assessment of the target volume , either through repeat cbct , 4d imaging , or both . ", "multiple studies have been performed examining causative factors of interfractional changes and the effect of these variations on dosimetry throughout treatment . ", "examined 10 patients to determine whether tumor excursion due to respiratory motion was stable when comparing images acquired at the time of simulation with those during treatment . ", "the authors found that while there was interfraction consistency in tumor excursion during treatment , the relationship between the gtv and other anatomic structures between respiratory changes varied with rescanning . ", "the authors therefore expressed caution in relying on surrogate anatomic markers to assess tumor motion throughout treatment . ", "bosmans et al . assessed 23 patients with locally advanced nsclc who underwent ct - pet and respiration - correlated imaging prior to treatment , and repeated at the first and second weeks after the start of radiation . ", "the authors observed that while changes in tumor motion were relatively small , there was a great deal of variation in tumor size during therapy . ", "these changes ranged from an increase in greater than 30% to a decrease of the same magnitude . ", "the authors concluded that the changes in tumor size warranted assessment for replanning during the course of therapy .", "van zwienen et al . found that clinically evident regression occurred in approximately 40% of patients undergoing definitive treatment for nsclc , with approximately 10% of patients undergoing > 25% reduction in size by week three and in 24 out of 114 patients by week four . ", "a study from johns hopkins university supported this finding of large variations in the gtv , and thus also recommended an adaptive approach in conventionally fractionated patients . ", "britton et al . analyzed the effect of gross tumor volume regression and motion changes during the course of radiation therapy in locally advanced nsclc at md anderson cancer center . ", "the authors found that in 8 patients with weekly 4dct data sets , the tumor volume was reduced by a range of 1571% . ", "there was also noted to be increased tumor mobility in the si and ap directions throughout treatment , without any clear trends in tumor motion . in a follow - up study at the same institution , the authors found that the dose to 95% of the ptv and itv with weekly ct scans changed by approximately 12% and 2.5% , respectively . while the lung v20 and mean lung dose only increased by a mean of 3.1% and 2.2% , respectively , the spinal cord dose changed by an average of 34.3% .", "these studies together imply that continued assessment of the target volume is recommended and is essential in tumors that lie near the spinal cord . ", "figure 3 demonstrates the significant reduction in size of a tumor throughout treatment , and the large effect that this reduction has on the dose distribution . a study from denmark demonstrated that anatomical and motion changes persist even with respiratory gating . in a study by juher - nottrup et al .", ", ten patients receiving 60 gy in 2 gy fractions underwent serial 4d ct scans during treatment . ", "the authors found that the interfractional overlap of lung tumors was only 80%87% when bony landmarks were used with a gating technique , and that this overlap decreased to 70%76% when skin tattoos were present . with mediastinal tumors ,", "it can therefore be concluded that gating does not preclude interfractional adaptive planning in patients with lung or mediastinal tumors . ", "weight loss can be a factor in changing anatomy and in turn the dose to target volume , such as in the case of a patient that loses a great deal of weight that causes the effective beam path to change . ", "some investigators have questioned whether weight loss in itself can be a cause of setup error during the course of treatment due to factors such as changes in the position of skin marks . ", "johansen et al . attempted to answer this question by evaluating the relationship of interfractional setup errors with body mass index and weight loss . ", "the authors assessed 34 head and neck cancer patients and 20 lung cancer patients who received serial cbct images to evaluate whether there was a change in 3d position between the initial cbct scans and those from the 10th and 20th treatment session . ", "the study did not find a statistically significant correlation between setup error and either patient body mass index or weight loss .", "therefore , while it is still recommended that replanning is performed on the basis of weight changes during treatment , interfractional changes as a result of weight loss may be more a function of subsequent anatomical variations rather than true setup error .", "similar studies as above quantitating interfractional dosimetric error have been performed in patients being treated with sbrt . ", "matsugi et al . examined 4d ct scans for 8 patients being treated with sbrt , to measure interfraction variations in position and the size of target volumes with this technique . ", "in contrast to a conventionally fractionated treatment which is several weeks in duration , the authors found that the size of the gtv did not change significantly during treatment and that variations in motion range and position were also small .", ". however , the target volume is usually small in sbrt and a large fraction size is delivered in less than 5 fractions ; therefore , missing a small volume of target even just in one fraction can cause significant underdosing of the tumor and/or overdosing surrounding normal tissues .", "we therefore reiterate that we recommend daily volumetric verification for target coverage in sbrt . with the advent of proton therapy ,", "investigators have recently begun to examine the effect of interfractional motion and anatomic changes on dose distribution with this technique . ", "this issue is particularly important with protons because the impact of motion and anatomy changes is more significant in proton compared with photon ( figure 3 ) . ", "hui and chang et al . acquired weekly 4d - ct scans on 8 patients with locally advanced nsclc treated with imrt .", "a conformal passive scattering plan was generated for each patient and compared with imrt plan over 7 weeks of radiotherapy . ", "the authors found that normal tissue doses were increased and ctv coverage was significantly compromised in one patient ( with a decrease in approximately 8% ) with proton therapy but much less significantly in the imrt plan . ", "the authors concluded that proton therapy is more sensitive to motion and anatomy changes compared with photon and interfractional adaptive planning and is indicated in select patients . at our institution , all patients undergo a 4d ct simulation to assess for internal motion . ", "patients are immobilized with an upper body cradleand t - bar , which has a daily setup uncertainty of approximately 7 mm . at the time of simulation ,", "whose tumor motion is less than 1 cm , a free - breathing ", "technique is typically used , with the creation of an itv or igtv and radiation treatment delivery in all phases of the breathing cycle . ", "if the target volume moves more than 1 cm and the patient can breathe reproducibly , then radiation is either timed with certain phases of the breathing cycle while the patient breathes freely ( ventilatory gated technique ) , or the patient is instructed to hold their breath for at least 15 seconds while radiation is delivered at deep inspiration ( disb ) . ", "we utilize visual and/or audio feedback guidance for patients who can comply with these devices . ", "patients undergoing standard fractionated regimens are initially set up daily with kv imaging for verification , which reduces the setup margin to 5 mm . ", "all patients are assessed for concordance between bony / skin landmarks and tumor setup . if it is found that these setup parameters are discordant , or that the tumor is changing rapidly , cbct is employed , which reduces the itv to ptv margin to 3 mm . ", "all patients receiving sbrt undergo volumetric verification of set up and motion such as cbct prior to each treatment . ", "repeat 4d simulations are performed selectively at the physician 's discretion in patients with nsclc undergoing conventional fractionation for 6 - 7 weeks and are routinely performed in patients with small cell lung carcinoma undergoing hyperfractionated ( twice a day ) or dose escalated / accelerated radiotherapy for 3 to 7 weeks .", "adaptive 4d replanning is performed if motion / anatomy changes may change the target coverage and/or increase dose to surrounding critical structures .", "interfractional adaptive 4d planning is not routinely utilized on patients undergoing sbrt ( 4 - 10 fractions ) , as these patients receive daily volumetric imaging . ", "it is notable that it is still unclear if target volume reductions are warranted in the scenario of gtv shrinkage during the course of radiotherapy [ 5052 ] .", "some physicians advocate an approach in which the target volume remains constant due to concerns for residual microscopic disease . to address these concerns , one option is to deliver at least 50 gy , the standard dose for microscopic disease , to the original target volume , and then to boost the reduced volume to the full dose . " ], [ "new technologies are evolving to improve adaptive radiation therapy , such that the high dose region is focused on the target while sparing critical normal tissues .", "treatment planning based on 4d ct images and on - board image - guided adaptive treatment delivery assists the radiation oncologist in tracking tumor motion and targeting the tumor precisely . as advancements in technology have allowed for both intra- and interfraction replanning , investigators have begun to assess whether or not the response of tumor during treatment can lead to changes in total dose to portions of the target volume based on the magnitude of tumor response to the initial phase of treatment ( image - guided adaptive treatment ) . ", "this strategy is particularly relevant because several studies have shown that dose escalation in lung cancer improves local control . furthermore , prior studies have demonstrated that regions with high suvs and hypoxic areas are more likely to exhibit local failure when treated with radiation [ 54 , 55 ] .", "the information thus suggests that the radioresistance and propensity for distant metastasis in gross disease varies based on factors other than histologic type , stage , and tumor grade . ", "it is in this realm that adaptive dose escalation may have a role in addition to the utility of imrt dose painting .", "performed a pilot study in which 14 patients with nsclc underwent repeat pet - ct scans prior to the start of radiation therapy and mid - way through treatment . ", "boost fields were then designed based on residual pet avidity . in this dosimetric study , the authors found that this method allowed for a mean dose escalation of 58 gy or a reduction in normal tissue complication probability ( ntcp ) of up to 3% in patients with a reduction in tumor size . in a similar study , gillham et al .", "performed an additional pet - ct scan in week 5 or 6 of radiotherapy for patients being treated with localized inoperable nsclc to 66 gy in 2 gy fractions . ", "the authors found that there was a median ptv reduction of 20% , and that in this dosimetric study , dose escalation to 78 gy was feasible in four out of ten patients without exceeding normal tissue constraints .", "future studies will determine whether this technique of adaptive dose escalation is feasible from a clinical standpoint without increasing toxicity and while maintaining tumor control .", "an additional technologic advancement in the context of adaptive radiotherapy is the development of real - time ", "tracking of tumor motion so that the treatment can be actively and variably adapted in concordance with intrafractional changes . ", "in addition to cyberknife , which has been used clinically as discussed above , another commercially available system is the calypso medical three- or four - dimensional electromagnetic tracking system . ", "the system has undergone initial assessment in a wide variety of tumor sites and several studies have been published describing the technique . ", "for example , smith et al . compared dosimetric results with and without real - time tracking . ", "the authors found that the dose profiles were comparable with an idealized gating algorithm while minimizing the uncertainties inherent in the use of anatomical surrogates for target location , with a high level of efficiency . in a follow - up study ,", "the same authors proposed a method for linear accelerator gating with wireless internal fiducial markers without the requirement for ionizing radiation for imaging , leading to improvements in the dose distribution . adaptive radiotherapy may improve tumor control in lung cancer by reducing target misses , escalating target dose , and minimizing side effects by avoiding critical structures over the course of radiotherapy ." ] ]

[ "lipomas are benign , well circumscribed , expansile connective tissue neoplasms predominantly composed of mature white fat cells .", "they may occur anywhere in the body and usually present as slow - growing , solitary and asymptomatic subcutaneous or superficial lesions .", "about 20% of cases of lipoma affect the head and neck region with only 1%-5% of these neoplasms involving the oral cavity .", "histologically , lipomas are composed of mature adipocytes arranged in lobules that are separated by fibrous connective tissue septa and are occasionally associated with one or more secondary mesenchymal elements . different variants of lipoma have been described , such as fibrolipoma , angiolipoma , myolipoma , spindle cell lipoma , chondroid lipoma , chondrolipoma and osteolipoma .", "the buccal mucosa is the most affected intra oral site ; and lipomas and fibrolipomas are the most frequently observed histological types in the oral cavity . among the histopathological variants , lipomas with cartilaginous or osseous metaplasia , called chondrolipomas or osteolipomas respectively , have been described in the subcutaneous and deep soft tissues , particularly in the parosteal localization but are rare in the oral cavity . as per pubmed database , till date only 14 cases of chondrolipoma in oral cavity have been reported in english literature .", "herein , we report a case of this rare intraoral variant in a 35-year - old male patient .", "a 35-year - old indian male patient presented with a painless mass on the postero - dorsal region of tongue since last 20 years .", "the lesion showed slow growth and the patient reported that there was no increase in size for past many years .", "clinically , the lesion was an oval , well - circumscribed nodule measuring approximately 1.0 1.0 cm .", "on palpation , the mass was firm in consistency with no tenderness or discharge [ figure 1 ] .", "a clinical diagnosis of fibroma was made and based on the long standing history and benign appearance , an excisional biopsy was performed .", "the surgical area healed uneventfully and six - month follow - up of the patient revealed no evidence of any recurrence .", "figure shows nodular growth on the posterior dorsal surface of tongue gross examination of the excised mass revealed a pale yellowish cut surface with foci of shiny white areas [ figure 2 ] .", "microscopically , the tumor was composed of well circumscribed , encapsulated lobular mass of mature adipose tissue separated by fibrous septae .", "foci of metaplastic hyaline cartilage were evident within the mass along with delicate capillaries [ figure 3 ] .", "the cartilage cells as well as the fat cells did not show any mitosis , pleomorphism or any other histological evidence of malignancy .", "no evidence of lipoblasts or hibernoma - like cells was seen [ figure 4 ] .", "note the yellowish color of the cut surface of the lesion with foci of white hyalinized areas ( arrows ) low power photomicrograph showing stratified squamous epithelium overlying a well - circumscribed mass of adipose tissue , surrounded by a fibrous capsule and containing a focus of hyaline cartilage .", "( h&e stain , 40 ) high - power photomicrograph showing cartilaginous foci made up of mature normal appearing chondroid tissue with peripheral rim of spindle shaped lining cells .", "the first case of oral chondrolipoma was reported by mcandrew and greenspan in 1976 in a 72-year - old male in lower lip and since then only a handful of cases been reported in oral cavity .", "many clinical and histopathological characteristics of chondrolipomas of the oral cavity continue to be poorly understood because of the rarity of this tumor , together with the lack of data in the literature . a review of the pubmed database revealed 14 such cases in english literature . we have included only those cases which presented strictly within the oral cavity and histologically showed presence of only cartilaginous tissue in a predominantly mature adipocyte proliferation", "the clinicopathological features of these cases including the present case have been summarized in table 1 .", "clinical features of 14 cases of oral chondrolipoma described in english literature cases of oral chondrolipoma have been diagnosed in patients aged from 14 to 72 years , but it seems to be a tumor of older individuals with majority of cases being diagnosed after the age of 50 years .", "while these lesions are usually diagnosed in older adults , they may originate at a younger age as some of these tumors including the present case have shown long duration of presentation", ". there may be an actual bimodal age predilection as substantial number of cases ( almost one - third including the present case ) seem to originate in the first two decades of life but a larger number of cases will be required to accept or refute this observation .", "this feature of chondrolipoma slightly differs from a conventional lipoma , which are rarely seen in the first two decades of life and are thought to make their appearance at the age when fat starts accumulating in inactive individuals .", "hence , most lipomas including those of oral cavity become apparent between 5 to 7 decades of life .", "in fact all of the cases of chondrolipoma involving young individuals have been reported in recent literature probably highlighting the fact that awareness about this variant is increasing among histopathologists and possibly some of these lesions may have earlier been overlooked .", "similar to other lipomas at other sites , oral lipomas are believed to be more common in men and this seems to be true even for chondrolipomas in the oral cavity .", "oral chondrolipomas seem to have a strong predilection for occurrence on tongue but interestingly none of the lesions have been described in buccal mucosa which is the preferential site for conventional lipomas .", "histologically , variants of lipoma differ from ordinary lipoma by characteristic microscopic picture and specific clinical setting .", "this group is chiefly represented by angiolipoma , myolipoma , angiomyolipoma , myelolipoma , chondroid lipoma , spindle cell and pleomorphic lipoma .", "chondro lipomas are characterized by the proliferation of mature adipocytes with additional mature cartilaginous tissue formation .", "though this term has been used in past , it was first defined by stout for tumors that are composed of two or more mesenchymal elements . on the other hand ,", ", described mesenchymomas as an unencapsulated soft tissue neoplasms composed of two or more mature mesenchymal tissues not normally associated with each other and no single mesenchymal tissue should predominate with respect to the other mesenchymal elements .", "usually the cartilage in lipoma represents only a small part of the tumor and hence would not strictly fit to the above criteria .", "mature cartilaginous areas in a chondrolipoma should be distinguished from chondroid lipoma , a lipoma variant that is uncommon in the oral cavity and consists of mature adipocytes admixed with multivacuolated lipoblast like cells in a myxohyaline and chondroid matrix .", "this newly described lesion , having an immature aspect may give a pseudosarcomatous appearance and may be mistaken for lipoblastic or chondroblastic malignancies . on the other hand , in true chondrolipoma the adipose component is entirely composed of mature tissue with lack of any lipoblastic cells .", "due to similarity in nomenclature , the two entities are often confused with each other and it is possible they may have been diagnosed interchangeably in past . in this review", ", we have included only those cases which show typical features of a chondrolipoma and all cases showing immature lipoblast like component or hibernoma - like areas have been excluded .", "in addition , chondrolipoma may sometimes need to be differentiated from extra - skeletal chondroma specially those occurring in deep submucosal areas and hence surrounded by adipose tissue .", "histologically , chondromas are characterized by greater proportion of cartilaginous tissue arranged usually in distinct lobular pattern . whereas the chondroid component of chondrolipoma is focal and lacks any lobular arrangement .", "several hypotheses have been tried to explain the occurrence of cartilage within the mass of adipose tissue .", "one of the possible explanations could be that this tumor represents a true mesenchymoma i.e. both the chondroid as well as the adipose components being neoplastic in nature .", "the initial neoplastic change may take place in the pleuripotent mesenchymal stem cells , present in oral mucosa and these neoplastic stem cells may then differentiate into adipogenic and chondrogenic cells .", "presence of pluripotent mesenchymal stem cells , capable of such differentiation , has been demonstrated in the connective tissue of skeletal muscles and dermis and it may be prudent to assume that similar cells may also be present in the lamina propria and submucosal tissues of oral cavity .", "ability for multipotential differentiation including chondrogenic potential has been demonstrated in adipose tissue derived stem cells .", "hence , it could be possible that some of the neoplastic adipocytes may differentiate into chondroblastic cells , the stimulus being either spontaneous or a metaplastic reaction triggered by long - term irritation .", "similar metaplastic chondrogenesis has been described in peripheral fibromas of gingiva . and may be attributed to chronic mechanical stress .", "an interesting , albeit a little farfetched possibility is that the neoplastic adipose tissue may develop in a pre - existing cartilaginous choristoma .", "such choristomas , though rare , are known to occur in the oral cavity with tongue being the most common location .", "this may also explain the rarity of this lesion and preferential occurrence in tongue as compared to the higher propensity of involvement of buccal mucosa for conventional lipoma .", "finally , the lesion may not be neoplastic at all and may result from a combination of hamartomatous and choristomatous growth .", "such combined lipomaotus hamartomas and choristomas made up of mature adipose tissue containing cartilage have been described in other sites of the body as well as in inbred laboratory mice .", "it was suggested that those cases containing additional tissue elements in these chiefly lipomatous growths should be considered choristomas . even in the murine lipomatous growths", "additional tissue elements were found more frequently in long standing cases and it was considered that these additional elements like cartilage could be present at an earlier stage but may constitute only a minor volume of the lesion , hence could be easily overlooked and thus require careful complete serial sectioning of the entire growth .", "recently , role of factors like sox-9 , runx-2 , tgf- , bone morphogenic protein ( bmp ) has been discussed in chondrogenic potential of lipomas .", "we believe that oral chondrolipomas constitute a heterogenous group of lesions , some of which may represent true neoplasms while others , especially those appearing at a younger age , may be hamartomatous / choristomatous proliferations .", "the pluripotentiality of adipose derived stem cells and preferential expression and interplay of prochondrogenic molecules either through some genetic alteration or environmental factors are responsible for chondrogenesis in these lesions . whatever the pathogenesis , lesion is essentially benign and surgical excision is the treatment of choice .", "the cartilage found in the tumor most likely represents a metaplastic change or harmartomatous / choristomatous proliferation and could be attributed to multipotentiality of mesenchymal stem cells .", "the characteristics of oral lesions tend to show few deviations from the classical oral lipoma , chiefly being the marked predilection for involvement of tongue and possibly bimodal age of onset and warrants reporting of such cases with detailed clinicopathological evaluation in future ." ]

[ "<S> chondrolipomas are benign mesenchymal tumors characterized by the proliferation of mature adipocytes associated with variable amounts of mature cartilaginous tissue . herein , we describe a case of chondrolipoma of the tongue in a 35-year - old indian male . </S>", "<S> the lesion presented as a nodular , sessile , pink mass on posterio - dorsal surface of left side of the tongue since approximately 20 years . </S>", "<S> histopathologically , the mass revealed a well circumscribed , encapsulated proliferation of mature adipocytes with islands of well formed mature cartilaginous tissue . </S>", "<S> chondrolipomas are uncommon in the oral cavity , with only 14 cases being reported in the english literature . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION" ]

[ [ "lipomas are benign , well circumscribed , expansile connective tissue neoplasms predominantly composed of mature white fat cells .", "they may occur anywhere in the body and usually present as slow - growing , solitary and asymptomatic subcutaneous or superficial lesions .", "about 20% of cases of lipoma affect the head and neck region with only 1%-5% of these neoplasms involving the oral cavity .", "histologically , lipomas are composed of mature adipocytes arranged in lobules that are separated by fibrous connective tissue septa and are occasionally associated with one or more secondary mesenchymal elements . different variants of lipoma have been described , such as fibrolipoma , angiolipoma , myolipoma , spindle cell lipoma , chondroid lipoma , chondrolipoma and osteolipoma .", "the buccal mucosa is the most affected intra oral site ; and lipomas and fibrolipomas are the most frequently observed histological types in the oral cavity . among the histopathological variants , lipomas with cartilaginous or osseous metaplasia , called chondrolipomas or osteolipomas respectively , have been described in the subcutaneous and deep soft tissues , particularly in the parosteal localization but are rare in the oral cavity . as per pubmed database , till date only 14 cases of chondrolipoma in oral cavity have been reported in english literature .", "herein , we report a case of this rare intraoral variant in a 35-year - old male patient ." ], [ "a 35-year - old indian male patient presented with a painless mass on the postero - dorsal region of tongue since last 20 years .", "the lesion showed slow growth and the patient reported that there was no increase in size for past many years .", "clinically , the lesion was an oval , well - circumscribed nodule measuring approximately 1.0 1.0 cm .", "on palpation , the mass was firm in consistency with no tenderness or discharge [ figure 1 ] .", "a clinical diagnosis of fibroma was made and based on the long standing history and benign appearance , an excisional biopsy was performed .", "the surgical area healed uneventfully and six - month follow - up of the patient revealed no evidence of any recurrence .", "figure shows nodular growth on the posterior dorsal surface of tongue gross examination of the excised mass revealed a pale yellowish cut surface with foci of shiny white areas [ figure 2 ] .", "microscopically , the tumor was composed of well circumscribed , encapsulated lobular mass of mature adipose tissue separated by fibrous septae .", "foci of metaplastic hyaline cartilage were evident within the mass along with delicate capillaries [ figure 3 ] .", "the cartilage cells as well as the fat cells did not show any mitosis , pleomorphism or any other histological evidence of malignancy .", "no evidence of lipoblasts or hibernoma - like cells was seen [ figure 4 ] .", "note the yellowish color of the cut surface of the lesion with foci of white hyalinized areas ( arrows ) low power photomicrograph showing stratified squamous epithelium overlying a well - circumscribed mass of adipose tissue , surrounded by a fibrous capsule and containing a focus of hyaline cartilage .", "( h&e stain , 40 ) high - power photomicrograph showing cartilaginous foci made up of mature normal appearing chondroid tissue with peripheral rim of spindle shaped lining cells ." ], [ "the first case of oral chondrolipoma was reported by mcandrew and greenspan in 1976 in a 72-year - old male in lower lip and since then only a handful of cases been reported in oral cavity .", "many clinical and histopathological characteristics of chondrolipomas of the oral cavity continue to be poorly understood because of the rarity of this tumor , together with the lack of data in the literature . a review of the pubmed database revealed 14 such cases in english literature . we have included only those cases which presented strictly within the oral cavity and histologically showed presence of only cartilaginous tissue in a predominantly mature adipocyte proliferation", "the clinicopathological features of these cases including the present case have been summarized in table 1 .", "clinical features of 14 cases of oral chondrolipoma described in english literature cases of oral chondrolipoma have been diagnosed in patients aged from 14 to 72 years , but it seems to be a tumor of older individuals with majority of cases being diagnosed after the age of 50 years .", "while these lesions are usually diagnosed in older adults , they may originate at a younger age as some of these tumors including the present case have shown long duration of presentation", ". there may be an actual bimodal age predilection as substantial number of cases ( almost one - third including the present case ) seem to originate in the first two decades of life but a larger number of cases will be required to accept or refute this observation .", "this feature of chondrolipoma slightly differs from a conventional lipoma , which are rarely seen in the first two decades of life and are thought to make their appearance at the age when fat starts accumulating in inactive individuals .", "hence , most lipomas including those of oral cavity become apparent between 5 to 7 decades of life .", "in fact all of the cases of chondrolipoma involving young individuals have been reported in recent literature probably highlighting the fact that awareness about this variant is increasing among histopathologists and possibly some of these lesions may have earlier been overlooked .", "similar to other lipomas at other sites , oral lipomas are believed to be more common in men and this seems to be true even for chondrolipomas in the oral cavity .", "oral chondrolipomas seem to have a strong predilection for occurrence on tongue but interestingly none of the lesions have been described in buccal mucosa which is the preferential site for conventional lipomas .", "histologically , variants of lipoma differ from ordinary lipoma by characteristic microscopic picture and specific clinical setting .", "this group is chiefly represented by angiolipoma , myolipoma , angiomyolipoma , myelolipoma , chondroid lipoma , spindle cell and pleomorphic lipoma .", "chondro lipomas are characterized by the proliferation of mature adipocytes with additional mature cartilaginous tissue formation .", "though this term has been used in past , it was first defined by stout for tumors that are composed of two or more mesenchymal elements . on the other hand ,", ", described mesenchymomas as an unencapsulated soft tissue neoplasms composed of two or more mature mesenchymal tissues not normally associated with each other and no single mesenchymal tissue should predominate with respect to the other mesenchymal elements .", "usually the cartilage in lipoma represents only a small part of the tumor and hence would not strictly fit to the above criteria .", "mature cartilaginous areas in a chondrolipoma should be distinguished from chondroid lipoma , a lipoma variant that is uncommon in the oral cavity and consists of mature adipocytes admixed with multivacuolated lipoblast like cells in a myxohyaline and chondroid matrix .", "this newly described lesion , having an immature aspect may give a pseudosarcomatous appearance and may be mistaken for lipoblastic or chondroblastic malignancies . on the other hand , in true chondrolipoma the adipose component is entirely composed of mature tissue with lack of any lipoblastic cells .", "due to similarity in nomenclature , the two entities are often confused with each other and it is possible they may have been diagnosed interchangeably in past . in this review", ", we have included only those cases which show typical features of a chondrolipoma and all cases showing immature lipoblast like component or hibernoma - like areas have been excluded .", "in addition , chondrolipoma may sometimes need to be differentiated from extra - skeletal chondroma specially those occurring in deep submucosal areas and hence surrounded by adipose tissue .", "histologically , chondromas are characterized by greater proportion of cartilaginous tissue arranged usually in distinct lobular pattern . whereas the chondroid component of chondrolipoma is focal and lacks any lobular arrangement .", "several hypotheses have been tried to explain the occurrence of cartilage within the mass of adipose tissue .", "one of the possible explanations could be that this tumor represents a true mesenchymoma i.e. both the chondroid as well as the adipose components being neoplastic in nature .", "the initial neoplastic change may take place in the pleuripotent mesenchymal stem cells , present in oral mucosa and these neoplastic stem cells may then differentiate into adipogenic and chondrogenic cells .", "presence of pluripotent mesenchymal stem cells , capable of such differentiation , has been demonstrated in the connective tissue of skeletal muscles and dermis and it may be prudent to assume that similar cells may also be present in the lamina propria and submucosal tissues of oral cavity .", "ability for multipotential differentiation including chondrogenic potential has been demonstrated in adipose tissue derived stem cells .", "hence , it could be possible that some of the neoplastic adipocytes may differentiate into chondroblastic cells , the stimulus being either spontaneous or a metaplastic reaction triggered by long - term irritation .", "similar metaplastic chondrogenesis has been described in peripheral fibromas of gingiva . and may be attributed to chronic mechanical stress .", "an interesting , albeit a little farfetched possibility is that the neoplastic adipose tissue may develop in a pre - existing cartilaginous choristoma .", "such choristomas , though rare , are known to occur in the oral cavity with tongue being the most common location .", "this may also explain the rarity of this lesion and preferential occurrence in tongue as compared to the higher propensity of involvement of buccal mucosa for conventional lipoma .", "finally , the lesion may not be neoplastic at all and may result from a combination of hamartomatous and choristomatous growth .", "such combined lipomaotus hamartomas and choristomas made up of mature adipose tissue containing cartilage have been described in other sites of the body as well as in inbred laboratory mice .", "it was suggested that those cases containing additional tissue elements in these chiefly lipomatous growths should be considered choristomas . even in the murine lipomatous growths", "additional tissue elements were found more frequently in long standing cases and it was considered that these additional elements like cartilage could be present at an earlier stage but may constitute only a minor volume of the lesion , hence could be easily overlooked and thus require careful complete serial sectioning of the entire growth .", "recently , role of factors like sox-9 , runx-2 , tgf- , bone morphogenic protein ( bmp ) has been discussed in chondrogenic potential of lipomas .", "we believe that oral chondrolipomas constitute a heterogenous group of lesions , some of which may represent true neoplasms while others , especially those appearing at a younger age , may be hamartomatous / choristomatous proliferations .", "the pluripotentiality of adipose derived stem cells and preferential expression and interplay of prochondrogenic molecules either through some genetic alteration or environmental factors are responsible for chondrogenesis in these lesions . whatever the pathogenesis , lesion is essentially benign and surgical excision is the treatment of choice ." ], [ "the cartilage found in the tumor most likely represents a metaplastic change or harmartomatous / choristomatous proliferation and could be attributed to multipotentiality of mesenchymal stem cells .", "the characteristics of oral lesions tend to show few deviations from the classical oral lipoma , chiefly being the marked predilection for involvement of tongue and possibly bimodal age of onset and warrants reporting of such cases with detailed clinicopathological evaluation in future ." ] ]

[ "a retrospective chart review was performed for cases of infectious scleritis seen from january 2007 to august 2011 .", "the diagnostic criteria for a case of infectious scleritis included the presence of single or multiple inflamed anterior scleral nodules or ulcerations , which revealed a causative organism on microbiological culture .", "microbial cultures were considered significant if growth of the same organism was present on more than one solid medium or if there was a confluent growth on inoculation on one solid medium or if growth of one medium was consistent with direct microscopy findings .", "data regarding patient demographics , predisposing factors , causative organisms , and clinical features for all 12 patients was extracted .", "all patients except one were followed up for a period of at least 6 months and , therefore , the treatment and outcome details were analyzed for 11 patients .", "data was analyzed by the chi - square test using spss version 11.0.1 to determine the prognostic factors .", "twelve eyes of 12 patients were identified to have culture - positive infectious scleritis . demographic data and clinical features", "preceding surgical procedures performed included manual small - incision cataract surgery ( 4 ) , pterygium excision ( 1 ) , and trabeculectomy ( 1 ) .", "none of the patients had a history of fever , joint pain , respiratory illness , skin rashes , etc .", ", one patient ( case 2 ) had progeria , another had chronic liver disease ( case 6 ) , and 2 patients had diabetes ( case 8 and 10 ) .", "patients with multifocal scleritis were associated with growth of either gram - negative bacilli ( gnb ) or nocardia on culture an example of which is shown in fig .", "2a and b. demographics , clinical features , organisms , management , and outcomes of infectious scleritis ( a ) multifocal scleral abscesses owing to pseudomonas sp .", "( b ) healed scleritis with minimal scleral thinning 2 months after treatment with topical fortified antibiotics and dexamethasone ( case 11 ) ( a ) nocardia asteroides : gram stain showing filamentous forms fragmented into bacillary and coccoid forms ( magnification = 1000 ) .", "( b ) nocardia asteroides : orange coloured , centrally heaped , dry colony all patients received topical antimicrobials based on the culture and sensitivity report .", "seven patients were also treated with topical steroids due to severe intra - ocular inflammation .", "in addition to medical treatment , 5 patients underwent various surgical procedures in an attempt to decrease the load of infective organism or to preserve the globe as shown in table 1 and fig .", "of these , only 2 patients had a good final visual acuity ( better than 6/18 ) on the last follow - up visit .", "six months after corneo - scleral patch graft following infectious scleritis post - cataract surgery ( case 8) we determined the prognostic factors of infectious scleritis using the chi - square test .", "a poor visual acuity at presentation ( less than counting fingers at 1 m ) usually resulted in a worse visual outcome ( p = 0.005 ) .", "the causes of poor visual acuity at last follow - up include corneal scarring following keratitis ( 1 ) , failed corneal graft ( 1 ) , secondary angle closure glaucoma and optic atrophy ( 1 ) , choroidal detachment ( 1 ) , and endophthalmitis ( 5 ) .", "the visual outcome was not dependant on any particular organism ( p = 0.099 ) or the presence of multifocal abscesses ( p = 0.209 ) .", "the addition of surgical intervention did not result in a significantly better visual outcome than medical management alone ( p = 0.209 ) , but resulted in a higher rate of globe preservation ( p = 0.045 ) .", "a clinical suspicion of infectious scleritis in all cases of scleral inflammation following trauma has been emphasized previously .", "however , in our series , 4 cases were preceded by cataract surgery and only 1 by pterygium surgery .", "another case series of indian eyes also showed maximum number of their cases had cataract surgery prior to an episode of infectious scleritis .", "this may be due to the large number of scleral - tunnel small incision cataract surgeries performed in india as compared to clear corneal phacoemulsification performed in developed countries .", "pseudomonas aeruginosa is the leading cause of microbial scleritis in reported literature . in our series , 33.33% had gnb , 25% had nocardia , and 25% had fungal infection .", "one of the fungi was identified to be rhodotorula spp , which we have reported elsewhere . in cases of scleritis following trauma", "where infection is suspected but routine microbial culture is negative , polymerase chain reaction tests may be used to confirm the diagnosis .", "if these tests are also negative , the possible diagnosis of surgically induced necrotizing scleritis ( sins ) should be considered .", "sins is a believed to be a delayed - type hypersensitivity response to any form of surgical trauma . up to 90% of patients who develop sins", "may have an undiagnosed systemic vasculitis and , therefore , a detailed systemic investigation is warranted .", "this is often seen as late as 9 months after the surgical intervention and , after ruling out an infectious cause , requires treatment with intensive systemic immunosuppression .", "when infectious scleritis occurs long after surgery , it has been hypothesized to be due to an underlying sins with secondary microbial infection , which can make diagnosis and treatment difficult .", "these patients may benefit from a systemic evaluation to rule out an autoimmune vasculitis , which will require appropriate management .", "some presented with scleritis and developed secondary intraocular spread , which has been described in other reports .", "others had scleritis , uveitis , and endophthalmitis at presentation ( e.g. , case 7 where the sclera in the area of the bleb was necrotic with vitreous inflammation ) . the wide use of anti - metabolites at the time of trabeculectomy increases risk of scleral melt and inflammation , which may have resulted in secondary infection and endophthalmitis .", "pseudomonas is most often associated with this presentation , which has been attributed to an intra - scleral dissemination facilitated by its proteolytic products and motility .", "our series had 6 cases of multifocal scleral abscesses , 3 of which were due to p. aeruginosa , 2 due to nocardia , and 1 due to klebsiella sp .", "a review of past literature suggests that multifocal scleritis can be caused by several organisms and may be related to factors such as the load of infection , rather than specific organisms .", "the visual acuity at presentation is the most important prognostic factor determining the final visual acuity , and this has been seen in other studies .", "this suggests that early diagnosis should help preserve the patient 's remaining vision if effective treatment is initiated .", "there is a lot of discrepancy among researchers regarding the effect of surgical debridement on the outcome of infectious scleritis .", "a large series showed that despite adjuvant surgery in 46 of 56 eyes , approximately 50% lost functional vision .", "however , unlike other studies , we did not find any difference in the final vision between the medically and surgically treated groups . in conclusion ,", "manual small incision cataract surgery is a common predisposing factor to infectious scleritis in developing countries .", "the visual acuity at presentation is the most important prognostic factor determining the final visual outcome .", "aggressive surgical intervention improves the chance of globe preservation but does not improve the visual outcome ." ]

[ "<S> in this retrospective case series , we studied the predisposing factors , causative organisms , clinical spectrum , and outcomes of 12 cases of culture - proven infectious scleritis . </S>", "<S> nine of 12 patients had a history of preceding trauma ( surgical or accidental ) . </S>", "<S> past surgical history included small - incision cataract surgery ( 4 ) , pterygium surgery ( 1 ) , and trabeculectomy ( 1 ) . </S>", "<S> six patients had multifocal scleral abscesses due to pseudomonas , klebsiella , or nocardia . only 2 patients retained useful vision ( > 6/18 ) . a poor visual acuity at presentation usually resulted in a worse visual outcome ( p = 0.005 ) . </S>", "<S> four eyes developed phthisis . </S>", "<S> the addition of surgical intervention did not result in a significantly better visual outcome than medical management alone ( p = 0.209 ) , but resulted in a higher globe preservation rate ( p = 0.045 ) . </S>", "<S> therefore , we concluded that infection must be ruled out in cases of scleritis with preceding history of trauma , and aggressive surgical intervention improves the anatomical outcome but does not change the visual outcome . </S>" ]

[ "Materials and Methods", "Results", "Discussion" ]

[ [ "a retrospective chart review was performed for cases of infectious scleritis seen from january 2007 to august 2011 .", "the diagnostic criteria for a case of infectious scleritis included the presence of single or multiple inflamed anterior scleral nodules or ulcerations , which revealed a causative organism on microbiological culture .", "microbial cultures were considered significant if growth of the same organism was present on more than one solid medium or if there was a confluent growth on inoculation on one solid medium or if growth of one medium was consistent with direct microscopy findings .", "data regarding patient demographics , predisposing factors , causative organisms , and clinical features for all 12 patients was extracted .", "all patients except one were followed up for a period of at least 6 months and , therefore , the treatment and outcome details were analyzed for 11 patients .", "data was analyzed by the chi - square test using spss version 11.0.1 to determine the prognostic factors ." ], [ "twelve eyes of 12 patients were identified to have culture - positive infectious scleritis . demographic data and clinical features", "preceding surgical procedures performed included manual small - incision cataract surgery ( 4 ) , pterygium excision ( 1 ) , and trabeculectomy ( 1 ) .", "none of the patients had a history of fever , joint pain , respiratory illness , skin rashes , etc .", ", one patient ( case 2 ) had progeria , another had chronic liver disease ( case 6 ) , and 2 patients had diabetes ( case 8 and 10 ) .", "patients with multifocal scleritis were associated with growth of either gram - negative bacilli ( gnb ) or nocardia on culture an example of which is shown in fig .", "2a and b. demographics , clinical features , organisms , management , and outcomes of infectious scleritis ( a ) multifocal scleral abscesses owing to pseudomonas sp .", "( b ) healed scleritis with minimal scleral thinning 2 months after treatment with topical fortified antibiotics and dexamethasone ( case 11 ) ( a ) nocardia asteroides : gram stain showing filamentous forms fragmented into bacillary and coccoid forms ( magnification = 1000 ) .", "( b ) nocardia asteroides : orange coloured , centrally heaped , dry colony all patients received topical antimicrobials based on the culture and sensitivity report .", "seven patients were also treated with topical steroids due to severe intra - ocular inflammation .", "in addition to medical treatment , 5 patients underwent various surgical procedures in an attempt to decrease the load of infective organism or to preserve the globe as shown in table 1 and fig .", "of these , only 2 patients had a good final visual acuity ( better than 6/18 ) on the last follow - up visit .", "six months after corneo - scleral patch graft following infectious scleritis post - cataract surgery ( case 8) we determined the prognostic factors of infectious scleritis using the chi - square test .", "a poor visual acuity at presentation ( less than counting fingers at 1 m ) usually resulted in a worse visual outcome ( p = 0.005 ) .", "the causes of poor visual acuity at last follow - up include corneal scarring following keratitis ( 1 ) , failed corneal graft ( 1 ) , secondary angle closure glaucoma and optic atrophy ( 1 ) , choroidal detachment ( 1 ) , and endophthalmitis ( 5 ) .", "the visual outcome was not dependant on any particular organism ( p = 0.099 ) or the presence of multifocal abscesses ( p = 0.209 ) .", "the addition of surgical intervention did not result in a significantly better visual outcome than medical management alone ( p = 0.209 ) , but resulted in a higher rate of globe preservation ( p = 0.045 ) ." ], [ "a clinical suspicion of infectious scleritis in all cases of scleral inflammation following trauma has been emphasized previously .", "however , in our series , 4 cases were preceded by cataract surgery and only 1 by pterygium surgery .", "another case series of indian eyes also showed maximum number of their cases had cataract surgery prior to an episode of infectious scleritis .", "this may be due to the large number of scleral - tunnel small incision cataract surgeries performed in india as compared to clear corneal phacoemulsification performed in developed countries .", "pseudomonas aeruginosa is the leading cause of microbial scleritis in reported literature . in our series , 33.33% had gnb , 25% had nocardia , and 25% had fungal infection .", "one of the fungi was identified to be rhodotorula spp , which we have reported elsewhere . in cases of scleritis following trauma", "where infection is suspected but routine microbial culture is negative , polymerase chain reaction tests may be used to confirm the diagnosis .", "if these tests are also negative , the possible diagnosis of surgically induced necrotizing scleritis ( sins ) should be considered .", "sins is a believed to be a delayed - type hypersensitivity response to any form of surgical trauma . up to 90% of patients who develop sins", "may have an undiagnosed systemic vasculitis and , therefore , a detailed systemic investigation is warranted .", "this is often seen as late as 9 months after the surgical intervention and , after ruling out an infectious cause , requires treatment with intensive systemic immunosuppression .", "when infectious scleritis occurs long after surgery , it has been hypothesized to be due to an underlying sins with secondary microbial infection , which can make diagnosis and treatment difficult .", "these patients may benefit from a systemic evaluation to rule out an autoimmune vasculitis , which will require appropriate management .", "some presented with scleritis and developed secondary intraocular spread , which has been described in other reports .", "others had scleritis , uveitis , and endophthalmitis at presentation ( e.g. , case 7 where the sclera in the area of the bleb was necrotic with vitreous inflammation ) . the wide use of anti - metabolites at the time of trabeculectomy increases risk of scleral melt and inflammation , which may have resulted in secondary infection and endophthalmitis .", "pseudomonas is most often associated with this presentation , which has been attributed to an intra - scleral dissemination facilitated by its proteolytic products and motility .", "our series had 6 cases of multifocal scleral abscesses , 3 of which were due to p. aeruginosa , 2 due to nocardia , and 1 due to klebsiella sp .", "a review of past literature suggests that multifocal scleritis can be caused by several organisms and may be related to factors such as the load of infection , rather than specific organisms .", "the visual acuity at presentation is the most important prognostic factor determining the final visual acuity , and this has been seen in other studies .", "this suggests that early diagnosis should help preserve the patient 's remaining vision if effective treatment is initiated .", "there is a lot of discrepancy among researchers regarding the effect of surgical debridement on the outcome of infectious scleritis .", "a large series showed that despite adjuvant surgery in 46 of 56 eyes , approximately 50% lost functional vision .", "however , unlike other studies , we did not find any difference in the final vision between the medically and surgically treated groups . in conclusion ,", "manual small incision cataract surgery is a common predisposing factor to infectious scleritis in developing countries .", "the visual acuity at presentation is the most important prognostic factor determining the final visual outcome .", "aggressive surgical intervention improves the chance of globe preservation but does not improve the visual outcome ." ] ]

[ "lipoma is a common benign soft tissue neoplasm that sometimes may have mixed tissue components .", "ossification of a lipoma was first described in 1959 , and it is rarely reported7,16 ) .", "several names have been used to describe ossification of lipomas , including secondary calcification , ossified lipoma , ossifying lipoma , or osteolipoma , and some authors have used these terms interchangeably . as with classic lipomas , lipomas with ossifications may be found in any part of the body , but are usually found in the head , neck , oral cavity , and extremities , adjacent to bone .", "only two cases of osteolipoma arising in connection with vertebrae have been described . here ,", "we present a case of an intramuscular osteolipoma on the posterior neck , independent of vertebrae , and review the clinicopathologic features of osteolipoma previously published in the literature .", "a 51-year - old female presented with a 5-year history of a painless , progressively enlarging mass on the posterior neck .", "physical examination revealed a soft , non - tender , uniform mass without surface abnormalities . computed tomography ( ct ) and magnetic resonance imaging ( mri ) showed a circumscribed mass involving the posterior neck muscles in the midline between the c2 and c6 spinous processes with a large calcified component ( fig .", "the ovoid mass was measured 4 cm in width , 6 cm in length , and 3 cm in height , without infiltration of the cervical spine .", "subsequently , the patient underwent a whole body bone scan , which revealed an amorphous calcification of the posterior neck without metastasis ( fig .", "the mass had a well - demarcated margin and the outer wall was very firm without adhesion to ligaments or paravertebral muscles .", "gross examination revealed a well - circumscribed mass composed of adipose and bony tissue , with red bone marrow visible on the cut surface ( fig .", "3 ) . histological study showed a lipomatous lesion with large foci of osseous metaplasia at the periphery of the mass ; the bony portion was surrounded by mature adipose tissue ( fig .", "microscopic examination of the red bone marrow showed a meshwork of bone trabeculae and hematopoietic marrow elements ( fig .", "the patient was diagnosed with an intramuscular osteolipoma of the posterior neck and no recurrence was observed at the 6-month follow - up .", "a lipoma is a common soft tissue neoplasm , but lipomas with distinct osseous components are rare2 - 4,8,10 - 12,14,15 ) . as noted by heffernan et al.11 ) , in allen 's review of 635 cases , less than 1% of lipomas were ossified .", "further , as reported by de castro , et al.5 ) , when furlong analyzed histological findings from a group of 125 lipomas of the oral and maxillofacial region , there was no variant that presented as osseous metaplasia .", "lipomas presenting with osseous components have been reported as secondary calcification of lipoma or ossified lipoma4,12,14 ) .", "secondary calcification of lipoma involves calcification that progresses from the periphery inwards as a result of necrosis after impaired blood supply or repeated microtrauma5,14 ) .", "an ossified lipoma is defined as a histologic variant of lipoma that has undergone osseous metaplasia , and can be classified into 2 groups according to the predominance of the osseous component : ossifying lipoma and osteolipoma .", "if the adipose component of the lipoma is more dominant than the osseous component , it is an ossifying lipoma , whereas an osteolipoma presents with a dominant osseous component14 ) .", "osteolipomas have been reported in middle aged or elderly patients presenting with large painless masses with a long duration , even years , and they may be found incidentally2,7 ) .", "they have been reported at various sites located adjacent to bone or periosteum , including upper and lower extremity , oral mucosa , soft tissue of neck , and intracranial regions , including tuber cinereum , hypothalamus , suprasellar cistern , and the interhemispheric area1 - 5,7,10 - 15,17,18 ) .", "one patient was an 8-year - old female who presented with a progressively enlarging swelling over the lumbar region with a dermal sinus tract12 ) , and other was 20-year - old female with a cervical osteolipoma contained within the spinal canal14 ) .", "presented here , the osseous lipoma was located in the deep neck musculature , independent of bony vertebral structures .", "there are several hypotheses regarding the pathogenetic mechanisms that influence the osseous metaplasia of lipomas , but the details are still not clear .", "first , these tumours may originate from multipotent mesenchymal cells that can differentiate into lipoblasts , chondroblasts , fibroblasts , angioblasts , and osteoblasts .", "this could account for the several lipomatous variations of classic lipomas , i.e. lipoblastoma , lipomatosis , fibrolipoma , angiolipoma , myolipoma , myelolipoma , hibernoma , and atypical lipoma , and mesenchymoma is defined as a rare soft tissue lesion that is composed of fibrous tissue associated with two or more types of well differentiated mesenchymal cells that would not normally be found in the same area 2,7 ) .", "second , ossification may also have been induced by poor nutritional supply in the centre of a large lipoma after repetitive trauma , metabolic changes , or ischemia leading to transformation of fibroblasts into osteoblasts2,8 ) .", "fritchie et al.8 ) reported cytogenetic analyses of three osteolipomas and reported that the translocations in all cases were consistent with the karyotypic features of lipoma .", "other benign tumors that may contain bone including teratomas or dermoids , masses with secondary ossification due to trauma , liposarcomas with metaplastic changes , or congenital malformations , should be considered in the differential diagnosis .", "in addition , tumour calcinosis , calcification in a bursa , and other conditions such as ossifying fibromas , myositis ossificans , and osteosarcomas should be taken into consideration .", "the use of ct scanning provides excellent visualization of the calcified or ossified components of a lipoma and confirmation of proximity to adjacent bone , and mr imaging can also provide detailed information that is useful for further evaluation2,9 ) .", "definitive diagnosis of osteolipoma can be made with histopathologic findings after surgical excision , which is usually the recommended treatment . a histopathologic appearance of diffuse , mature ossification within adipose tissue and gross features demonstrating a dominant osseous component confirm the diagnosis .", "histologically confirmed osteolipomas are benign neoplasms , as with classic lipomas , and do not recur2,4 - 6,11 ) .", "to the best of our knowledge , it is the first case report of an intramuscular osteolipoma within the posterior neck independent of adjacent bone .", "we suggest that although osteolipoma is a rare variant , it is important to keep it in mind when a soft tissue mass of the posterior neck mixed with osseous component is encountered ." ]

[ "<S> ossified lipoma or osteolipoma are rarely reported . </S>", "<S> it is defined as a histologic variant of lipoma that has undergone osseous metaplasia . </S>", "<S> osteolipoma presents with a dominant osseous component within a lipoma . </S>", "<S> we report a case of a histologically confirmed osteolipoma on the nuchal ligament independent of bone . the patient was a 51-year - old female who presented with a 5-year history of a painless , progressively enlarging mass on the posterior neck . </S>", "<S> computed tomography and magnetic resonance imaging showed a circumscribed mass compatible with fat between the c2 and c6 spinous processes with a large calcified irregular component . </S>", "<S> the mass with dual components was totally removed under general anesthesia and no recurrence was observed after 6 months of follow - up . </S>", "<S> we also reviewed the clinicopathologic features of previously reported osteolipomas in the literature and suggest that although osteolipoma is a rare variant of lipoma , it should be considered in the differential diagnosis when a lipoma of the posterior neck mixed with a bony component is encountered . </S>" ]

[ "INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION" ]

[ [ "lipoma is a common benign soft tissue neoplasm that sometimes may have mixed tissue components .", "ossification of a lipoma was first described in 1959 , and it is rarely reported7,16 ) .", "several names have been used to describe ossification of lipomas , including secondary calcification , ossified lipoma , ossifying lipoma , or osteolipoma , and some authors have used these terms interchangeably . as with classic lipomas , lipomas with ossifications may be found in any part of the body , but are usually found in the head , neck , oral cavity , and extremities , adjacent to bone .", "only two cases of osteolipoma arising in connection with vertebrae have been described . here ,", "we present a case of an intramuscular osteolipoma on the posterior neck , independent of vertebrae , and review the clinicopathologic features of osteolipoma previously published in the literature ." ], [ "a 51-year - old female presented with a 5-year history of a painless , progressively enlarging mass on the posterior neck .", "physical examination revealed a soft , non - tender , uniform mass without surface abnormalities . computed tomography ( ct ) and magnetic resonance imaging ( mri ) showed a circumscribed mass involving the posterior neck muscles in the midline between the c2 and c6 spinous processes with a large calcified component ( fig .", "the ovoid mass was measured 4 cm in width , 6 cm in length , and 3 cm in height , without infiltration of the cervical spine .", "subsequently , the patient underwent a whole body bone scan , which revealed an amorphous calcification of the posterior neck without metastasis ( fig .", "the mass had a well - demarcated margin and the outer wall was very firm without adhesion to ligaments or paravertebral muscles .", "gross examination revealed a well - circumscribed mass composed of adipose and bony tissue , with red bone marrow visible on the cut surface ( fig .", "3 ) . histological study showed a lipomatous lesion with large foci of osseous metaplasia at the periphery of the mass ; the bony portion was surrounded by mature adipose tissue ( fig .", "microscopic examination of the red bone marrow showed a meshwork of bone trabeculae and hematopoietic marrow elements ( fig .", "the patient was diagnosed with an intramuscular osteolipoma of the posterior neck and no recurrence was observed at the 6-month follow - up ." ], [ "a lipoma is a common soft tissue neoplasm , but lipomas with distinct osseous components are rare2 - 4,8,10 - 12,14,15 ) . as noted by heffernan et al.11 ) , in allen 's review of 635 cases , less than 1% of lipomas were ossified .", "further , as reported by de castro , et al.5 ) , when furlong analyzed histological findings from a group of 125 lipomas of the oral and maxillofacial region , there was no variant that presented as osseous metaplasia .", "lipomas presenting with osseous components have been reported as secondary calcification of lipoma or ossified lipoma4,12,14 ) .", "secondary calcification of lipoma involves calcification that progresses from the periphery inwards as a result of necrosis after impaired blood supply or repeated microtrauma5,14 ) .", "an ossified lipoma is defined as a histologic variant of lipoma that has undergone osseous metaplasia , and can be classified into 2 groups according to the predominance of the osseous component : ossifying lipoma and osteolipoma .", "if the adipose component of the lipoma is more dominant than the osseous component , it is an ossifying lipoma , whereas an osteolipoma presents with a dominant osseous component14 ) .", "osteolipomas have been reported in middle aged or elderly patients presenting with large painless masses with a long duration , even years , and they may be found incidentally2,7 ) .", "they have been reported at various sites located adjacent to bone or periosteum , including upper and lower extremity , oral mucosa , soft tissue of neck , and intracranial regions , including tuber cinereum , hypothalamus , suprasellar cistern , and the interhemispheric area1 - 5,7,10 - 15,17,18 ) .", "one patient was an 8-year - old female who presented with a progressively enlarging swelling over the lumbar region with a dermal sinus tract12 ) , and other was 20-year - old female with a cervical osteolipoma contained within the spinal canal14 ) .", "presented here , the osseous lipoma was located in the deep neck musculature , independent of bony vertebral structures .", "there are several hypotheses regarding the pathogenetic mechanisms that influence the osseous metaplasia of lipomas , but the details are still not clear .", "first , these tumours may originate from multipotent mesenchymal cells that can differentiate into lipoblasts , chondroblasts , fibroblasts , angioblasts , and osteoblasts .", "this could account for the several lipomatous variations of classic lipomas , i.e. lipoblastoma , lipomatosis , fibrolipoma , angiolipoma , myolipoma , myelolipoma , hibernoma , and atypical lipoma , and mesenchymoma is defined as a rare soft tissue lesion that is composed of fibrous tissue associated with two or more types of well differentiated mesenchymal cells that would not normally be found in the same area 2,7 ) .", "second , ossification may also have been induced by poor nutritional supply in the centre of a large lipoma after repetitive trauma , metabolic changes , or ischemia leading to transformation of fibroblasts into osteoblasts2,8 ) .", "fritchie et al.8 ) reported cytogenetic analyses of three osteolipomas and reported that the translocations in all cases were consistent with the karyotypic features of lipoma .", "other benign tumors that may contain bone including teratomas or dermoids , masses with secondary ossification due to trauma , liposarcomas with metaplastic changes , or congenital malformations , should be considered in the differential diagnosis .", "in addition , tumour calcinosis , calcification in a bursa , and other conditions such as ossifying fibromas , myositis ossificans , and osteosarcomas should be taken into consideration .", "the use of ct scanning provides excellent visualization of the calcified or ossified components of a lipoma and confirmation of proximity to adjacent bone , and mr imaging can also provide detailed information that is useful for further evaluation2,9 ) .", "definitive diagnosis of osteolipoma can be made with histopathologic findings after surgical excision , which is usually the recommended treatment . a histopathologic appearance of diffuse , mature ossification within adipose tissue and gross features demonstrating a dominant osseous component confirm the diagnosis .", "histologically confirmed osteolipomas are benign neoplasms , as with classic lipomas , and do not recur2,4 - 6,11 ) ." ], [ "to the best of our knowledge , it is the first case report of an intramuscular osteolipoma within the posterior neck independent of adjacent bone .", "we suggest that although osteolipoma is a rare variant , it is important to keep it in mind when a soft tissue mass of the posterior neck mixed with osseous component is encountered ." ] ]

[ "the most appropriate way to diagnose airway obstruction is currently the subject of heated debate.110 most national and international chronic obstructive pulmonary disease ( copd ) guidelines recommend to use a forced expiratory volume in one second / forced vital capacity ratio ( fev1/fvc ) of 0.70 as a suitable threshold value to define the presence of an obstructive ventilatory defect .", "commonly used guidelines include those of the global initiative for obstructive lung disease ( gold),11 the american thoracic society / european respiratory society,12 the british thoracic society,13 the canadian thoracic society,14 and the national institute for health and clinical excellence.15 however , there is evidence that the fixed cut - off value of 0.70 for fev1/fvc becomes less specific in males aged > 40 years and females > 50 years , implying a risk of overestimation of airway obstruction .", "this is due to an age - related decline in pulmonary volumes , particularly in fev1 , which is observed even in healthy people with no history of exposure to noxious particles or gases.1619 proposed strategies for reducing the misclassification of airway obstruction include use of the lower limit of normal for fev1/fvc , calculated as the fifth percentile of the normal distribution in a healthy population,2022 or the use of different fev1/fvc thresholds for different age groups ( eg , 0.70 for subjects aged < 70 years , 0.65 for those aged 7080 years , and 0.60 for those aged > 80 years).19 on this basis , a group of colleagues involved in respiratory research and/or the diagnosis and treatment of lung diseases recently wrote an open letter to members of the gold committee inviting them to change the method by which airway obstruction is defined and asking for retraction of the fixed ratio in favor of the lower limit of normal.1 the aim of the present study was to provide additional information for determination of the most appropriate spirometric criteria for confirming airway obstruction in the elderly , by describing lung function and calculating the lower limit of normal for fev1/fvc in healthy nonsmoking elderly subjects ( age > 65 years ) who participated in the italian multicenter sara ( acronym of salute respiratoria nellanziano , italian for respiratory health in the elderly ) study .", "the degree of potential misclassification relative to use of 0.70 or other proposed fixed thresholds for fev1/fvc was also evaluated .", "we performed a cross - sectional analysis of data from the sara study , the design of which , along with technical characteristics of instruments as well as training of operators and results of quality control of spirometry , have been described in detail elsewhere.23 briefly , the study involved 24 pulmonary or geriatric institutions distributed throughout italy .", "a total of 1971 subjects aged 65100 years were recruited as consecutive outpatients referred between january 1996 and december 1997 to the participating centers ( see appendix ) .", "lung function was measured using an identical fully computerized water - sealed stead - wells spirometer ( baires system , biomedin , padua , italy ) by specifically trained and certified personnel supervised by a rigorous real - time control of acceptability and repeatability23 according to american thoracic society recommendations.24 fvc maneuvers were performed with the patient sitting .", "the fev1/fvc ratio was calculated on the basis of the highest values of individual parameters obtained in the acceptable curves for each subject .", "analyses were conducted only on patients with good repeatability of the above - mentioned indices ( difference between two best values < 150 ml).25 of the 1870 subjects who performed spirometry , we selected only those without any previous or present diagnosis or any sign or symptom suggestive of respiratory diseases according to the modified international union against tuberculosis and lung disease bronchial symptoms questionnaire.25,26 current smokers and previous smokers with a smoking exposure > 5 pack / year were excluded , since the < 5 pack / year smoking exposure was not significantly associated with decreased lung function.27 additional exclusion criteria were : severe hepatic failure ; severe renal failure ; severe cardiac failure ; cognitive and/or sensory impairment severe enough to affect a multidimensional assessment ; severe kyphoscoliosis with occiput wall distance ( distance between the occiput and the wall when the patient stands with heels and shoulder against the wall with the back straight ) > 10 ; occurrence of a major psychosocial event ( eg , bereavement ) within the past 6 months ; and hospitalization for any reason within the past 6 months .", "we further excluded individuals who had hypertension ( diastolic pressure 90 mmhg and/or systolic pressure 160 mmhg ) , diabetes , and/or major electrocardiographic abnormalities .", "statistical analyses were performed using spss ( spss inc , chicago , il ) and stata ( stata corporation , college station , tx ) software packages .", "regression models were used for testing the relationship between spirometric indices and anthropometric variables . based on the recommendations from the american thoracic society / european respiratory society task force,22 the lower limit of normal for fev1/fvc", "was estimated as the fifth percentile of its frequency distribution . to evaluate the effect of aging on spirometric measures independently of body height , fev1 and fvc were normalized for height at the third power.28,29", "after applying the above - mentioned selection criteria , our final data set consisted of 367 healthy , nonsmoking subjects .", "one hundred and one subjects were excluded for lack of availability of lung function testing , a further 709 subjects because of a history of respiratory disease , 445 for significant smoking exposure ( > 5 pack / year ) , 262 for inadequate quality of spirometry , and 87 for the above - mentioned additional exclusion criteria . tables 1 and 2 show anthropometric and functional data for the sample and distribution of the participants according to age and gender .", "although the most advanced ages were less represented , a total of 73 subjects aged 80 years and over were included .", "the sample consisted of 314 never - smokers ( 85.6% ) and 53 former - smokers with a smoking exposure from 0.15 to 5 pack - years ( mean standard deviation , 2.75 1.5 ) .", "as shown in figure 1 , the values of fev1/fvc showed a normal frequency distribution .", "the mean fev1/fvc was 0.75 0.6 in males and 0.78 0.6 in females , whereas the corresponding fifth percentiles were 0.65 and 0.67 , respectively , in males and females .", "fev1 and fvc significantly decreased with age ( r = 0.38 and 0.35 ; p < 0.001 ) , while fev1/fvc , was not significantly correlated with age or height in either gender group over the considered range of age ( lowest p = 0.103 ) .", "as a consequence , there was no rationale to develop reference equations for fev1/fvc , with age or height as independent variables in this restricted range of age .", "figure 2 shows the decline in fev1 and fvc with increasing age observed in the study sample in men and women , after normalization for height . in the sample of healthy subjects ,", "15% had a fev1/fvc < 0.70 , and would have been inappropriately classified as obstructed according to gold criteria .", "table 3 describes the proportion of participants with the ratio < 70 in the different age groups . by applying the fev1/fvc thresholds proposed by hardie et al19 for different age groups ( ie , 0.70 for < 70 years", ", 0.65 for 7080 years , and 0.60 for > 80 years ) , the percentage of obstructed subjects decreased to 6% ( men 11% , women 4% ) .", "in particular , the proportion of subjects aged 6570 years with a ratio below 0.70 was 11% ( men 16% , women 8% ) ; the proportion of subjects between 70 and 80 years of age with the ratio below 0.65 was 5% ( men 12% , women 2% ) , whereas none of the subjects aged 80 years or more had fev1/fvc < 0.60 .", "this study provides additional evidence helpful for determining the most appropriate spirometric criteria to define airway obstruction in elderly subjects .", "the fifth percentile of fev1/fvc observed in the considered sample of healthy subjects aged > 65 years was lower than 0.70 in both men and women , thus confirming the inadequacy of this threshold for fev1/fvc after the age of 65 years .", "moreover , the findings of the present study suggest that , in such a population , fev1/fvc < 0.65 and < 0.67 ( for males and females , respectively ) could represent valid criteria that are simple to use and incorporate the known physiological decline in lung function with aging .", "the present findings are in agreement with observations made by other authors,1618 who have emphasized that the use of the fev1/fvc threshold proposed by gold leads to a risk of overdiagnosis of copd in geriatric subjects .", "furthermore , the current results suggest that the inaccuracy of using a threshold of 0.70 for fev1/fvc already exists in subjects aged 65 years and older . this differs to some extent from what has been suggested by the most recent gold guidelines11 ( that recognize some imprecision of the threshold of 0.70 for fev1/fvc in people over 70 years of age ) and by medbo et al,10 who suggested the use of a threshold of fev1/fvc < 0.65 in subjects over the age of 70 years on the basis of prebronchodilator spirometry data from a population - based study in norway .", "interestingly , in our study , the rate of decline in the fev1/fvc ratio with aging was not statistically significant in men or women older than 65 years , because of a concomitant decline in both fev1 and fvc .", "thus , in contrast with the findings of hardie et al,19 our results do not support the need to decrease the lower limit of normal for fev1/fvc to 0.60 in elderly subjects ( > 80 years ) .", "for the same reason , no predictive equation for fev1/fvc could be derived ; accordingly , the mean value and the fifth percentile of fev1/fvc from this healthy population can be used as the predicted value and lower limit of normal , respectively , for people aged 65 years and over .", "all reference equations derived from samples with a wide age range describe a progressive decline in the fev1/fvc ratio with aging ; the age - related decrease involves both fev1 and fvc , seems to be nonlinear , and accelerates with aging.3032 in 1982 , crapo et al33 found that between 20 and 70 years of age , vital capacity decreases to approximately 75% of the best values achieved previously . according to the present observations , presumably in the oldest people the decline of vital capacity accelerates more than in the younger age groups and such a decline is similar to the reduction of fev1 , so that the fev1/fvc ratio could undergo minimal variations in the last decades .", "recently , langhammer et al34 and falaschetti et al35 observed that fev1/fvc reaches a near plateau phase in elderly subjects .", "the authors emphasized that , although the sample consisted of subjects with a wide range of height and age , the extremes did not influence the equations . in a reference study specifically designed for elderly residents in madrid ( age range 6585 years ) , garcia et al36 found a significant relationship between fev1/fvc and age in men and between fev1/fvc , age and height in women . even in this study ,", "the predicted equation for fev1/fvc had a very low r ( 0.048 and 0.083 for men and women , respectively ) and the authors highlighted the strong negative relationship of fvc with age . by applying predictive equations recently derived from kuster et al37 in a swiss population , the lower limit of normal for fev1/fvc does not show important decreases with aging ; for example , in men with a height of 170 cm , the lower limit of normal for fev1/fvc ranges from 0.66 at the age of 65 years to 0.64 for people aged 95 years .", "all these results support a position in favor of almost stable predicted values and lower limits of normal for fev1/fvc in elderly people aged 65 years and over . a clear position in favor of the fixed threshold of fev1/fvc <", "0.70 has been recently reported by other authors , with the claim that it is easy to use , thus helping to remove barriers to widespread use of spirometry.3,68 probably the use of the two lower fixed values for the elderly , suggested by data from the sara study , does not add elements of particular complexity for physicians involved in interpretation of pulmonary function tests .", "however , this approach would diminish the number of false diagnoses of copd , with significant cost savings due to reduction of inappropriately prescribed drugs .", "thus , more resources could be redirected to primary prevention of copd ( smoking cessation ) and treatment of more severe copd .", "authors advocating retaining fev1/fvc < 0.70 often quote the results of mannino et al , who found that such a threshold is very good for identifying patients at risk of death and copd - related hospitalizations.38 however , although this indicates that fev1/fvc < 0.70 may recognize a proportion of individuals at risk , it does not mean that this is the best way to diagnose the disease . on the other hand ,", "vas fragoso et al39 found elevated risk of death and respiratory symptoms in adults with fev1/fvc less than the lower limit of normal .", "sorino et al40 recently confirmed that fev1/fvc less than the lower limit of normal , fev1/fev6 less than the lower limit of normal , and fev1 less than the lower limit of normal are all significant predictors of all - cause and cardiopulmonary mortality in older individuals .", "the strongest spirometric predictor of all - cause mortality remains the appropriately named vital capacity , because the majority of deaths in adult smokers , with or without copd , are caused by cardiovascular disease .", "two recent studies investigated subjects in between the two definitions of airway obstruction ( ie , fev1/fvc < 0.70 but lower limit of normal ) , showing that their clinical profile is characterized by relevant comorbid disease and poor health - related quality of life , but similar exercise , frequency of exacerbations , and indices of systemic effects.41,42 the investigators emphasized that these subjects might be at risk and should be followed carefully ; we should be aware that fewer than one in five smokers with mild airway obstruction ever develop clinically important copd , and that today we are not yet able to identify which smokers will be rapid fallers.2,4 the present study has some limitations .", "first , the cohort was recruited for a cross - sectional investigation , whereas the effect of aging on respiratory function would be better assessed in a longitudinal study .", "it is plausible that fev1/fvc decreases significantly even after the age of 65 years , but this can be less reliable when derived by a cross - sectional observation of older people .", "in fact , they could represent individuals who had higher spirometric values at a younger age , and fev1/fvc similar to those of younger subjects at the time of recruitment .", "second , subjects participating in the sara project were not randomly selected from the population , but consisted mainly of subjects with nonrespiratory illnesses attending outpatient clinics ; this might have resulted in some selection bias , although it would not explain the higher pulmonary volumes than in other studies .", "the authors wish to emphasize that fev1/fvc less than the lower limit of normal should not be the only criterion used for diagnosis of airway obstruction , but should always be combined with evaluation of fev1 as percent of predicted .", "indeed , patients with severe airflow limitation could have an important reduction both in fev1 and fvc , with a sustained fev1/fvc ratio .", "thus , in doubtful cases , measurement of residual volume and total lung capacity is recommended . in conclusion ,", "the present findings confirm the inadequacy of fev1/fvc < 0.70 for diagnosis of airway obstruction in elderly people , and we propose other easy to remember thresholds for fev1/fvc after 65 years of age , ie , 0.65 in men and 0.67 women .", "further studies are needed to assess both the classificatory and prognostic properties of such a threshold as well as epidemiological surveys to confirm it .", "coordinators : v bellia ( palermo ) and f rengo ( napoli ) . scientific committee members : r antonelli incalzi ( taranto ) , v grassi ( brescia ) , s maggi ( padua ) , g masotti ( florence ) , g melillo ( naples ) , d olivieri ( parma ) , m palleschi ( rome ) , r pistelli ( rome ) , m trabucchi ( rome ) , s zuccaro ( rome ) . participating centers , principal investigator , and associated investigators ( the latter in brackets ) : divisione di medicina i , ospedale geriatrici inrca , ancona , dl consales ( d lo nardo , p paggi ) ; divisione di geriatria , ospedale civile , asti , f goria ( p fea , g iraldi , r corradi ) ; cattedra di gerontologia e geriatria , policlinico universitario , bari , a capurso ( r flora , s torres , g venezia , m mesto ) ; divisione di geriatria , ospedale malpighi , bologna , s semeraro ( l bellotti , a tansella ) ; divisione di medicina generale , ospedale civile , brescia , v grassi ( s cossi , g guerini , c fantoni , m de martinis , l pini ) ; clinica pneumologica , fondazione ", "e maugeri , telese , g melillo ( r battiloro , c gaudiosi , s de angelis ) ; istituto di medicina interna e geriatria , ospedale cannizzaro , catania , l motta ( i alessandria , s savia ) ; istituto di gerontologia e geriatrici , ospedale ponte nuovo , universit florence , florence , g masotti ( m chiarlone , s zacchei ) ; divisione di geriatria , ospedale morgagni , forli , v pedone ( d angelini , d cilla ) ; divisione di geriatria , ospedale galliera , genova , e palummeri ( m agretti , p costelli , d torriglia ) ; grouppo ricerca geriatrica ricerca geriatrica , ospedale richiedei , gussago , m trabucchi ( p barbisoni , f guerini , p ranieri ) ; divisione di geriatria , ospedale generale , laquila , f caione ( d caione , m la chiara ) ; divisione di geriatria , ospedale san gerardo , monza , g galetti ( a cantatore , d casarotti , g anni ) ; cattedra di gerontologia e geriatria , universit federico ii , napoli , f rengo ( f cacciatore , ai pisacreta , c calabrese ) ; istituto di medicina interna , ospedale geriatrico , padova , g enzi ( p dalla mont , s peruzza , p albanese , f tiozzo ) ; istituto di clinica delle malattie dellapparato respiratorio , ospedale rasori , parma , d olivieri ( v bocchino , a comel , n barbarito ) ; istituto di gerontologia e geriatria , policlinico monteluce , perugia , u senin ( f arnone , l camilli , s peretti ) ; divisione di geriatria , ospedale israelitico , roma , sm zuccaro ( m marchetti , l palleschi ) ; divisione di geriatria , ospedale generale addolorata , roma , m palleschi ( c cieri , f vetta ) ; istituto di medicina interna e geriatria , policlinico gemelli , roma , pu carbonin ( f pagano , p ranieri ) ; istituto di semeiotica medicina e geriatria , policlinico le scotte , siena , s forconi ( g abate , g marotta , e pagni ) ; fondazione san raffaele , cittadella della carit , taranto , r antonelli - incalzi ( c imperiale , c spada ) ; cattedra di gerontologia e geriatria , ospedale maggiore , milano , c vergani ( g giardini , mc sandrini , i dallera ) ; cattedra di malattie dellapparato respiratorio , ospedale v cervello , palermo , v bellia ( f catalano , n scichilone , s battaglia ) . coordinating center : dipartimento di medicina , pneumologia , fisiologia e nutrizione umana , universit degli studi di palermo ." ]

[ "<S> backgroundthe choice between lower limit of normal or fixed value of forced expiratory volume in one second / forced vital capacity ratio ( fev1/fvc ) < 0.70 as the criterion for confirming airway obstruction is an open issue . in this study , we compared the criteria of lower limit of normal and fixed fev1/fvc for diagnosis of airway obstruction , with a focus on healthy elderly people.methodswe selected 367 healthy nonsmoking subjects aged 6593 years from 1971 participants in the population - based sara ( salute respiratoria nellanziano , italian for respiratory health in the elderly ) study , analyzed their spirometric data , and tested the relationship between spirometric indices and anthropometric variables . </S>", "<S> the lower limit of normal for fev1/fvc was calculated as the fifth percentile of the normal distribution for selected subjects.resultswhile fev1 and fvc decreased significantly with aging , the relationship between fev1/fvc and age was not statistically significant in men or women . </S>", "<S> the lower limit of normal for fev1/fvc was 0.65 in men and 0.67 in women . </S>", "<S> fifty - five participants ( 15% ) had fev1/fvc < 0.70 and would have been inappropriately classified as obstructed according to the global initiative for obstructive lung disease , american thoracic society / european respiratory society , and canadian guidelines on chronic obstructive pulmonary disease . by applying different fev1/fvc thresholds for the different age groups , as previously proposed in the literature , ( 0.70 for < 70 years </S>", "<S> , 0.65 for 7080 years , and 0.60 for > 80 years ) the percentage of patients classified as obstructed decreased to 6% . </S>", "<S> no subjects older than 80 years had an fev1/fvc < 0.60.conclusionthe present results confirm the inadequacy of fev1/fvc < 0.70 as a diagnostic criterion for airway obstruction after the age of 65 years . </S>", "<S> fev1/fvc < 0.65 and < 0.67 ( for men and women , respectively ) could identify subjects with airway obstruction in such a population . </S>", "<S> further reduction of the threshold after 80 years is not justified . </S>" ]

[ "Introduction", "Materials and methods", "Results", "Discussion", "Respiratory Health in the Elderly study group" ]

[ [ "the most appropriate way to diagnose airway obstruction is currently the subject of heated debate.110 most national and international chronic obstructive pulmonary disease ( copd ) guidelines recommend to use a forced expiratory volume in one second / forced vital capacity ratio ( fev1/fvc ) of 0.70 as a suitable threshold value to define the presence of an obstructive ventilatory defect .", "commonly used guidelines include those of the global initiative for obstructive lung disease ( gold),11 the american thoracic society / european respiratory society,12 the british thoracic society,13 the canadian thoracic society,14 and the national institute for health and clinical excellence.15 however , there is evidence that the fixed cut - off value of 0.70 for fev1/fvc becomes less specific in males aged > 40 years and females > 50 years , implying a risk of overestimation of airway obstruction .", "this is due to an age - related decline in pulmonary volumes , particularly in fev1 , which is observed even in healthy people with no history of exposure to noxious particles or gases.1619 proposed strategies for reducing the misclassification of airway obstruction include use of the lower limit of normal for fev1/fvc , calculated as the fifth percentile of the normal distribution in a healthy population,2022 or the use of different fev1/fvc thresholds for different age groups ( eg , 0.70 for subjects aged < 70 years , 0.65 for those aged 7080 years , and 0.60 for those aged > 80 years).19 on this basis , a group of colleagues involved in respiratory research and/or the diagnosis and treatment of lung diseases recently wrote an open letter to members of the gold committee inviting them to change the method by which airway obstruction is defined and asking for retraction of the fixed ratio in favor of the lower limit of normal.1 the aim of the present study was to provide additional information for determination of the most appropriate spirometric criteria for confirming airway obstruction in the elderly , by describing lung function and calculating the lower limit of normal for fev1/fvc in healthy nonsmoking elderly subjects ( age > 65 years ) who participated in the italian multicenter sara ( acronym of salute respiratoria nellanziano , italian for respiratory health in the elderly ) study .", "the degree of potential misclassification relative to use of 0.70 or other proposed fixed thresholds for fev1/fvc was also evaluated ." ], [ "we performed a cross - sectional analysis of data from the sara study , the design of which , along with technical characteristics of instruments as well as training of operators and results of quality control of spirometry , have been described in detail elsewhere.23 briefly , the study involved 24 pulmonary or geriatric institutions distributed throughout italy .", "a total of 1971 subjects aged 65100 years were recruited as consecutive outpatients referred between january 1996 and december 1997 to the participating centers ( see appendix ) .", "lung function was measured using an identical fully computerized water - sealed stead - wells spirometer ( baires system , biomedin , padua , italy ) by specifically trained and certified personnel supervised by a rigorous real - time control of acceptability and repeatability23 according to american thoracic society recommendations.24 fvc maneuvers were performed with the patient sitting .", "the fev1/fvc ratio was calculated on the basis of the highest values of individual parameters obtained in the acceptable curves for each subject .", "analyses were conducted only on patients with good repeatability of the above - mentioned indices ( difference between two best values < 150 ml).25 of the 1870 subjects who performed spirometry , we selected only those without any previous or present diagnosis or any sign or symptom suggestive of respiratory diseases according to the modified international union against tuberculosis and lung disease bronchial symptoms questionnaire.25,26 current smokers and previous smokers with a smoking exposure > 5 pack / year were excluded , since the < 5 pack / year smoking exposure was not significantly associated with decreased lung function.27 additional exclusion criteria were : severe hepatic failure ; severe renal failure ; severe cardiac failure ; cognitive and/or sensory impairment severe enough to affect a multidimensional assessment ; severe kyphoscoliosis with occiput wall distance ( distance between the occiput and the wall when the patient stands with heels and shoulder against the wall with the back straight ) > 10 ; occurrence of a major psychosocial event ( eg , bereavement ) within the past 6 months ; and hospitalization for any reason within the past 6 months .", "we further excluded individuals who had hypertension ( diastolic pressure 90 mmhg and/or systolic pressure 160 mmhg ) , diabetes , and/or major electrocardiographic abnormalities .", "statistical analyses were performed using spss ( spss inc , chicago , il ) and stata ( stata corporation , college station , tx ) software packages .", "regression models were used for testing the relationship between spirometric indices and anthropometric variables . based on the recommendations from the american thoracic society / european respiratory society task force,22 the lower limit of normal for fev1/fvc", "was estimated as the fifth percentile of its frequency distribution . to evaluate the effect of aging on spirometric measures independently of body height , fev1 and fvc were normalized for height at the third power.28,29" ], [ "after applying the above - mentioned selection criteria , our final data set consisted of 367 healthy , nonsmoking subjects .", "one hundred and one subjects were excluded for lack of availability of lung function testing , a further 709 subjects because of a history of respiratory disease , 445 for significant smoking exposure ( > 5 pack / year ) , 262 for inadequate quality of spirometry , and 87 for the above - mentioned additional exclusion criteria . tables 1 and 2 show anthropometric and functional data for the sample and distribution of the participants according to age and gender .", "although the most advanced ages were less represented , a total of 73 subjects aged 80 years and over were included .", "the sample consisted of 314 never - smokers ( 85.6% ) and 53 former - smokers with a smoking exposure from 0.15 to 5 pack - years ( mean standard deviation , 2.75 1.5 ) .", "as shown in figure 1 , the values of fev1/fvc showed a normal frequency distribution .", "the mean fev1/fvc was 0.75 0.6 in males and 0.78 0.6 in females , whereas the corresponding fifth percentiles were 0.65 and 0.67 , respectively , in males and females .", "fev1 and fvc significantly decreased with age ( r = 0.38 and 0.35 ; p < 0.001 ) , while fev1/fvc , was not significantly correlated with age or height in either gender group over the considered range of age ( lowest p = 0.103 ) .", "as a consequence , there was no rationale to develop reference equations for fev1/fvc , with age or height as independent variables in this restricted range of age .", "figure 2 shows the decline in fev1 and fvc with increasing age observed in the study sample in men and women , after normalization for height . in the sample of healthy subjects ,", "15% had a fev1/fvc < 0.70 , and would have been inappropriately classified as obstructed according to gold criteria .", "table 3 describes the proportion of participants with the ratio < 70 in the different age groups . by applying the fev1/fvc thresholds proposed by hardie et al19 for different age groups ( ie , 0.70 for < 70 years", ", 0.65 for 7080 years , and 0.60 for > 80 years ) , the percentage of obstructed subjects decreased to 6% ( men 11% , women 4% ) .", "in particular , the proportion of subjects aged 6570 years with a ratio below 0.70 was 11% ( men 16% , women 8% ) ; the proportion of subjects between 70 and 80 years of age with the ratio below 0.65 was 5% ( men 12% , women 2% ) , whereas none of the subjects aged 80 years or more had fev1/fvc < 0.60 ." ], [ "this study provides additional evidence helpful for determining the most appropriate spirometric criteria to define airway obstruction in elderly subjects .", "the fifth percentile of fev1/fvc observed in the considered sample of healthy subjects aged > 65 years was lower than 0.70 in both men and women , thus confirming the inadequacy of this threshold for fev1/fvc after the age of 65 years .", "moreover , the findings of the present study suggest that , in such a population , fev1/fvc < 0.65 and < 0.67 ( for males and females , respectively ) could represent valid criteria that are simple to use and incorporate the known physiological decline in lung function with aging .", "the present findings are in agreement with observations made by other authors,1618 who have emphasized that the use of the fev1/fvc threshold proposed by gold leads to a risk of overdiagnosis of copd in geriatric subjects .", "furthermore , the current results suggest that the inaccuracy of using a threshold of 0.70 for fev1/fvc already exists in subjects aged 65 years and older . this differs to some extent from what has been suggested by the most recent gold guidelines11 ( that recognize some imprecision of the threshold of 0.70 for fev1/fvc in people over 70 years of age ) and by medbo et al,10 who suggested the use of a threshold of fev1/fvc < 0.65 in subjects over the age of 70 years on the basis of prebronchodilator spirometry data from a population - based study in norway .", "interestingly , in our study , the rate of decline in the fev1/fvc ratio with aging was not statistically significant in men or women older than 65 years , because of a concomitant decline in both fev1 and fvc .", "thus , in contrast with the findings of hardie et al,19 our results do not support the need to decrease the lower limit of normal for fev1/fvc to 0.60 in elderly subjects ( > 80 years ) .", "for the same reason , no predictive equation for fev1/fvc could be derived ; accordingly , the mean value and the fifth percentile of fev1/fvc from this healthy population can be used as the predicted value and lower limit of normal , respectively , for people aged 65 years and over .", "all reference equations derived from samples with a wide age range describe a progressive decline in the fev1/fvc ratio with aging ; the age - related decrease involves both fev1 and fvc , seems to be nonlinear , and accelerates with aging.3032 in 1982 , crapo et al33 found that between 20 and 70 years of age , vital capacity decreases to approximately 75% of the best values achieved previously . according to the present observations , presumably in the oldest people the decline of vital capacity accelerates more than in the younger age groups and such a decline is similar to the reduction of fev1 , so that the fev1/fvc ratio could undergo minimal variations in the last decades .", "recently , langhammer et al34 and falaschetti et al35 observed that fev1/fvc reaches a near plateau phase in elderly subjects .", "the authors emphasized that , although the sample consisted of subjects with a wide range of height and age , the extremes did not influence the equations . in a reference study specifically designed for elderly residents in madrid ( age range 6585 years ) , garcia et al36 found a significant relationship between fev1/fvc and age in men and between fev1/fvc , age and height in women . even in this study ,", "the predicted equation for fev1/fvc had a very low r ( 0.048 and 0.083 for men and women , respectively ) and the authors highlighted the strong negative relationship of fvc with age . by applying predictive equations recently derived from kuster et al37 in a swiss population , the lower limit of normal for fev1/fvc does not show important decreases with aging ; for example , in men with a height of 170 cm , the lower limit of normal for fev1/fvc ranges from 0.66 at the age of 65 years to 0.64 for people aged 95 years .", "all these results support a position in favor of almost stable predicted values and lower limits of normal for fev1/fvc in elderly people aged 65 years and over . a clear position in favor of the fixed threshold of fev1/fvc <", "0.70 has been recently reported by other authors , with the claim that it is easy to use , thus helping to remove barriers to widespread use of spirometry.3,68 probably the use of the two lower fixed values for the elderly , suggested by data from the sara study , does not add elements of particular complexity for physicians involved in interpretation of pulmonary function tests .", "however , this approach would diminish the number of false diagnoses of copd , with significant cost savings due to reduction of inappropriately prescribed drugs .", "thus , more resources could be redirected to primary prevention of copd ( smoking cessation ) and treatment of more severe copd .", "authors advocating retaining fev1/fvc < 0.70 often quote the results of mannino et al , who found that such a threshold is very good for identifying patients at risk of death and copd - related hospitalizations.38 however , although this indicates that fev1/fvc < 0.70 may recognize a proportion of individuals at risk , it does not mean that this is the best way to diagnose the disease . on the other hand ,", "vas fragoso et al39 found elevated risk of death and respiratory symptoms in adults with fev1/fvc less than the lower limit of normal .", "sorino et al40 recently confirmed that fev1/fvc less than the lower limit of normal , fev1/fev6 less than the lower limit of normal , and fev1 less than the lower limit of normal are all significant predictors of all - cause and cardiopulmonary mortality in older individuals .", "the strongest spirometric predictor of all - cause mortality remains the appropriately named vital capacity , because the majority of deaths in adult smokers , with or without copd , are caused by cardiovascular disease .", "two recent studies investigated subjects in between the two definitions of airway obstruction ( ie , fev1/fvc < 0.70 but lower limit of normal ) , showing that their clinical profile is characterized by relevant comorbid disease and poor health - related quality of life , but similar exercise , frequency of exacerbations , and indices of systemic effects.41,42 the investigators emphasized that these subjects might be at risk and should be followed carefully ; we should be aware that fewer than one in five smokers with mild airway obstruction ever develop clinically important copd , and that today we are not yet able to identify which smokers will be rapid fallers.2,4 the present study has some limitations .", "first , the cohort was recruited for a cross - sectional investigation , whereas the effect of aging on respiratory function would be better assessed in a longitudinal study .", "it is plausible that fev1/fvc decreases significantly even after the age of 65 years , but this can be less reliable when derived by a cross - sectional observation of older people .", "in fact , they could represent individuals who had higher spirometric values at a younger age , and fev1/fvc similar to those of younger subjects at the time of recruitment .", "second , subjects participating in the sara project were not randomly selected from the population , but consisted mainly of subjects with nonrespiratory illnesses attending outpatient clinics ; this might have resulted in some selection bias , although it would not explain the higher pulmonary volumes than in other studies .", "the authors wish to emphasize that fev1/fvc less than the lower limit of normal should not be the only criterion used for diagnosis of airway obstruction , but should always be combined with evaluation of fev1 as percent of predicted .", "indeed , patients with severe airflow limitation could have an important reduction both in fev1 and fvc , with a sustained fev1/fvc ratio .", "thus , in doubtful cases , measurement of residual volume and total lung capacity is recommended . in conclusion ,", "the present findings confirm the inadequacy of fev1/fvc < 0.70 for diagnosis of airway obstruction in elderly people , and we propose other easy to remember thresholds for fev1/fvc after 65 years of age , ie , 0.65 in men and 0.67 women .", "further studies are needed to assess both the classificatory and prognostic properties of such a threshold as well as epidemiological surveys to confirm it ." ], [ "coordinators : v bellia ( palermo ) and f rengo ( napoli ) . scientific committee members : r antonelli incalzi ( taranto ) , v grassi ( brescia ) , s maggi ( padua ) , g masotti ( florence ) , g melillo ( naples ) , d olivieri ( parma ) , m palleschi ( rome ) , r pistelli ( rome ) , m trabucchi ( rome ) , s zuccaro ( rome ) . participating centers , principal investigator , and associated investigators ( the latter in brackets ) : divisione di medicina i , ospedale geriatrici inrca , ancona , dl consales ( d lo nardo , p paggi ) ; divisione di geriatria , ospedale civile , asti , f goria ( p fea , g iraldi , r corradi ) ; cattedra di gerontologia e geriatria , policlinico universitario , bari , a capurso ( r flora , s torres , g venezia , m mesto ) ; divisione di geriatria , ospedale malpighi , bologna , s semeraro ( l bellotti , a tansella ) ; divisione di medicina generale , ospedale civile , brescia , v grassi ( s cossi , g guerini , c fantoni , m de martinis , l pini ) ; clinica pneumologica , fondazione ", "e maugeri , telese , g melillo ( r battiloro , c gaudiosi , s de angelis ) ; istituto di medicina interna e geriatria , ospedale cannizzaro , catania , l motta ( i alessandria , s savia ) ; istituto di gerontologia e geriatrici , ospedale ponte nuovo , universit florence , florence , g masotti ( m chiarlone , s zacchei ) ; divisione di geriatria , ospedale morgagni , forli , v pedone ( d angelini , d cilla ) ; divisione di geriatria , ospedale galliera , genova , e palummeri ( m agretti , p costelli , d torriglia ) ; grouppo ricerca geriatrica ricerca geriatrica , ospedale richiedei , gussago , m trabucchi ( p barbisoni , f guerini , p ranieri ) ; divisione di geriatria , ospedale generale , laquila , f caione ( d caione , m la chiara ) ; divisione di geriatria , ospedale san gerardo , monza , g galetti ( a cantatore , d casarotti , g anni ) ; cattedra di gerontologia e geriatria , universit federico ii , napoli , f rengo ( f cacciatore , ai pisacreta , c calabrese ) ; istituto di medicina interna , ospedale geriatrico , padova , g enzi ( p dalla mont , s peruzza , p albanese , f tiozzo ) ; istituto di clinica delle malattie dellapparato respiratorio , ospedale rasori , parma , d olivieri ( v bocchino , a comel , n barbarito ) ; istituto di gerontologia e geriatria , policlinico monteluce , perugia , u senin ( f arnone , l camilli , s peretti ) ; divisione di geriatria , ospedale israelitico , roma , sm zuccaro ( m marchetti , l palleschi ) ; divisione di geriatria , ospedale generale addolorata , roma , m palleschi ( c cieri , f vetta ) ; istituto di medicina interna e geriatria , policlinico gemelli , roma , pu carbonin ( f pagano , p ranieri ) ; istituto di semeiotica medicina e geriatria , policlinico le scotte , siena , s forconi ( g abate , g marotta , e pagni ) ; fondazione san raffaele , cittadella della carit , taranto , r antonelli - incalzi ( c imperiale , c spada ) ; cattedra di gerontologia e geriatria , ospedale maggiore , milano , c vergani ( g giardini , mc sandrini , i dallera ) ; cattedra di malattie dellapparato respiratorio , ospedale v cervello , palermo , v bellia ( f catalano , n scichilone , s battaglia ) . coordinating center : dipartimento di medicina , pneumologia , fisiologia e nutrizione umana , universit degli studi di palermo ." ] ]

[ "proteomics aims to identify large sets of proteins in defined biological fractions , e.g. , tissue or cell extracts , isolated organelles , or protein fractions generated by biochemical preparation procedures . as a first step ,", "protein identifications today are nearly exclusively carried out by mass spectrometry ( ms ) . however , since the mass of a native protein normally does not allow any conclusion on its identity , proteins have to be fragmented by specific endopeptidases before mass analyses to generate defined peptides .", "these peptide fragments are then searched against a database of theoretically digested proteins using a software package such as mascot , sequest , or x !", "many proteome projects use one- or two - dimensional ( 2d ) gel electrophoresis for protein separation . following this experimental strategy ,", "proteins are first separated , then individually picked from gels , treated with an endopeptidase ( trypsin ) and finally identified by ms and database interrogation . as a result , proteome reference maps ", "proteome maps were developed in several fields of biology and are greatly appreciated by the scientific community ( see for instance gallardo et al .", "originally , maps were exclusively presented in scientific publications in the form of images and long lists of identified proteins which are assigned to the spots on the image using arrows and numbers . about a decade ago , the first web - based resources were developed which offer interactive features in the maps : upon clicking on a spot , information on a protein is presented in a small pop - up window which sometimes is linked to further background information in databases ( examples at http://seed.proteome.free.fr/ , http://gabi.rzpd.de/projects/arabidopsis_proteomics/ , http://gene64.dna.affrc.go.jp/rpd/ ) .", "recently , the gelmap software package for annotating gel - based proteome data was developed .", "it offers several new possibilities for the web - based publication of reference maps , e.g. , functional annotation of proteins and assignment of multiple proteins to individual spots on a gel ( rode et al . , 2011a ) .", "in the following sections , we summarize the characteristics of gelmap and introduce novel features which were newly implemented into the gelmap software package ( gelmap 2.0 ) .", "gelmap is offered as a web tool free of charge at www.gelmap.de . minimal requirement for building a new map", "are ( a ) an image file and ( b ) a protein table which includes the coordinates of the spots on the gel .", "( by reading the spot s coordinates from graphics software like photoshop , gimp , or even ms paint ) but is usually generated automatically by specialized proteomics software tools like delta2d ( decodon , greifswald , germany ) , decyder ( ge healthcare , munich , germany ) , or progenesis samespots ( non - linear dynamics , newcastle , uk ) .", "to use gelmap to full capacity , the table should be extended with information on ( 1 ) the spot identification number ( corresponding to the number on the gel image ) , ( 2 ) the mascot probability score ( or the score of another matching software package ) , ( 3 ) coverage of a protein by identified peptides , ( 4 ) the accession number of a protein , ( 5 ) the database used for protein identification , ( 6 ) assignment of a protein to functional categories like a protein complex , a physiological area or subcellular localization , ( 7 ) molecular mass ( calculated and/or apparent mass on the gel ) , etc .", "( table 1 ) . to ensure that gelmap recognizes ids , coordinates , scores , accession numbers , and filters ,", "a second line beneath the line with the column names has to include special keywords ( table 1 ) .", "the third line can contain commentaries for the tooltips in gelmaps spreadsheet view ( figure 1 ) . to mark the columns of high importance", "since gelmap provides a three - level filter tree , the fifth line can define if this column should be used as a ( 1 ) root level , ( 2 ) second level , or ( 3 ) third level filter .", "this control information is required for a fully functional map and is documented with examples on www.gelmap.de/howto .", "the two icons given in the header allow access to the protein spreadsheet and the peptide spreadsheet . ", "the image file and the protein spreadsheet file can be directly uploaded at www.gelmap.de/create .", "the resulting map consists of the gel image , a side menu for functional categories of the identified proteins ( right side ) , and frames for search options ( figure 1 ) .", ", information automatically is displayed in the following way : ( i ) upon hovering over a spot , all proteins identified within this spot are displayed within a tooltip .", "proteins are ordered according to their mascot probability scores ( this , on a semi - quantitative level , reflects abundance of the identified proteins within a protein spot ) .", "( ii ) after clicking a spot , the protein names included in the tooltip are transformed into links , which can be used to get protein - specific information within a second info frame .", "in addition to the information on the protein accession and the calculated molecular mass of the protein this frame can offer links to an external database ( via the accession number ) and to the protein spreadsheet ( link more protein details ) .", "the protein spreadsheet also can be accessed directly by using an icon in the header ( figure 1 ) .", "( iii ) proteins assigned to functional categories are accessible via the three - level tree in the side menu . upon clicking onto a topic included in this menu ,", "all proteins forming part of this category automatically become highlighted by circles on the gel image . at the same time , subcategories become visible for many topics of the main menu , which allow to differentially visualize proteins assigned to the next functional level . for details", "we recommend visiting one of the established gelmaps , e.g. , http://gelmap.de/47 . to display primary ms data for all identified proteins , a second table called peptide spreadsheet was introduced in gelmap 2.0 ( figure 1 ) .", "this table may include information on all identified peptides within a spot . in the case of the arabidopsis thaliana bn / sds map ,", "the table includes information on the amino acid sequences of the peptides , peptide modifications , and positions of peptides with respect to the complete protein sequences ( column range : number of the first and the last amino acid of a peptide with respect to the n - terminal methionine ) .", "meta portals which are realized on the basis of peptide position data ( see integration of gelmap into the mascp gator ) .", "the linked icon for the peptide spreadsheet is located next to the protein spreadsheet icon at the top ( figure 1 ) .", "alternatively , the peptide spreadsheet is accessible through a text link in the information frames for individual proteins on the gel image .", "the content of this table can be freely defined as long as the first column contains the hit s i d to link this information to the spots ( see http://www.gelmap.de/47 for example ) . in summary", ", gelmap offers the following features : proteins of interest can easily be found using the different search options or by browsing / navigating through / using the categories in the side menu . since all proteins are annotated according to functional criteria , sets of proteins involved in metabolic processes or other functional categories are easily visualized .", "detailed information on the identified proteins is provided by the information frames which are accessible via the gel image .", "background information and raw data on all proteins is given in the protein spreadsheet and the peptide spreadsheet ", "gelmap allows complete annotation of ms data for all analyzed protein spots : not only the protein with the highest mascot probability score is given , but all identified proteins above a lower boundary score to be defined by the user .", "the gelmap portal was established for creation and presentation of reference maps . upon finishing a new map", ", a user can decide to make it accessible via the official gelmap site and/or via a direct link which can be presented in publications .", "gelmap also allows private ( password protected ) projects to share proteome data in a closed group or analyzing results without sharing them . currently ( february 28th , 2012 ) , four projects are publicly available at the gelmap portal : the cyclamen persicum seed proteome ( rode et al . , 2011b ) . in the frame of this project ,", "identified proteins are assigned to 30 different metabolic pathways within 10 metabolic divisions . furthermore .", "in addition to the features mentioned above , this gelmap allows to differentially display proteins of changed abundance between the two compared protein fractions .", "this illustrates the various extensions which easily can be realized based on the gelmap software package .", "the a. thaliana mitochondrial proteome separated by 2d blue native / sds page ( klodmann et al . , 2011", "this map is based on a special protein separation procedure : protein complexes are separated under native conditions on the first gel dimension and the subunits of the protein complexes under denaturing conditions on the second dimension , which is carried out in the presence of sds . on the resulting gels , proteins belonging to the same protein complex form a vertical row of spots .", "for 2d blue native / sds page , gelmap offers special advantages : selective display of functional categories allows identifying vertical positioning of proteins of low abundance .", "this led to the discovery of new protein complexes in arabidopsis mitochondria . in the frame of this study ,", "471 non - redundant proteins were identified and assigned to more than 35 protein complexes .", "the a. thaliana mitochondrial proteome separated by 2d ief / sds page ( taylor et al . , 2011 ) .", "recently , the proteomic aggregation utility mascp gator has been established to link proteome databases for the model plant a. thaliana ( joshi et al . , 2011 ) .", "in this platform , protein data is simultaneous displayed from many different international repositories to allow assessment of the global knowledge of a protein or several proteins .", "so far , projects integrated in the mascp gator exclusively represented shot - gun approaches not based on 2d page . since september 2011 , gelmap offers a limited application programming interface ( api ) which allows other projects ( such as gator ) to run automated search queries on the gelmap database and collect the results .", "the two gelmaps dedicated to the mitochondrial proteome of arabidopsis very recently were the first gel - based projects integrated into the mascp gator . upon submission of a protein accession", ", the user of the mascp gator directly can access the reference maps at www.gelmap.de ( figure 2 ) .", "information on peptides available at gelmap is graphically displayed at the mascp gator site together with peptide information of other databases . in the future", "integration of the mitochondrial reference maps at gelmap into mascp gator ( http://gator.masc-proteomics.org/ ) . upon submission of a protein accession ( here : at2g47260 ) , matching peptides from gelmap are graphically displayed at the gator site .", "direct access to gelmap is provided by links in the left side menu of the gator .", "the gelmap software package allows complete annotation of ms data corresponding to 2d protein separations . based on its central features functional annotation of proteins and at the same time annotation of complete sets of proteins identified within each spot it allows the comprehensive evaluation of gel - based proteome data sets . in the future , the gelmap platform will be used to annotate further projects .", "integration of these projects into the mascp gator might soon allow extensive coverage of the arabidopsis proteome by gel - based analyses .", "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 ." ]

[ "<S> in classical proteome analyses , final experimental data are ( a ) images of 2d protein separations obtained by gel electrophoresis and ( b ) corresponding lists of proteins which were identified by mass spectrometry ( ms ) . for data </S>", "<S> annotation , software tools were developed which allow the linking of protein identity data directly to 2d gels ( clickable gels ) . </S>", "<S> gelmap is a new online software tool to annotate 2d protein maps . </S>", "<S> it allows ( i ) functional annotation of all identified proteins according to biological categories defined by the user , e.g. , subcellular localization , metabolic pathway , or assignment to a protein complex and ( ii ) annotation of several proteins per analyzed protein spot according to ms primary data . </S>", "<S> options to differentially display proteins of functional categories offer new opportunities for data evaluation . </S>", "<S> for instance , if used for the annotation of 2d blue native / sds gels , gelmap allows the identification of protein complexes of low abundance . </S>", "<S> a web portal has been established for presentation and evaluation of protein identity data related to 2d gels and is freely accessible at http://www.gelmap.de/. </S>" ]

[ "Introduction", "The GelMap Software Package", "The GelMap Portal", "Integration of GelMap Into MASCP Gator", "Perspective", "Conflict of Interest Statement" ]

[ [ "proteomics aims to identify large sets of proteins in defined biological fractions , e.g. , tissue or cell extracts , isolated organelles , or protein fractions generated by biochemical preparation procedures . as a first step ,", "protein identifications today are nearly exclusively carried out by mass spectrometry ( ms ) . however , since the mass of a native protein normally does not allow any conclusion on its identity , proteins have to be fragmented by specific endopeptidases before mass analyses to generate defined peptides .", "these peptide fragments are then searched against a database of theoretically digested proteins using a software package such as mascot , sequest , or x !", "many proteome projects use one- or two - dimensional ( 2d ) gel electrophoresis for protein separation . following this experimental strategy ,", "proteins are first separated , then individually picked from gels , treated with an endopeptidase ( trypsin ) and finally identified by ms and database interrogation . as a result , proteome reference maps ", "proteome maps were developed in several fields of biology and are greatly appreciated by the scientific community ( see for instance gallardo et al .", "originally , maps were exclusively presented in scientific publications in the form of images and long lists of identified proteins which are assigned to the spots on the image using arrows and numbers . about a decade ago , the first web - based resources were developed which offer interactive features in the maps : upon clicking on a spot , information on a protein is presented in a small pop - up window which sometimes is linked to further background information in databases ( examples at http://seed.proteome.free.fr/ , http://gabi.rzpd.de/projects/arabidopsis_proteomics/ , http://gene64.dna.affrc.go.jp/rpd/ ) .", "recently , the gelmap software package for annotating gel - based proteome data was developed .", "it offers several new possibilities for the web - based publication of reference maps , e.g. , functional annotation of proteins and assignment of multiple proteins to individual spots on a gel ( rode et al . , 2011a ) .", "in the following sections , we summarize the characteristics of gelmap and introduce novel features which were newly implemented into the gelmap software package ( gelmap 2.0 ) ." ], [ "gelmap is offered as a web tool free of charge at www.gelmap.de . minimal requirement for building a new map", "are ( a ) an image file and ( b ) a protein table which includes the coordinates of the spots on the gel .", "( by reading the spot s coordinates from graphics software like photoshop , gimp , or even ms paint ) but is usually generated automatically by specialized proteomics software tools like delta2d ( decodon , greifswald , germany ) , decyder ( ge healthcare , munich , germany ) , or progenesis samespots ( non - linear dynamics , newcastle , uk ) .", "to use gelmap to full capacity , the table should be extended with information on ( 1 ) the spot identification number ( corresponding to the number on the gel image ) , ( 2 ) the mascot probability score ( or the score of another matching software package ) , ( 3 ) coverage of a protein by identified peptides , ( 4 ) the accession number of a protein , ( 5 ) the database used for protein identification , ( 6 ) assignment of a protein to functional categories like a protein complex , a physiological area or subcellular localization , ( 7 ) molecular mass ( calculated and/or apparent mass on the gel ) , etc .", "( table 1 ) . to ensure that gelmap recognizes ids , coordinates , scores , accession numbers , and filters ,", "a second line beneath the line with the column names has to include special keywords ( table 1 ) .", "the third line can contain commentaries for the tooltips in gelmaps spreadsheet view ( figure 1 ) . to mark the columns of high importance", "since gelmap provides a three - level filter tree , the fifth line can define if this column should be used as a ( 1 ) root level , ( 2 ) second level , or ( 3 ) third level filter .", "this control information is required for a fully functional map and is documented with examples on www.gelmap.de/howto .", "the two icons given in the header allow access to the protein spreadsheet and the peptide spreadsheet . ", "the image file and the protein spreadsheet file can be directly uploaded at www.gelmap.de/create .", "the resulting map consists of the gel image , a side menu for functional categories of the identified proteins ( right side ) , and frames for search options ( figure 1 ) .", ", information automatically is displayed in the following way : ( i ) upon hovering over a spot , all proteins identified within this spot are displayed within a tooltip .", "proteins are ordered according to their mascot probability scores ( this , on a semi - quantitative level , reflects abundance of the identified proteins within a protein spot ) .", "( ii ) after clicking a spot , the protein names included in the tooltip are transformed into links , which can be used to get protein - specific information within a second info frame .", "in addition to the information on the protein accession and the calculated molecular mass of the protein this frame can offer links to an external database ( via the accession number ) and to the protein spreadsheet ( link more protein details ) .", "the protein spreadsheet also can be accessed directly by using an icon in the header ( figure 1 ) .", "( iii ) proteins assigned to functional categories are accessible via the three - level tree in the side menu . upon clicking onto a topic included in this menu ,", "all proteins forming part of this category automatically become highlighted by circles on the gel image . at the same time , subcategories become visible for many topics of the main menu , which allow to differentially visualize proteins assigned to the next functional level . for details", "we recommend visiting one of the established gelmaps , e.g. , http://gelmap.de/47 . to display primary ms data for all identified proteins , a second table called peptide spreadsheet was introduced in gelmap 2.0 ( figure 1 ) .", "this table may include information on all identified peptides within a spot . in the case of the arabidopsis thaliana bn / sds map ,", "the table includes information on the amino acid sequences of the peptides , peptide modifications , and positions of peptides with respect to the complete protein sequences ( column range : number of the first and the last amino acid of a peptide with respect to the n - terminal methionine ) .", "meta portals which are realized on the basis of peptide position data ( see integration of gelmap into the mascp gator ) .", "the linked icon for the peptide spreadsheet is located next to the protein spreadsheet icon at the top ( figure 1 ) .", "alternatively , the peptide spreadsheet is accessible through a text link in the information frames for individual proteins on the gel image .", "the content of this table can be freely defined as long as the first column contains the hit s i d to link this information to the spots ( see http://www.gelmap.de/47 for example ) . in summary", ", gelmap offers the following features : proteins of interest can easily be found using the different search options or by browsing / navigating through / using the categories in the side menu . since all proteins are annotated according to functional criteria , sets of proteins involved in metabolic processes or other functional categories are easily visualized .", "detailed information on the identified proteins is provided by the information frames which are accessible via the gel image .", "background information and raw data on all proteins is given in the protein spreadsheet and the peptide spreadsheet ", "gelmap allows complete annotation of ms data for all analyzed protein spots : not only the protein with the highest mascot probability score is given , but all identified proteins above a lower boundary score to be defined by the user ." ], [ "the gelmap portal was established for creation and presentation of reference maps . upon finishing a new map", ", a user can decide to make it accessible via the official gelmap site and/or via a direct link which can be presented in publications .", "gelmap also allows private ( password protected ) projects to share proteome data in a closed group or analyzing results without sharing them . currently ( february 28th , 2012 ) , four projects are publicly available at the gelmap portal : the cyclamen persicum seed proteome ( rode et al . , 2011b ) . in the frame of this project ,", "identified proteins are assigned to 30 different metabolic pathways within 10 metabolic divisions . furthermore .", "in addition to the features mentioned above , this gelmap allows to differentially display proteins of changed abundance between the two compared protein fractions .", "this illustrates the various extensions which easily can be realized based on the gelmap software package .", "the a. thaliana mitochondrial proteome separated by 2d blue native / sds page ( klodmann et al . , 2011", "this map is based on a special protein separation procedure : protein complexes are separated under native conditions on the first gel dimension and the subunits of the protein complexes under denaturing conditions on the second dimension , which is carried out in the presence of sds . on the resulting gels , proteins belonging to the same protein complex form a vertical row of spots .", "for 2d blue native / sds page , gelmap offers special advantages : selective display of functional categories allows identifying vertical positioning of proteins of low abundance .", "this led to the discovery of new protein complexes in arabidopsis mitochondria . in the frame of this study ,", "471 non - redundant proteins were identified and assigned to more than 35 protein complexes .", "the a. thaliana mitochondrial proteome separated by 2d ief / sds page ( taylor et al . , 2011 ) ." ], [ "recently , the proteomic aggregation utility mascp gator has been established to link proteome databases for the model plant a. thaliana ( joshi et al . , 2011 ) .", "in this platform , protein data is simultaneous displayed from many different international repositories to allow assessment of the global knowledge of a protein or several proteins .", "so far , projects integrated in the mascp gator exclusively represented shot - gun approaches not based on 2d page . since september 2011 , gelmap offers a limited application programming interface ( api ) which allows other projects ( such as gator ) to run automated search queries on the gelmap database and collect the results .", "the two gelmaps dedicated to the mitochondrial proteome of arabidopsis very recently were the first gel - based projects integrated into the mascp gator . upon submission of a protein accession", ", the user of the mascp gator directly can access the reference maps at www.gelmap.de ( figure 2 ) .", "information on peptides available at gelmap is graphically displayed at the mascp gator site together with peptide information of other databases . in the future", "integration of the mitochondrial reference maps at gelmap into mascp gator ( http://gator.masc-proteomics.org/ ) . upon submission of a protein accession ( here : at2g47260 ) , matching peptides from gelmap are graphically displayed at the gator site .", "direct access to gelmap is provided by links in the left side menu of the gator ." ], [ "the gelmap software package allows complete annotation of ms data corresponding to 2d protein separations . based on its central features functional annotation of proteins and at the same time annotation of complete sets of proteins identified within each spot it allows the comprehensive evaluation of gel - based proteome data sets . in the future , the gelmap platform will be used to annotate further projects .", "integration of these projects into the mascp gator might soon allow extensive coverage of the arabidopsis proteome by gel - based analyses ." ], [ "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 ." ] ]

[ "brain structure segmentation is the apportionment of brain tissue into gray matter and white matter , based on the appearance of tissue in images produced by magnetic resonance imaging ( mri ) .", "because manual tracing of the boundaries between tissues in the brain is labor intensive , difficult , error - prone , and unrealistic for large amounts of data , an automated or semiautomated segmentation technique is needed for either visualization or diagnosis .", "different imaging modalities , such as t1-weighted , t2-weighted , or proton density ( pd ) images , have been used for different segmentation methods .", "t1-weighted images , because of their good contrast , have been widely tested for various segmentation methods [ 26 ] .", "a t1-map is a parametric image of pure t1 ( spin lattice relaxation time ) , derived from the solution of an equation describing tissue relaxation , and a parametric t1-map which is different from a t1-weighted image .", "the relationship between t1 and several diseases , such as schizophrenia or sickle cell disease , has been studied [ 710 ] , and t1 may be used as a possible indicator of pathology .", "change in t1 of certain voxels in the brain over time may be an early indicator of possible pathology .", "therefore , the segmentation of a parametric brain t1-map may highlight pathology unseen by other imaging approaches . ", "the three tissues white matter ( wm ) , gray matter ( gm ) , and cerebrospinal fluid ( csf ) constitute the main parts in the brain .", "different methods have been proposed to achieve this goal , and they may be classified into various categories : fuzzy segmentation methods [ 4 , 11 ] , markov random field ( mrf ) methods [ 12 , 13 ] , bayesian methods , active contour methods [ 1 , 5 , 6 ] , or the combinations of two or more techniques", ". some of these combinations are as follows : leemput et al . used expectation maximization ( em ) and mrf , xu et al", "it is an adaptive version of mumford - shah 's model to systematically segment different tissues in the brain . before the segmentation ,", "a t1-map may be calculated by a rapid method known as the variable nutation method which provides comparable precision but much faster speed over conventional methods .", "this method requires the acquisition of a set of flip angle images and the t1 information can be extracted therein .", "[ 18 , 19 ] proposed methods to determine the optimal flip angles by basically maximizing the signal - to - noise ratio ( snr ) of a t1-map .", "their first work required the knowledge of average tr / t1 in advance though , and their second work introduced a weighted least square method to estimate the angles .", "we take another approach to determine the flip angles to achieve the trade - off between acquisition time and t1 accuracy . ", "since a t1-map is generated by a set of flip angle images , alignment of the images is important in order to obtain an accurate t1-map .", "the registration of flip angle images is usually ignored though because the movement of the head in the coil is minute .", "however , slight registration errors affect the resulting t1-map dramatically , as will be shown . ", "radiofrequency ( rf)-inhomogeneity is another unavoidable problem encountered in mr imaging . the nonuniform distribution of the rf field can cause the resulting images to have low contrast and inhomogeneous intensity , which makes quantitative description and segmentation of the image difficult .", "rf inhomogeneity affects the generation of t1-map in the sense that the spins are not tilted by the predefined nominal angles [ 17 , 2022 ] .", "hence , we focus on calibrating the rf nonuniformity in order to generate an accurate t1-map .", "cheng and wright calculated an analytical form of t1 errors induced by rf nonuniformity and allowed simple correction of t1 measurements .", "we propose a method , which assumes the average t1 value among a transverse plane is the same across the central brain slices , to jointly segment a t1-map and calibrate the rf nonuniformity along the direction perpendicular to the transverse plane .", "taking into account that we average out the heterogeneity of t1 , and doing so only within few central brain slices , this assumption help calibrate rf - inhomogeneity and generate a quantitative t1-map for segmentation . ", "section 2 we state our adapted model , with a fast implementation method . in section 3", "we first illustrate some necessary preprocessing to generate an accurate t1-map , which includes the registration of flip angle images with the generation of a brain mask and the determination of optimal flip angles , to achieve a trade - off between quality and efficiency .", "we propose a novel method to jointly segment a t1-map and calibrate rf - inhomogeneity , and in section 5 we show the results of registering the flip angle images , determining optimal flip angles , and segmenting the t1-maps .", "we also show validations , the resulting t1-map after rf - calibration , and the 3d segmentation for two sets of t1-weighted data . at last in section 6", "active contour methods comprise a popular segmentation technique in which an initialized contour is driven by a partial differential equation ( pde ) to minimize an energy functional designed to attract the contour toward image edges .", "active contour methods can be classified into two categories : edge - based [ 2427 , 2734 ] and region - based [ 16 , 3544 ] methods .", "edge - based methods examine the gradient information of the image , and stop the contour whenever the gradient is high .", "however , there are many situations when the edge is not clearly characterized by the gradient , and it has been shown that region - based methods outperform edge - based methods [ 3537 , 41 , 43 , 44 ] . by examining some statistics of the region inside and outside of the active contour , and optimizing the separation of these two statistics , we may achieve a better segmentation performance , thus making region - based methods more attractive . ", "our proposed model is a modified momford and shah functional , and falls in the region - based category .", "two adaptations of mumford - shah 's model constitute the novelty of our proposed technique .", "first is the incorporation of an information - theoretic view , which characterizes the statistical property of data .", "the second is a selective weighting , similar to the weight parameter in , which favors erring towards one tissue type or another , and thus make 3-tissue segmentation possible .", "we use a modified mumford - shah energy functional : \n\t\t\t\t\t\t\t\t ( 1)e(frin , frout , c)=i = in , outiri{(frig(i))2+fri2}dx+cds , \n\t\t\t\t\t\t\t where fri approximate a function g( ) applied of the image i for region ri , i = in or out .", "fri is smooth within each region ri , but not across the boundaries ; c denotes the region boundaries , which is the contour of interest in this paper , and 0 i 1 are weights such that in + out = 1 . by minimizing this functional", "we obtain a function fri which is faithful to the image ( first term ) and smooth within each region but not across the boundaries ( second term ) , while penalizing excessive length of the boundaries ( last term ) . ", "the first adaptation is the function g( ) applied on to image i instead of the image itself .", "specifically , an information - theoretic approach for maximizing a probabilistic disparity measure , jensen - shannon ( js ) divergence , was proposed .", "a constructed function g( ) characterizes the property of the probability density function ( pdf ) of the image intensity such as skewness ( g(i ) = i ) , or kurtosis ( g(i ) = i ) , relative to a gaussian . a proper choice of g( )", "will capture the statistical characteristics of the data and will hence achieve a good segmentation . ", "the second adaptation , similar to that in , is the selective weight i applied on the inside / outside energy terms .", "this provides a probabilistic assignment to the segmented regions , and also makes 3-tissue segmentation possible , as will be shown in section 3 . enhancing the weight in is tantamount to penalizing both the error of the difference between the approximated function frin and the data fidelity term g(i ) , as well as the degree of smoothness of frin", "this would yield a smaller segmented region which is likely to be more faithful to the image , and of purer tissues .", "figure 1 shows examples of white matter ( wm ) segmentation with different weights . with a larger inside - weight in ,", "the segmented region is smaller and the segmented tissues are purer . minimizing the mumford - shah energy", "functional involves solving for the approximating functions frin / out and for the contour c. the joint search for these infinite dimensional unknowns usually entails gradient descent flows .", "in particular , the approximated functions are typically modeled as a linear combination of a basis set whose dimension equals the number of pixels in the image , that is , each pixel is assumed to be independent .", "alvino and yezzi proposed a fast implementation using a significantly smaller basis number to model the approximated functions , while still achieving sufficient resemblance to the obtained functions when using the pixel - by - pixel basis . ", "we adopt their so - called linear heat equation basis with the change from i to g(i ) to incorporate the statistical properties of the data from an information - theoretic point of view in the previous section .", "we hence have , \n\t\t\t\t\t\t\t\t ( 2)fri=1,ig(i)+2,imean(g(i ) ) , \n\t\t\t\t\t\t\t where mean( ) is the average function , and the coefficients j , in(out ) , j = 1,2 , may be similarly derived in .", "our derivations are included in the appendix . substituting ( 2 ) into ( 1 ) , and using a classical methods of variational calculus", ", the gradient descent evolution of the curve may be derived as \n\t\t\t\t\t\t\t\t ( 3)ct={out[(froutg(i))2+frout2 ] in[(fring(i))2+frin2]}nn,(4)=(outfoutinfin)nn , \n\t\t\t\t\t\t\t where denotes the curvature of the contour c , t an artificial time evolution parameter , and n the outward normal of the contour .", "fin / out are derived by substituting frin / out from ( 2 ) into ( 3 ) as shown in the appendix .", "a brain parametric t1-map is the calculated result ( variable nutation method ) from several flip angle images . because the flip angle images are acquired at different times , and because the subject may move during image acquisition , registration should be carried out first to obtain an accurate t1-map .", "moreover , in order to achieve a balance between the acquisition time and the resulting t1-map quality , we propose a method to determine optimal flip angles . in the following subsections", "we will first illustrate how we register the flip angle images and obtain a brain mask as a byproduct , then describe our method to determine a set of optimal flip angles , and at last describe our proposed procedure to segment a t1-map .", "the value of t1 is traditionally determined by acquisition methods such as inversion recovery ( ir ) or saturation - recovery ( sr ) .", "other rapid methods , such as variable nutation ( the despot method ) have been proposed , and require acquisition of several flip angle images , and calculation of t1 .", "since these flip angle images are acquired at different times , registration must first be accomplished . even though the interval between consecutive scans may be as short as two minutes , and movement of the subject 's head inside the receiver coil", "may be a few pixels ( under the resolution of a 256 256 image ) , the effects of such off - registration can be significant , as shown ( figure 5 ) . here", "we describe a method to register flip angle images and jointly obtain a mask , as a byproduct , to get rid of the skull and other structures around the brain . ", "we use a joint segmentation and registration ( jsr ) technique proposed by yezzi et al . with an additional tuning weight , to achieve registration and to obtain the mask .", "the theory of jsr technique consists of evolving two contours , with a enforced relationship ( ex : rigid or affine transform ) between them , in two images according to a partial differential equation ( pde ) which is a result of optimizing , for example , the sum of two energy functionals . for our particular task", ", we may choose a region - based energy , such as chan and vese 's model incorporating weights , which arises as a special case of ( 1 ) with the data term g(i ) = i and the approximating function frin / out = mean(i ) inside and outside the contour .", "this model approximates the image i by a simple piecewise constant function , which suits our goal here because it creates a mask that divides the image into two parts- brain region and nonbrain region . ", "we observe that the boundary between the brain and nonbrain is visually more easily distinguished than the boundary between different tissues within the brain , for every flip angle image .", "we therefore put a very small weight in on the inside energy in ( 1 ) to penalize very little the difference between the data inside the contour , i , and the approximated function mean(i ) .", "we carry out this registration technique pairwise for all flip angle images , and once the flip - angel images are registered to each other , they are used to generate a t1-map .", "the determination of the flip angles that yields qualitative t1-map and saves time , is mainly by comparing the difference between the gold standard and the t1-maps generated from different combinatorial flip angles .", "we begin by acquiring images at a set of 19 flip angles which spans the range of standard angles and will give an optimized t1-map .", "we call this particular t1-map the gold standard , or t1 g , and denote this set of angles as 19 = [ 2 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 ] .", "we then compare the t1-map generated from all combinations of the subset of 19 with t1 g . out of these combinations", "since the data acquisition is slice - based , this study of optimal flip angle focuses on the central slice , which is least affected by rf - inhomogeneity and is routinely selected at the level of the basal ganglia , including both the genu and the splenium of the corpus callosum , and generally shows the putamen and lateral ventricle .", "we denote the optimal n - angles opt , n , which is a subset of 19 , as those that exhibit the smallest difference between t1 g and the t1-map generated by n flip angles : \n\t\t\t\t\t\t\t\t ( 5)opt , n = arg min n(x , y)brain|t1g(x , y)t1n(x , y)| , \n\t\t\t\t\t\t\t where n { 2,3 , , 19 } , n19 , t1n denotes the t1-map generated by n , and the summation of ( x , y ) is over the whole brain region at the central slice . equation ( 5 ) therefore gives 18 sets , with the number of elements ranging from 2 to 19 , of optimal angles . for the determination of the optimal set of flip angles , in reaching a compromise between efficiency and t1-map quality", ", we examine the error between t1opt , n and t1 g and compute the error rate .", "the error rate is defined as \n\t\t\t\t\t\t\t\t ( 6)e=(x , y)braine(x , y)a(brain) , \n\t\t\t\t\t\t\t where a(brain ) is the area of the brain , and e(x , y ) is the error , defined as 1 if the difference between t1g(x , y ) and t1opt , n(x , y ) is greater than some threshold , and 0 otherwise . the threshold is defined as the minimum of the standard deviation among the t1 values of three brain tissues ( wm , gm , and csf ) manually segmented by an expert .", "the summation of e(x , y ) is over the whole brain at the central slice . ", "the plot of the error rate versus the number of flip angles is shown in figure 6 , where the inflection point of the fitted curve is at 10 flip angles ( opt,10 = [ 2 , 5 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 ] ) .", "once the brain mask is obtained ( section 3.1 ) , it is used to segment away the skull , leaving only three major tissues in the image : wm , gm , and csf .", "notice that the curve evolution corresponding to the energy introduced in section 2 always results in a binary segmentation , where we will have regions inside ( foreground ) and outside ( background ) the contour(s ) .", "we can not simultaneously segment the three tissues , even though we will later talk about multiphase segmentation .", "we may , however , tune the weights ( section 2.1 ) , to penalize the error between the data term and the approximated function ( 1 ) to segment one tissue at a time , with a progression analogous to that of peeling an onion . ", "we illustrate our t1-map segmentation procedure for a hard segmentation of three tissues , and the probabilistic segmentation is obtained by varying the weights around the value of the trained weight ( section 5.4 ) .", "a t1-map segmentation procedure consists of three steps , where the first two steps are to evolve the contours by minimizing the energy in ( 1 ) with different weights , and the third step is just a simple subtraction .", "the procedure is as follows : treat wm as the foreground , everything else as the background ; let in in ( 1 ) be the trained in , wm , and use it to segment wm in the interior region of the contour . ", "treat wm and gm as the foreground , csf and everything else as the background ; let in in ( 1 ) be the trained in , csf , and use it to obtain csf in the exterior region of the curve filtered by the brain mask . ", "the procedure is based on the anatomical observation that gm is enclosed by csf , and that csf is separated from wm [ 1 , 3 ] , such that we may peel off one layer at a time . ", "the choice of function g(i ) in ( 1 ) , which is chosen to better capture the statistical property of t1-map ( and for other modality ) , will be shown in section 5 .", "the values of in , wm and in , csf are determined through a training process .", "if re denotes the segmentation region by the expert , and rin denotes the segmentation region by the weight in with some fixed g(i ) , for some tissue , then the value of in , tissue is determined by minimizing \n\t\t\t\t\t\t\t\t ( 7)in , tissue = argmin in{1|rerin||rin|+|re||rerin| } , \n\t\t\t\t\t\t\t where |r| denotes the number of pixels in region r. the first term inside the bracket is an overlap metric ( om ) which will be discussed again in section 4 .", "the strength of the rf field varies within the mr scanner , such that the resulting image may be of low contrast or of nonuniform intensity . in what follows", ", we will first describe how a t1-map is calculated from a set of flip angle images , then how the t1-map is affected by rf - inhomogeneity and then show our proposed correction method . a t1-map is calculated by variable nutation .", "flip - angle images are acquired using a flash sequence at different flip angles , and t1 is calculated from the slope of a least square fitted line to the pair of data [ s()/sin , s()/tan ] , where s( ) is the signal strength of the flip angle image expressed as a function of the flip angle . rf - inhomogeneity affects the computation of t1 in that the spins are not tilted by the nominal angle because the strength of the rf field is not as predefined .", "calibration of rf - inhomogeneity , or , the correction of flip angles , produces a more accurate t1-map . a spatially dependent scaling factor , therefore , is introduced to adjust the tilted angle [ 20 , 22 ] , that is , replacing by (8)[s()sin(),s()tan( ) ] , \n\t\t\t\t\t\t\t and t1 is extracted from the slope of the line fitted to the above space .", "our imaging setting is slice - based across different transverse planes ( figure 2 ) , thus our proposed joint segmentation and rf - inhomogeneity calibration ( jsric ) method is as an initial step to easily calibrate rf - inhomogeneity vertically .", "our method is based on the assumption that the average t1 of wm should be roughly the same across central brain transverse slices .", "we however average out the heterogeneity of t1 , and carry out the method only within few central brain slices .", "the advantage is that as the segmentation delineates more precisely the wm region , flip angle correction is therefore enhanced , and as flip angle correction is carried out more precisely , which gives better quality t1-map , segmentation is facilitated as well . ", "our joint segmentation and rf - inhomogeneity calibration ( jsric ) method requires first taking the average t1 value for segmented wm at the central slice ( which has relatively uniform b1 field [ 51 , 53 ] ) as the reference , and then iteratively segmenting and searching for the scaling factor in ( 8) for all other slices .", "this is a three - step iterative process ( as shown inside the dashed box in flowchart 3 ) . ", "segment wm for the central slice by the method proposed in section 3.3 , compute the average t1 value of wm , and denote the average as m. for slice m , varies in ( 8) , and calculate the corresponding t1-map , denoted as t1( ) .", "if goes beyond max , claim the current slice as uncalibratable and repeat this step for the next slice m + 1 .", "segment wm for t1( ) , compute the average t1 of wm , and denote it by l( ) .", "if m l( ) , go back to step 2 and increase , otherwise claim it is done for the current slice . ", "the variables min , max , and in flowchart 3q are all predefined parameters .", "in this section we show a series of results from segmenting a parametric brain t1-map , which includes the registration of flip angle images , generation of a brain mask as a byproduct , determination of the optimal flip angles , and joint t1-map segmentation and rf - inhomogeneity calibration .", "in addition , we will apply our proposed segmentation method to another modality , t1-weighted images , in a 3d setting , to show the generality of our segmentation method .", "the subjects being scanned were recruited from a clinic in the department of psychiatry at the university of north carolina , under an irb - approved protocol to image the brain .", "a transverse 3d flash sequence using different flip angles was acquired in a 3 t siemens mri scanner with a quadrature head coil .", "the scan parameters were : tr = 25 msec , te = 2.83 msec , 16 slices , and slice thickness = 5 mm .", "the center slice used for optimal flip angle study was routinely selected as described ( section 3.2 ) .", "the registration of a set of flip angle images is carried out pair wise , and figure 4 shows two flip angle images and the resulting brain mask .", "note that even though the flip angle images are off - registered by no more than four pixels in both the x- and y - directions , the impact is obvious .", "figure 5 shows two t1-maps generated by registered and unregistered flip angle images , and we can see that after registration the anatomical structure of the t1-map is more readily distinguished , and the high signal intensity artifact in the upper left of the unregistered t1-map map is reduced .", "the plot of the error rate e ( defined in ( 6 ) ) versus the number of flip angle is shown in figure 6 , and section 3.2 already concluded that a set of 10 flip angles ( opt,10 = [ 2 , 5 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 ] ) is a good compromise between t1 quality and efficiency .", "figure 7 shows a comparison of generated t1-maps when using 2 optimized flip angles ( [ 5 , 55 ] ) , 6 optimized flip angles ( [ 2 , 5 , 60 , 65 , 70 , 75 ] ) and 10 optimized flip angles , with an error map ", "the t1-map generated by 2 angles exhibits substantially more error than the maps using 6 or 10 angles , as shown by the bright pixels in the error map .", "we conclude that 10 flip angles is an acceptable choice considering the trade - off between accuracy and scan time . ", "out of a total of 16 slices , the bottom 4 slices were discarded because they did not cover sufficient wm for segmentation by our jsric method .", "the method follows the flowchart in the dashed box shown in figure 3 from slice 5 to slice 15 .", "slice 16 could not be calculated , possibly because of its proximity to the boundary of the rf field and hence degraded . ", "the function g(i ) introduced in ( 1 ) was empirically chosen as the cubic function i , which characterizes the skewness of a pdf .", "moreover , in , wm and in , csf are obtained by training according to ( 7 ) based on an expert 's manual segmentation of one subject ( training subject ) .", "these values are in , wm = 0.93 and in , csf = 0.53 . the same values are then applied to the other subject ( testing subject ) .", "the segmentation of a t1-map is achieved by evolving contours according to ( 4 ) .", "since the curve evolution is based on a gradient flow , the final result may vary depending upon the initialization .", "a t1-weighted image histogram is shown ( figure 8(a ) ) , and a spectral analysis can be carried out to threshold the image as an initialization [ 5 , 55 ] , to achieve a good segmentation after fine tuning of the contour .", "the t1-map does not have the same level of contrast as a t1-weighted image ( figure 8(b ) ) .", "we therefore initialize the contours by either placing uniformly spaced squares or by manually seeding ( by mouse clicking and dragging on the image ) .", "( versus slice number ) for two subjects , where the parameter is as defined ( section 4.1 ) . the strength of the rf field drops significantly at the top slices ( slice 14 and 15 ) such that the corresponding flip angles have to be rectified by a scaling factor much smaller than 1 .", "figure 10 shows two t1-maps generated with and without flip angle rectification for the same subject at slice number 5 , 6 , 14 and 15 .", "it is clearly seen that the top 2 slices of the t1-maps with calibration have much better quality than those without . \n", "figure 11 shows some selective segmentation results of wm and gm for the test subject .", "the commonly examined metrics which determine the performance of a segmentation are tp ( true positive ) , fp ( false positive ) , and om ( overlap metric ) [ 1 , 5 ] .", "the overlap metric is defined for a class assignment as the sum of the number of pixels that both have the class assignment in each segmentation divided by the sum of pixels where either segmentation has the class assignment .", "this metric approaches 1 for segmentations that are very similar , and is near 0 when they share no similarly classified pixels .", "figure 12 shows three overlap metric curves ( om versus slice number ) of wm and gm segmentation for the training subject ( the test subject exhibits a similar result ) .", "the first curve corresponds to the segmentation of calibrated t1-maps , with our tuned weights and cubic function g(i ) = i , the second corresponds to the t1-maps generated by nonrectified flip angles ( using the same segmentation parameters ) , and the third corresponds to the calibrated t1-map , with function g(i ) = i and tuned weights ( in , wm = 0.9 and in , csf = 0.7 ) .", "these results show that calibrated t1-maps enhance the segmentation performance compared to un - calibrated t1-maps , especially at the top two slices ( slice 14 and 15 ) which are most seriously affected by rf - inhomogeneity .", "the comparison of different functions g(i ) shows that the performance of wm segmentation is comparable for the two functions .", "there is , however , a significant difference for csf segmentation , thus affecting gm segmentation . the cubic function", "it also demonstrates that the cubic function better characterizes the statistical properties , the skewness , of the data .", "figure 13 shows tp and fp for wm segmentation with different weights in around the value of in , wm , to demonstrate the notion of our probabilistic segmentation .", "tp = |rinre|/|re| , and fp = ( |rin| |rinre|)/|re| , where re and rin are the expert segmented regions and ours using in respectively , and |r| denotes the area of region r. when the weight increases , so does the penalty for the error between the data term and the approximating function ( 1 ) . therefore tp and fp decrease correspondingly , and vice versa . in this section we show the generality of our proposed segmentation method in a 3d setting by applying it to a commonly exploited modality t1-weighted images .", "the same procedures are carried out as in section 3.3 , except that now the images are collated into volumes and the active contour is replaced by an active surface .", "we test it on two open databases accessible online brainweb ( http://www.bic.mni.mcgill.ca/brainweb/ ) and ibsr ( http://www.cma.mgh.harvard.edu/ibsr/ ) .", "the former is a simulated brain mri database , therefore ground truth is provided and the latter is genuine brain mri data which also has been manually segmented by experts .", "we preprocessed the data to filter out everything except for the three main tissues , wm , gm , and csf . ", "we first tested our segmentation method on two brainweb subjects ( a training and a testing subject ) using simulated brain t1-weighted data .", "the images are 1 mm slice thick , with 3% noise level , and 20% rf intensity nonuniformity ( inu ) and each data set was 217 181 106 pixels . because the contrast between different tissues was high , we did a histogram analysis and applied the threshold method similar to for initialization .", "as i , and in , wm and in , csf are obtained by training as 0.3 and 0.2 respectively .", "the results show that it achieves a high performance of om being around 0.8 for three tissues .", "the computational time for one subject is less then 1 minute on a laptop with a 1.73 ghz cpu and 1 gb memory .", "we then tested our segmentation method on 20 data sets of t1-weighted images provided by the center for morphometric analysis at massachusetts general hospital on the ibsr website .", "the data set for each subject was 256 256 l , where l ranges from 58 to 63 pixels .", "we arbitrarily chose one subject ( subject 1_24 ) as the training data , empirically chose the function g(i ) = i for wm and g(i ) = i for csf , frin(out ) = meanin(out)(g(i ) ) , in , wm = 0.75 , and in , csf = 0.2 .", "we did not carry out any preprocessing to denoise the data or to decrease the rf - inhomogeneity effect which seriously degraded the data .", "therefore the spectral analysis could not be carried out and we used uniformly spaced cubes as the initialization .", "out of 20 subjects we however still have 3 particularly unsuccessful cases ( specifically subject 2_4 , 16_3 , and 111_2 ) due to rf - inhomogeneity which we did not rectify . including these three subjects", ", we have an averaged overlap metric around 0.651 , and if excluding these 3 outliers , we achieved an averaged om of around 0.707 . the computational time is less then 5 minutes on the same pc as above .", "the statistics show that our method outperforms most other methods even if the outliers are included ; if excluding those , we have wm segmentation slightly poorer but gm segmentation slightly better than the current best segmentation method .", "to register flip angle images we used the jsr technique , which has the advantage of jointly segmenting , registering , and generating a brain mask .", "other techniques such as the information - theoretic method only register the images , and the brain mask has to be otherwise generated .", "our jsr has been carried out pair wise on 10 flip angle images , registering 9 images to a reference image . in theory , however , it should be possible to integrate multiple - image in jsr 's formulation . by summing the energy functionals of multiple images with a relationship enforced between each contour , that is , e(c1 , g2 , , gn ) = e1(c1 ) + e2(g2(c1 ) ) + + en(gn(c1 ) ) , where ci = gi(c1 ) , i = 1 , , n", ", the evolution of the contours ci and registration parameters gi may be derived similarly .", "our segmentation of three brain tissues is based on the tuning of weights , to penalize differently the error of the approximated function , to obtain different regions of tissue .", "segmentation uses the anatomical nature of brain tissue which has a layered structure such that we may peel off one layer at a time .", "our method uses an active contour that is able to separate an image into two parts : the inside and the outside of the contour . however , multiphase active contour techniques exist [ 36 , 58 ] and are able to evolve multiple contours simultaneously and segment multiple regions at once .", "our method is different in the sense that it has a probabilistic flavor that the tuning weights determine a purity - level of segmented tissues .", "our jsric method works by enforcing the average of white matter t1 value to be homogeneous across different transverse planes in the central brain region , to find the scaling factors which affects the flip angles . even though t1 has regional heterogeneity , by taking the average of wm for the transverse plane we average out this heterogeneity , and doing so only in the central brain region .", "wm segmentation and rf - calibration enhance the precision of one another , as shown in the performance plot in the previous section .", "in conclusion , we propose an adapted mumford - shah type energy functional for segmentation .", "the two adaptations are : ( 1 ) a function g(i ) is able to characterize the statistical properties of the data to achieve better segmentation results , and ( 2 ) the tuning weights in(out ) are able to segment brain tissues in a probabilistic fashion and achieve three - tissue segmentation .", "we also propose a novel method ( jsric ) to jointly segment a t1-map and calibrate rf - inhomogeneity .", "the whole procedure moreover includes the determination of optimal flip angles in achieving the balance between accuracy and efficiency , and joint registration of flip angle images and generation of brain mask .", "after rf - calibration , the top and bottom slices of t1-maps show better contrast and enhance the segmentation performance . the segmentation method has also been applied to t1-weighted data , to show the generality of our segmentation method , and the results are validated by ground truth and by expert manual segmentations .", "to derive the gradient flow of our energy in ( 1 ) with alvino et al .", "'s fast mumford - shah implementation , we first restate their result for the approximated function in scale space .", "suppose the approximated function fri , i = in or out , is expressed as a linear combination of basis j : \n\t\t\t\t\t\t\t\t ( a.1)fri=j=1nj , ij , \n\t\t\t\t\t\t\t then the coefficients j , i can be solved by a linear system \n\t\t\t\t\t\t\t\t ( a.2)i+ii=i , \n\t\t\t\t\t\t\t where \n\t\t\t\t\t\t\t\t ( a.3)i=[ri11rin1ri12rin2ri1nrinn],i=[ri1,1rin,1ri1,2rin,2ri1,nrin,n ] , \n\t\t\t\t\t\t\t and each n n matrices must be computed for each region .", "also , \n\t\t\t\t\t\t\t\t ( a.4)i=[1,i2,in , i ] , i=[ri1g(i)ring(i ) ] \n\t\t\t\t\t\t\t are each n 1 vectors . ", "therefore we may replace the basis j by g(i ) and mean(g(i ) ) as in ( 2 ) , and solve for the coefficients i : \n\t\t\t\t\t\t\t\t ( a.5)1i = timisiti+wimisi , 2i = witi+wimisi \n\t\t\t\t\t\t\t where \n\t\t\t\t\t\t\t\t ( a.6)si=rig(i)dxdy , mi = siridxdy , ti=rig(i)2dxdy , wi=rig(i)2dxdy . then substituting the coefficients in ( 2 ) by the above results and then pluging frin(out ) into ( 3 )" ]

[ "<S> we propose a constrained version of mumford and shah 's ( 1989 ) segmentation model with an information - theoretic \n point of view in order to devise a systematic procedure to \n segment brain magnetic resonance imaging ( mri ) data for \n parametric t1-map and t1-weighted images , in both 2-d and \n 3d settings . </S>", "<S> incorporation of a tuning weight in particular adds \n a probabilistic flavor to our segmentation method , and makes \n the 3-tissue segmentation possible . </S>", "<S> moreover , we proposed a \n novel method to jointly segment the t1-map and calibrate rf inhomogeneity \n ( jsric ) . </S>", "<S> this method assumes the average t1 value of white matter is the same across transverse slices in \n the central brain region , and jsric is able to rectify the flip angles \n to generate calibrated t1-maps . in order to generate an \n accurate t1-map , </S>", "<S> the determination of optimal flip - angles and \n the registration of flip - angle images are examined . </S>", "<S> our jsric \n method is validated on two human subjects in the 2d t1-map \n modality and our segmentation method is validated by two public \n databases , brainweb and ibsr , of t1-weighted modality in \n the 3d setting . </S>" ]

[ "1. Introduction", "2. Proposed Model for Segmentation", "3. Segmentation of a Parametric Brain T", "4. Joint T", "5. Experimental Results", "6. Discussion", "7. Conclusion", " Derivation of Gradient Flows for Our Proposed Energy" ]

[ [ "brain structure segmentation is the apportionment of brain tissue into gray matter and white matter , based on the appearance of tissue in images produced by magnetic resonance imaging ( mri ) .", "because manual tracing of the boundaries between tissues in the brain is labor intensive , difficult , error - prone , and unrealistic for large amounts of data , an automated or semiautomated segmentation technique is needed for either visualization or diagnosis .", "different imaging modalities , such as t1-weighted , t2-weighted , or proton density ( pd ) images , have been used for different segmentation methods .", "t1-weighted images , because of their good contrast , have been widely tested for various segmentation methods [ 26 ] .", "a t1-map is a parametric image of pure t1 ( spin lattice relaxation time ) , derived from the solution of an equation describing tissue relaxation , and a parametric t1-map which is different from a t1-weighted image .", "the relationship between t1 and several diseases , such as schizophrenia or sickle cell disease , has been studied [ 710 ] , and t1 may be used as a possible indicator of pathology .", "change in t1 of certain voxels in the brain over time may be an early indicator of possible pathology .", "therefore , the segmentation of a parametric brain t1-map may highlight pathology unseen by other imaging approaches . ", "the three tissues white matter ( wm ) , gray matter ( gm ) , and cerebrospinal fluid ( csf ) constitute the main parts in the brain .", "different methods have been proposed to achieve this goal , and they may be classified into various categories : fuzzy segmentation methods [ 4 , 11 ] , markov random field ( mrf ) methods [ 12 , 13 ] , bayesian methods , active contour methods [ 1 , 5 , 6 ] , or the combinations of two or more techniques", ". some of these combinations are as follows : leemput et al . used expectation maximization ( em ) and mrf , xu et al", "it is an adaptive version of mumford - shah 's model to systematically segment different tissues in the brain . before the segmentation ,", "a t1-map may be calculated by a rapid method known as the variable nutation method which provides comparable precision but much faster speed over conventional methods .", "this method requires the acquisition of a set of flip angle images and the t1 information can be extracted therein .", "[ 18 , 19 ] proposed methods to determine the optimal flip angles by basically maximizing the signal - to - noise ratio ( snr ) of a t1-map .", "their first work required the knowledge of average tr / t1 in advance though , and their second work introduced a weighted least square method to estimate the angles .", "we take another approach to determine the flip angles to achieve the trade - off between acquisition time and t1 accuracy . ", "since a t1-map is generated by a set of flip angle images , alignment of the images is important in order to obtain an accurate t1-map .", "the registration of flip angle images is usually ignored though because the movement of the head in the coil is minute .", "however , slight registration errors affect the resulting t1-map dramatically , as will be shown . ", "radiofrequency ( rf)-inhomogeneity is another unavoidable problem encountered in mr imaging . the nonuniform distribution of the rf field can cause the resulting images to have low contrast and inhomogeneous intensity , which makes quantitative description and segmentation of the image difficult .", "rf inhomogeneity affects the generation of t1-map in the sense that the spins are not tilted by the predefined nominal angles [ 17 , 2022 ] .", "hence , we focus on calibrating the rf nonuniformity in order to generate an accurate t1-map .", "cheng and wright calculated an analytical form of t1 errors induced by rf nonuniformity and allowed simple correction of t1 measurements .", "we propose a method , which assumes the average t1 value among a transverse plane is the same across the central brain slices , to jointly segment a t1-map and calibrate the rf nonuniformity along the direction perpendicular to the transverse plane .", "taking into account that we average out the heterogeneity of t1 , and doing so only within few central brain slices , this assumption help calibrate rf - inhomogeneity and generate a quantitative t1-map for segmentation . ", "section 2 we state our adapted model , with a fast implementation method . in section 3", "we first illustrate some necessary preprocessing to generate an accurate t1-map , which includes the registration of flip angle images with the generation of a brain mask and the determination of optimal flip angles , to achieve a trade - off between quality and efficiency .", "we propose a novel method to jointly segment a t1-map and calibrate rf - inhomogeneity , and in section 5 we show the results of registering the flip angle images , determining optimal flip angles , and segmenting the t1-maps .", "we also show validations , the resulting t1-map after rf - calibration , and the 3d segmentation for two sets of t1-weighted data . at last in section 6" ], [ "active contour methods comprise a popular segmentation technique in which an initialized contour is driven by a partial differential equation ( pde ) to minimize an energy functional designed to attract the contour toward image edges .", "active contour methods can be classified into two categories : edge - based [ 2427 , 2734 ] and region - based [ 16 , 3544 ] methods .", "edge - based methods examine the gradient information of the image , and stop the contour whenever the gradient is high .", "however , there are many situations when the edge is not clearly characterized by the gradient , and it has been shown that region - based methods outperform edge - based methods [ 3537 , 41 , 43 , 44 ] . by examining some statistics of the region inside and outside of the active contour , and optimizing the separation of these two statistics , we may achieve a better segmentation performance , thus making region - based methods more attractive . ", "our proposed model is a modified momford and shah functional , and falls in the region - based category .", "two adaptations of mumford - shah 's model constitute the novelty of our proposed technique .", "first is the incorporation of an information - theoretic view , which characterizes the statistical property of data .", "the second is a selective weighting , similar to the weight parameter in , which favors erring towards one tissue type or another , and thus make 3-tissue segmentation possible .", "we use a modified mumford - shah energy functional : \n\t\t\t\t\t\t\t\t ( 1)e(frin , frout , c)=i = in , outiri{(frig(i))2+fri2}dx+cds , \n\t\t\t\t\t\t\t where fri approximate a function g( ) applied of the image i for region ri , i = in or out .", "fri is smooth within each region ri , but not across the boundaries ; c denotes the region boundaries , which is the contour of interest in this paper , and 0 i 1 are weights such that in + out = 1 . by minimizing this functional", "we obtain a function fri which is faithful to the image ( first term ) and smooth within each region but not across the boundaries ( second term ) , while penalizing excessive length of the boundaries ( last term ) . ", "the first adaptation is the function g( ) applied on to image i instead of the image itself .", "specifically , an information - theoretic approach for maximizing a probabilistic disparity measure , jensen - shannon ( js ) divergence , was proposed .", "a constructed function g( ) characterizes the property of the probability density function ( pdf ) of the image intensity such as skewness ( g(i ) = i ) , or kurtosis ( g(i ) = i ) , relative to a gaussian . a proper choice of g( )", "will capture the statistical characteristics of the data and will hence achieve a good segmentation . ", "the second adaptation , similar to that in , is the selective weight i applied on the inside / outside energy terms .", "this provides a probabilistic assignment to the segmented regions , and also makes 3-tissue segmentation possible , as will be shown in section 3 . enhancing the weight in is tantamount to penalizing both the error of the difference between the approximated function frin and the data fidelity term g(i ) , as well as the degree of smoothness of frin", "this would yield a smaller segmented region which is likely to be more faithful to the image , and of purer tissues .", "figure 1 shows examples of white matter ( wm ) segmentation with different weights . with a larger inside - weight in ,", "the segmented region is smaller and the segmented tissues are purer . minimizing the mumford - shah energy", "functional involves solving for the approximating functions frin / out and for the contour c. the joint search for these infinite dimensional unknowns usually entails gradient descent flows .", "in particular , the approximated functions are typically modeled as a linear combination of a basis set whose dimension equals the number of pixels in the image , that is , each pixel is assumed to be independent .", "alvino and yezzi proposed a fast implementation using a significantly smaller basis number to model the approximated functions , while still achieving sufficient resemblance to the obtained functions when using the pixel - by - pixel basis . ", "we adopt their so - called linear heat equation basis with the change from i to g(i ) to incorporate the statistical properties of the data from an information - theoretic point of view in the previous section .", "we hence have , \n\t\t\t\t\t\t\t\t ( 2)fri=1,ig(i)+2,imean(g(i ) ) , \n\t\t\t\t\t\t\t where mean( ) is the average function , and the coefficients j , in(out ) , j = 1,2 , may be similarly derived in .", "our derivations are included in the appendix . substituting ( 2 ) into ( 1 ) , and using a classical methods of variational calculus", ", the gradient descent evolution of the curve may be derived as \n\t\t\t\t\t\t\t\t ( 3)ct={out[(froutg(i))2+frout2 ] in[(fring(i))2+frin2]}nn,(4)=(outfoutinfin)nn , \n\t\t\t\t\t\t\t where denotes the curvature of the contour c , t an artificial time evolution parameter , and n the outward normal of the contour .", "fin / out are derived by substituting frin / out from ( 2 ) into ( 3 ) as shown in the appendix ." ], [ "a brain parametric t1-map is the calculated result ( variable nutation method ) from several flip angle images . because the flip angle images are acquired at different times , and because the subject may move during image acquisition , registration should be carried out first to obtain an accurate t1-map .", "moreover , in order to achieve a balance between the acquisition time and the resulting t1-map quality , we propose a method to determine optimal flip angles . in the following subsections", "we will first illustrate how we register the flip angle images and obtain a brain mask as a byproduct , then describe our method to determine a set of optimal flip angles , and at last describe our proposed procedure to segment a t1-map .", "the value of t1 is traditionally determined by acquisition methods such as inversion recovery ( ir ) or saturation - recovery ( sr ) .", "other rapid methods , such as variable nutation ( the despot method ) have been proposed , and require acquisition of several flip angle images , and calculation of t1 .", "since these flip angle images are acquired at different times , registration must first be accomplished . even though the interval between consecutive scans may be as short as two minutes , and movement of the subject 's head inside the receiver coil", "may be a few pixels ( under the resolution of a 256 256 image ) , the effects of such off - registration can be significant , as shown ( figure 5 ) . here", "we describe a method to register flip angle images and jointly obtain a mask , as a byproduct , to get rid of the skull and other structures around the brain . ", "we use a joint segmentation and registration ( jsr ) technique proposed by yezzi et al . with an additional tuning weight , to achieve registration and to obtain the mask .", "the theory of jsr technique consists of evolving two contours , with a enforced relationship ( ex : rigid or affine transform ) between them , in two images according to a partial differential equation ( pde ) which is a result of optimizing , for example , the sum of two energy functionals . for our particular task", ", we may choose a region - based energy , such as chan and vese 's model incorporating weights , which arises as a special case of ( 1 ) with the data term g(i ) = i and the approximating function frin / out = mean(i ) inside and outside the contour .", "this model approximates the image i by a simple piecewise constant function , which suits our goal here because it creates a mask that divides the image into two parts- brain region and nonbrain region . ", "we observe that the boundary between the brain and nonbrain is visually more easily distinguished than the boundary between different tissues within the brain , for every flip angle image .", "we therefore put a very small weight in on the inside energy in ( 1 ) to penalize very little the difference between the data inside the contour , i , and the approximated function mean(i ) .", "we carry out this registration technique pairwise for all flip angle images , and once the flip - angel images are registered to each other , they are used to generate a t1-map .", "the determination of the flip angles that yields qualitative t1-map and saves time , is mainly by comparing the difference between the gold standard and the t1-maps generated from different combinatorial flip angles .", "we begin by acquiring images at a set of 19 flip angles which spans the range of standard angles and will give an optimized t1-map .", "we call this particular t1-map the gold standard , or t1 g , and denote this set of angles as 19 = [ 2 , 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 ] .", "we then compare the t1-map generated from all combinations of the subset of 19 with t1 g . out of these combinations", "since the data acquisition is slice - based , this study of optimal flip angle focuses on the central slice , which is least affected by rf - inhomogeneity and is routinely selected at the level of the basal ganglia , including both the genu and the splenium of the corpus callosum , and generally shows the putamen and lateral ventricle .", "we denote the optimal n - angles opt , n , which is a subset of 19 , as those that exhibit the smallest difference between t1 g and the t1-map generated by n flip angles : \n\t\t\t\t\t\t\t\t ( 5)opt , n = arg min n(x , y)brain|t1g(x , y)t1n(x , y)| , \n\t\t\t\t\t\t\t where n { 2,3 , , 19 } , n19 , t1n denotes the t1-map generated by n , and the summation of ( x , y ) is over the whole brain region at the central slice . equation ( 5 ) therefore gives 18 sets , with the number of elements ranging from 2 to 19 , of optimal angles . for the determination of the optimal set of flip angles , in reaching a compromise between efficiency and t1-map quality", ", we examine the error between t1opt , n and t1 g and compute the error rate .", "the error rate is defined as \n\t\t\t\t\t\t\t\t ( 6)e=(x , y)braine(x , y)a(brain) , \n\t\t\t\t\t\t\t where a(brain ) is the area of the brain , and e(x , y ) is the error , defined as 1 if the difference between t1g(x , y ) and t1opt , n(x , y ) is greater than some threshold , and 0 otherwise . the threshold is defined as the minimum of the standard deviation among the t1 values of three brain tissues ( wm , gm , and csf ) manually segmented by an expert .", "the summation of e(x , y ) is over the whole brain at the central slice . ", "the plot of the error rate versus the number of flip angles is shown in figure 6 , where the inflection point of the fitted curve is at 10 flip angles ( opt,10 = [ 2 , 5 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 ] ) .", "once the brain mask is obtained ( section 3.1 ) , it is used to segment away the skull , leaving only three major tissues in the image : wm , gm , and csf .", "notice that the curve evolution corresponding to the energy introduced in section 2 always results in a binary segmentation , where we will have regions inside ( foreground ) and outside ( background ) the contour(s ) .", "we can not simultaneously segment the three tissues , even though we will later talk about multiphase segmentation .", "we may , however , tune the weights ( section 2.1 ) , to penalize the error between the data term and the approximated function ( 1 ) to segment one tissue at a time , with a progression analogous to that of peeling an onion . ", "we illustrate our t1-map segmentation procedure for a hard segmentation of three tissues , and the probabilistic segmentation is obtained by varying the weights around the value of the trained weight ( section 5.4 ) .", "a t1-map segmentation procedure consists of three steps , where the first two steps are to evolve the contours by minimizing the energy in ( 1 ) with different weights , and the third step is just a simple subtraction .", "the procedure is as follows : treat wm as the foreground , everything else as the background ; let in in ( 1 ) be the trained in , wm , and use it to segment wm in the interior region of the contour . ", "treat wm and gm as the foreground , csf and everything else as the background ; let in in ( 1 ) be the trained in , csf , and use it to obtain csf in the exterior region of the curve filtered by the brain mask . ", "the procedure is based on the anatomical observation that gm is enclosed by csf , and that csf is separated from wm [ 1 , 3 ] , such that we may peel off one layer at a time . ", "the choice of function g(i ) in ( 1 ) , which is chosen to better capture the statistical property of t1-map ( and for other modality ) , will be shown in section 5 .", "the values of in , wm and in , csf are determined through a training process .", "if re denotes the segmentation region by the expert , and rin denotes the segmentation region by the weight in with some fixed g(i ) , for some tissue , then the value of in , tissue is determined by minimizing \n\t\t\t\t\t\t\t\t ( 7)in , tissue = argmin in{1|rerin||rin|+|re||rerin| } , \n\t\t\t\t\t\t\t where |r| denotes the number of pixels in region r. the first term inside the bracket is an overlap metric ( om ) which will be discussed again in section 4 ." ], [ "the strength of the rf field varies within the mr scanner , such that the resulting image may be of low contrast or of nonuniform intensity . in what follows", ", we will first describe how a t1-map is calculated from a set of flip angle images , then how the t1-map is affected by rf - inhomogeneity and then show our proposed correction method . a t1-map is calculated by variable nutation .", "flip - angle images are acquired using a flash sequence at different flip angles , and t1 is calculated from the slope of a least square fitted line to the pair of data [ s()/sin , s()/tan ] , where s( ) is the signal strength of the flip angle image expressed as a function of the flip angle . rf - inhomogeneity affects the computation of t1 in that the spins are not tilted by the nominal angle because the strength of the rf field is not as predefined .", "calibration of rf - inhomogeneity , or , the correction of flip angles , produces a more accurate t1-map . a spatially dependent scaling factor , therefore , is introduced to adjust the tilted angle [ 20 , 22 ] , that is , replacing by (8)[s()sin(),s()tan( ) ] , \n\t\t\t\t\t\t\t and t1 is extracted from the slope of the line fitted to the above space .", "our imaging setting is slice - based across different transverse planes ( figure 2 ) , thus our proposed joint segmentation and rf - inhomogeneity calibration ( jsric ) method is as an initial step to easily calibrate rf - inhomogeneity vertically .", "our method is based on the assumption that the average t1 of wm should be roughly the same across central brain transverse slices .", "we however average out the heterogeneity of t1 , and carry out the method only within few central brain slices .", "the advantage is that as the segmentation delineates more precisely the wm region , flip angle correction is therefore enhanced , and as flip angle correction is carried out more precisely , which gives better quality t1-map , segmentation is facilitated as well . ", "our joint segmentation and rf - inhomogeneity calibration ( jsric ) method requires first taking the average t1 value for segmented wm at the central slice ( which has relatively uniform b1 field [ 51 , 53 ] ) as the reference , and then iteratively segmenting and searching for the scaling factor in ( 8) for all other slices .", "this is a three - step iterative process ( as shown inside the dashed box in flowchart 3 ) . ", "segment wm for the central slice by the method proposed in section 3.3 , compute the average t1 value of wm , and denote the average as m. for slice m , varies in ( 8) , and calculate the corresponding t1-map , denoted as t1( ) .", "if goes beyond max , claim the current slice as uncalibratable and repeat this step for the next slice m + 1 .", "segment wm for t1( ) , compute the average t1 of wm , and denote it by l( ) .", "if m l( ) , go back to step 2 and increase , otherwise claim it is done for the current slice . ", "the variables min , max , and in flowchart 3q are all predefined parameters ." ], [ "in this section we show a series of results from segmenting a parametric brain t1-map , which includes the registration of flip angle images , generation of a brain mask as a byproduct , determination of the optimal flip angles , and joint t1-map segmentation and rf - inhomogeneity calibration .", "in addition , we will apply our proposed segmentation method to another modality , t1-weighted images , in a 3d setting , to show the generality of our segmentation method .", "the subjects being scanned were recruited from a clinic in the department of psychiatry at the university of north carolina , under an irb - approved protocol to image the brain .", "a transverse 3d flash sequence using different flip angles was acquired in a 3 t siemens mri scanner with a quadrature head coil .", "the scan parameters were : tr = 25 msec , te = 2.83 msec , 16 slices , and slice thickness = 5 mm .", "the center slice used for optimal flip angle study was routinely selected as described ( section 3.2 ) .", "the registration of a set of flip angle images is carried out pair wise , and figure 4 shows two flip angle images and the resulting brain mask .", "note that even though the flip angle images are off - registered by no more than four pixels in both the x- and y - directions , the impact is obvious .", "figure 5 shows two t1-maps generated by registered and unregistered flip angle images , and we can see that after registration the anatomical structure of the t1-map is more readily distinguished , and the high signal intensity artifact in the upper left of the unregistered t1-map map is reduced .", "the plot of the error rate e ( defined in ( 6 ) ) versus the number of flip angle is shown in figure 6 , and section 3.2 already concluded that a set of 10 flip angles ( opt,10 = [ 2 , 5 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 ] ) is a good compromise between t1 quality and efficiency .", "figure 7 shows a comparison of generated t1-maps when using 2 optimized flip angles ( [ 5 , 55 ] ) , 6 optimized flip angles ( [ 2 , 5 , 60 , 65 , 70 , 75 ] ) and 10 optimized flip angles , with an error map ", "the t1-map generated by 2 angles exhibits substantially more error than the maps using 6 or 10 angles , as shown by the bright pixels in the error map .", "we conclude that 10 flip angles is an acceptable choice considering the trade - off between accuracy and scan time . ", "out of a total of 16 slices , the bottom 4 slices were discarded because they did not cover sufficient wm for segmentation by our jsric method .", "the method follows the flowchart in the dashed box shown in figure 3 from slice 5 to slice 15 .", "slice 16 could not be calculated , possibly because of its proximity to the boundary of the rf field and hence degraded . ", "the function g(i ) introduced in ( 1 ) was empirically chosen as the cubic function i , which characterizes the skewness of a pdf .", "moreover , in , wm and in , csf are obtained by training according to ( 7 ) based on an expert 's manual segmentation of one subject ( training subject ) .", "these values are in , wm = 0.93 and in , csf = 0.53 . the same values are then applied to the other subject ( testing subject ) .", "the segmentation of a t1-map is achieved by evolving contours according to ( 4 ) .", "since the curve evolution is based on a gradient flow , the final result may vary depending upon the initialization .", "a t1-weighted image histogram is shown ( figure 8(a ) ) , and a spectral analysis can be carried out to threshold the image as an initialization [ 5 , 55 ] , to achieve a good segmentation after fine tuning of the contour .", "the t1-map does not have the same level of contrast as a t1-weighted image ( figure 8(b ) ) .", "we therefore initialize the contours by either placing uniformly spaced squares or by manually seeding ( by mouse clicking and dragging on the image ) .", "( versus slice number ) for two subjects , where the parameter is as defined ( section 4.1 ) . the strength of the rf field drops significantly at the top slices ( slice 14 and 15 ) such that the corresponding flip angles have to be rectified by a scaling factor much smaller than 1 .", "figure 10 shows two t1-maps generated with and without flip angle rectification for the same subject at slice number 5 , 6 , 14 and 15 .", "it is clearly seen that the top 2 slices of the t1-maps with calibration have much better quality than those without . \n", "figure 11 shows some selective segmentation results of wm and gm for the test subject .", "the commonly examined metrics which determine the performance of a segmentation are tp ( true positive ) , fp ( false positive ) , and om ( overlap metric ) [ 1 , 5 ] .", "the overlap metric is defined for a class assignment as the sum of the number of pixels that both have the class assignment in each segmentation divided by the sum of pixels where either segmentation has the class assignment .", "this metric approaches 1 for segmentations that are very similar , and is near 0 when they share no similarly classified pixels .", "figure 12 shows three overlap metric curves ( om versus slice number ) of wm and gm segmentation for the training subject ( the test subject exhibits a similar result ) .", "the first curve corresponds to the segmentation of calibrated t1-maps , with our tuned weights and cubic function g(i ) = i , the second corresponds to the t1-maps generated by nonrectified flip angles ( using the same segmentation parameters ) , and the third corresponds to the calibrated t1-map , with function g(i ) = i and tuned weights ( in , wm = 0.9 and in , csf = 0.7 ) .", "these results show that calibrated t1-maps enhance the segmentation performance compared to un - calibrated t1-maps , especially at the top two slices ( slice 14 and 15 ) which are most seriously affected by rf - inhomogeneity .", "the comparison of different functions g(i ) shows that the performance of wm segmentation is comparable for the two functions .", "there is , however , a significant difference for csf segmentation , thus affecting gm segmentation . the cubic function", "it also demonstrates that the cubic function better characterizes the statistical properties , the skewness , of the data .", "figure 13 shows tp and fp for wm segmentation with different weights in around the value of in , wm , to demonstrate the notion of our probabilistic segmentation .", "tp = |rinre|/|re| , and fp = ( |rin| |rinre|)/|re| , where re and rin are the expert segmented regions and ours using in respectively , and |r| denotes the area of region r. when the weight increases , so does the penalty for the error between the data term and the approximating function ( 1 ) . therefore tp and fp decrease correspondingly , and vice versa . in this section we show the generality of our proposed segmentation method in a 3d setting by applying it to a commonly exploited modality t1-weighted images .", "the same procedures are carried out as in section 3.3 , except that now the images are collated into volumes and the active contour is replaced by an active surface .", "we test it on two open databases accessible online brainweb ( http://www.bic.mni.mcgill.ca/brainweb/ ) and ibsr ( http://www.cma.mgh.harvard.edu/ibsr/ ) .", "the former is a simulated brain mri database , therefore ground truth is provided and the latter is genuine brain mri data which also has been manually segmented by experts .", "we preprocessed the data to filter out everything except for the three main tissues , wm , gm , and csf . ", "we first tested our segmentation method on two brainweb subjects ( a training and a testing subject ) using simulated brain t1-weighted data .", "the images are 1 mm slice thick , with 3% noise level , and 20% rf intensity nonuniformity ( inu ) and each data set was 217 181 106 pixels . because the contrast between different tissues was high , we did a histogram analysis and applied the threshold method similar to for initialization .", "as i , and in , wm and in , csf are obtained by training as 0.3 and 0.2 respectively .", "the results show that it achieves a high performance of om being around 0.8 for three tissues .", "the computational time for one subject is less then 1 minute on a laptop with a 1.73 ghz cpu and 1 gb memory .", "we then tested our segmentation method on 20 data sets of t1-weighted images provided by the center for morphometric analysis at massachusetts general hospital on the ibsr website .", "the data set for each subject was 256 256 l , where l ranges from 58 to 63 pixels .", "we arbitrarily chose one subject ( subject 1_24 ) as the training data , empirically chose the function g(i ) = i for wm and g(i ) = i for csf , frin(out ) = meanin(out)(g(i ) ) , in , wm = 0.75 , and in , csf = 0.2 .", "we did not carry out any preprocessing to denoise the data or to decrease the rf - inhomogeneity effect which seriously degraded the data .", "therefore the spectral analysis could not be carried out and we used uniformly spaced cubes as the initialization .", "out of 20 subjects we however still have 3 particularly unsuccessful cases ( specifically subject 2_4 , 16_3 , and 111_2 ) due to rf - inhomogeneity which we did not rectify . including these three subjects", ", we have an averaged overlap metric around 0.651 , and if excluding these 3 outliers , we achieved an averaged om of around 0.707 . the computational time is less then 5 minutes on the same pc as above .", "the statistics show that our method outperforms most other methods even if the outliers are included ; if excluding those , we have wm segmentation slightly poorer but gm segmentation slightly better than the current best segmentation method ." ], [ "to register flip angle images we used the jsr technique , which has the advantage of jointly segmenting , registering , and generating a brain mask .", "other techniques such as the information - theoretic method only register the images , and the brain mask has to be otherwise generated .", "our jsr has been carried out pair wise on 10 flip angle images , registering 9 images to a reference image . in theory , however , it should be possible to integrate multiple - image in jsr 's formulation . by summing the energy functionals of multiple images with a relationship enforced between each contour , that is , e(c1 , g2 , , gn ) = e1(c1 ) + e2(g2(c1 ) ) + + en(gn(c1 ) ) , where ci = gi(c1 ) , i = 1 , , n", ", the evolution of the contours ci and registration parameters gi may be derived similarly .", "our segmentation of three brain tissues is based on the tuning of weights , to penalize differently the error of the approximated function , to obtain different regions of tissue .", "segmentation uses the anatomical nature of brain tissue which has a layered structure such that we may peel off one layer at a time .", "our method uses an active contour that is able to separate an image into two parts : the inside and the outside of the contour . however , multiphase active contour techniques exist [ 36 , 58 ] and are able to evolve multiple contours simultaneously and segment multiple regions at once .", "our method is different in the sense that it has a probabilistic flavor that the tuning weights determine a purity - level of segmented tissues .", "our jsric method works by enforcing the average of white matter t1 value to be homogeneous across different transverse planes in the central brain region , to find the scaling factors which affects the flip angles . even though t1 has regional heterogeneity , by taking the average of wm for the transverse plane we average out this heterogeneity , and doing so only in the central brain region .", "wm segmentation and rf - calibration enhance the precision of one another , as shown in the performance plot in the previous section ." ], [ "in conclusion , we propose an adapted mumford - shah type energy functional for segmentation .", "the two adaptations are : ( 1 ) a function g(i ) is able to characterize the statistical properties of the data to achieve better segmentation results , and ( 2 ) the tuning weights in(out ) are able to segment brain tissues in a probabilistic fashion and achieve three - tissue segmentation .", "we also propose a novel method ( jsric ) to jointly segment a t1-map and calibrate rf - inhomogeneity .", "the whole procedure moreover includes the determination of optimal flip angles in achieving the balance between accuracy and efficiency , and joint registration of flip angle images and generation of brain mask .", "after rf - calibration , the top and bottom slices of t1-maps show better contrast and enhance the segmentation performance . the segmentation method has also been applied to t1-weighted data , to show the generality of our segmentation method , and the results are validated by ground truth and by expert manual segmentations ." ], [ "to derive the gradient flow of our energy in ( 1 ) with alvino et al .", "'s fast mumford - shah implementation , we first restate their result for the approximated function in scale space .", "suppose the approximated function fri , i = in or out , is expressed as a linear combination of basis j : \n\t\t\t\t\t\t\t\t ( a.1)fri=j=1nj , ij , \n\t\t\t\t\t\t\t then the coefficients j , i can be solved by a linear system \n\t\t\t\t\t\t\t\t ( a.2)i+ii=i , \n\t\t\t\t\t\t\t where \n\t\t\t\t\t\t\t\t ( a.3)i=[ri11rin1ri12rin2ri1nrinn],i=[ri1,1rin,1ri1,2rin,2ri1,nrin,n ] , \n\t\t\t\t\t\t\t and each n n matrices must be computed for each region .", "also , \n\t\t\t\t\t\t\t\t ( a.4)i=[1,i2,in , i ] , i=[ri1g(i)ring(i ) ] \n\t\t\t\t\t\t\t are each n 1 vectors . ", "therefore we may replace the basis j by g(i ) and mean(g(i ) ) as in ( 2 ) , and solve for the coefficients i : \n\t\t\t\t\t\t\t\t ( a.5)1i = timisiti+wimisi , 2i = witi+wimisi \n\t\t\t\t\t\t\t where \n\t\t\t\t\t\t\t\t ( a.6)si=rig(i)dxdy , mi = siridxdy , ti=rig(i)2dxdy , wi=rig(i)2dxdy . then substituting the coefficients in ( 2 ) by the above results and then pluging frin(out ) into ( 3 )" ] ]

[ "knowledge about dysphonia manifestations and the degree of commitment that this change \n causes in laryngeal and vocal behavior are important for defining rehabilitation \n direction . in order to do that , procedures are necessary for clinical assessment . \n perceptual and acoustic assessments , among other procedures , are commonly \n performed .", "oral and vocal fold diadochokinesis ( ddk ) is one of the tests that can be applied to \n perform acoustic analysis .", "the ddk , or syllable alternating motion rate , is the ability to perform rapidly \n opposite contractions of relatively simple patterns .", "vocal fold ddk analysis allows evaluation of \n neuromuscular integrity of the vocal folds and oral ddk analyzes the ability to perform quick repetitions of \n speech segments .", "some studies try to \n understand the vocal manifestation of dysphonic patients through the perceptual and \n acoustic analysis of voice .", "( 2008 ) evaluated voice acoustic parameter changes in patients with \n parkinson 's disease ( pd ) who were searching for their relationship with motor control . \n", "the voices perceptual and acoustic ratings evaluations of 20 pd patients , 12 men and 8 \n women , were compared with the voices of 20 control subjects of corresponding ages and \n genders .", "patients with pd had smaller values for maximum phonation time and ddk compared to the \n control group , while jitter , shimmer and average fundamental frequency ( f0 ) were similar \n between the two groups .", "although men have poorer performance in ddk than women , these \n values were not statistically significant .", "thus , only a few significant correlations \n between vocal parameters and motor control were found .", "the vocal fold ddk measurement indicates laryngeal gestures of opening and closing of \n the vocal folds , being that changes in extension and speed movement of vocal folds will \n reproduce changes in the production rate in patterns of duration and transglottic \n airflow rate .", "therefore , the presence \n of organic and functional changes in dysphonia cases may relate to ddk results .", "the ddk evaluation associated with other clinical assessment procedures is an important \n resource in understanding the manifestations of individual communication disorders .", "the \n assessment of oral and vocal fold diadochokinesis ( ddk ) in individuals with voice \n disorders may contribute to the understanding of factors that affect balanced vocal \n production .", "scientific studies that make use of this assessment tool support advances in \n knowledge in this area and reflect the development of more appropriate therapeutic \n planning .", "consequently , the objective of this study was to compare the results of oral and vocal \n fold ddk of dysphonic women to the results of oral and vocal fold ddk in women without \n vocal disorders .", "this study has been approved by the research ethical committee of the school of \n dentistry of the university of so paulo , bauru campus .", "the recordings were obtained in an acoustically treated studio , and the patients \n remained seated with a headset microphone ( akg , model - c444pp ) positioned laterally \n between 60 degrees and 5 cm from the labial commissure .", "the emissions were recorded on a \n intel pentium 4 computer ( cpu 2.040 ghz and 256 mb ram ) with \n a 17 \" lg flatron e7015 monitor and a creative audigy ii sound card .", "the system also used \n sound forge 7.0 professional audio software ( madison , wi , usa ) , with a sampling rate of \n 44.100 hz and a 16 bit mono channel .", "the experimental group was composed of 28 women ranging in age from 19 to 54 years \n old , diagnosed with dysphonia and referred for a voice assessment by a speech \n pathologist and otorhinolaryngologist .", "the control group included 30 nondysphonic \n women age - matched with the experimental group who were evaluated in previous research \n with normal adults .", "the presence of central neurological diseases and the presence of communication \n disorders were considered exclusion factors for both groups , with the exception of \n dysphonia for the experimental group . during the oral and vocal fold ddk test isolated emissions", "were requested ; namely , \n the syllables \" pa , \" \" ta \" and \" ka , \" the sequence \" pataka \" and then the vowels \" a \" and \n \" i. \" before the beginning of the test , the patient was requested to repeat the sound \n as rapidly as possible .", "the patients practiced the sounds before each recording by \n repeating as fast as possible , after deep breathing , in a clear and precise way the \n emissions with a comfortable frequency and intensity in order to understand how to \n perform the test . soon thereafter", ", the subjects were guided to perform the tasks , by \n receiving a signal with the thumb to begin and to stop the repetition .", "the \" pa , \" \" ta , \" \" ka , \" \" pataka , \" \" a \" and \" i \" emissions were edited by sound forge 7.0 \n professional audio editing professional .", "software ( madison , wi , usa ) . the beginning \n and end of each sample were excluded , except for four seconds from the third to the \n sixth second . monosyllabic emissions \" pa , \" \" ta \" and \" ka \" and the vowels \" a \" and \" i \" were filed at \n sampling rate of 11.025 hz , 16 bit mono channel and analyzed by advanced motor speech \n profile program ( msp ) model 5141 version 2.5.2 ( kay 's elemetrics corp . , lincoln park , \n nj , usa ) , whose parameters are described in figure \n 1 .", "the msp program displays a graphical record of emissions showing the \n horizontal axis ( time in seconds ) and the vertical axis ( energy in db ) . to do the ddk \n count ,", "the program drew a line in the central point at the energy scale of the \n vertical axis in db .", "the line of analysis was manually positioned in the \n corresponding value to the intensity average of the ddk sample , provided by the msp \n program , and moved up or down , so the program could count all emissions when \n necessary ( figure 2 ) .", "parameters for diadochokinesis ( ddk ) analyzed by advanced motor speech profile \n ( msp ) program advanced motor speech profile ( msp ) program graphic , where time ( seconds ) on \n the horizontal axis and energy ( db ) on the vertical axis can be observed the ddk of the \" pataka \" sequence was analyzed quantitatively through the sound forge \n 7.0 program .", "the count of trisyllables per second was performed manually with the \n auditory and visual waveform support displayed by the program .", "average values of oral and vocal fold ddk in dysphonic and nondysphonic women were \n correlated using the \" t student \" test and were considered significant when \n p<0.05 .", "the experimental group was composed of 28 women ranging in age from 19 to 54 years \n old , diagnosed with dysphonia and referred for a voice assessment by a speech \n pathologist and otorhinolaryngologist .", "the control group included 30 nondysphonic \n women age - matched with the experimental group who were evaluated in previous research \n with normal adults .", "the presence of central neurological diseases and the presence of communication \n disorders were considered exclusion factors for both groups , with the exception of \n dysphonia for the experimental group .", "during the oral and vocal fold ddk test isolated emissions were requested ; namely , \n the syllables \" pa , \" \" ta \" and \" ka , \" the sequence \" pataka \" and then the vowels \" a \" and \n \" i. \" before the beginning of the test , the patient was requested to repeat the sound \n as rapidly as possible .", "the patients practiced the sounds before each recording by \n repeating as fast as possible , after deep breathing , in a clear and precise way the \n emissions with a comfortable frequency and intensity in order to understand how to \n perform the test . soon thereafter", ", the subjects were guided to perform the tasks , by \n receiving a signal with the thumb to begin and to stop the repetition .", "the \" pa , \" \" ta , \" \" ka , \" \" pataka , \" \" a \" and \" i \" emissions were edited by sound forge 7.0 \n professional audio editing professional .", "the beginning \n and end of each sample were excluded , except for four seconds from the third to the \n sixth second .", "monosyllabic emissions \" pa , \" \" ta \" and \" ka \" and the vowels \" a \" and \" i \" were filed at \n sampling rate of 11.025 hz , 16 bit mono channel and analyzed by advanced motor speech \n profile program ( msp ) model 5141 version 2.5.2 ( kay 's elemetrics corp .", ", lincoln park , \n nj , usa ) , whose parameters are described in figure \n 1 .", "the msp program displays a graphical record of emissions showing the \n horizontal axis ( time in seconds ) and the vertical axis ( energy in db ) . to do the ddk \n count", ", the program drew a line in the central point at the energy scale of the \n vertical axis in db .", "the line of analysis was manually positioned in the \n corresponding value to the intensity average of the ddk sample , provided by the msp \n program , and moved up or down , so the program could count all emissions when \n necessary ( figure 2 ) .", "parameters for diadochokinesis ( ddk ) analyzed by advanced motor speech profile \n ( msp ) program advanced motor speech profile ( msp ) program graphic , where time ( seconds ) on \n the horizontal axis and energy ( db ) on the vertical axis can be observed the ddk of the \" pataka \" sequence was analyzed quantitatively through the sound forge \n 7.0 program .", "the count of trisyllables per second was performed manually with the \n auditory and visual waveform support displayed by the program .", "average values of oral and vocal fold ddk in dysphonic and nondysphonic women were \n correlated using the \" t student \" test and were considered significant when \n p<0.05 .", "subjects were classified by the types of dysphonia , according to behlau , et \n al .", "it \n was considered primary functional dysphonia when there were no injuries or structural \n alterations of vocal folds identified by nasolaryngoscopy ; secondary functional \n dysphonia to anatomical inadequacy , in the presence of structural alterations of vocal \n folds , such as epidermoid cyst , varicosity , stria sulcus ; organic functional dysphonia , \n in the presence of edema , leucoplast or bilateral nodules ; and organic dysphonia in \n cases of gastroesophageal reflux disease or unilateral vocal fold paralysis .", "thus , the \n sample was composed of a participant with primary functional dysphonia , thirteen with \n secondary functional dysphonia by anatomical inadequacy , seven with organic and \n functional dysphonia , and three with organic dysphonia , because three women in the \n sample had no otorhinolaryngologic diagnosis .", "the results concerning the oral and vocal fold ddk of the two groups were compared using \n the \" t student \" test and are described in table \n 1 .", "comparison of oral and vocal fold diadochokinesis ( ddk ) average and standard \n deviation , between dysphonic and nondysphonic women , in the emissions of \" pa , \" \n \" ta , \" \" ka , \" \" pataka \" , \" a , \" and \" i. \"", "although the findings did not show a statistically significant correlation of oral and \n vocal fold ddk between the group of women with and without voice disorders ( table 1 ) , some aspects about the performance of \n diadochokinetic tasks are relevant and should be discussed .", "the results showed that the ddk speed , the average time between the vocalizations , the \n ability to keep constant vocalizations and keeping the intensity of these vocalizations \n constant did not correlate with the presence of dysphonia ( table 1 ) .", "it was expected that no neurological changes related to \n vocal production would interfere with oral and vocal fold ddk , since for both tasks \n there was participation of the opening and closing of vocal folds , especially related to \n \" a \" and \" i \" emissions , that involve specifically alternating motions of glottic \n adduction and abduction , since organic and functional factors may also interfere with \n the results of the ddk , including not necessarily expressing a disturbance in oral \n communication .", "it was observed that in the \" pa \" emission , the parameter cvi ( p=0.061 ) , although not \n statistically significant , showed a tendency for nondysphonic women to keep the \n vocalizations more constant as compared to the dysphonic group .", "this can be explained by \n the fact that the glottic changes found in dysphonic women , make intensity control \n difficult , since vocal balance depends on correct coordination between the myoelastic \n and aerodynamic forces of the larynx ; \n", "this finding is possibly related to the size of the \n sample , thus , statistically significant values can be found if the number of subjects \n were more expressive .", "the impairment of phonoarticulatory \n organs can generate vocal consequences , such as changes in vocal quality , impaired \n laryngeal mobility , and extrinsic muscle tension of the larynx .", "the interference of articulation with phonation shows \n that such factors might affect the motor performance of these structures and thus , in \n ddk .", "thus , there is a physiological and \n anatomical inter - connecting of the structures common to phonation and \n articulation .", "thus , individuals who \n have dysphonia due to a myofunctional alteration that initiates larynx overload could \n have poorer ddk performance .", "some minor structural alterations have morphological characteristics that change the \n vibration pattern of vocal folds .", "therefore , the sulcus vocalis shows \n an arched vocal fold , and cysts \n appear on a bulged vocal fold .", "this \n could interfere with the speed of the vocal folds alternating motion rate ( ddk ) . \n", "leeper , heeneman and reynolds ( 1990 ) reported that , when ddk involves vowel emission , refined \n control of vocal fold opening and closing is required .", "factors such as rigidity and mass can interfere with the fundamental frequency of \n voice , beyond subglottic \n pressure , which reflects on vocal intensity .", "there are significant differences in the \n effect of frequency and vocal intensity upon the airflow through the vocal \n folds .", "furthermore , vocal \n intensity seems to be the primary factor controlling the speed of the ddk and airflow \n during the production of the vowel \" a \" . in this study , although no statistically significant correlation was observed , an \n average frequency of disturbances in the ddk \" a \" ( ddcjiit ) were higher in dysphonic \n subjects than in the control group , and this value was approximately doubled .", "high \n frequency values of ddk disturbance may indicate a lower ability to control the vocal \n folds ' motion during phonation .", "vocal fold ddk has not been investigated in detail like \n an analysis of laryngeal subsystem , as has already occurred with oral ddk .", "nondysphonic individuals have appropriate adjustments in their laryngeal mechanism that \n allows for quick glottal opening and closing during the production of the consonants and \n vowels of syllables .", "this suggests \n that these adjustments in laryngeal diseases are inadequate , resulting in an increased \n effort to compensate .", "therefore , information obtained with the ddk can be reference for \n the interpretation of complex tasks .", "there is a need for more studies that compare ddk in a control group and in dysphonic \n individuals , the latter split into distinct groups according to the vocal alteration , \n thus making it possible to compare diadochokinetic task performance differences in \n different dysphonias .", "although the results do n't indicate any difficulties in oral and laryngeal motor control \n in the dysphonic group , the major instability in vocal fold ddk in the experimental \n group should be considered , and studies of this ability in individuals with \n communication disorders must be intensified ." ]

[ "<S> the evaluation of oral and vocal fold diadochokinesis ( ddk ) in individuals with voice \n disorders may contribute to the understanding of factors that affect the balanced \n vocal production . </S>", "<S> scientific studies that make use of this assessment tool support \n the knowledge advance of this area , reflecting the development of more appropriate \n therapeutic planning.objectiveto compare the results of oral and vocal fold ddk in dysphonic women and in women \n without vocal disorders . </S>", "<S> material and methodsfor this study , 28 voice recordings of women from 19 to 54 years old , diagnosed \n with dysphonia and submitted to a voice assessment from speech pathologist and \n otorhinolaryngologist , were used . </S>", "<S> the control group included 30 nondysphonic women \n evaluated in prior research from normal adults . </S>", "<S> the analysis parameters like \n number and duration of emissions , as well as the regularity of the repetition of \n syllables \" pa \" , \" ta \" , \" ka \" and the vowels \" a \" and \" i , \" were provided by the \n advanced motor speech profile program ( msp ) model-5141 , version-2.5.2 ( kaypentax ) . \n </S>", "<S> the ddk sequence \" pataka \" was analyzed quantitatively through the sound forge 7.0 \n program , as well as manually with the audio - visual help of sound waves . </S>", "<S> average \n values of oral and vocal fold ddk dysphonic and nondysphonic women were compared \n using the \" t student \" test and were considered significant when p<0.05 . </S>", "<S> resultsthe findings showed no significant differences between populations ; however , the \n coefficient of variation of period ( cvp ) and jitter of period ( jittp ) average of \n the \" ka , \" \" a \" and \" i \" emissions , respectively , were higher in dysphonic women \n ( cvp=10.42% , 12.79% , 12.05% ; jittp=2.05% , 6.05% , 3.63% ) compared to the control \n group ( cvp=8.86% ; 10.95% , 11.20% ; jittp=1.82% , 2.98% , 3.15% ) . </S>", "<S> conclusionalthough the results do not indicate any difficulties in oral and laryngeal motor \n control in the dysphonic group , the largest instability in vocal fold ddk in the \n experimental group should be considered , and studies of this ability in \n individuals with communication disorders must be intensified . </S>" ]

[ "INTRODUCTION", "MATERIAL AND METHODS", "Patients", "Procedures", "Recording Editing", "Oral and vocal fold DDK analysis", "Data analysis", "RESULTS", "DISCUSSION", "CONCLUSION" ]

[ [ "knowledge about dysphonia manifestations and the degree of commitment that this change \n causes in laryngeal and vocal behavior are important for defining rehabilitation \n direction . in order to do that , procedures are necessary for clinical assessment . \n perceptual and acoustic assessments , among other procedures , are commonly \n performed .", "oral and vocal fold diadochokinesis ( ddk ) is one of the tests that can be applied to \n perform acoustic analysis .", "the ddk , or syllable alternating motion rate , is the ability to perform rapidly \n opposite contractions of relatively simple patterns .", "vocal fold ddk analysis allows evaluation of \n neuromuscular integrity of the vocal folds and oral ddk analyzes the ability to perform quick repetitions of \n speech segments .", "some studies try to \n understand the vocal manifestation of dysphonic patients through the perceptual and \n acoustic analysis of voice .", "( 2008 ) evaluated voice acoustic parameter changes in patients with \n parkinson 's disease ( pd ) who were searching for their relationship with motor control . \n", "the voices perceptual and acoustic ratings evaluations of 20 pd patients , 12 men and 8 \n women , were compared with the voices of 20 control subjects of corresponding ages and \n genders .", "patients with pd had smaller values for maximum phonation time and ddk compared to the \n control group , while jitter , shimmer and average fundamental frequency ( f0 ) were similar \n between the two groups .", "although men have poorer performance in ddk than women , these \n values were not statistically significant .", "thus , only a few significant correlations \n between vocal parameters and motor control were found .", "the vocal fold ddk measurement indicates laryngeal gestures of opening and closing of \n the vocal folds , being that changes in extension and speed movement of vocal folds will \n reproduce changes in the production rate in patterns of duration and transglottic \n airflow rate .", "therefore , the presence \n of organic and functional changes in dysphonia cases may relate to ddk results .", "the ddk evaluation associated with other clinical assessment procedures is an important \n resource in understanding the manifestations of individual communication disorders .", "the \n assessment of oral and vocal fold diadochokinesis ( ddk ) in individuals with voice \n disorders may contribute to the understanding of factors that affect balanced vocal \n production .", "scientific studies that make use of this assessment tool support advances in \n knowledge in this area and reflect the development of more appropriate therapeutic \n planning .", "consequently , the objective of this study was to compare the results of oral and vocal \n fold ddk of dysphonic women to the results of oral and vocal fold ddk in women without \n vocal disorders ." ], [ "this study has been approved by the research ethical committee of the school of \n dentistry of the university of so paulo , bauru campus .", "the recordings were obtained in an acoustically treated studio , and the patients \n remained seated with a headset microphone ( akg , model - c444pp ) positioned laterally \n between 60 degrees and 5 cm from the labial commissure .", "the emissions were recorded on a \n intel pentium 4 computer ( cpu 2.040 ghz and 256 mb ram ) with \n a 17 \" lg flatron e7015 monitor and a creative audigy ii sound card .", "the system also used \n sound forge 7.0 professional audio software ( madison , wi , usa ) , with a sampling rate of \n 44.100 hz and a 16 bit mono channel .", "the experimental group was composed of 28 women ranging in age from 19 to 54 years \n old , diagnosed with dysphonia and referred for a voice assessment by a speech \n pathologist and otorhinolaryngologist .", "the control group included 30 nondysphonic \n women age - matched with the experimental group who were evaluated in previous research \n with normal adults .", "the presence of central neurological diseases and the presence of communication \n disorders were considered exclusion factors for both groups , with the exception of \n dysphonia for the experimental group . during the oral and vocal fold ddk test isolated emissions", "were requested ; namely , \n the syllables \" pa , \" \" ta \" and \" ka , \" the sequence \" pataka \" and then the vowels \" a \" and \n \" i. \" before the beginning of the test , the patient was requested to repeat the sound \n as rapidly as possible .", "the patients practiced the sounds before each recording by \n repeating as fast as possible , after deep breathing , in a clear and precise way the \n emissions with a comfortable frequency and intensity in order to understand how to \n perform the test . soon thereafter", ", the subjects were guided to perform the tasks , by \n receiving a signal with the thumb to begin and to stop the repetition .", "the \" pa , \" \" ta , \" \" ka , \" \" pataka , \" \" a \" and \" i \" emissions were edited by sound forge 7.0 \n professional audio editing professional .", "software ( madison , wi , usa ) . the beginning \n and end of each sample were excluded , except for four seconds from the third to the \n sixth second . monosyllabic emissions \" pa , \" \" ta \" and \" ka \" and the vowels \" a \" and \" i \" were filed at \n sampling rate of 11.025 hz , 16 bit mono channel and analyzed by advanced motor speech \n profile program ( msp ) model 5141 version 2.5.2 ( kay 's elemetrics corp . , lincoln park , \n nj , usa ) , whose parameters are described in figure \n 1 .", "the msp program displays a graphical record of emissions showing the \n horizontal axis ( time in seconds ) and the vertical axis ( energy in db ) . to do the ddk \n count ,", "the program drew a line in the central point at the energy scale of the \n vertical axis in db .", "the line of analysis was manually positioned in the \n corresponding value to the intensity average of the ddk sample , provided by the msp \n program , and moved up or down , so the program could count all emissions when \n necessary ( figure 2 ) .", "parameters for diadochokinesis ( ddk ) analyzed by advanced motor speech profile \n ( msp ) program advanced motor speech profile ( msp ) program graphic , where time ( seconds ) on \n the horizontal axis and energy ( db ) on the vertical axis can be observed the ddk of the \" pataka \" sequence was analyzed quantitatively through the sound forge \n 7.0 program .", "the count of trisyllables per second was performed manually with the \n auditory and visual waveform support displayed by the program .", "average values of oral and vocal fold ddk in dysphonic and nondysphonic women were \n correlated using the \" t student \" test and were considered significant when \n p<0.05 ." ], [ "the experimental group was composed of 28 women ranging in age from 19 to 54 years \n old , diagnosed with dysphonia and referred for a voice assessment by a speech \n pathologist and otorhinolaryngologist .", "the control group included 30 nondysphonic \n women age - matched with the experimental group who were evaluated in previous research \n with normal adults .", "the presence of central neurological diseases and the presence of communication \n disorders were considered exclusion factors for both groups , with the exception of \n dysphonia for the experimental group ." ], [ "during the oral and vocal fold ddk test isolated emissions were requested ; namely , \n the syllables \" pa , \" \" ta \" and \" ka , \" the sequence \" pataka \" and then the vowels \" a \" and \n \" i. \" before the beginning of the test , the patient was requested to repeat the sound \n as rapidly as possible .", "the patients practiced the sounds before each recording by \n repeating as fast as possible , after deep breathing , in a clear and precise way the \n emissions with a comfortable frequency and intensity in order to understand how to \n perform the test . soon thereafter", ", the subjects were guided to perform the tasks , by \n receiving a signal with the thumb to begin and to stop the repetition ." ], [ "the \" pa , \" \" ta , \" \" ka , \" \" pataka , \" \" a \" and \" i \" emissions were edited by sound forge 7.0 \n professional audio editing professional .", "the beginning \n and end of each sample were excluded , except for four seconds from the third to the \n sixth second ." ], [ "monosyllabic emissions \" pa , \" \" ta \" and \" ka \" and the vowels \" a \" and \" i \" were filed at \n sampling rate of 11.025 hz , 16 bit mono channel and analyzed by advanced motor speech \n profile program ( msp ) model 5141 version 2.5.2 ( kay 's elemetrics corp .", ", lincoln park , \n nj , usa ) , whose parameters are described in figure \n 1 .", "the msp program displays a graphical record of emissions showing the \n horizontal axis ( time in seconds ) and the vertical axis ( energy in db ) . to do the ddk \n count", ", the program drew a line in the central point at the energy scale of the \n vertical axis in db .", "the line of analysis was manually positioned in the \n corresponding value to the intensity average of the ddk sample , provided by the msp \n program , and moved up or down , so the program could count all emissions when \n necessary ( figure 2 ) .", "parameters for diadochokinesis ( ddk ) analyzed by advanced motor speech profile \n ( msp ) program advanced motor speech profile ( msp ) program graphic , where time ( seconds ) on \n the horizontal axis and energy ( db ) on the vertical axis can be observed the ddk of the \" pataka \" sequence was analyzed quantitatively through the sound forge \n 7.0 program .", "the count of trisyllables per second was performed manually with the \n auditory and visual waveform support displayed by the program ." ], [ "average values of oral and vocal fold ddk in dysphonic and nondysphonic women were \n correlated using the \" t student \" test and were considered significant when \n p<0.05 ." ], [ "subjects were classified by the types of dysphonia , according to behlau , et \n al .", "it \n was considered primary functional dysphonia when there were no injuries or structural \n alterations of vocal folds identified by nasolaryngoscopy ; secondary functional \n dysphonia to anatomical inadequacy , in the presence of structural alterations of vocal \n folds , such as epidermoid cyst , varicosity , stria sulcus ; organic functional dysphonia , \n in the presence of edema , leucoplast or bilateral nodules ; and organic dysphonia in \n cases of gastroesophageal reflux disease or unilateral vocal fold paralysis .", "thus , the \n sample was composed of a participant with primary functional dysphonia , thirteen with \n secondary functional dysphonia by anatomical inadequacy , seven with organic and \n functional dysphonia , and three with organic dysphonia , because three women in the \n sample had no otorhinolaryngologic diagnosis .", "the results concerning the oral and vocal fold ddk of the two groups were compared using \n the \" t student \" test and are described in table \n 1 .", "comparison of oral and vocal fold diadochokinesis ( ddk ) average and standard \n deviation , between dysphonic and nondysphonic women , in the emissions of \" pa , \" \n \" ta , \" \" ka , \" \" pataka \" , \" a , \" and \" i. \"" ], [ "although the findings did not show a statistically significant correlation of oral and \n vocal fold ddk between the group of women with and without voice disorders ( table 1 ) , some aspects about the performance of \n diadochokinetic tasks are relevant and should be discussed .", "the results showed that the ddk speed , the average time between the vocalizations , the \n ability to keep constant vocalizations and keeping the intensity of these vocalizations \n constant did not correlate with the presence of dysphonia ( table 1 ) .", "it was expected that no neurological changes related to \n vocal production would interfere with oral and vocal fold ddk , since for both tasks \n there was participation of the opening and closing of vocal folds , especially related to \n \" a \" and \" i \" emissions , that involve specifically alternating motions of glottic \n adduction and abduction , since organic and functional factors may also interfere with \n the results of the ddk , including not necessarily expressing a disturbance in oral \n communication .", "it was observed that in the \" pa \" emission , the parameter cvi ( p=0.061 ) , although not \n statistically significant , showed a tendency for nondysphonic women to keep the \n vocalizations more constant as compared to the dysphonic group .", "this can be explained by \n the fact that the glottic changes found in dysphonic women , make intensity control \n difficult , since vocal balance depends on correct coordination between the myoelastic \n and aerodynamic forces of the larynx ; \n", "this finding is possibly related to the size of the \n sample , thus , statistically significant values can be found if the number of subjects \n were more expressive .", "the impairment of phonoarticulatory \n organs can generate vocal consequences , such as changes in vocal quality , impaired \n laryngeal mobility , and extrinsic muscle tension of the larynx .", "the interference of articulation with phonation shows \n that such factors might affect the motor performance of these structures and thus , in \n ddk .", "thus , there is a physiological and \n anatomical inter - connecting of the structures common to phonation and \n articulation .", "thus , individuals who \n have dysphonia due to a myofunctional alteration that initiates larynx overload could \n have poorer ddk performance .", "some minor structural alterations have morphological characteristics that change the \n vibration pattern of vocal folds .", "therefore , the sulcus vocalis shows \n an arched vocal fold , and cysts \n appear on a bulged vocal fold .", "this \n could interfere with the speed of the vocal folds alternating motion rate ( ddk ) . \n", "leeper , heeneman and reynolds ( 1990 ) reported that , when ddk involves vowel emission , refined \n control of vocal fold opening and closing is required .", "factors such as rigidity and mass can interfere with the fundamental frequency of \n voice , beyond subglottic \n pressure , which reflects on vocal intensity .", "there are significant differences in the \n effect of frequency and vocal intensity upon the airflow through the vocal \n folds .", "furthermore , vocal \n intensity seems to be the primary factor controlling the speed of the ddk and airflow \n during the production of the vowel \" a \" . in this study , although no statistically significant correlation was observed , an \n average frequency of disturbances in the ddk \" a \" ( ddcjiit ) were higher in dysphonic \n subjects than in the control group , and this value was approximately doubled .", "high \n frequency values of ddk disturbance may indicate a lower ability to control the vocal \n folds ' motion during phonation .", "vocal fold ddk has not been investigated in detail like \n an analysis of laryngeal subsystem , as has already occurred with oral ddk .", "nondysphonic individuals have appropriate adjustments in their laryngeal mechanism that \n allows for quick glottal opening and closing during the production of the consonants and \n vowels of syllables .", "this suggests \n that these adjustments in laryngeal diseases are inadequate , resulting in an increased \n effort to compensate .", "therefore , information obtained with the ddk can be reference for \n the interpretation of complex tasks .", "there is a need for more studies that compare ddk in a control group and in dysphonic \n individuals , the latter split into distinct groups according to the vocal alteration , \n thus making it possible to compare diadochokinetic task performance differences in \n different dysphonias ." ], [ "although the results do n't indicate any difficulties in oral and laryngeal motor control \n in the dysphonic group , the major instability in vocal fold ddk in the experimental \n group should be considered , and studies of this ability in individuals with \n communication disorders must be intensified ." ] ]

[ "restless legs syndrome ( rls ) is a sensorimotor disorder characterized by an urge to move the legs that are associated with unpleasant paresthesias .", "usually , the symptoms worsen when at rest and at night and are relieved by movement .", "various studies suggest that genetic component , iron - deficiency , disturbances in the dopaminergic neurotransmitter system and abnormality in spinal conduction pathways are associated with the disorder .", "transcranial magnetic stimulation ( tms ) , developed in 1985 , is a noninvasive technique with the ability to stimulate neurons in the cerebral cortex through the scalp safely and with minimal discomfort .", "repetitive tms ( rtms ) is a technique that delivers long trains of closely spaced pulses to specific brain areas in order to alter cortical activity and connectivity .", "previous studies have suggested that low - frequency rtms decreases the excitability of the cortex while high - frequency rtms increases it .", "recently , rtms has been widely applied in the treatment of patients with psychiatric disorders , epilepsy , migraine , chronic pain , and neurodegenerative disorders , including parkinson 's disease ( pd ) , although its mechanism is not yet well understood .", "numerous reports have demonstrated that rtms of the human primary motor cortex induces the release of dopamine in the putamen , which indicates that rtms probably modulates striatal dopaminergic neurotransmission .", "currently , an increasing number of studies have provided support for a link between rls and pd .", "some studies have found that the prevalence of rls is higher in patients with pd than in the general population .", "based on the common mechanism involving disturbances in the dopaminergic neurotransmitter system between rls and pd , we investigated whether rtms application to the cortex was beneficial in patients with rls .", "we included fourteen idiopathic rls patients treated at the sleep clinic of our hospital between 2011 and 2012 that were diagnosed according to the international criteria of the international restless legs syndrome study group set in 2003 .", "the exclusion criteria were as follows : ( i ) all secondary rls stemming from a vitamin deficiency , iron - deficiency anemia , pregnancy , diabetes mellitus , severe metabolic disorders , liver dysfunction , or renal disease ; ( ii ) peripheral neuropathy and radiculopathy ; ( iii ) a history of psychiatric disease ; ( iv ) neuropathic pain ; ( v ) leg cramps or epilepsy ; ( vi ) use of a cardiac pacemaker , vagal nerve stimulator , or any metal implants ; ( vii ) other severe medical diseases . patients were not placed on any new medications , including dopaminergic agonists , psycholeptics , or benzodiazepines ; if they were already taking them for at least 4 weeks prior to the initiation of the study , they continued the medicine throughout the study at the prescribed dosage . all patients provided written informed consent , and the study had the approval of hospital ethics committee .", "we administered rtms at 15 hz using a magstim system ( magstim super rapid stimulator , magstim company , whitland , dyfed , uk ) with a figure - eight coil .", "one rtms train consisted of 75 pulses delivered at 15 hz with an intertrain interval of 10 min . in one session , 600 pulses ( 8 rtms trains ) were delivered to each hemisphere .", "one session was performed per day for 5 continuous days and stopped for 2 days .", "the patients were seated in a comfortable chair , and the coil was positioned at the leg representation in the motor cortex of frontal lobe . the optimal stimulation position for the tibialis anterior muscle", "was located by stimulating the presumed motor cortex at every 1 cm in a 6-cm .", "the resting motor threshold ( rmt ) was defined as the minimal stimulus intensity that produced a motor evoked potential in the relaxed muscle with a peak - to - peak amplitude of > 50 mv on 50% of 10 trials .", "the international rls rating scale ( irls - rs ) , pittsburgh sleep quality index ( psqi ) , hamilton anxiety scale ( hama ) and hamilton depression scale ( hamd ) were used to evaluate the severity of rls , quality of sleep , and the severity of anxiety and depression , respectively .", "the assessments were taken at the baseline ( prior to stimulation ) , at end of 14 session , and at 1- and 2-month posttreatment by a trained clinical neurologist .", "one - way analysis of variance was used to compare the means of scale scores at different time points . a p < 0.05 was considered as statistically significant .", "we included fourteen idiopathic rls patients treated at the sleep clinic of our hospital between 2011 and 2012 that were diagnosed according to the international criteria of the international restless legs syndrome study group set in 2003 .", "the exclusion criteria were as follows : ( i ) all secondary rls stemming from a vitamin deficiency , iron - deficiency anemia , pregnancy , diabetes mellitus , severe metabolic disorders , liver dysfunction , or renal disease ; ( ii ) peripheral neuropathy and radiculopathy ; ( iii ) a history of psychiatric disease ; ( iv ) neuropathic pain ; ( v ) leg cramps or epilepsy ; ( vi ) use of a cardiac pacemaker , vagal nerve stimulator , or any metal implants ; ( vii ) other severe medical diseases . patients were not placed on any new medications , including dopaminergic agonists , psycholeptics , or benzodiazepines ; if they were already taking them for at least 4 weeks prior to the initiation of the study , they continued the medicine throughout the study at the prescribed dosage . all patients provided written informed consent , and the study had the approval of hospital ethics committee .", "we administered rtms at 15 hz using a magstim system ( magstim super rapid stimulator , magstim company , whitland , dyfed , uk ) with a figure - eight coil .", "one rtms train consisted of 75 pulses delivered at 15 hz with an intertrain interval of 10 min . in one session , 600 pulses ( 8 rtms trains )", "one session was performed per day for 5 continuous days and stopped for 2 days .", "then , another 4 days of stimulation were given . in total , 14 sessions were performed for each patient in our study . the patients were seated in a comfortable chair , and the coil was positioned at the leg representation in the motor cortex of frontal lobe .", "the optimal stimulation position for the tibialis anterior muscle was located by stimulating the presumed motor cortex at every 1 cm in a 6-cm .", "the resting motor threshold ( rmt ) was defined as the minimal stimulus intensity that produced a motor evoked potential in the relaxed muscle with a peak - to - peak amplitude of > 50 mv on 50% of 10 trials .", "the international rls rating scale ( irls - rs ) , pittsburgh sleep quality index ( psqi ) , hamilton anxiety scale ( hama ) and hamilton depression scale ( hamd ) were used to evaluate the severity of rls , quality of sleep , and the severity of anxiety and depression , respectively .", "the assessments were taken at the baseline ( prior to stimulation ) , at end of 14 session , and at 1- and 2-month posttreatment by a trained clinical neurologist .", "one - way analysis of variance was used to compare the means of scale scores at different time points . a p < 0.05 was considered as statistically significant .", "among 14 patients , there were 4 males and 10 females with a mean age of 59.22 10.10 years and a range of 4673 years .", "the irls - rs scores at the four - time points assessed ( baseline , end of 14 session , 1- and 2-month posttreatment ) are summarized in table 1 .", "all of the irls - rs , psqi and hama scores showed continuous and significant improvement posttreatment compared to baseline .", "the irls - rs , psqi , hama , and hamd scores in the fourteen idiopathic rls patients irls - rs : international rls rating scale ; psqi : pittsburgh sleep quality index ; hama : hamilton anxiety scale ; hamd : hamilton depression scale . * different from baseline , p<0.05 .", "our results indicated a significant improvement in irls - rs scores after 14 sessions , from 23.86 5.88 to 11.21 7.23 .", "this proved the effect of rtms in treating rls and that the effect could last for some time ( at least 2 months , according to our study ) after the stimulation .", "khedr et al . found that repeated sessions of rtms could produce prolonged changes in enhanced dopamine function that may be responsible for long - lasting clinical effects .", "after rtms treatment , psqi scores decreased from 15.00 4.88 to 9.29 3.91 and persisted at least 2 months after treatment .", "the long - lasting change in psqi scores was consistent with the improvement in irls - rs scores .", "found rls patients had significantly higher levels of anxiety and depression . in our study ,", "rls patients showed obvious improvements in anxiety , and further improvements occurred in the 2 months after treatment .", "in addition , the patients depression improved after treatment , although this change was not statistically significant .", "it is likely that rtms improved symptoms of rls directly rather than its associated symptoms , such as mood disorders , which in turn led to an improvement in symptoms of rls .", "many studies have indicated that rls is not associated with the use of antidepressants , and some studies have suggested that antidepressants might exacerbate rls symptoms .", "in addition , depression did not improve as significantly as the symptoms of rls in our study .", "thus , we believe that rtms alleviates rls directly rather than through the treatment of associated symptoms .", "found that high - frequency rtms over the supplementary motor area ( sma ) improved irls - rs scores significantly after 5 and 10 sessions of stimulation .", "this result is consistent with ours , although the configuration of stimulation parameters differed , and they did not evaluate sleep quality , anxiety or depression . in burcu 's study ,", "the stimulus frequency was 5 hz , and the stimulation was centered at points 3 cm anterior to the leg motor area at the sagittal midline .", "their parameters were taken from a previous study of pd , in which 5 hz rtms was administered over the sma , resulting in a modest improvement of motor symptoms .", "a recent study verified connectivity between the sma and the primary motor cortex . because stimulating either the sma or the primary motor cortex can alleviate rls syndrome , we speculate a common effect of rtms on upstream sma and downstream primary motor cortex .", "the exact mechanism underlying the treatment of rls with rtms is complex and not clearly understood .", "previous studies have revealed a shortened cortical silent period in the anterior tibialis muscle in patients with rls , which indicated a disturbed supraspinal inhibition mediated by decreased excitation of cortical inhibitory interneurons , thus leading to the hyperexcitability of spinal pathways .", "another probable mechanism involves the release of endogenous dopamine in the striatum , based on single photon emission computed tomography studies .", "however , a study conducted in japan suggested that chronic rtms had a limited effect on the dopaminergic system .", "hence , there may be multiple mechanisms of action involved in modulating symptoms of rls .", "revealing the exact mechanism is interesting and valuable and should be the subject of future study .", "in addition , no adverse effects were observed during stimulation or after treatment , and all patients showed good compliance .", "thus , 15 hz rtms delivered over the leg representation area of motor cortex is a safe treatment for rls .", "medications traditionally used to treat rls provide dramatic immediate benefits but may augment rls symptoms over time .", "thus , rtms and medications each possess advantages and provide patients with a variety of treatment options .", "however , our study had a limited sample size and no control group , therefore , a large case - control study is necessary to provide more convincing evidence . in addition , an optimized rtms paradigm should be established . in the future", ", we plan to compare cortical excitement before and after rtms treatment using neurophysiological and imaging measurements , such as paired pulse tms and functional magnetic resonance imaging , to identify the mechanism underlying rls . in conclusion", ", our study proves the utility of rtms for the treatment of rls patients despite the study 's limited sample size .", "in the future , a large case - control study should be performed , and the rtms protocol should be optimized ." ]

[ "<S> background : repetitive transcranial magnetic stimulation ( rtms ) is a noninvasive technique used to alter cortex excitability that has been proposed as an efficient method for treating brain hyperexcitability or hypoexcitability disorders . the aim of this study was to investigate whether high - frequency rtms could have any beneficial effects in restless legs syndrome ( rls).methods : fourteen patients with rls were given high - frequency rtms ( 15 hz , 100% motor threshold ) to the leg representation motor cortex area of the frontal lobe for 14 sessions over 18 days . </S>", "<S> patients were diagnosed according to the international criteria proposed by the international restless legs syndrome study group in 2003 . </S>", "<S> the international rls rating scale ( irls - rs ) , pittsburgh sleep quality index ( psqi ) , hamilton anxiety scale ( hama ) and hamilton depression scale were used to evaluate the severity of rls , sleep quality , anxiety and depression , respectively . </S>", "<S> the scale scores were evaluated at four - time points ( baseline , end of the 14th session , and at 1- and 2-month posttreatment ) . </S>", "<S> one - way analysis of variance was used to compare scale scores at different time points.results:there was significant improvement in the irls - rs ( from 23.86 5.88 to 11.21 7.23 , p < 0.05 ) , psqi ( from 15.00 4.88 to 9.29 3.91 , p < 0.05 ) , and hama ( from 17.93 7.11 to 10.36 7.13 , p < 0.05 ) scale scores at the end of 14th session , with ongoing effects lasting for at least 2 months.conclusions:high-frequency rtms can markedly alleviate the motor system symptoms , sleep disturbances , and anxiety in rls patients . </S>", "<S> these results suggest that rtms might be an option for treating rls . </S>" ]

[ "I", "M", "Patients", "Repetitive transcranial magnetic stimulation procedure", "Assessment", "Statistical analysis", "R", "D" ]

[ [ "restless legs syndrome ( rls ) is a sensorimotor disorder characterized by an urge to move the legs that are associated with unpleasant paresthesias .", "usually , the symptoms worsen when at rest and at night and are relieved by movement .", "various studies suggest that genetic component , iron - deficiency , disturbances in the dopaminergic neurotransmitter system and abnormality in spinal conduction pathways are associated with the disorder .", "transcranial magnetic stimulation ( tms ) , developed in 1985 , is a noninvasive technique with the ability to stimulate neurons in the cerebral cortex through the scalp safely and with minimal discomfort .", "repetitive tms ( rtms ) is a technique that delivers long trains of closely spaced pulses to specific brain areas in order to alter cortical activity and connectivity .", "previous studies have suggested that low - frequency rtms decreases the excitability of the cortex while high - frequency rtms increases it .", "recently , rtms has been widely applied in the treatment of patients with psychiatric disorders , epilepsy , migraine , chronic pain , and neurodegenerative disorders , including parkinson 's disease ( pd ) , although its mechanism is not yet well understood .", "numerous reports have demonstrated that rtms of the human primary motor cortex induces the release of dopamine in the putamen , which indicates that rtms probably modulates striatal dopaminergic neurotransmission .", "currently , an increasing number of studies have provided support for a link between rls and pd .", "some studies have found that the prevalence of rls is higher in patients with pd than in the general population .", "based on the common mechanism involving disturbances in the dopaminergic neurotransmitter system between rls and pd , we investigated whether rtms application to the cortex was beneficial in patients with rls ." ], [ "we included fourteen idiopathic rls patients treated at the sleep clinic of our hospital between 2011 and 2012 that were diagnosed according to the international criteria of the international restless legs syndrome study group set in 2003 .", "the exclusion criteria were as follows : ( i ) all secondary rls stemming from a vitamin deficiency , iron - deficiency anemia , pregnancy , diabetes mellitus , severe metabolic disorders , liver dysfunction , or renal disease ; ( ii ) peripheral neuropathy and radiculopathy ; ( iii ) a history of psychiatric disease ; ( iv ) neuropathic pain ; ( v ) leg cramps or epilepsy ; ( vi ) use of a cardiac pacemaker , vagal nerve stimulator , or any metal implants ; ( vii ) other severe medical diseases . patients were not placed on any new medications , including dopaminergic agonists , psycholeptics , or benzodiazepines ; if they were already taking them for at least 4 weeks prior to the initiation of the study , they continued the medicine throughout the study at the prescribed dosage . all patients provided written informed consent , and the study had the approval of hospital ethics committee .", "we administered rtms at 15 hz using a magstim system ( magstim super rapid stimulator , magstim company , whitland , dyfed , uk ) with a figure - eight coil .", "one rtms train consisted of 75 pulses delivered at 15 hz with an intertrain interval of 10 min . in one session , 600 pulses ( 8 rtms trains ) were delivered to each hemisphere .", "one session was performed per day for 5 continuous days and stopped for 2 days .", "the patients were seated in a comfortable chair , and the coil was positioned at the leg representation in the motor cortex of frontal lobe . the optimal stimulation position for the tibialis anterior muscle", "was located by stimulating the presumed motor cortex at every 1 cm in a 6-cm .", "the resting motor threshold ( rmt ) was defined as the minimal stimulus intensity that produced a motor evoked potential in the relaxed muscle with a peak - to - peak amplitude of > 50 mv on 50% of 10 trials .", "the international rls rating scale ( irls - rs ) , pittsburgh sleep quality index ( psqi ) , hamilton anxiety scale ( hama ) and hamilton depression scale ( hamd ) were used to evaluate the severity of rls , quality of sleep , and the severity of anxiety and depression , respectively .", "the assessments were taken at the baseline ( prior to stimulation ) , at end of 14 session , and at 1- and 2-month posttreatment by a trained clinical neurologist .", "one - way analysis of variance was used to compare the means of scale scores at different time points . a p < 0.05 was considered as statistically significant ." ], [ "we included fourteen idiopathic rls patients treated at the sleep clinic of our hospital between 2011 and 2012 that were diagnosed according to the international criteria of the international restless legs syndrome study group set in 2003 .", "the exclusion criteria were as follows : ( i ) all secondary rls stemming from a vitamin deficiency , iron - deficiency anemia , pregnancy , diabetes mellitus , severe metabolic disorders , liver dysfunction , or renal disease ; ( ii ) peripheral neuropathy and radiculopathy ; ( iii ) a history of psychiatric disease ; ( iv ) neuropathic pain ; ( v ) leg cramps or epilepsy ; ( vi ) use of a cardiac pacemaker , vagal nerve stimulator , or any metal implants ; ( vii ) other severe medical diseases . patients were not placed on any new medications , including dopaminergic agonists , psycholeptics , or benzodiazepines ; if they were already taking them for at least 4 weeks prior to the initiation of the study , they continued the medicine throughout the study at the prescribed dosage . all patients provided written informed consent , and the study had the approval of hospital ethics committee ." ], [ "we administered rtms at 15 hz using a magstim system ( magstim super rapid stimulator , magstim company , whitland , dyfed , uk ) with a figure - eight coil .", "one rtms train consisted of 75 pulses delivered at 15 hz with an intertrain interval of 10 min . in one session , 600 pulses ( 8 rtms trains )", "one session was performed per day for 5 continuous days and stopped for 2 days .", "then , another 4 days of stimulation were given . in total , 14 sessions were performed for each patient in our study . the patients were seated in a comfortable chair , and the coil was positioned at the leg representation in the motor cortex of frontal lobe .", "the optimal stimulation position for the tibialis anterior muscle was located by stimulating the presumed motor cortex at every 1 cm in a 6-cm .", "the resting motor threshold ( rmt ) was defined as the minimal stimulus intensity that produced a motor evoked potential in the relaxed muscle with a peak - to - peak amplitude of > 50 mv on 50% of 10 trials ." ], [ "the international rls rating scale ( irls - rs ) , pittsburgh sleep quality index ( psqi ) , hamilton anxiety scale ( hama ) and hamilton depression scale ( hamd ) were used to evaluate the severity of rls , quality of sleep , and the severity of anxiety and depression , respectively .", "the assessments were taken at the baseline ( prior to stimulation ) , at end of 14 session , and at 1- and 2-month posttreatment by a trained clinical neurologist ." ], [ "one - way analysis of variance was used to compare the means of scale scores at different time points . a p < 0.05 was considered as statistically significant ." ], [ "among 14 patients , there were 4 males and 10 females with a mean age of 59.22 10.10 years and a range of 4673 years .", "the irls - rs scores at the four - time points assessed ( baseline , end of 14 session , 1- and 2-month posttreatment ) are summarized in table 1 .", "all of the irls - rs , psqi and hama scores showed continuous and significant improvement posttreatment compared to baseline .", "the irls - rs , psqi , hama , and hamd scores in the fourteen idiopathic rls patients irls - rs : international rls rating scale ; psqi : pittsburgh sleep quality index ; hama : hamilton anxiety scale ; hamd : hamilton depression scale . * different from baseline , p<0.05 ." ], [ "our results indicated a significant improvement in irls - rs scores after 14 sessions , from 23.86 5.88 to 11.21 7.23 .", "this proved the effect of rtms in treating rls and that the effect could last for some time ( at least 2 months , according to our study ) after the stimulation .", "khedr et al . found that repeated sessions of rtms could produce prolonged changes in enhanced dopamine function that may be responsible for long - lasting clinical effects .", "after rtms treatment , psqi scores decreased from 15.00 4.88 to 9.29 3.91 and persisted at least 2 months after treatment .", "the long - lasting change in psqi scores was consistent with the improvement in irls - rs scores .", "found rls patients had significantly higher levels of anxiety and depression . in our study ,", "rls patients showed obvious improvements in anxiety , and further improvements occurred in the 2 months after treatment .", "in addition , the patients depression improved after treatment , although this change was not statistically significant .", "it is likely that rtms improved symptoms of rls directly rather than its associated symptoms , such as mood disorders , which in turn led to an improvement in symptoms of rls .", "many studies have indicated that rls is not associated with the use of antidepressants , and some studies have suggested that antidepressants might exacerbate rls symptoms .", "in addition , depression did not improve as significantly as the symptoms of rls in our study .", "thus , we believe that rtms alleviates rls directly rather than through the treatment of associated symptoms .", "found that high - frequency rtms over the supplementary motor area ( sma ) improved irls - rs scores significantly after 5 and 10 sessions of stimulation .", "this result is consistent with ours , although the configuration of stimulation parameters differed , and they did not evaluate sleep quality , anxiety or depression . in burcu 's study ,", "the stimulus frequency was 5 hz , and the stimulation was centered at points 3 cm anterior to the leg motor area at the sagittal midline .", "their parameters were taken from a previous study of pd , in which 5 hz rtms was administered over the sma , resulting in a modest improvement of motor symptoms .", "a recent study verified connectivity between the sma and the primary motor cortex . because stimulating either the sma or the primary motor cortex can alleviate rls syndrome , we speculate a common effect of rtms on upstream sma and downstream primary motor cortex .", "the exact mechanism underlying the treatment of rls with rtms is complex and not clearly understood .", "previous studies have revealed a shortened cortical silent period in the anterior tibialis muscle in patients with rls , which indicated a disturbed supraspinal inhibition mediated by decreased excitation of cortical inhibitory interneurons , thus leading to the hyperexcitability of spinal pathways .", "another probable mechanism involves the release of endogenous dopamine in the striatum , based on single photon emission computed tomography studies .", "however , a study conducted in japan suggested that chronic rtms had a limited effect on the dopaminergic system .", "hence , there may be multiple mechanisms of action involved in modulating symptoms of rls .", "revealing the exact mechanism is interesting and valuable and should be the subject of future study .", "in addition , no adverse effects were observed during stimulation or after treatment , and all patients showed good compliance .", "thus , 15 hz rtms delivered over the leg representation area of motor cortex is a safe treatment for rls .", "medications traditionally used to treat rls provide dramatic immediate benefits but may augment rls symptoms over time .", "thus , rtms and medications each possess advantages and provide patients with a variety of treatment options .", "however , our study had a limited sample size and no control group , therefore , a large case - control study is necessary to provide more convincing evidence . in addition , an optimized rtms paradigm should be established . in the future", ", we plan to compare cortical excitement before and after rtms treatment using neurophysiological and imaging measurements , such as paired pulse tms and functional magnetic resonance imaging , to identify the mechanism underlying rls . in conclusion", ", our study proves the utility of rtms for the treatment of rls patients despite the study 's limited sample size .", "in the future , a large case - control study should be performed , and the rtms protocol should be optimized ." ] ]

[ "malaria control programmes utilising indoor residual spraying are only effective if a high coverage of targeted structures is achieved and an insecticide that is effective against the specific mosquito vector is correctly applied .", "ongoing monitoring of spraying operations is essential to assure optimal programme performance and early corrective action , where indicated .", "successful development and application of a computerised spraying operations management system in mpumalanga province , south africa during 1998 resulted in its adaptation and introduction in neighbouring maputo province , southern mozambique during 2000 .", "the structure and components of this computerised management system are described , and its ' operational benefit in southern mozambique , where community - based spray operators apply intradomiciliary insecticide , are reviewed .", "the computerised management system allowed malaria programme management and field supervisors to monitor spraying coverage , insecticide consumption and application rates on an ongoing basis .", "the system supported a successful transition to community - based spraying , while assuring correct insecticide application and spraying completion according to schedule .", "in 1946 , south africa introduced intradomiciliary spraying with residual insecticides , ddt ( dichlorodiphenyltrichloroethane ) and bhc ( benzene hexachloride ) , to kill indoor - resting vector mosquitoes and thereby control malaria .", "this resulted in a 75 percent reduction in the geographic extent of the malaria - affected area , with malaria occurrence limited to summer epidemics in the low - lying northern and eastern border areas with botswana , zimbabwe and mozambique .", "similar malaria control programmes were initiated in other southern african countries . in mozambique , spraying operations for malaria control collapsed during the 1970s due to the protracted civil war with a resulting high burden of endemic malaria throughout the country .", "recently there has been a resurgence of malaria in southern africa , attributed to a number of factors , including parasite drug - resistance , mosquito insecticide - resistance , climate changes and large - scale population migration .", "the hiv / aids epidemic has resulted in a simultaneous dwindling of resources available for public health programmes , thus placing an onus on programme managers to ensure optimal efficiency of their activities .", "an efficient spraying programme is characterized by application of the correct volumes of insecticide on surfaces suitable for mosquito resting .", "this should be achieved before the onset of peak malaria transmission with a high coverage of targeted structures .", "a formal spraying management system was introduced in south africa for the first time in 1973 , with activity forms being completed by the field officials responsible for each spraying team and a \" hut - card \" left under the eave of each sprayed dwelling .", "this was expanded to a set of seven different spraying record forms in 1975 under the auspices of the world health organization .", "this set consisted of a spray operator record , a daily spray - team report , a weekly report , a monthly report , a locality completion report , a sector completion report and a spraying completion report .", "although this manual system was cumbersome it became entrenched , and remained essentially unaltered in south africa for more than 20 years .", "a major flaw of this data system was that field staff were not trained to use the data for programme monitoring .", "thus serious programme deficiencies usually only became apparent at the end of the spraying season when the data were centrally analysed .", "mpumalanga province , located on the border with mozambique in the north - east of south africa , began to replace permanently employed spray personnel with temporary spray personnel recruited from each malaria - affected community .", "one hundred community - based spray personnel were appointed , each accountable to their local community , and the team was responsible for spraying almost 170,000 structures during each annual spraying round . with supervisory support limited to one field manager and six field officers , it was postulated that unless an improved spraying management system was introduced , programme performance and efficiency might deteriorate .", "a computerised management system was developed in mpumalanga province to enable malaria programme management and field supervisors to monitor , even on a daily basis when required , spraying coverage , individual spray operator 's performance , and insecticide consumption and application rates .", "the success of this computerised management system resulted in it being extended to neighbouring swaziland and also to southern mozambique , where a malaria vector control programme was recently re - introduced through the multilateral lubombo spatial development initiative ( lsdi ) , a partnership between the governments of mozambique , swaziland and south africa .", "we describe the structure and components of this computerised management system , and provide operational evidence of its value in the southern mozambique malaria control programme .", "the computerised spraying management system in southern mozambique was designed prior to the first round of spraying early in 2000 .", "the area targeted for spraying included the rural districts of namaacha and matutuine , and sections of the peri - urban districts of matola and boane in maputo province , covering an area of 7,962 square kilometres with an estimated 200,000 homes .", "the management system comprises two platforms , a spraying database and a spatial mapping platform .", "a microsoft access 2000 database was used with front - end data entry and output screens .", "the data entry screen mirrors the manually completed weekly spraying form , which collates data from daily spraying report forms that are submitted each week by spray operators to their supervisor ( figure 1 ) .", "\" drop - down \" menus ensure speed , ease and accuracy of data entry .", "a number of datasets have been integrated to form the basis of the spraying data base , including a comprehensive listing of place names to be sprayed , number of homes ( ministry of health database ) , demographic data for each electoral area ( 1995 electoral census by the department of planning and cooperation ) , spray operator identification codes and details of insecticides used during spraying operations .", "each record is automatically allocated a unique number and all information can be linked through a relational database to a specific spray operator , team , time - period or administrative area .", "additional information collected includes the number of visits made to a specific house , reasons for not spraying ( where applicable ) , insecticide used by each spray operator and number of spray - can refills . data is automatically backed up after each day of data entry onto an external zip drive .", "data entry screen of the malaria information system the geographic information system ( gis ) platform required both spatial ( roads , clinics , locality boundaries ) and attribute data ( locality or clinic names , population figures ) .", "the gis software package mapinfo professional[mapinfo corporation , 4 global view , troy , new york , 12189 , usa .", "] was used and maps of southern mozambique ( scale 1:250,000 ) were obtained from the direco nacional de geografia e cadastro in maputo .", "the south african medical research council digitally captured relevant geographical and cadastral features , including roads , rivers , towns and different administrative level boundaries .", "the smallest administrative unit boundaries at which data was captured ( localities ) were drawn onto local maps by the staff of the ministry of health involved in the project and then digitally captured for inclusion in the gis display .", "preset queries , to generate standardized management reports , were planned and designed in partnership with field supervisors and programme management staff to meet their specific needs .", "however , the system was designed to allow user - friendly production of customized additional reports where required .", "specific training was provided for programme supervisors on making sense of data and interpreting reports , with a spraying database manual developed for ready reference .", "a major emphasis in training has been on identifying operational problems and implementing timely remedial action .", "as field managers enter the summary data at the namaacha field office on a weekly basis , necessary remedial action is taken within a week .", "the ongoing monitoring of individual spray operator 's productivity , in terms of number of structures sprayed each day and volume of insecticide sprayed per structure , has facilitated early detection of operational problems , leading to prompt investigation and supportive corrective action by supervisors .", "a standardized aid for evaluating individual operators ' spraying quality during field visits was designed and implemented .", "field managers discuss evaluation results with spray operators on - site in order to eliminate mistakes .", "the frequency of required supervisory corrective interventions has steadily decreased since the beginning of the programme and it is likely that some of this effect is due to the enhanced monitoring and response made possible by the computer management system .", "insecticide application rates ( grams per square meter ) are constantly monitored for under- and over - application", ". under - application reduces insecticide residual activity and thus effectiveness , while over - application is wasteful . where sub - optimal application rates were detected , investigation included scrutiny of insecticide preparation , nozzle condition , application pressure , distance of nozzle tip from sprayed surface , and spraying rhythm .", "spraying coverage exceeded 90% of 222,000 structures in southern mozambique , a level that is considered more than adequate . in areas where the management system repeatedly identified lower coverage than expected , investigations were conducted to determine whether this was the result of diminished community support , absenteeism or poor motivation of the specific spray operator .", "maps depicting the coverage achieved by each team proved useful to malaria control programme management for tracking teams ' progress , providing ongoing updates to senior health management and encouraging spray personnel to attain their target ( figure 2 ) .", "an example of a spraying progress map by locality in maputo province , mozambique , 2003 an additional benefit was that data captured during the first spraying round during 2000 was used to verify official data sources and provide actual household figures to replace official government estimates .", "this provided more precise information for malaria control planning and demographic data needed by other government agencies for planning .", "competition for ever - dwindling public health resources is a major challenge for malaria control programmes in sub - saharan africa .", "constant monitoring and evaluation of spraying activities is obligatory where indoor residual spraying is an important component of malaria control , to ensure effective application and prevent wastage .", "computerised management systems are proving useful tools for providing timely information to address potentially deleterious human , social and technical factors that could negatively impact on malaria control .", "the computerised management system has been in operation for three years supporting the mainly rural vector control programme that now covers an area of 13,770 square kilometres in southern mozambique .", "not only has it supported a successful and cost - effective transition to a community - based spraying approach , but it has also ensured that spraying was completed to schedule . the ability to immediately identify problems at the level of an individual spray operator and institute appropriate remedial action assured good coverage and programme efficiency .", "the computerised management system is a useful method for enhancing malaria control by improving application of existing tools .", "note : a powerpoint presentation providing more detail on the malaria management information system and database is available at .", "mb , bs , cm , jlg , dd conceptualised , developed and implemented the mpumalanga computerised management system prototype ; mb , bs , cm , bm adapted this system for application in maputo province , mozambique .", "all authors contributed to the review of the system and the preparation of this manuscript ." ]

[ "<S> backgroundmalaria control programmes utilising indoor residual spraying are only effective if a high coverage of targeted structures is achieved and an insecticide that is effective against the specific mosquito vector is correctly applied . </S>", "<S> ongoing monitoring of spraying operations is essential to assure optimal programme performance and early corrective action , where indicated.methodssuccessful development and application of a computerised spraying operations management system in mpumalanga province , south africa during 1998 resulted in its adaptation and introduction in neighbouring maputo province , southern mozambique during 2000 . </S>", "<S> the structure and components of this computerised management system are described , and its ' operational benefit in southern mozambique , where community - based spray operators apply intradomiciliary insecticide , are reviewed.conclusionsthe computerised management system allowed malaria programme management and field supervisors to monitor spraying coverage , insecticide consumption and application rates on an ongoing basis . </S>", "<S> the system supported a successful transition to community - based spraying , while assuring correct insecticide application and spraying completion according to schedule . </S>" ]

[ "Background", "Methods", "Conclusions", "Background", "Description of the management information system", "Application for enhanced malaria control", "Conclusions", "Authors' contributions" ]

[ [ "malaria control programmes utilising indoor residual spraying are only effective if a high coverage of targeted structures is achieved and an insecticide that is effective against the specific mosquito vector is correctly applied .", "ongoing monitoring of spraying operations is essential to assure optimal programme performance and early corrective action , where indicated ." ], [ "successful development and application of a computerised spraying operations management system in mpumalanga province , south africa during 1998 resulted in its adaptation and introduction in neighbouring maputo province , southern mozambique during 2000 .", "the structure and components of this computerised management system are described , and its ' operational benefit in southern mozambique , where community - based spray operators apply intradomiciliary insecticide , are reviewed ." ], [ "the computerised management system allowed malaria programme management and field supervisors to monitor spraying coverage , insecticide consumption and application rates on an ongoing basis .", "the system supported a successful transition to community - based spraying , while assuring correct insecticide application and spraying completion according to schedule ." ], [ "in 1946 , south africa introduced intradomiciliary spraying with residual insecticides , ddt ( dichlorodiphenyltrichloroethane ) and bhc ( benzene hexachloride ) , to kill indoor - resting vector mosquitoes and thereby control malaria .", "this resulted in a 75 percent reduction in the geographic extent of the malaria - affected area , with malaria occurrence limited to summer epidemics in the low - lying northern and eastern border areas with botswana , zimbabwe and mozambique .", "similar malaria control programmes were initiated in other southern african countries . in mozambique , spraying operations for malaria control collapsed during the 1970s due to the protracted civil war with a resulting high burden of endemic malaria throughout the country .", "recently there has been a resurgence of malaria in southern africa , attributed to a number of factors , including parasite drug - resistance , mosquito insecticide - resistance , climate changes and large - scale population migration .", "the hiv / aids epidemic has resulted in a simultaneous dwindling of resources available for public health programmes , thus placing an onus on programme managers to ensure optimal efficiency of their activities .", "an efficient spraying programme is characterized by application of the correct volumes of insecticide on surfaces suitable for mosquito resting .", "this should be achieved before the onset of peak malaria transmission with a high coverage of targeted structures .", "a formal spraying management system was introduced in south africa for the first time in 1973 , with activity forms being completed by the field officials responsible for each spraying team and a \" hut - card \" left under the eave of each sprayed dwelling .", "this was expanded to a set of seven different spraying record forms in 1975 under the auspices of the world health organization .", "this set consisted of a spray operator record , a daily spray - team report , a weekly report , a monthly report , a locality completion report , a sector completion report and a spraying completion report .", "although this manual system was cumbersome it became entrenched , and remained essentially unaltered in south africa for more than 20 years .", "a major flaw of this data system was that field staff were not trained to use the data for programme monitoring .", "thus serious programme deficiencies usually only became apparent at the end of the spraying season when the data were centrally analysed .", "mpumalanga province , located on the border with mozambique in the north - east of south africa , began to replace permanently employed spray personnel with temporary spray personnel recruited from each malaria - affected community .", "one hundred community - based spray personnel were appointed , each accountable to their local community , and the team was responsible for spraying almost 170,000 structures during each annual spraying round . with supervisory support limited to one field manager and six field officers , it was postulated that unless an improved spraying management system was introduced , programme performance and efficiency might deteriorate .", "a computerised management system was developed in mpumalanga province to enable malaria programme management and field supervisors to monitor , even on a daily basis when required , spraying coverage , individual spray operator 's performance , and insecticide consumption and application rates .", "the success of this computerised management system resulted in it being extended to neighbouring swaziland and also to southern mozambique , where a malaria vector control programme was recently re - introduced through the multilateral lubombo spatial development initiative ( lsdi ) , a partnership between the governments of mozambique , swaziland and south africa .", "we describe the structure and components of this computerised management system , and provide operational evidence of its value in the southern mozambique malaria control programme ." ], [ "the computerised spraying management system in southern mozambique was designed prior to the first round of spraying early in 2000 .", "the area targeted for spraying included the rural districts of namaacha and matutuine , and sections of the peri - urban districts of matola and boane in maputo province , covering an area of 7,962 square kilometres with an estimated 200,000 homes .", "the management system comprises two platforms , a spraying database and a spatial mapping platform .", "a microsoft access 2000 database was used with front - end data entry and output screens .", "the data entry screen mirrors the manually completed weekly spraying form , which collates data from daily spraying report forms that are submitted each week by spray operators to their supervisor ( figure 1 ) .", "\" drop - down \" menus ensure speed , ease and accuracy of data entry .", "a number of datasets have been integrated to form the basis of the spraying data base , including a comprehensive listing of place names to be sprayed , number of homes ( ministry of health database ) , demographic data for each electoral area ( 1995 electoral census by the department of planning and cooperation ) , spray operator identification codes and details of insecticides used during spraying operations .", "each record is automatically allocated a unique number and all information can be linked through a relational database to a specific spray operator , team , time - period or administrative area .", "additional information collected includes the number of visits made to a specific house , reasons for not spraying ( where applicable ) , insecticide used by each spray operator and number of spray - can refills . data is automatically backed up after each day of data entry onto an external zip drive .", "data entry screen of the malaria information system the geographic information system ( gis ) platform required both spatial ( roads , clinics , locality boundaries ) and attribute data ( locality or clinic names , population figures ) .", "the gis software package mapinfo professional[mapinfo corporation , 4 global view , troy , new york , 12189 , usa .", "] was used and maps of southern mozambique ( scale 1:250,000 ) were obtained from the direco nacional de geografia e cadastro in maputo .", "the south african medical research council digitally captured relevant geographical and cadastral features , including roads , rivers , towns and different administrative level boundaries .", "the smallest administrative unit boundaries at which data was captured ( localities ) were drawn onto local maps by the staff of the ministry of health involved in the project and then digitally captured for inclusion in the gis display .", "preset queries , to generate standardized management reports , were planned and designed in partnership with field supervisors and programme management staff to meet their specific needs .", "however , the system was designed to allow user - friendly production of customized additional reports where required .", "specific training was provided for programme supervisors on making sense of data and interpreting reports , with a spraying database manual developed for ready reference .", "a major emphasis in training has been on identifying operational problems and implementing timely remedial action .", "as field managers enter the summary data at the namaacha field office on a weekly basis , necessary remedial action is taken within a week ." ], [ "the ongoing monitoring of individual spray operator 's productivity , in terms of number of structures sprayed each day and volume of insecticide sprayed per structure , has facilitated early detection of operational problems , leading to prompt investigation and supportive corrective action by supervisors .", "a standardized aid for evaluating individual operators ' spraying quality during field visits was designed and implemented .", "field managers discuss evaluation results with spray operators on - site in order to eliminate mistakes .", "the frequency of required supervisory corrective interventions has steadily decreased since the beginning of the programme and it is likely that some of this effect is due to the enhanced monitoring and response made possible by the computer management system .", "insecticide application rates ( grams per square meter ) are constantly monitored for under- and over - application", ". under - application reduces insecticide residual activity and thus effectiveness , while over - application is wasteful . where sub - optimal application rates were detected , investigation included scrutiny of insecticide preparation , nozzle condition , application pressure , distance of nozzle tip from sprayed surface , and spraying rhythm .", "spraying coverage exceeded 90% of 222,000 structures in southern mozambique , a level that is considered more than adequate . in areas where the management system repeatedly identified lower coverage than expected , investigations were conducted to determine whether this was the result of diminished community support , absenteeism or poor motivation of the specific spray operator .", "maps depicting the coverage achieved by each team proved useful to malaria control programme management for tracking teams ' progress , providing ongoing updates to senior health management and encouraging spray personnel to attain their target ( figure 2 ) .", "an example of a spraying progress map by locality in maputo province , mozambique , 2003 an additional benefit was that data captured during the first spraying round during 2000 was used to verify official data sources and provide actual household figures to replace official government estimates .", "this provided more precise information for malaria control planning and demographic data needed by other government agencies for planning ." ], [ "competition for ever - dwindling public health resources is a major challenge for malaria control programmes in sub - saharan africa .", "constant monitoring and evaluation of spraying activities is obligatory where indoor residual spraying is an important component of malaria control , to ensure effective application and prevent wastage .", "computerised management systems are proving useful tools for providing timely information to address potentially deleterious human , social and technical factors that could negatively impact on malaria control .", "the computerised management system has been in operation for three years supporting the mainly rural vector control programme that now covers an area of 13,770 square kilometres in southern mozambique .", "not only has it supported a successful and cost - effective transition to a community - based spraying approach , but it has also ensured that spraying was completed to schedule . the ability to immediately identify problems at the level of an individual spray operator and institute appropriate remedial action assured good coverage and programme efficiency .", "the computerised management system is a useful method for enhancing malaria control by improving application of existing tools .", "note : a powerpoint presentation providing more detail on the malaria management information system and database is available at ." ], [ "mb , bs , cm , jlg , dd conceptualised , developed and implemented the mpumalanga computerised management system prototype ; mb , bs , cm , bm adapted this system for application in maputo province , mozambique .", "all authors contributed to the review of the system and the preparation of this manuscript ." ] ]

[ "tuberculosis ( tb ) is still a major cause of morbidity and mortality around the world and the second leading infectious cause of death among adult globally .", "the risk factors for the development of tuberculosis include immigration , low income populations , immunosuppression , human immunodeficiency virus , and living in close contact with patients suffering from tuberculosis [ 2 , 3 ] .", "the precise incidence of genital tb can not be determined with certainty as some cases are asymptomatic and uncovered accidentally during investigation of infertility . in developing countries , genital", "the symptoms and signs of abdominopelvic tb can mimic peritoneal carcinosis or ovarian malignancy . based on iranian national health program ,", "it is more than 50 years that all iranian newborn will receive vaccination against tb after delivery and before releasing from the hospital . ", "but in cities close to the country borders , cases of tb have been reported mainly due to immigration from neighborhood countries particularly afghanistan , pakistan and iraq .", "although pulmonary tb can be diagnosed by its particular signs and symptoms as well as laboratory and imaging assessment , peritoneal tb has common symptoms with advanced ovarian carcinoma , including pelvic pain , mass , ascites , and elevated serum ca125 levels . based on literature review some other case reports showed ambiguity in the differentiation between peritoneal tuberculosis and advanced ovarian cancer before operation [ 2 , 57 ] . therefore , preoperative diagnosis between these two distinct diseases continues to be a dilemma .", "we presented three cases of peritoneal tuberculosis mimicking ovarian malignancy to point out the importance of the histopathologic diagnosis before chemotherapy in women suspected having advanced ovarian cancer , especially if there is a family history of pulmonary tb or infertility .", "three women with abdominopelvic tb were operated during one year in taleghani hospital with preoperative suspicious of ovarian malignancy .", "case 1 a 24-year - old married , nulligravid woman was referred from gasteroentrology ward to the gynecologic ward with the complain of abdominal pain and distension , nausea , on and off vomiting in the preceding 3 months .", "her past medical history was unremarkable but in gynecologic history hypomenorrhea was highlighted ( figure 1 ) .", "family history revealed that her father had been diagnosed with pulmory tb , also her cousin had been involved with genital tb . during physical examination", ", she was a pale young lady with a distended abdomen and a fixed mass palpated up to the umbilicus . in her paraclinic investigations , among all serum tumor markers related to ovarian cancer , there was only a mild elevation of ca-125 ( 45 u / ml ) hemoglobin was 9.0 mg / dl . as she had a positive family history for tb a chest x - ray and skin tuberculin test have been performed and both were negative for tb.pelvic ultrasonography showed a normal size uterus with endometrial thickness equal to 5 mm . there was a septated multicystic mass of 134 140 81 mm cube in right adnexa with extension toward the left adnexa", ". left ovary was also bigger than normal ( 70 35 ) mm containing multiple follicles .", "sonographic impression was mucinous cystadenoma of ovary or hydatid cyst.abdominopelvic computed tomography showed large amount of ascites in peritoneal cavity with cystic and solid vegetative mass , mild thickening of peritoneal layers in lower abdomen suggesting ovarian mucinous cystadenocarcinoma with peritoneal metastasis was noted .", "other possibilities suggested were primary carcinoma of mesenteric , peritoneal like mesothelium , or papillary carcinoma of peritoneum.because of highly suggestion of ovarian malignancy preoperation , the patient underwent exploratory laparatomy with midline incision", ". total volume of 400cc ascitic fluid was aspirated and sent for cytology and culture .", "adhesions were released . then left ovary 5 5 6 cm appeared with hemorrhagic cyst and adhesion to the left fallopian tube .", "a 24-year - old married , nulligravid woman was referred from gasteroentrology ward to the gynecologic ward with the complain of abdominal pain and distension , nausea , on and off vomiting in the preceding 3 months .", "her past medical history was unremarkable but in gynecologic history hypomenorrhea was highlighted ( figure 1 ) .", "family history revealed that her father had been diagnosed with pulmory tb , also her cousin had been involved with genital tb . during physical examination", ", she was a pale young lady with a distended abdomen and a fixed mass palpated up to the umbilicus . in her paraclinic investigations , among all serum tumor markers related to ovarian cancer", ", there was only a mild elevation of ca-125 ( 45 u / ml ) hemoglobin was 9.0 mg / dl .", "as she had a positive family history for tb a chest x - ray and skin tuberculin test have been performed and both were negative for tb .", "pelvic ultrasonography showed a normal size uterus with endometrial thickness equal to 5 mm .", "there was a septated multicystic mass of 134 140 81 mm cube in right adnexa with extension toward the left adnexa", ". left ovary was also bigger than normal ( 70 35 ) mm containing multiple follicles .", "abdominopelvic computed tomography showed large amount of ascites in peritoneal cavity with cystic and solid vegetative mass , mild thickening of peritoneal layers in lower abdomen suggesting ovarian mucinous cystadenocarcinoma with peritoneal metastasis was noted .", "other possibilities suggested were primary carcinoma of mesenteric , peritoneal like mesothelium , or papillary carcinoma of peritoneum . because of highly suggestion of ovarian malignancy preoperation , the patient underwent exploratory laparatomy with midline incision .", "adhesions were released . then left ovary 5 5 6 cm appeared with hemorrhagic cyst and adhesion to the left fallopian tube .", "case 2 a 39 year old married woman gravida 6 , para 6 has been transferred to the gynecologic ward from the gasteroentrology ward with the complain of abdominal pain , weight loss and fever in the last 4 months ( figure 2 ) .", "her past medical history was unremarkable , and in her surgical history , last 2 deliveries were through cesarean section and she performed the tubal ligation .", "it is worth to mention that her normal menstrual pattern has been changed to oligomenorrhea since last 6 months .", "she had also a positive family history ; her mother had been diagnosed with pulmonary tb .", "as case 1 , due to positive family history for tb , a chest x - ray and skin tuberculin test has been performed and both were negative for tb.profiling of serum tumor markers and revealed a high level of ca-125 : 207 u / ml .", "hemoglobin was 10 mg%.pelvic ultrasonography disclosed uterus and adnexa as a large complex cystic mass shifting to the right with extensive adhesion to the adjacent structures . as the preoperation assessment was in favour of ovarian malignancy , the patient underwent exploratory laparatomy with a midline incision .", "there was no ascites , but miliary seeding all over the peritoneum and small bowel and large bowel , uterus , fallopian tubes and ovaries were seen , which were biopsied . a confluent mass containing uterus , ovaries and fallopian tube was seen which was necrotic .", "the frozen sections of the biopsied specimens showed granulation tissue , but as the family was completed and the mass was necrotic , hysterectomy and bilateral salpingo oophorectomy have been done .", "the permanent result of pathology showed many granulomatous lesion comprised epithelial cells , histiocytes , giant cells .", "right ovary and right tube were unremarkable , left ovary , fallopian tube and uterus had granulomatous lesion consistent with tuberculosis .", "old married woman gravida 6 , para 6 has been transferred to the gynecologic ward from the gasteroentrology ward with the complain of abdominal pain , weight loss and fever in the last 4 months ( figure 2 ) .", "her past medical history was unremarkable , and in her surgical history , last 2 deliveries were through cesarean section and she performed the tubal ligation .", "it is worth to mention that her normal menstrual pattern has been changed to oligomenorrhea since last 6 months .", "she had also a positive family history ; her mother had been diagnosed with pulmonary tb . her abdominopelvic examination revealed a fixed and tender mass in the pelvis .", "as case 1 , due to positive family history for tb , a chest x - ray and skin tuberculin test has been performed and both were negative for tb .", "profiling of serum tumor markers and revealed a high level of ca-125 : 207 u / ml .", "pelvic ultrasonography disclosed uterus and adnexa as a large complex cystic mass shifting to the right with extensive adhesion to the adjacent structures . as the preoperation assessment was in favour of ovarian malignancy , the patient underwent exploratory laparatomy with a midline incision .", "there was no ascites , but miliary seeding all over the peritoneum and small bowel and large bowel , uterus , fallopian tubes and ovaries were seen , which were biopsied . a confluent mass containing uterus , ovaries and fallopian tube was seen which was necrotic .", "the frozen sections of the biopsied specimens showed granulation tissue , but as the family was completed and the mass was necrotic , hysterectomy and bilateral salpingo oophorectomy have been done .", "the permanent result of pathology showed many granulomatous lesion comprised epithelial cells , histiocytes , giant cells .", "right ovary and right tube were unremarkable , left ovary , fallopian tube and uterus had granulomatous lesion consistent with tuberculosis .", "case 3 a 57-year - old married woman , gravida 9 , para 8 , consulted with the gynecologic ward by gasteroentrology consultant and then admitted complaining abdominal pain and distension since last two months ( figure 3 ) .", "she lost 10 kg during these 2 months which is highly suggestive of a serious problem .", "ultrasonography showed normal liver , spleen , kidneys , and uterus , but showed cystic masses in both adnexa with septation in favour of malignancy .", "so laparatomy with midline incision was done with impression of ovarian malignancy.about one liter ascites aspirated and omental cake with miliary seeding , which were seen all over the peritoneum and pelvic organs and over the liver and spleen , has been biopsied .", "ovarian masses were removed and sent for frozen sections along with the other biopsied specimens with the result of granulomatosis . because of necrotic and hemorrhagic appearance of uterus and ovaries as well as the patient 's age and condition total abdominal hysterectomy and bilateral salpingooophorectomy were done to prevent postoperation complications .", "right ovary with granulomatous inflammation in favour of tb ascitic fluid cytology showed inflammatory cells .", "patient was discharged with antituberculosis drugs.all three cases were in very good status in 6 months followup with normal pelvic exam , normal pelvic ultrasonography and ca125 measurement . \n a 57-year - old married woman , gravida 9 , para 8 , consulted with the gynecologic ward by gasteroentrology consultant and then admitted complaining abdominal pain and distension since last two months ( figure 3 )", ". she lost 10 kg during these 2 months which is highly suggestive of a serious problem .", "ultrasonography showed normal liver , spleen , kidneys , and uterus , but showed cystic masses in both adnexa with septation in favour of malignancy .", "about one liter ascites aspirated and omental cake with miliary seeding , which were seen all over the peritoneum and pelvic organs and over the liver and spleen , has been biopsied .", "ovarian masses were removed and sent for frozen sections along with the other biopsied specimens with the result of granulomatosis .", "because of necrotic and hemorrhagic appearance of uterus and ovaries as well as the patient 's age and condition total abdominal hysterectomy and bilateral salpingooophorectomy were done to prevent postoperation complications .", "right ovary with granulomatous inflammation in favour of tb ascitic fluid cytology showed inflammatory cells .", "all three cases were in very good status in 6 months followup with normal pelvic exam , normal pelvic ultrasonography and ca125 measurement .", "peritoneal tb with nonspecific symptoms mimicking ovarian malignancy is a serious problem especially in developing countries .", "diagnosis of peritoneal tb before operation is not easy , there is no particular laboratory or imaging assessment to differentiate this disease from advanced ovarian cancer . in our 3 cases diagnosed with abdominopelvic tb ,", "two were in reproductive age ( cases 1 and 2 ) and one postmenopausal ( case 3 ) .", "all the cases were admitted firstly to gasteroentrology due to abdominopelvic pain and distension and then transferred to gynecology oncology ward with the diagnosis of advanced ovarian malignancy .", "adnexal mass suspicious of malignancy in ct scan and/or sonography and elevated ca125 were seen in all cases .", "family history for pulmonary tb was positive in cases 1 and 2 and genital tb for case 1 .", "the first two cases had a history of oligomenorrhea and hypomenorrhea in the last year before surgery . due to the positive family history , peritoneal", "as previous case reports [ 7 , 8 ] in all our 3 cases non - invasive tests , such as acid - fast stain and culture of the ascitic fluid , ultrasonographic features of the abdomen and pelvis , ct , as well as serum ca125 level were nonspecific for differentiating abdominopelvic tb from ovarian malignancy . in our 3 cases , because of ambiguity of the results of non invasive tests , explorative laparotomy with a midline incision has been done before treatment . ", "ascites or peritoneal washings were sent for cytology as well as bacteriologic examination which was negative for both malignant cell as well as tb miliary deposits all over .", "the omentum or peritoneal surfaces were seen and assessed by frozen - section analysis , which detected granulamotous inflammation with central necrosis highly suggestive of tuberculous peritonitis . ", "therefore , although tuberculin skin test may be helpful but might be negative , as in our cases , it might be positive because vaccination is a national program in our country .", "the ca125 level , which is elevated in more than half of early and two thirds of advanced epithelial ovarian malignancy , can be increased in peritoneal tb [ 9 , 10 ] . ", "predictive value , specificity , and sensitivity of this marker to reveal epithelial ovarian malignancy are less in premenopausal than postmenopausal due to other benign conditions [ 11 , 12 ] .", "therefore , in areas where tb is endemic , in premenopausal women with elevated ca125 , this infection should be considered . ", "some studies assessed new diagnostic tools to differentiate abdominopelvic tb from malignamcy before treatment . ", "( 1998 ) studied the polymerase chain reaction for mycobacteria but they reported a negative results in cases of peritoneal tuberculosis mimicking advanced ovarian cancer .", "tinelli ( 2008 ) suggested that abdominopelvic laparoscopy with histopathological findings plus enzyme - linked immuno - spot can confirm the suspect of ap - tb .", "although we were unable to perform the last two investigations , their validity is still under investigation .", "consequently , we suggest a laparatomy and biopsy of involved area as the gold standard method for definite differentiation between advanced ovarian malignancy and peritoneal tb especially in endemic areas for tb with limitations of using new or expensive technology . ", "although some studies suggest laparoscopic biopsy for histologic diagnosis , we preferred a minilaparatomy to decrease the complications of laparoscopic entrance to the abdomen with extensive adhesions between loop of intestines , abdominal wall , and pelvic organs seen in abdominopelvic tb .", "although this report was not a case control study but it seems that removing the infected pelvic masses from the field of the operation before beginning the medical therapy is beneficial . in conclusion ,", "cytology or histopathologic verification of ovarian malignancy before beginning of aggressive therapy including debulking and or adjuvant chemotherapy is mandatory . ", "based on these cases , even in the presence of symptoms and signs highly suggestive of ovarian malignancy , the presence of familial history of pulmonary and/or peritoneal tb along with complaining of oligomenorrhea or hypomenorrhea with low socioeconomic situation are enough markers for the physician to rule out abdominopelvic tb . women living on the border of our country , close to immigrants campus are mostly at risk to develop the tb .", "therefore , taking a precise personal and family history as well as geographic and socioeconomic status of the patients will be helpful . ", "considering this , we suggest that all families , friends , and people living in the vicinity of women diagnosed with tb should be called through a national health program to be tested for tb and treated accordingly ." ]

[ "<S> genital tb may present as on abdominopelvic mass mimicking ovarian malignancy because clinical and laboratory findings are similar . </S>", "<S> family history is very important and should be considered for differential diagnosis . </S>", "<S> three cases of genital tb with presentation of abdominopelvic masses and with no signs and symptoms of tb were presented . </S>", "<S> two of them had positive family history of pulmonary tb . </S>", "<S> tissue diagnosis was the best method for diagnosis of genital tb , but it should be reminded that if positive family history of tb was present , mini laparotomy should be done to take biopsy and to make rapid diagnosis before treatment . </S>" ]

[ "1. Introduction", "2. Case Reports", "3. Discussion" ]

[ [ "tuberculosis ( tb ) is still a major cause of morbidity and mortality around the world and the second leading infectious cause of death among adult globally .", "the risk factors for the development of tuberculosis include immigration , low income populations , immunosuppression , human immunodeficiency virus , and living in close contact with patients suffering from tuberculosis [ 2 , 3 ] .", "the precise incidence of genital tb can not be determined with certainty as some cases are asymptomatic and uncovered accidentally during investigation of infertility . in developing countries , genital", "the symptoms and signs of abdominopelvic tb can mimic peritoneal carcinosis or ovarian malignancy . based on iranian national health program ,", "it is more than 50 years that all iranian newborn will receive vaccination against tb after delivery and before releasing from the hospital . ", "but in cities close to the country borders , cases of tb have been reported mainly due to immigration from neighborhood countries particularly afghanistan , pakistan and iraq .", "although pulmonary tb can be diagnosed by its particular signs and symptoms as well as laboratory and imaging assessment , peritoneal tb has common symptoms with advanced ovarian carcinoma , including pelvic pain , mass , ascites , and elevated serum ca125 levels . based on literature review some other case reports showed ambiguity in the differentiation between peritoneal tuberculosis and advanced ovarian cancer before operation [ 2 , 57 ] . therefore , preoperative diagnosis between these two distinct diseases continues to be a dilemma .", "we presented three cases of peritoneal tuberculosis mimicking ovarian malignancy to point out the importance of the histopathologic diagnosis before chemotherapy in women suspected having advanced ovarian cancer , especially if there is a family history of pulmonary tb or infertility ." ], [ "three women with abdominopelvic tb were operated during one year in taleghani hospital with preoperative suspicious of ovarian malignancy .", "case 1 a 24-year - old married , nulligravid woman was referred from gasteroentrology ward to the gynecologic ward with the complain of abdominal pain and distension , nausea , on and off vomiting in the preceding 3 months .", "her past medical history was unremarkable but in gynecologic history hypomenorrhea was highlighted ( figure 1 ) .", "family history revealed that her father had been diagnosed with pulmory tb , also her cousin had been involved with genital tb . during physical examination", ", she was a pale young lady with a distended abdomen and a fixed mass palpated up to the umbilicus . in her paraclinic investigations , among all serum tumor markers related to ovarian cancer , there was only a mild elevation of ca-125 ( 45 u / ml ) hemoglobin was 9.0 mg / dl . as she had a positive family history for tb a chest x - ray and skin tuberculin test have been performed and both were negative for tb.pelvic ultrasonography showed a normal size uterus with endometrial thickness equal to 5 mm . there was a septated multicystic mass of 134 140 81 mm cube in right adnexa with extension toward the left adnexa", ". left ovary was also bigger than normal ( 70 35 ) mm containing multiple follicles .", "sonographic impression was mucinous cystadenoma of ovary or hydatid cyst.abdominopelvic computed tomography showed large amount of ascites in peritoneal cavity with cystic and solid vegetative mass , mild thickening of peritoneal layers in lower abdomen suggesting ovarian mucinous cystadenocarcinoma with peritoneal metastasis was noted .", "other possibilities suggested were primary carcinoma of mesenteric , peritoneal like mesothelium , or papillary carcinoma of peritoneum.because of highly suggestion of ovarian malignancy preoperation , the patient underwent exploratory laparatomy with midline incision", ". total volume of 400cc ascitic fluid was aspirated and sent for cytology and culture .", "adhesions were released . then left ovary 5 5 6 cm appeared with hemorrhagic cyst and adhesion to the left fallopian tube .", "a 24-year - old married , nulligravid woman was referred from gasteroentrology ward to the gynecologic ward with the complain of abdominal pain and distension , nausea , on and off vomiting in the preceding 3 months .", "her past medical history was unremarkable but in gynecologic history hypomenorrhea was highlighted ( figure 1 ) .", "family history revealed that her father had been diagnosed with pulmory tb , also her cousin had been involved with genital tb . during physical examination", ", she was a pale young lady with a distended abdomen and a fixed mass palpated up to the umbilicus . in her paraclinic investigations , among all serum tumor markers related to ovarian cancer", ", there was only a mild elevation of ca-125 ( 45 u / ml ) hemoglobin was 9.0 mg / dl .", "as she had a positive family history for tb a chest x - ray and skin tuberculin test have been performed and both were negative for tb .", "pelvic ultrasonography showed a normal size uterus with endometrial thickness equal to 5 mm .", "there was a septated multicystic mass of 134 140 81 mm cube in right adnexa with extension toward the left adnexa", ". left ovary was also bigger than normal ( 70 35 ) mm containing multiple follicles .", "abdominopelvic computed tomography showed large amount of ascites in peritoneal cavity with cystic and solid vegetative mass , mild thickening of peritoneal layers in lower abdomen suggesting ovarian mucinous cystadenocarcinoma with peritoneal metastasis was noted .", "other possibilities suggested were primary carcinoma of mesenteric , peritoneal like mesothelium , or papillary carcinoma of peritoneum . because of highly suggestion of ovarian malignancy preoperation , the patient underwent exploratory laparatomy with midline incision .", "adhesions were released . then left ovary 5 5 6 cm appeared with hemorrhagic cyst and adhesion to the left fallopian tube .", "case 2 a 39 year old married woman gravida 6 , para 6 has been transferred to the gynecologic ward from the gasteroentrology ward with the complain of abdominal pain , weight loss and fever in the last 4 months ( figure 2 ) .", "her past medical history was unremarkable , and in her surgical history , last 2 deliveries were through cesarean section and she performed the tubal ligation .", "it is worth to mention that her normal menstrual pattern has been changed to oligomenorrhea since last 6 months .", "she had also a positive family history ; her mother had been diagnosed with pulmonary tb .", "as case 1 , due to positive family history for tb , a chest x - ray and skin tuberculin test has been performed and both were negative for tb.profiling of serum tumor markers and revealed a high level of ca-125 : 207 u / ml .", "hemoglobin was 10 mg%.pelvic ultrasonography disclosed uterus and adnexa as a large complex cystic mass shifting to the right with extensive adhesion to the adjacent structures . as the preoperation assessment was in favour of ovarian malignancy , the patient underwent exploratory laparatomy with a midline incision .", "there was no ascites , but miliary seeding all over the peritoneum and small bowel and large bowel , uterus , fallopian tubes and ovaries were seen , which were biopsied . a confluent mass containing uterus , ovaries and fallopian tube was seen which was necrotic .", "the frozen sections of the biopsied specimens showed granulation tissue , but as the family was completed and the mass was necrotic , hysterectomy and bilateral salpingo oophorectomy have been done .", "the permanent result of pathology showed many granulomatous lesion comprised epithelial cells , histiocytes , giant cells .", "right ovary and right tube were unremarkable , left ovary , fallopian tube and uterus had granulomatous lesion consistent with tuberculosis .", "old married woman gravida 6 , para 6 has been transferred to the gynecologic ward from the gasteroentrology ward with the complain of abdominal pain , weight loss and fever in the last 4 months ( figure 2 ) .", "her past medical history was unremarkable , and in her surgical history , last 2 deliveries were through cesarean section and she performed the tubal ligation .", "it is worth to mention that her normal menstrual pattern has been changed to oligomenorrhea since last 6 months .", "she had also a positive family history ; her mother had been diagnosed with pulmonary tb . her abdominopelvic examination revealed a fixed and tender mass in the pelvis .", "as case 1 , due to positive family history for tb , a chest x - ray and skin tuberculin test has been performed and both were negative for tb .", "profiling of serum tumor markers and revealed a high level of ca-125 : 207 u / ml .", "pelvic ultrasonography disclosed uterus and adnexa as a large complex cystic mass shifting to the right with extensive adhesion to the adjacent structures . as the preoperation assessment was in favour of ovarian malignancy , the patient underwent exploratory laparatomy with a midline incision .", "there was no ascites , but miliary seeding all over the peritoneum and small bowel and large bowel , uterus , fallopian tubes and ovaries were seen , which were biopsied . a confluent mass containing uterus , ovaries and fallopian tube was seen which was necrotic .", "the frozen sections of the biopsied specimens showed granulation tissue , but as the family was completed and the mass was necrotic , hysterectomy and bilateral salpingo oophorectomy have been done .", "the permanent result of pathology showed many granulomatous lesion comprised epithelial cells , histiocytes , giant cells .", "right ovary and right tube were unremarkable , left ovary , fallopian tube and uterus had granulomatous lesion consistent with tuberculosis .", "case 3 a 57-year - old married woman , gravida 9 , para 8 , consulted with the gynecologic ward by gasteroentrology consultant and then admitted complaining abdominal pain and distension since last two months ( figure 3 ) .", "she lost 10 kg during these 2 months which is highly suggestive of a serious problem .", "ultrasonography showed normal liver , spleen , kidneys , and uterus , but showed cystic masses in both adnexa with septation in favour of malignancy .", "so laparatomy with midline incision was done with impression of ovarian malignancy.about one liter ascites aspirated and omental cake with miliary seeding , which were seen all over the peritoneum and pelvic organs and over the liver and spleen , has been biopsied .", "ovarian masses were removed and sent for frozen sections along with the other biopsied specimens with the result of granulomatosis . because of necrotic and hemorrhagic appearance of uterus and ovaries as well as the patient 's age and condition total abdominal hysterectomy and bilateral salpingooophorectomy were done to prevent postoperation complications .", "right ovary with granulomatous inflammation in favour of tb ascitic fluid cytology showed inflammatory cells .", "patient was discharged with antituberculosis drugs.all three cases were in very good status in 6 months followup with normal pelvic exam , normal pelvic ultrasonography and ca125 measurement . \n a 57-year - old married woman , gravida 9 , para 8 , consulted with the gynecologic ward by gasteroentrology consultant and then admitted complaining abdominal pain and distension since last two months ( figure 3 )", ". she lost 10 kg during these 2 months which is highly suggestive of a serious problem .", "ultrasonography showed normal liver , spleen , kidneys , and uterus , but showed cystic masses in both adnexa with septation in favour of malignancy .", "about one liter ascites aspirated and omental cake with miliary seeding , which were seen all over the peritoneum and pelvic organs and over the liver and spleen , has been biopsied .", "ovarian masses were removed and sent for frozen sections along with the other biopsied specimens with the result of granulomatosis .", "because of necrotic and hemorrhagic appearance of uterus and ovaries as well as the patient 's age and condition total abdominal hysterectomy and bilateral salpingooophorectomy were done to prevent postoperation complications .", "right ovary with granulomatous inflammation in favour of tb ascitic fluid cytology showed inflammatory cells .", "all three cases were in very good status in 6 months followup with normal pelvic exam , normal pelvic ultrasonography and ca125 measurement ." ], [ "peritoneal tb with nonspecific symptoms mimicking ovarian malignancy is a serious problem especially in developing countries .", "diagnosis of peritoneal tb before operation is not easy , there is no particular laboratory or imaging assessment to differentiate this disease from advanced ovarian cancer . in our 3 cases diagnosed with abdominopelvic tb ,", "two were in reproductive age ( cases 1 and 2 ) and one postmenopausal ( case 3 ) .", "all the cases were admitted firstly to gasteroentrology due to abdominopelvic pain and distension and then transferred to gynecology oncology ward with the diagnosis of advanced ovarian malignancy .", "adnexal mass suspicious of malignancy in ct scan and/or sonography and elevated ca125 were seen in all cases .", "family history for pulmonary tb was positive in cases 1 and 2 and genital tb for case 1 .", "the first two cases had a history of oligomenorrhea and hypomenorrhea in the last year before surgery . due to the positive family history , peritoneal", "as previous case reports [ 7 , 8 ] in all our 3 cases non - invasive tests , such as acid - fast stain and culture of the ascitic fluid , ultrasonographic features of the abdomen and pelvis , ct , as well as serum ca125 level were nonspecific for differentiating abdominopelvic tb from ovarian malignancy . in our 3 cases , because of ambiguity of the results of non invasive tests , explorative laparotomy with a midline incision has been done before treatment . ", "ascites or peritoneal washings were sent for cytology as well as bacteriologic examination which was negative for both malignant cell as well as tb miliary deposits all over .", "the omentum or peritoneal surfaces were seen and assessed by frozen - section analysis , which detected granulamotous inflammation with central necrosis highly suggestive of tuberculous peritonitis . ", "therefore , although tuberculin skin test may be helpful but might be negative , as in our cases , it might be positive because vaccination is a national program in our country .", "the ca125 level , which is elevated in more than half of early and two thirds of advanced epithelial ovarian malignancy , can be increased in peritoneal tb [ 9 , 10 ] . ", "predictive value , specificity , and sensitivity of this marker to reveal epithelial ovarian malignancy are less in premenopausal than postmenopausal due to other benign conditions [ 11 , 12 ] .", "therefore , in areas where tb is endemic , in premenopausal women with elevated ca125 , this infection should be considered . ", "some studies assessed new diagnostic tools to differentiate abdominopelvic tb from malignamcy before treatment . ", "( 1998 ) studied the polymerase chain reaction for mycobacteria but they reported a negative results in cases of peritoneal tuberculosis mimicking advanced ovarian cancer .", "tinelli ( 2008 ) suggested that abdominopelvic laparoscopy with histopathological findings plus enzyme - linked immuno - spot can confirm the suspect of ap - tb .", "although we were unable to perform the last two investigations , their validity is still under investigation .", "consequently , we suggest a laparatomy and biopsy of involved area as the gold standard method for definite differentiation between advanced ovarian malignancy and peritoneal tb especially in endemic areas for tb with limitations of using new or expensive technology . ", "although some studies suggest laparoscopic biopsy for histologic diagnosis , we preferred a minilaparatomy to decrease the complications of laparoscopic entrance to the abdomen with extensive adhesions between loop of intestines , abdominal wall , and pelvic organs seen in abdominopelvic tb .", "although this report was not a case control study but it seems that removing the infected pelvic masses from the field of the operation before beginning the medical therapy is beneficial . in conclusion ,", "cytology or histopathologic verification of ovarian malignancy before beginning of aggressive therapy including debulking and or adjuvant chemotherapy is mandatory . ", "based on these cases , even in the presence of symptoms and signs highly suggestive of ovarian malignancy , the presence of familial history of pulmonary and/or peritoneal tb along with complaining of oligomenorrhea or hypomenorrhea with low socioeconomic situation are enough markers for the physician to rule out abdominopelvic tb . women living on the border of our country , close to immigrants campus are mostly at risk to develop the tb .", "therefore , taking a precise personal and family history as well as geographic and socioeconomic status of the patients will be helpful . ", "considering this , we suggest that all families , friends , and people living in the vicinity of women diagnosed with tb should be called through a national health program to be tested for tb and treated accordingly ." ] ]

[ "stroke is the second leading cause of death and causes permanent disability in one - third of \n survivors1 , 2 .", "many stroke patients suffer from motor impairments that limit \n muscle control , activities of daily living , and locomotion activity .", "in addition , stroke \n patients have increased double support duration , decreased cadence , shorter stride , \n decreased stance time , and shorter lower limb joint range of motion compared to able - bodied \n persons3,4,5 . rhythmic auditory stimulation ( ras )", "uses musical stimuli to improve the gait performance of \n patients who have neurological conditions ( e.g. , parkinson s disease , cerebral palsy , \n traumatic brain injury , stroke)6 . because \n music includes temporal characteristics such as anticipation and predictability7 , ras may act on the indicators that inform \n the gait - phase sequence .", "stroke patients have shown better performance when engaging in \n conventional gait training with ras versus conventional gait training only6 .", "for example , a stroke group that received \n ras combined with gait training showed greater improvements in gait velocity , cadence , \n stride length8 , and hip joint motion \n symmetry and decreased vertical center of mass displacement9 .", "lee et al.10 \n reported improvements in gait symmetry , velocity , and cadence in stroke patients when gait \n training by matching the footfall of the affected side with ras and applying baseline and \n > 30% baseline speed .", "compared to the control group , the chronic stroke patients who had \n trained with home - based auditory stimulation showed significant improvements in affected \n side stride length , unaffected side stride length , stride length ratio , affected side single \n support time , single support time ratio , and gait velocity11 .", "recently , the results of studies with auditory cueing systems for gait rehabilitation \n training have been reported .", "nagano , japan)12 , 13 , which was developed to simultaneously support locomotion \n compensation and gait rehabilitation training using ras methods .", "this device consists of a \n transmitter that pairs with three - axis acceleration sensors worn on each ankle and wireless \n headphones .", "muto \n et al.13 reported a significant decrease \n in ground - contact timing asymmetries and gait tempo fluctuation during gait training with \n the walk - mate device in stroke hemiplegic patients . in another study , casamassima et \n", "al.14 described inertial measurement \n units ( imu ) based on a wearable auditory feedback system that monitors gait training with \n parkinson s disease patients .", "this system provides real - time audio feedback and estimates \n spatiotemporal gait parameters via gait event data detected from imu sensors mounted on both \n shoes .", "the advantages of the application include its \n portability , simple configuration , inclusion of various auditory stimuli , and ease of saving \n real - time data .", "this device type must have the capacity to be used during gait training \n anywhere at any time .", "in this study , two aspects of our smartphone - based ras application \n were investigated : ( 1 ) the effects of various ras tempos on stroke gait patterns and ( 2 ) the \n effects of short - term gait training on spatiotemporal gait parameters in stroke \n patients .", "the inclusion criteria \n were as follows : at least 6 months since stroke , no visual or auditory deficits , and \n independent gait > 10 meters .", "the general characteristics of the study participants are \n detailed in table 1table 1.participant informationparticipants ( n=15)gender ( m / f)11/4age ( years)56.0 ( 7.4)height ( cm)167.7 ( 7.7)weight ( kg)68.3 ( 8.2)paretic side ( left / right)8/7since onset ( months)81.9 ( 87.8)mmse - k28.4 ( 2.1)mbi96.3 ( 4.4)values are means ( sd ) .", "mmse - k : mini mental state examination - korean version , mbi : \n modified barthel index .", "the institutional review board of the korea national rehabilitation center \n approved the study protocol .", "the purpose and procedures of the study were fully explained , \n and all participants were required to provide informed consent .", "mmse - k : mini mental state examination - korean version , mbi : \n modified barthel index gait training with ras was performed in an unobstructed space and additional auditory \n stimuli were blocked in order for subjects to concentrate on the ras sound .", "each ras \n condition was practiced for 10 minutes . a 3-minute adaptation period and a 7-minute \n gait - training period", "all subjects walked at a comfortable speed three times , \n after which their mean cadence ( baseline ) was calculated and converted to 10% , 5% , 0% , \n + 5% , and + 10% of baseline .", "the mean cadence was calculated by averaging three 10-meter walk \n individual times , while subtracting the acceleration and deceleration periods .", "the ras application has seven sound sources ( c - e - g , c - f - a , a - d - g , clap , click , gun , and \n robot ) that could be selected and applied based on the user s preference . to adapt to the \n ras sounds", ", participants listened to the rhythm of the application and were instructed to \n tap their fingers in their lap or stamp their feet when in a seated position .", "then , subjects were asked to \n listen carefully to the rhythm of the ras application and heel strike to the sounds . \n", "ten - minute intervals between trials eliminated any remaining effects that could influence \n the next trial .", "imu sensors ( shimmer3 , shimmer , dublin , ireland ) were attached above both ankle joints for \n heel - strike detection .", "these sensors were connected to the ras smartphone application via \n bluetooth , and heel - strike detection information was shown on the display as a red dot . \n", "thus , gait training focused on the timing of heel strikes on the ground and patients were \n asked to execute heel strikes in tandem with the ras sound . a validated wireless inertial sensing device ( g - walk , bts s.p.a .", "the sensor was attached \n to subjects waists ( l45 intervertebral space ) using a semi - elastic belt to determine the \n acceleration values for three anatomical axes : anterior - posterior , medial - lateral , and \n vertical17 .", "park and woo18 suggested that the g - walk represents a \n valid method for assessing the effectiveness of clinical therapeutic interventions of gait \n analysis .", "their comparative study proved that there is a significant and high correlation \n between gait velocity and cadence and the foot pressure sensor system ( gaitrite , cir \n systems , inc . ,", "spatiotemporal parameters such as gait \n speed , cadence , stride length , gait cycle duration , and step length on the affected and \n unaffected sides were recorded .", "the spatial symmetry ratio was calculated by recording the \n step length on the affected and unaffected sides .", "for each trial , the subjects \n were asked to walk a level gait way ( 16 m ) . to eliminate acceleration and deceleration \n effects , only the middle distance ( 10 m ) was used for calculations .", "data collection was \n repeated three times with 3 minutes of rest between trials . to determine the effect of \n various ras tempos on gait patterns ,", "the kolmogorov - smirnov test was used firstly to check \n if the data were normally distributed . secondly , a one - way repeated measures analysis of \n variance ( anova ) and least significant difference ( lsd ) post - hoc analysis were performed . \n", "data were analyzed using spss statistics for windows , version 21.0 ( ibm corp . , armonk , ny , \n usa )", "parametersbaseline10%5%0%+5%+10%after ras trainingspeed(m / min)70.53(12.36)57.84(8.27)64.30(9.74)68.53(10.89)77.22(16.07)77.53(15.21)79.53(15.85)cadence(steps / min)51.89(6.23)46.30(5.35)48.99(6.03)51.44(6.07)53.73(6.50)55.32(6.79)53.30 ( 7.22)stride length(m)1.37(0.22)1.27(0.17)1.32(0.18)1.35(0.18)1.44(0.22)1.42(0.24)1.50(0.22)gait cycleduration ( s)1.18(0.16)1.32(0.17)1.25(0.17)1.18(0.16)1.13(0.16)1.10(0.15)1.15(0.18)step lengthaffected side ( m)0.69(0.11)0.65(0.09)0.67(0.10)0.67(0.09)0.73(0.12)0.72(0.12)0.74(0.09)step lengthunaffected side ( m)0.68(0.13)0.61(0.10)0.65(0.11)0.67(0.10)0.70(0.13)0.68(0.13)0.75(0.15)symmetry ratio(step length)1.04(0.17)1.06(0.14)1.04(0.15)1.02(0.15)1.06(0.16)1.08(0.15)1.02(0.14)values are means ( sd ) .", "* significantly different from baseline ( without ras ) \n ( p<0.05 ) . significantly different from the 10% ras condition \n ( p<0.05 ) . significantly different from the 5% ras condition \n ( p<0.05 )", "significantly different from the 0% ras condition \n ( p<0.05 ) . significantly different from the + 5% ras condition \n ( p<0.05 ) .", "* * significantly different from the + 10% ras condition ( p<0.05 ) . \n significantly different from after ras training ( without ras ) \n ( p<0.05 ) . in the 10% ras condition ,", "m / min , f=34.219 ) , cadence \n ( 46.30 5.35 steps / min , f=114.253 ) , stride length ( 1.27 0.17 m , f=12.082 ) , step length on \n the affected side ( 0.65 0.09 m , f=14.026 ) , and step length on the unaffected side ( 0.61 \n 0.1 m , f=13.066 ) decreased significantly compared to baseline ( p < 0.05 ) .", "furthermore , \n gait cycle duration ( 1.32 0.17 s , f=150.236 ) increased significantly compared to baseline \n ( p < 0.05 ) . in the 5% ras condition ,", "m / min , f=34.219 ) and \n cadence ( 48.99 6.03 steps / min , f=114.253 ) decreased significantly , while gait cycle \n duration ( 1.25 0.17 s , f=150.236 ) increased significantly compared to baseline ( p < \n 0.05 ) . in the + 5% ras condition , gait speed ( 77.22 16.07", "m / min , f=34.219 ) and cadence \n ( 53.73 6.5 steps / min , f=114.253 ) increased significantly , while gait cycle duration ( 1.13 \n 0.16 s , f=150.236 ) decreased significantly compared to baseline ( p < 0.05 ) . in the + 10% \n ras condition , gait speed ( 77.53 15.21", "m / min , f=34.219 ) and cadence ( 55.32 6.79 \n steps / min , f=114.253 ) increased significantly , while gait cycle duration ( 1.10 0.15 s , \n f=150.236 ) decreased significantly compared to baseline ( p < 0.05 ) .", "m / min , f=34.219 ) , cadence ( 53.30 7.22 steps / min , \n f=114.253 ) , stride length ( 1.50 0.22 m , f=12.082 ) , step length on the affected side ( 0.74 \n 0.09 m , f=14.026 ) , and step length on the unaffected side ( 0.75 0.15 m , f=13.066 ) \n increased significantly compared to baseline ( p < 0.05 ) .", "gait cycle duration ( 0.75 \n 0.15 s , f=150.236 ) decreased significantly compared to baseline ( p < 0.05 ) .", "values are means ( sd ) . * significantly different from baseline ( without ras ) \n ( p<0.05 ) . ", "significantly different from the 5% ras condition \n ( p<0.05 ) . significantly different from the 0% ras condition \n ( p<0.05 ) . significantly different from the + 5% ras condition \n ( p<0.05 )", "* * significantly different from the + 10% ras condition ( p<0.05 ) . \n significantly different from after ras training ( without ras ) \n ( p<0.05 )", "ras is an increasingly popular form of rehabilitative exercise therapy for neurological \n gait disorders .", "auditory - motor synchronization in the reticulospinal tract is the key \n concept of ras19 .", "timing cues in gait \n motion have the capacity to improve movement control and change temporal gait \n parameters7 .", "several studies have \n demonstrated the effects of ras on neurological gait disorders such as stroke , parkinson s \n disease , and cerebral palsy .", "these studies reported improvements in gait velocity , stride \n length , and cadence in response to ras rehabilitative therapeutic methods8 , 19 , 20 .", "however , most studies of stroke patients \n have used ras with constant speed- or time - based stimulation increases .", "cha et al.21 studied the immediate effects of four ras \n tempos ( 10% , 0% , + 10% , and + 20% ) on the gait patterns of hemiplegic stroke patients .", "the \n authors reported that a faster ras tempo increased gait velocity , which indicated that there \n was potential to immediately improve walking abilities .", "suh et al.22 reported improvements in gait velocity , stride length , \n cadence , and standing balance in a group of hemiplegic stroke patients who received gait \n training with 5% and 10% increased ras compared to a group that did not receive ras . in this study ,", "the effect of various ras tempos on gait patterns was examined and the \n effectiveness of short - term gait training with a smartphone - based ras application was \n confirmed .", "the results showed significant differences in spatiotemporal gait parameters \n after ras training compared with those before training . in the 10% ras condition ,", "gait \n speed , cadence , stride length , and step length on the affected and unaffected sides \n decreased significantly , while gait cycle duration increased significantly . in the 5% ras \n condition ,", "gait speed and cadence decreased significantly , while gait cycle duration \n increased significantly . in the + 5% and + 10% ras conditions , gait speed and cadence \n increased significantly , while gait cycle duration decreased significantly .", "when the ras \n tempo increased , all spatiotemporal gait parameters appeared to increase gradually , while \n the gait cycle duration decreased .", "gait speed , cadence , and gait cycle duration showed the \n highest significant improvement in the + 10% ras condition .", "after gait training with ras , \n gait speed , cadence , stride length , and step length on the affected and unaffected sides \n increased significantly ; gait cycle duration decreased significantly compared to that before \n ras gait training .", "it is especially noteworthy that step length increased significantly not \n only on the affected side but also on the unaffected side .", "increased gait speed , which is determined by cadence and stride length23 , is often an outcome measure when evaluating gait \n function .", "the improvement in movement patterns activates an internal time - keeping mechanism , \n which leads to movement synchronization24 . in our study ,", "therefore , the spinal motor system was optimized for movement \n patterns following the reticulospinal pathway at the brainstem level25 .", "suteerawattananon et al.26 reported that auditory cues can increase spinal motor neuron \n excitability via the reticulospinal tract and decrease muscle reaction time in response to \n motor commands , thus improving gait speed .", "therefore , the auditory information provides \n stimulation that bypasses the damaged anatomy , leading to good muscle performance or timing \n control , and modulates a motor timing - control signal that is received by the brain s \n compensatory network .", "this has a useful effect on the neurological function and \n movement - processing centers of the brain27 . in the 0% ras condition", "prassas et al.9 reported no \n significant changes in stride length when baseline speed was applied with stroke patients . \n", "another study also reported no significant differences in gait parameters such as gait \n velocity , cadence , or stride length in hemiplegic stroke patients when 0% ras was \n applied21 .", "therefore , it may be \n inferred that rhythmical tempo variations have a positive impact on the gait of stroke \n patients .", "however , the equivalent rhythm to gait speed did not significantly affect the gait \n of stroke patients .", "symmetry is an important factor during exercise or therapeutic treatment , particularly \n after stroke .", "patterson et al . recommended that step length , stance time , and swing time be \n used as gait parameters when determining the symmetry ratio28 . in the present study , step length as it pertained to spatial \n symmetry was examined .", "symmetry ratio appeared to improve at 0% and around the 5% ras \n condition as well as after ras training .", "these trends are likely related to the fact that \n patients walked with symmetrical auditory cueing that adjusted to a comfortable walking \n cadence .", "although statistically significant results were not obtained , the tendencies of the \n spatial symmetry ratio were confirmed .", "further studies involving more subjects and temporal \n symmetry factors such as stance time and swing time will be needed .", "the present study showed that hemiplegic stroke patients spatiotemporal gait parameters \n change with various ras tempos .", "moreover , significant improvements in gait speed , cadence , \n stride length , and step length on the affected and unaffected sides were observed after gait \n training .", "previous studies of gait training with ras over the course of three weeks reported \n significant improvements in gait velocity , stride length , cadence , and symmetry8 as well as standing balance22 for stroke patients .", "future studies need \n to evaluate more patients and the long - term effects after a certain period of ras gait \n training . a longer period of intervention could result in a higher number of positive \n results . in the future , we plan to combine our ras system with a wearable lower - limb rehabilitation \n robot .", "zanotto et al.29 examined how \n different auditory feedback modalities in healthy subjects may affect gait modification \n through short - term gait training using an exoskeleton robot .", "the authors asserted that a \n combination of kinetic and visual guidance might be as effective as combined kinetic \n guidance and rhythmic cues .", "we also hope to conduct comparative analyses with robotic \n devices for various tempos . in this study", ", imu sensors were attached to both ankle joints to gather heel - strike \n information .", "the acquired information , which was displayed on a smartphone , was used by the \n participants to time their heel strikes to match the ras sound during gait training .", "we plan \n to update the ras application for use not only in gait training but also during real - time \n gait evaluation ( via calculations based on heel - strike information , including gait \n symmetry ) .", "we expect the application to be useful during gait rehabilitative training and \n that chronic stroke survivors in any environment may find it useful ." ]

[ "<S> [ purpose ] the effects of various rhythmic auditory stimulation tempos on stroke gait \n pattern changes when training patients with a smartphone - based rhythmic auditory \n stimulation application were investigated . </S>", "<S> [ subjects and methods ] fifteen patients with \n chronic stroke were included . </S>", "<S> cadence during comfortable walking was measured ( baseline ) . \n </S>", "<S> after the baseline findings were recorded , rhythmic auditory stimulation with five \n different tempos ( i.e. , 10% , 5% , 0% , + 5% , and + 10% change from baseline ) was randomly \n applied . </S>", "<S> finally , comfortable walking without rhythmic auditory stimulation was initiated \n to evaluate gait pattern changes . </S>", "<S> [ results ] as the tempo increased , the spatiotemporal \n gait parameters of the stroke patients changed significantly . </S>", "<S> gait speed , cadence , and \n gait cycle duration showed the greatest improvement in the + 10% rhythmic auditory \n stimulation condition compared to baseline . </S>", "<S> after gait training with rhythmic auditory \n stimulation , gait speed , cadence , stride length , gait cycle duration , and step length of \n the affected and unaffected sides improved significantly compared to baseline . \n </S>", "<S> [ conclusion ] significant changes in the gait pattern of stroke patients were noted for \n various tempos after training with rhythmic auditory stimulation . </S>", "<S> these findings could be \n used to customize rehabilitative gait training for patients who experience stroke with \n hemiplegia . </S>" ]

[ "INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "stroke is the second leading cause of death and causes permanent disability in one - third of \n survivors1 , 2 .", "many stroke patients suffer from motor impairments that limit \n muscle control , activities of daily living , and locomotion activity .", "in addition , stroke \n patients have increased double support duration , decreased cadence , shorter stride , \n decreased stance time , and shorter lower limb joint range of motion compared to able - bodied \n persons3,4,5 . rhythmic auditory stimulation ( ras )", "uses musical stimuli to improve the gait performance of \n patients who have neurological conditions ( e.g. , parkinson s disease , cerebral palsy , \n traumatic brain injury , stroke)6 . because \n music includes temporal characteristics such as anticipation and predictability7 , ras may act on the indicators that inform \n the gait - phase sequence .", "stroke patients have shown better performance when engaging in \n conventional gait training with ras versus conventional gait training only6 .", "for example , a stroke group that received \n ras combined with gait training showed greater improvements in gait velocity , cadence , \n stride length8 , and hip joint motion \n symmetry and decreased vertical center of mass displacement9 .", "lee et al.10 \n reported improvements in gait symmetry , velocity , and cadence in stroke patients when gait \n training by matching the footfall of the affected side with ras and applying baseline and \n > 30% baseline speed .", "compared to the control group , the chronic stroke patients who had \n trained with home - based auditory stimulation showed significant improvements in affected \n side stride length , unaffected side stride length , stride length ratio , affected side single \n support time , single support time ratio , and gait velocity11 .", "recently , the results of studies with auditory cueing systems for gait rehabilitation \n training have been reported .", "nagano , japan)12 , 13 , which was developed to simultaneously support locomotion \n compensation and gait rehabilitation training using ras methods .", "this device consists of a \n transmitter that pairs with three - axis acceleration sensors worn on each ankle and wireless \n headphones .", "muto \n et al.13 reported a significant decrease \n in ground - contact timing asymmetries and gait tempo fluctuation during gait training with \n the walk - mate device in stroke hemiplegic patients . in another study , casamassima et \n", "al.14 described inertial measurement \n units ( imu ) based on a wearable auditory feedback system that monitors gait training with \n parkinson s disease patients .", "this system provides real - time audio feedback and estimates \n spatiotemporal gait parameters via gait event data detected from imu sensors mounted on both \n shoes .", "the advantages of the application include its \n portability , simple configuration , inclusion of various auditory stimuli , and ease of saving \n real - time data .", "this device type must have the capacity to be used during gait training \n anywhere at any time .", "in this study , two aspects of our smartphone - based ras application \n were investigated : ( 1 ) the effects of various ras tempos on stroke gait patterns and ( 2 ) the \n effects of short - term gait training on spatiotemporal gait parameters in stroke \n patients ." ], [ "the inclusion criteria \n were as follows : at least 6 months since stroke , no visual or auditory deficits , and \n independent gait > 10 meters .", "the general characteristics of the study participants are \n detailed in table 1table 1.participant informationparticipants ( n=15)gender ( m / f)11/4age ( years)56.0 ( 7.4)height ( cm)167.7 ( 7.7)weight ( kg)68.3 ( 8.2)paretic side ( left / right)8/7since onset ( months)81.9 ( 87.8)mmse - k28.4 ( 2.1)mbi96.3 ( 4.4)values are means ( sd ) .", "mmse - k : mini mental state examination - korean version , mbi : \n modified barthel index .", "the institutional review board of the korea national rehabilitation center \n approved the study protocol .", "the purpose and procedures of the study were fully explained , \n and all participants were required to provide informed consent .", "mmse - k : mini mental state examination - korean version , mbi : \n modified barthel index gait training with ras was performed in an unobstructed space and additional auditory \n stimuli were blocked in order for subjects to concentrate on the ras sound .", "each ras \n condition was practiced for 10 minutes . a 3-minute adaptation period and a 7-minute \n gait - training period", "all subjects walked at a comfortable speed three times , \n after which their mean cadence ( baseline ) was calculated and converted to 10% , 5% , 0% , \n + 5% , and + 10% of baseline .", "the mean cadence was calculated by averaging three 10-meter walk \n individual times , while subtracting the acceleration and deceleration periods .", "the ras application has seven sound sources ( c - e - g , c - f - a , a - d - g , clap , click , gun , and \n robot ) that could be selected and applied based on the user s preference . to adapt to the \n ras sounds", ", participants listened to the rhythm of the application and were instructed to \n tap their fingers in their lap or stamp their feet when in a seated position .", "then , subjects were asked to \n listen carefully to the rhythm of the ras application and heel strike to the sounds . \n", "ten - minute intervals between trials eliminated any remaining effects that could influence \n the next trial .", "imu sensors ( shimmer3 , shimmer , dublin , ireland ) were attached above both ankle joints for \n heel - strike detection .", "these sensors were connected to the ras smartphone application via \n bluetooth , and heel - strike detection information was shown on the display as a red dot . \n", "thus , gait training focused on the timing of heel strikes on the ground and patients were \n asked to execute heel strikes in tandem with the ras sound . a validated wireless inertial sensing device ( g - walk , bts s.p.a .", "the sensor was attached \n to subjects waists ( l45 intervertebral space ) using a semi - elastic belt to determine the \n acceleration values for three anatomical axes : anterior - posterior , medial - lateral , and \n vertical17 .", "park and woo18 suggested that the g - walk represents a \n valid method for assessing the effectiveness of clinical therapeutic interventions of gait \n analysis .", "their comparative study proved that there is a significant and high correlation \n between gait velocity and cadence and the foot pressure sensor system ( gaitrite , cir \n systems , inc . ,", "spatiotemporal parameters such as gait \n speed , cadence , stride length , gait cycle duration , and step length on the affected and \n unaffected sides were recorded .", "the spatial symmetry ratio was calculated by recording the \n step length on the affected and unaffected sides .", "for each trial , the subjects \n were asked to walk a level gait way ( 16 m ) . to eliminate acceleration and deceleration \n effects , only the middle distance ( 10 m ) was used for calculations .", "data collection was \n repeated three times with 3 minutes of rest between trials . to determine the effect of \n various ras tempos on gait patterns ,", "the kolmogorov - smirnov test was used firstly to check \n if the data were normally distributed . secondly , a one - way repeated measures analysis of \n variance ( anova ) and least significant difference ( lsd ) post - hoc analysis were performed . \n", "data were analyzed using spss statistics for windows , version 21.0 ( ibm corp . , armonk , ny , \n usa )" ], [ "parametersbaseline10%5%0%+5%+10%after ras trainingspeed(m / min)70.53(12.36)57.84(8.27)64.30(9.74)68.53(10.89)77.22(16.07)77.53(15.21)79.53(15.85)cadence(steps / min)51.89(6.23)46.30(5.35)48.99(6.03)51.44(6.07)53.73(6.50)55.32(6.79)53.30 ( 7.22)stride length(m)1.37(0.22)1.27(0.17)1.32(0.18)1.35(0.18)1.44(0.22)1.42(0.24)1.50(0.22)gait cycleduration ( s)1.18(0.16)1.32(0.17)1.25(0.17)1.18(0.16)1.13(0.16)1.10(0.15)1.15(0.18)step lengthaffected side ( m)0.69(0.11)0.65(0.09)0.67(0.10)0.67(0.09)0.73(0.12)0.72(0.12)0.74(0.09)step lengthunaffected side ( m)0.68(0.13)0.61(0.10)0.65(0.11)0.67(0.10)0.70(0.13)0.68(0.13)0.75(0.15)symmetry ratio(step length)1.04(0.17)1.06(0.14)1.04(0.15)1.02(0.15)1.06(0.16)1.08(0.15)1.02(0.14)values are means ( sd ) .", "* significantly different from baseline ( without ras ) \n ( p<0.05 ) . significantly different from the 10% ras condition \n ( p<0.05 ) . significantly different from the 5% ras condition \n ( p<0.05 )", "significantly different from the 0% ras condition \n ( p<0.05 ) . significantly different from the + 5% ras condition \n ( p<0.05 ) .", "* * significantly different from the + 10% ras condition ( p<0.05 ) . \n significantly different from after ras training ( without ras ) \n ( p<0.05 ) . in the 10% ras condition ,", "m / min , f=34.219 ) , cadence \n ( 46.30 5.35 steps / min , f=114.253 ) , stride length ( 1.27 0.17 m , f=12.082 ) , step length on \n the affected side ( 0.65 0.09 m , f=14.026 ) , and step length on the unaffected side ( 0.61 \n 0.1 m , f=13.066 ) decreased significantly compared to baseline ( p < 0.05 ) .", "furthermore , \n gait cycle duration ( 1.32 0.17 s , f=150.236 ) increased significantly compared to baseline \n ( p < 0.05 ) . in the 5% ras condition ,", "m / min , f=34.219 ) and \n cadence ( 48.99 6.03 steps / min , f=114.253 ) decreased significantly , while gait cycle \n duration ( 1.25 0.17 s , f=150.236 ) increased significantly compared to baseline ( p < \n 0.05 ) . in the + 5% ras condition , gait speed ( 77.22 16.07", "m / min , f=34.219 ) and cadence \n ( 53.73 6.5 steps / min , f=114.253 ) increased significantly , while gait cycle duration ( 1.13 \n 0.16 s , f=150.236 ) decreased significantly compared to baseline ( p < 0.05 ) . in the + 10% \n ras condition , gait speed ( 77.53 15.21", "m / min , f=34.219 ) and cadence ( 55.32 6.79 \n steps / min , f=114.253 ) increased significantly , while gait cycle duration ( 1.10 0.15 s , \n f=150.236 ) decreased significantly compared to baseline ( p < 0.05 ) .", "m / min , f=34.219 ) , cadence ( 53.30 7.22 steps / min , \n f=114.253 ) , stride length ( 1.50 0.22 m , f=12.082 ) , step length on the affected side ( 0.74 \n 0.09 m , f=14.026 ) , and step length on the unaffected side ( 0.75 0.15 m , f=13.066 ) \n increased significantly compared to baseline ( p < 0.05 ) .", "gait cycle duration ( 0.75 \n 0.15 s , f=150.236 ) decreased significantly compared to baseline ( p < 0.05 ) .", "values are means ( sd ) . * significantly different from baseline ( without ras ) \n ( p<0.05 ) . ", "significantly different from the 5% ras condition \n ( p<0.05 ) . significantly different from the 0% ras condition \n ( p<0.05 ) . significantly different from the + 5% ras condition \n ( p<0.05 )", "* * significantly different from the + 10% ras condition ( p<0.05 ) . \n significantly different from after ras training ( without ras ) \n ( p<0.05 )" ], [ "ras is an increasingly popular form of rehabilitative exercise therapy for neurological \n gait disorders .", "auditory - motor synchronization in the reticulospinal tract is the key \n concept of ras19 .", "timing cues in gait \n motion have the capacity to improve movement control and change temporal gait \n parameters7 .", "several studies have \n demonstrated the effects of ras on neurological gait disorders such as stroke , parkinson s \n disease , and cerebral palsy .", "these studies reported improvements in gait velocity , stride \n length , and cadence in response to ras rehabilitative therapeutic methods8 , 19 , 20 .", "however , most studies of stroke patients \n have used ras with constant speed- or time - based stimulation increases .", "cha et al.21 studied the immediate effects of four ras \n tempos ( 10% , 0% , + 10% , and + 20% ) on the gait patterns of hemiplegic stroke patients .", "the \n authors reported that a faster ras tempo increased gait velocity , which indicated that there \n was potential to immediately improve walking abilities .", "suh et al.22 reported improvements in gait velocity , stride length , \n cadence , and standing balance in a group of hemiplegic stroke patients who received gait \n training with 5% and 10% increased ras compared to a group that did not receive ras . in this study ,", "the effect of various ras tempos on gait patterns was examined and the \n effectiveness of short - term gait training with a smartphone - based ras application was \n confirmed .", "the results showed significant differences in spatiotemporal gait parameters \n after ras training compared with those before training . in the 10% ras condition ,", "gait \n speed , cadence , stride length , and step length on the affected and unaffected sides \n decreased significantly , while gait cycle duration increased significantly . in the 5% ras \n condition ,", "gait speed and cadence decreased significantly , while gait cycle duration \n increased significantly . in the + 5% and + 10% ras conditions , gait speed and cadence \n increased significantly , while gait cycle duration decreased significantly .", "when the ras \n tempo increased , all spatiotemporal gait parameters appeared to increase gradually , while \n the gait cycle duration decreased .", "gait speed , cadence , and gait cycle duration showed the \n highest significant improvement in the + 10% ras condition .", "after gait training with ras , \n gait speed , cadence , stride length , and step length on the affected and unaffected sides \n increased significantly ; gait cycle duration decreased significantly compared to that before \n ras gait training .", "it is especially noteworthy that step length increased significantly not \n only on the affected side but also on the unaffected side .", "increased gait speed , which is determined by cadence and stride length23 , is often an outcome measure when evaluating gait \n function .", "the improvement in movement patterns activates an internal time - keeping mechanism , \n which leads to movement synchronization24 . in our study ,", "therefore , the spinal motor system was optimized for movement \n patterns following the reticulospinal pathway at the brainstem level25 .", "suteerawattananon et al.26 reported that auditory cues can increase spinal motor neuron \n excitability via the reticulospinal tract and decrease muscle reaction time in response to \n motor commands , thus improving gait speed .", "therefore , the auditory information provides \n stimulation that bypasses the damaged anatomy , leading to good muscle performance or timing \n control , and modulates a motor timing - control signal that is received by the brain s \n compensatory network .", "this has a useful effect on the neurological function and \n movement - processing centers of the brain27 . in the 0% ras condition", "prassas et al.9 reported no \n significant changes in stride length when baseline speed was applied with stroke patients . \n", "another study also reported no significant differences in gait parameters such as gait \n velocity , cadence , or stride length in hemiplegic stroke patients when 0% ras was \n applied21 .", "therefore , it may be \n inferred that rhythmical tempo variations have a positive impact on the gait of stroke \n patients .", "however , the equivalent rhythm to gait speed did not significantly affect the gait \n of stroke patients .", "symmetry is an important factor during exercise or therapeutic treatment , particularly \n after stroke .", "patterson et al . recommended that step length , stance time , and swing time be \n used as gait parameters when determining the symmetry ratio28 . in the present study , step length as it pertained to spatial \n symmetry was examined .", "symmetry ratio appeared to improve at 0% and around the 5% ras \n condition as well as after ras training .", "these trends are likely related to the fact that \n patients walked with symmetrical auditory cueing that adjusted to a comfortable walking \n cadence .", "although statistically significant results were not obtained , the tendencies of the \n spatial symmetry ratio were confirmed .", "further studies involving more subjects and temporal \n symmetry factors such as stance time and swing time will be needed .", "the present study showed that hemiplegic stroke patients spatiotemporal gait parameters \n change with various ras tempos .", "moreover , significant improvements in gait speed , cadence , \n stride length , and step length on the affected and unaffected sides were observed after gait \n training .", "previous studies of gait training with ras over the course of three weeks reported \n significant improvements in gait velocity , stride length , cadence , and symmetry8 as well as standing balance22 for stroke patients .", "future studies need \n to evaluate more patients and the long - term effects after a certain period of ras gait \n training . a longer period of intervention could result in a higher number of positive \n results . in the future , we plan to combine our ras system with a wearable lower - limb rehabilitation \n robot .", "zanotto et al.29 examined how \n different auditory feedback modalities in healthy subjects may affect gait modification \n through short - term gait training using an exoskeleton robot .", "the authors asserted that a \n combination of kinetic and visual guidance might be as effective as combined kinetic \n guidance and rhythmic cues .", "we also hope to conduct comparative analyses with robotic \n devices for various tempos . in this study", ", imu sensors were attached to both ankle joints to gather heel - strike \n information .", "the acquired information , which was displayed on a smartphone , was used by the \n participants to time their heel strikes to match the ras sound during gait training .", "we plan \n to update the ras application for use not only in gait training but also during real - time \n gait evaluation ( via calculations based on heel - strike information , including gait \n symmetry ) .", "we expect the application to be useful during gait rehabilitative training and \n that chronic stroke survivors in any environment may find it useful ." ] ]

[ "the ultimate goal of periodontal therapy includes not only the arrest of progressive \n periodontal disease but also the restitution of those parts of the supporting apparatus \n which have been destroyed by disease .", "regenerative procedures have been evaluated in \n several studies using grafting materials , root surface conditioning , guided tissue \n regeneration and application of growth factors .", "different materials have been introduced for bone defect filling , i.e. , autografts , \n xenografts , allografts , and alloplasts . among the various subgroups of alloplastic bone \n grafts ,", "bioactive glass ( bg ) is a type of bioactive ceramic with the ability to improve \n bone formation that has been assessed in experimental models .", "previous studies have shown that bg particles , when implanted in vivo , \n incorporate into the connective tissue and undergo chemical transformation increasing \n the ph of the site to approximately 10 .", "this process leads to \n the formation of a silica gel on the surface of the particles and subsequently an \n amorphous calcium phosphate layer is formed by the interaction between calcium and \n phosphate from the bg and the surrounding medium .", "hench and polak ( 2002 ) have shown that the release of ions ( na , ca , and si ) from bg \n materials controls the cell cycle leading to the differentiation and proliferation of \n bone cells , modulating the expression of genes that regulate osteogenesis , and the \n synthesis of growth factors .", "growth factors are vital modulators during this process which can induce the migration , \n attachment , proliferation and differentiation of periodontal progenitor cells . \n", "platelet - rich plasma ( prp ) is a preparation of autologous plasma that contains a higher \n platelet concentration , allowing it to deliver a greater concentration of autologous \n growth factors such us platelet - derived growth factor ( pdgf ) , transforming growth factor \n beta ( tgf- ) , vascular endothelial growth factor ( vegf ) , insulin - like growth \n factor ( igf - i ) , and epithelial growth factor ( egf ) , that regulate cell proliferation , \n chemotaxis and differentiation . some specific platelet growth factors , like pdgf and tgf- ,", "could promote the \n growth and differentiation of the periodontal ligament and alveolar bone cells and could \n be responsible for the clinical improvement observed in experimental sites", ". other interesting feature of prp is its \n sticky consistency due to its high fibrin content .", "the fibrin component of prp may work \n as a haemostatic agent aiding in stabilizing the graft material and the blood \n clot .", "blood clot \n immobilization has been suggested as an important event in the early phases of wound \n healing of periodontal regenerative procedures .", "studies using histological techniques suggested that prp preparations may enhance local \n bone formation , while \n others did not confirm these \n findings .", "clinical studies have reported that treatments with a combination of prp and \n demineralized freeze - dried bone graft allograft ( dfdba ) improve clinical parameters in \n intrabony periodontal defects . in \n the other hand", "did nt find additional \n benefit in the reduction of pocket depth , clinical attachment gain and defect filling \n using prp with bg in the treatment of intrabony defects .", "the use of prp associated with biomaterials is an alternative treatment approach that \n may favor the process of bone tissue regeneration and should be investigated .", "therefore , \n the goal of this study was to evaluate , histomorphometrically , the healing of surgically \n created intrabony defects in dogs treated with prp+bg .", "nine adult female mongrel dogs ( mean weight=15 kg ) were included in the experiment .", "the \n study protocol was approved by the institutional animal research committee , state \n university of campinas ( protocol # 686 - 1 ) .", "the surgical procedures were performed under \n general anesthesia with intravenous injection of a 3% sodium pentobarbital solution ( 0.5 \n ml / kg ) .", "the mandibular second and fourth premolars had been previously extracted and the \n extraction sites had been allowed to heal for 2 months . after this period , buccal and \n lingual mucoperiosteal flaps were elevated and 3-wall intrabony defects ( 4x4x4 mm ) were \n created at the mesial and distal aspect of the left and right mandibular first molar \n ( figure 1 ) . following root planning , aiming cementum removal ,", "a reference \n notch was made with curettes on the root surface at the cement - enamel junction .", "clinical view of the surgically created 3-wall intrabony defect ( 4x4x4 mm ) at the \n mesial aspect of the first mandibular molar before that the gingival flaps have been positioned and sutured , a tofflemire matrix \n involved by cotton ligature was adapted over the tooth surface and the remaining defect \n was filled with gutta - percha ( figure 2 ) .", "so , the \n defects were exposed to plaque accumulation for a period of one month before the \n treatment .", "after that , scaling and root planing was performed and a regimen of daily \n brushing plus topical application of 0.1% chlorhexidine gluconate was instituted for 7 \n days prior to the surgical procedures .", "clinical view of the insertion of gutta - percha in the defect blood was obtained several min before the administration of anesthesia .", "four 5-ml \n tubes containing 0.5 ml of 3.2% sodium citrate solution ( an anticoagulant ) were drawn \n from each dog .", "the blood was thus separated into 3 basic parts : red blood cells ( at the \n bottom of the tube ) , platelet enriched plasma ( a discrete grey line in the middle of \n the tube ) and platelet poor plasma ( at the top of the tube ) .", "the portion \n corresponding to the platelet poor plasma was discarded from each tube and the \n remaining content was centrifuged again at 1,200 rpm for 15 min .", "four hundred \n microliters of the middle portion , corresponding to the platelet enriched plasma , \n were pipetted from each tube . in order to obtain the gel ,", "30 l of 10% calcium \n chloride was added to prp and heated in a water bath at 37c .", "the gel was achieved \n after a period of 10 to 15 min . a tube with a sample of whole blood \n as well as a sample of prp , retrieved before adding calcium chloride of 10% , was sent \n to a clinical veterinary laboratory to perform a platelet counting .", "in the moment of the defect treatment , an apical reference notch was made with \n curettes on the root surface at the remaining bone level .", "each defect in each animal \n ( a total of 4 defects/ animal ) was randomly assigned to one of the following \n treatments ( the treatments with prp were located at the same side that was randomly \n determined ) : control : the defect was naturally filled with coagulum ; \n bg ( perioglas , us biomaterials , alachua , fl , usa ) : bg particles \n were applied , filling the defect ; prp : after obtaining the prp gel , \n it was immediately applied on the defect ; prp+bg : bg was \n incorporated to the prp ( 1:1 ) and this assemblage was immediately taken to a water \n bath at 37c for formation of a mixture of the gel and the bg graft .", "primary , tension - free wound closure was accomplished following defect treatment , with \n the gingival flaps positioned and sutured with interrupted sutures ( vicryl , ethicon \n inc , so jos dos campos , sp , brazil ) at their presurgical position . \n", "immediately after the surgeries , an intramuscular injection of penicillin ( 1.5 ml \n 150,000 iu ) was given to the animal .", "postoperative plaque control was performed by irrigation with a solution of 1% \n chlorhexidine gluconate ( every other day ) .", "after 90 days , under general anesthesia , \n the animals were sacrificed with a perfusion of 10% neutral formalin solution .", "the \n jaws were dissected and the blocks containing the experimental specimens were \n removed , and decalcified in a solution of equal parts of 50% formic acid and 20% \n sodium citrate for 5 months .", "the decalcified specimens were washed in running water , \n dehydrated and embedded in paraffin .", "five sections representing the midbuccal portion of each defect were selected for the \n histometric procedures .", "the \n following distances were measured : total epithelium ( sulcular and junctional epithelium ) : from the \n gingival margin to the apical border of the junctional epithelium . connective tissue adaptation ( without cementum ) : from the apical \n border of the junctional epithelium to the coronal end of new cementum .", "new cementum : from the apical notch to the most coronal part of new \n cementum . new bone ( bone position ) : from the apical notch to the most coronal \n extent of new bone .", "negative values would be assigned if the bony crest was located \n apically to the apical notch .", "new bone area : area of newly formed bone filling the defect area \n ( limited by the distance between the two notches in an apico - coronal direction and 4 \n mm away from the root surface in a lateral direction ) .", "the area of new bone was \n measured with an image analysis system that allowed the placement of a lattice on the \n image of the created bone defect .", "the intersection lines localized on bone were \n marked to calculate the new bone area whereas intersections lines localized on bg \n particles were not marked and therefore were not included for this purpose .", "the measurements were performed using a microscope ( zeiss axioskop 2 , carl zeiss \n instruments , gottingen , lower saxony , germany ) with a 1.25/.035 objective associated \n with a video camera / computer / software ( image pro plus version 3.0 , media cybernetics , \n silver spring , md , usa ) .", "the mean values for all \n groups were determined using the individual means from the 9 dogs .", "the hypothesis \n that there were no differences among the groups was tested by one - way anova .", "if a statistical \n difference was detected , tukey s pairwise multiple comparison test was used .", "four 5-ml \n tubes containing 0.5 ml of 3.2% sodium citrate solution ( an anticoagulant ) were drawn \n from each dog .", "the blood was thus separated into 3 basic parts : red blood cells ( at the \n bottom of the tube ) , platelet enriched plasma ( a discrete grey line in the middle of \n the tube ) and platelet poor plasma ( at the top of the tube ) .", "the portion \n corresponding to the platelet poor plasma was discarded from each tube and the \n remaining content was centrifuged again at 1,200 rpm for 15 min .", "four hundred \n microliters of the middle portion , corresponding to the platelet enriched plasma , \n were pipetted from each tube . in order to obtain the gel ,", "30 l of 10% calcium \n chloride was added to prp and heated in a water bath at 37c .", "the gel was achieved \n after a period of 10 to 15 min . a tube with a sample of whole blood \n as well as a sample of prp , retrieved before adding calcium chloride of 10% ,", "in the moment of the defect treatment , an apical reference notch was made with \n curettes on the root surface at the remaining bone level . each defect in each animal \n", "( a total of 4 defects/ animal ) was randomly assigned to one of the following \n treatments ( the treatments with prp were located at the same side that was randomly \n determined ) : control : the defect was naturally filled with coagulum ; \n bg ( perioglas , us biomaterials , alachua , fl , usa ) : bg particles \n were applied , filling the defect ; prp : after obtaining the prp gel , \n it was immediately applied on the defect ; prp+bg : bg was \n incorporated to the prp ( 1:1 ) and this assemblage was immediately taken to a water \n bath at 37c for formation of a mixture of the gel and the bg graft .", "primary , tension - free wound closure was accomplished following defect treatment , with \n the gingival flaps positioned and sutured with interrupted sutures ( vicryl , ethicon \n inc , so jos dos campos , sp , brazil ) at their presurgical position . \n", "immediately after the surgeries , an intramuscular injection of penicillin ( 1.5 ml \n 150,000 iu ) was given to the animal .", "postoperative plaque control was performed by irrigation with a solution of 1% \n chlorhexidine gluconate ( every other day ) .", "after 90 days , under general anesthesia , \n the animals were sacrificed with a perfusion of 10% neutral formalin solution .", "the \n jaws were dissected and the blocks containing the experimental specimens were \n removed , and decalcified in a solution of equal parts of 50% formic acid and 20% \n sodium citrate for 5 months .", "the decalcified specimens were washed in running water , \n dehydrated and embedded in paraffin .", "five sections representing the midbuccal portion of each defect were selected for the \n histometric procedures .", "the \n following distances were measured : total epithelium ( sulcular and junctional epithelium ) : from the \n gingival margin to the apical border of the junctional epithelium . connective tissue adaptation ( without cementum ) : from the apical \n border of the junctional epithelium to the coronal end of new cementum .", "new cementum : from the apical notch to the most coronal part of new \n cementum . new bone ( bone position ) : from the apical notch to the most coronal \n extent of new bone .", "negative values would be assigned if the bony crest was located \n apically to the apical notch .", "new bone area : area of newly formed bone filling the defect area \n ( limited by the distance between the two notches in an apico - coronal direction and 4 \n mm away from the root surface in a lateral direction ) .", "the area of new bone was \n measured with an image analysis system that allowed the placement of a lattice on the \n image of the created bone defect .", "the intersection lines localized on bone were \n marked to calculate the new bone area whereas intersections lines localized on bg \n particles were not marked and therefore were not included for this purpose .", "the measurements were performed using a microscope ( zeiss axioskop 2 , carl zeiss \n instruments , gottingen , lower saxony , germany ) with a 1.25/.035 objective associated \n with a video camera / computer / software ( image pro plus version 3.0 , media cybernetics , \n silver spring , md , usa ) .", "mean values for each parameter were obtained per defect . the mean values for all \n groups", "the hypothesis \n that there were no differences among the groups was tested by one - way anova .", "if a statistical \n difference was detected , tukey s pairwise multiple comparison test was used .", "all \n groups presented a similar healing pattern characterized mainly by the presence of \n new cementum covering most of the root surface associated with the defect and new \n bone , up to the top of the defect , with a small amount of connective tissue and \n epithelium ( figure 3 ) .", "the periodontal ligament \n was characterized by the presence of collagen fibers obliquely oriented to the root \n surface , extending between the new cementum and the newly formed bone .", "photomicrographs of the treated 3-wall intrabony defect showing new bone and \n cementum formation extending coronally to the apical notch ( h&e , original \n magnification x20 ) .", "a ) control ; b ) prp ; c ) bg ; d ) bg+prp .", "note the remaining \n particles of bg surrounded by new bone and connective tissue ( c and d ) .", "arrows \n indicate areas with new bone in contact with bg particles in the defects treated with bg , it was possible to observe that part of the material \n seemed to be resorbed and replaced by bone , while some of the remaining bg particles \n showed dissolution of their central part with newly formed bone inside of them . some \n defects presented bone in direct contact and/or surrounding the bg particles ( figure 4 ) .", "almost all defects that received bg \n presented a dense connective tissue surrounding the remaining particles and areas \n suggesting the formation of new bone .", "new bone formation observed in the 3-wall intrabony defect treated with bg \n ( h&e , original magnification x100 ) .", "arrows indicate bone in direct contact \n with bg particles the histometric results are shown in table 1 . \n", "a superior area of new bone was observed in the sites treated with prp+bg and bg \n ( 13.802.32 mm and 15.632.64 mm , \n respectively ) . in the other groups ,", "the new bone area was 8.191.46 \n mm ( control group ) and 8.811.47 mm ( prp ) . although \n the difference in the new bone area , no significant difference was found in the \n linear parameter new bone , e.g. bone position : from the apical notch to the most \n coronal extent of new bone ( 4.370.44 mm , 4.240.68 mm , \n 4.370.46 mm and 4.440.42 mm , for control group , bg , prp and prp+bg , \n respectively ; p=0.85 ) . mean and standard deviation ( sd ) for the parameters evaluated after all the \n treatments ( control , bg , prp and bg+prp ) .", "means followed by different letters \n are statistically different at 5% data analysis showed no significant differences among the groups regarding the \n initial defect extension ( 4.550.17 mm , 4.480.20 mm , 4.480.28 \n mm and 4.570.32 mm , for control group , bg , prp and prp+bg , respectively ; \n p=0.74 ) .", "in addition , intergroup analysis demonstrated that no significant difference \n was observed among the groups in the following parameters : total epithelium extension \n ( 2.240.58 mm , 1.940.37 mm , 1.970.37 mm and 1.810.61 , \n for control group , bg , prp and prp+bg , respectively ; p=0.45 ) , connective tissue \n extension ( 0.900.28 mm , 0.840.41 mm , 1.070.27 mm and \n 1.150.32 mm , for control group , bg , prp and prp+bg , respectively ; p=0.59 ) and \n new cementum extension ( 2.630.70 mm , 2.560.36 mm , 2.370.38 mm \n and 3.100.47 mm , for control group , bg , prp and prp+bg , respectively ; \n p=0.85 ) .", "the results from the histometric measurements , i.e. , epithelial \n length ( the portion of epithelium located on the root ) , connective tissue adaptation \n and new cementum formation , expressed as the percentage of the distance between the \n coronal and the apical notch are presented in figure \n 5 .", "no undesirable events like inflammation and foreign body reactions were observed . all \n groups presented a similar healing pattern characterized mainly by the presence of \n new cementum covering most of the root surface associated with the defect and new \n bone , up to the top of the defect , with a small amount of connective tissue and \n epithelium ( figure 3 ) .", "the periodontal ligament \n was characterized by the presence of collagen fibers obliquely oriented to the root \n surface , extending between the new cementum and the newly formed bone .", "photomicrographs of the treated 3-wall intrabony defect showing new bone and \n cementum formation extending coronally to the apical notch ( h&e , original \n magnification x20 ) .", "a ) control ; b ) prp ; c ) bg ; d ) bg+prp .", "note the remaining \n particles of bg surrounded by new bone and connective tissue ( c and d ) .", "arrows \n indicate areas with new bone in contact with bg particles in the defects treated with bg , it was possible to observe that part of the material \n seemed to be resorbed and replaced by bone , while some of the remaining bg particles \n showed dissolution of their central part with newly formed bone inside of them . some \n defects presented bone in direct contact and/or surrounding the bg particles ( figure 4 ) .", "almost all defects that received bg \n presented a dense connective tissue surrounding the remaining particles and areas \n suggesting the formation of new bone .", "new bone formation observed in the 3-wall intrabony defect treated with bg \n ( h&e , original magnification x100 ) .", "a superior area of new bone was observed in the sites treated with prp+bg and bg \n ( 13.802.32 mm and 15.632.64 mm , \n respectively ) . in the other groups , the new bone area was 8.191.46 \n mm ( control group ) and 8.811.47 mm ( prp ) . although \n the difference in the new bone area , no significant difference was found in the \n linear parameter new bone , e.g. bone position : from the apical notch to the most \n coronal extent of new bone ( 4.370.44 mm , 4.240.68 mm , \n 4.370.46 mm and 4.440.42 mm , for control group , bg , prp and prp+bg , \n respectively ; p=0.85 ) . mean and standard deviation ( sd ) for the parameters evaluated after all the \n treatments ( control , bg , prp and bg+prp ) .", "means followed by different letters \n are statistically different at 5% data analysis showed no significant differences among the groups regarding the \n initial defect extension ( 4.550.17 mm , 4.480.20 mm , 4.480.28 \n mm and 4.570.32 mm , for control group , bg , prp and prp+bg , respectively ; \n p=0.74 ) .", "in addition , intergroup analysis demonstrated that no significant difference \n was observed among the groups in the following parameters : total epithelium extension \n ( 2.240.58 mm , 1.940.37 mm , 1.970.37 mm and 1.810.61 , \n for control group , bg , prp and prp+bg , respectively ; p=0.45 ) , connective tissue \n extension ( 0.900.28 mm , 0.840.41 mm , 1.070.27 mm and \n 1.150.32 mm , for control group , bg , prp and prp+bg , respectively ; p=0.59 ) and \n new cementum extension ( 2.630.70 mm , 2.560.36 mm , 2.370.38 mm \n and 3.100.47 mm , for control group , bg , prp and prp+bg , respectively ; \n p=0.85 ) .", "the results from the histometric measurements , i.e. , epithelial \n length ( the portion of epithelium located on the root ) , connective tissue adaptation \n and new cementum formation , expressed as the percentage of the distance between the \n coronal and the apical notch are presented in figure \n 5 .", "the use of different regenerative approaches in the treatment of periodontal defects has \n demonstrated a variable clinical result .", "some techniques have been used in association , aiming to increase \n their effect on periodontal regeneration .", "the present study was \n designed to evaluate , histomorphometrically , the healing process of 3-wall intrabony \n defects treated with bg , prp and their association .", "prp has become a focus of current studies due to its potential to accelerate wound \n healing .", "it has been previously shown that prp stimulated pdl \n cells and fibroblastic cell proliferationbut suppressed \n epithelial cell division in vitro .", "consequently , by ordering these cellular responses into a series \n of related events , prp may facilitate wound - healing and tissue regeneration .", "the claimed \n ability to suppress epithelial cell proliferation seems advantageous for periodontal \n regeneration by favoring the formation of a new connective tissue attachment and new \n cementum on the root surface .", "the \n results of the present study failed to demonstrate that the use of prp can promote an \n additional effect in terms of cementum formation when treating 3-wall intrabony defects . \n", "cementum formation for bg and prp followed the same trend observed for the control group \n ( bg=2.5 mm/57% , prp=2.4 mm/53% , control=58% ) while the prp+bg showed a slightly greater \n cementum formation ( prp+bg=3.10 mm/68% ) , but the differences among the groups were not \n statistically significant . in this respect", ", it should be recognized that a non - contained \n osseous defect might have favored the discrimination of quantitative differences among \n the treatments . in a previous histological study", ", kim , et al ( 2004 ) evaluated the healing patterns following flap surgery in \n experimentally induced 1- , 2- and 3-wall intrabony defects . according to the authors , \n one- and 3-wall intrabony defects", "can be considered pre - clinical models that are \n relatively easy to manage and from which homogeneous healing outcomes may be expected . \n", "they suggested that these defects appear to be reproducible models to evaluate candidate \n technologies for periodontal regeneration .", "regarding the 3-wall intrabony defect , they reported a mean \n cementum formation of approximately 70% of the initial defect extension . in the present \n study ,", "a period of 1 month of plaque accumulation was allowed as a way to obtain some \n chronification of the defect .", "the control group showed 2.6 mm ( 58% ) of the original \n defect covered with new cementum .", "this result confirms the high regenerative potential \n of this type of defect that provides favorable anatomic characteristics for clot \n stabilization and protection .", "it is well accepted that healing of intrabony defects is positively correlated to the \n number of bone walls limiting the defect .", "therefore , the 3-wall intrabony defect used in \n the present study provides ideal extent and location of tissue resources , cells and \n vascularity circumscribing the defect , which might have favored bone formation .", "the \n linear measurement of the new bone height revealed an impressive coronal bone formation \n for all the groups , with no significant differences among the treatments .", "the claimed osteoinductive properties of prp could not be supported by the results of the present study .", "it \n has been suggested that prp could be used to increase the bone deposition rate and the \n quality of bone regeneration when augmenting sites prior to or in conjunction with \n dental implant placement .", "nevertheless , more recent animal studies could not find \n increased bone regeneration when prp was used .", "recently , bioactive glass has been used as a way to achieve bone and periodontal \n regeneration based on its osteoconductive properties . the need for an \n alternative to autologous and allogenic bone grafts", "have encouraged the search of a \n material that could be safe , surgically convenient and predictably promote regeneration \n with a reduced morbidity . in this study ,", "the use of bg was not associated with any \n foreign body reaction , showing biocompatibility with periodontal tissues .", "histological observations after treatment of periodontal defects with bg in nonhuman \n primates showed a significant increase of new cementum and inhibition of downgrowth of \n junctional epithelium .", "when bg is \n implanted in vivo , the ph of the site increases , a layer rich in silica \n gel is formed on the surface of the particles and subsequently an amorphous calcium \n phosphate layer is formed by the interaction between calcium and phosphate from the bg \n and the surrounding medium .", "its \n superficial bioactivity may stimulate a rapid formation of a connective tissue seal that \n is supposed to have the ability to block the epithelium migration and allow for the \n repopulation of the previously contaminated area by periodontal ligament cells . in the present study ,", "almost all defects \n that received bg presented a dense connective tissue surrounding the remaining particles \n in accordance with observations of previous studies . in the groups", "treated with bg , the area of new bone was 13.802.32 mm \n ( prp+bg ) and 15.632.64 mm ( bg ) , respectively . in the groups that did \n", "not receive this material , the new bone area was 8.191.46 mm \n ( control group ) and 8.811.47 mm ( prp ) . based on the significant \n differences observed in the area of newly formed bone when groups with and without bg \n were compared the present study corroborates the hypothesis that this material could act \n as an osteoconductive graft .", "in addition , it was observed that some remaining bg \n granules showed dissolution of their core with newly formed bone , as described in the \n literature . aiming to increase the periodontal and bone regeneration", ", some studies evaluated the \n additional effect of prp , combined with different types of grafting materials , on the \n treatment of intrabony defects . in spite of some clinical results showing a greater \n reduction of pocket depth and", "gain in clinical attachment level , other clinical studies \n failed to demonstrate an additional effect . in 3-wall periimplant defects in dogs \n treated with prp associated to xenogenic bone graft ,", "the same \n authors also reported no significant effect with the addition of prp to xenogenic bone \n grafts on bone mineral density or graft maturity .", "these observations are in agreement with the results of the \n present study , which could not demonstrate an additional effect of prp in periodontal \n and bone regeneration of 3 wall intrabony defects .", "the histomorphometric findings \n demonstrated that the sites treated with prp+bg showed a similar amount of new bone \n ( 13.802.32 mm ) when compared with the sites that only received bg \n ( 15.632.64 mm ) .", "the ideal platelet and growth factor concentration to promote periodontal regeneration \n has not yet been established . to the date , there is insufficient information about \n growth factor interactions and how they influence the activations of gene expression and \n protein production . in the present study ,", "the achieved percentage mean of platelet \n concentration was 227.02% ( mean of 463.015 platelets / ml ) in relation to blood platelets \n count .", "( 2003 ) \n reported that growth factors may act at specific times and at appropriate concentrations \n and this may be other explanation for the different results obtained in studies \n evaluating the use of prp .", "the great variability in the outcomes of the studies with prp \n may also be explained by the diversity of methods to obtain it .", "the technique to produce \n prp in the present study was used in other animal studies by our research \n group .", "this technique enables the production of a platelet \n concentration generally four times greater than the whole blood . according to landsberg , \n et al .", "( 1998 ) , thrombin promotes the \n release of antibodies to coagulation factors v and vi , which could be a potentially \n life - threatening event .", "these antibodies cross - react with human factor v thereby , \n causing a factor v deficiency that can be sufficiently severe to induce excessive \n bleeding and even death . considering that , the use of thrombin in this type of study is \n not allowed in our country . in the present study , instead of thrombin", ", 10% calcium \n chloride was added to prp and heated in a water bath at 37c .", "( 2003 ) measured the concentrations of \n bfgf , vegf , pdgf - bb and tgf-1 released from platelet concentrate and whole \n blood , before and after the addition of calcium alone , thrombin alone and various \n concentrations of calcium and thrombin .", "those authors observed that calcium chloride , \n regardless of the use of thrombin , released platelet growth factors . in this \n study ,", "the concentration of \n growth factors that was released was sufficient to promote endothelial cell \n proliferation in vitro .", "recently , the tissue engineering science has provided a new possibility of cell therapy \n as a future alternative for the use of conventional regenerative techniques .", "this strategy that exploits the \n regenerative capacity of stem cells grown in a three - dimensional scaffold and \n subsequently implanted into the defect , in the presence of molecule modulators , may help \n overcoming several limitations of current regeneration modalities .", "in an in vitro study , \n the application of growth factors found in prp ( tgf-1 and igf - i ) in cultures of \n periodontal ligament cells , increased their mitotic activity and their capacity for \n adhesion on the root fragments surface . the use of platelet \n growth factors associated with cell therapy could be an alternative favoring the process \n of periodontal regeneration .", "within the limits of this animal model study , it may be concluded that the association \n of prp with bg did not exert an additional effect to periodontal regeneration of 3-wall \n intrabony defects in dogs .", "further studies are necessary to investigate the advantages \n and limitations of these materials in the treatment of periodontal defects ." ]

[ "<S> objectivethis study was designed to evaluate , histomorphometrically , the association of \n platelet - rich plasma ( prp ) and bioactive glass ( bg ) in the treatment of \n periodontal intrabony defects.material and methodsnine mongrel dogs were included in the study . </S>", "<S> three - wall intrabony defects were \n surgically created at the mesial and distal aspect of first mandibular molar and \n exposed to plaque accumulation for 1 month . </S>", "<S> the defects were randomly assigned to \n the groups : control , bg , prp , prp+bg . </S>", "<S> dogs were sacrificed 90 days after the \n surgeries . </S>", "<S> the histometric parameters evaluated were : length of sulcular and \n junctional epithelium , connective tissue adaptation , new cementum , new bone , \n defect extension and area of new bone filling the defect.resultsa superior area of new bone was observed in prp+bg and bg ( 13.802.32 \n mm2 and 15.632.64 mm2 , respectively ) when \n compared to the other groups ( 8.191.46 mm2 and 8.811.47 \n mm2 for control and prp , respectively ) . </S>", "<S> no statistically significant \n differences were observed in the remaining parameters.conclusionwithin the limits of this study , it may be concluded that prp failed to provide \n statistically significant improvements in the histometric parameters . </S>" ]

[ "INTRODUCTION", "MATERIAL AND METHODS", "PRP Preparation", "Defect Treatment", "Statistical analysis", "RESULTS", "I - Clinical Observations", "II - Histological Observations", "III Histometric Measurements", "DISCUSSION", "CONCLUSIONS" ]

[ [ "the ultimate goal of periodontal therapy includes not only the arrest of progressive \n periodontal disease but also the restitution of those parts of the supporting apparatus \n which have been destroyed by disease .", "regenerative procedures have been evaluated in \n several studies using grafting materials , root surface conditioning , guided tissue \n regeneration and application of growth factors .", "different materials have been introduced for bone defect filling , i.e. , autografts , \n xenografts , allografts , and alloplasts . among the various subgroups of alloplastic bone \n grafts ,", "bioactive glass ( bg ) is a type of bioactive ceramic with the ability to improve \n bone formation that has been assessed in experimental models .", "previous studies have shown that bg particles , when implanted in vivo , \n incorporate into the connective tissue and undergo chemical transformation increasing \n the ph of the site to approximately 10 .", "this process leads to \n the formation of a silica gel on the surface of the particles and subsequently an \n amorphous calcium phosphate layer is formed by the interaction between calcium and \n phosphate from the bg and the surrounding medium .", "hench and polak ( 2002 ) have shown that the release of ions ( na , ca , and si ) from bg \n materials controls the cell cycle leading to the differentiation and proliferation of \n bone cells , modulating the expression of genes that regulate osteogenesis , and the \n synthesis of growth factors .", "growth factors are vital modulators during this process which can induce the migration , \n attachment , proliferation and differentiation of periodontal progenitor cells . \n", "platelet - rich plasma ( prp ) is a preparation of autologous plasma that contains a higher \n platelet concentration , allowing it to deliver a greater concentration of autologous \n growth factors such us platelet - derived growth factor ( pdgf ) , transforming growth factor \n beta ( tgf- ) , vascular endothelial growth factor ( vegf ) , insulin - like growth \n factor ( igf - i ) , and epithelial growth factor ( egf ) , that regulate cell proliferation , \n chemotaxis and differentiation . some specific platelet growth factors , like pdgf and tgf- ,", "could promote the \n growth and differentiation of the periodontal ligament and alveolar bone cells and could \n be responsible for the clinical improvement observed in experimental sites", ". other interesting feature of prp is its \n sticky consistency due to its high fibrin content .", "the fibrin component of prp may work \n as a haemostatic agent aiding in stabilizing the graft material and the blood \n clot .", "blood clot \n immobilization has been suggested as an important event in the early phases of wound \n healing of periodontal regenerative procedures .", "studies using histological techniques suggested that prp preparations may enhance local \n bone formation , while \n others did not confirm these \n findings .", "clinical studies have reported that treatments with a combination of prp and \n demineralized freeze - dried bone graft allograft ( dfdba ) improve clinical parameters in \n intrabony periodontal defects . in \n the other hand", "did nt find additional \n benefit in the reduction of pocket depth , clinical attachment gain and defect filling \n using prp with bg in the treatment of intrabony defects .", "the use of prp associated with biomaterials is an alternative treatment approach that \n may favor the process of bone tissue regeneration and should be investigated .", "therefore , \n the goal of this study was to evaluate , histomorphometrically , the healing of surgically \n created intrabony defects in dogs treated with prp+bg ." ], [ "nine adult female mongrel dogs ( mean weight=15 kg ) were included in the experiment .", "the \n study protocol was approved by the institutional animal research committee , state \n university of campinas ( protocol # 686 - 1 ) .", "the surgical procedures were performed under \n general anesthesia with intravenous injection of a 3% sodium pentobarbital solution ( 0.5 \n ml / kg ) .", "the mandibular second and fourth premolars had been previously extracted and the \n extraction sites had been allowed to heal for 2 months . after this period , buccal and \n lingual mucoperiosteal flaps were elevated and 3-wall intrabony defects ( 4x4x4 mm ) were \n created at the mesial and distal aspect of the left and right mandibular first molar \n ( figure 1 ) . following root planning , aiming cementum removal ,", "a reference \n notch was made with curettes on the root surface at the cement - enamel junction .", "clinical view of the surgically created 3-wall intrabony defect ( 4x4x4 mm ) at the \n mesial aspect of the first mandibular molar before that the gingival flaps have been positioned and sutured , a tofflemire matrix \n involved by cotton ligature was adapted over the tooth surface and the remaining defect \n was filled with gutta - percha ( figure 2 ) .", "so , the \n defects were exposed to plaque accumulation for a period of one month before the \n treatment .", "after that , scaling and root planing was performed and a regimen of daily \n brushing plus topical application of 0.1% chlorhexidine gluconate was instituted for 7 \n days prior to the surgical procedures .", "clinical view of the insertion of gutta - percha in the defect blood was obtained several min before the administration of anesthesia .", "four 5-ml \n tubes containing 0.5 ml of 3.2% sodium citrate solution ( an anticoagulant ) were drawn \n from each dog .", "the blood was thus separated into 3 basic parts : red blood cells ( at the \n bottom of the tube ) , platelet enriched plasma ( a discrete grey line in the middle of \n the tube ) and platelet poor plasma ( at the top of the tube ) .", "the portion \n corresponding to the platelet poor plasma was discarded from each tube and the \n remaining content was centrifuged again at 1,200 rpm for 15 min .", "four hundred \n microliters of the middle portion , corresponding to the platelet enriched plasma , \n were pipetted from each tube . in order to obtain the gel ,", "30 l of 10% calcium \n chloride was added to prp and heated in a water bath at 37c .", "the gel was achieved \n after a period of 10 to 15 min . a tube with a sample of whole blood \n as well as a sample of prp , retrieved before adding calcium chloride of 10% , was sent \n to a clinical veterinary laboratory to perform a platelet counting .", "in the moment of the defect treatment , an apical reference notch was made with \n curettes on the root surface at the remaining bone level .", "each defect in each animal \n ( a total of 4 defects/ animal ) was randomly assigned to one of the following \n treatments ( the treatments with prp were located at the same side that was randomly \n determined ) : control : the defect was naturally filled with coagulum ; \n bg ( perioglas , us biomaterials , alachua , fl , usa ) : bg particles \n were applied , filling the defect ; prp : after obtaining the prp gel , \n it was immediately applied on the defect ; prp+bg : bg was \n incorporated to the prp ( 1:1 ) and this assemblage was immediately taken to a water \n bath at 37c for formation of a mixture of the gel and the bg graft .", "primary , tension - free wound closure was accomplished following defect treatment , with \n the gingival flaps positioned and sutured with interrupted sutures ( vicryl , ethicon \n inc , so jos dos campos , sp , brazil ) at their presurgical position . \n", "immediately after the surgeries , an intramuscular injection of penicillin ( 1.5 ml \n 150,000 iu ) was given to the animal .", "postoperative plaque control was performed by irrigation with a solution of 1% \n chlorhexidine gluconate ( every other day ) .", "after 90 days , under general anesthesia , \n the animals were sacrificed with a perfusion of 10% neutral formalin solution .", "the \n jaws were dissected and the blocks containing the experimental specimens were \n removed , and decalcified in a solution of equal parts of 50% formic acid and 20% \n sodium citrate for 5 months .", "the decalcified specimens were washed in running water , \n dehydrated and embedded in paraffin .", "five sections representing the midbuccal portion of each defect were selected for the \n histometric procedures .", "the \n following distances were measured : total epithelium ( sulcular and junctional epithelium ) : from the \n gingival margin to the apical border of the junctional epithelium . connective tissue adaptation ( without cementum ) : from the apical \n border of the junctional epithelium to the coronal end of new cementum .", "new cementum : from the apical notch to the most coronal part of new \n cementum . new bone ( bone position ) : from the apical notch to the most coronal \n extent of new bone .", "negative values would be assigned if the bony crest was located \n apically to the apical notch .", "new bone area : area of newly formed bone filling the defect area \n ( limited by the distance between the two notches in an apico - coronal direction and 4 \n mm away from the root surface in a lateral direction ) .", "the area of new bone was \n measured with an image analysis system that allowed the placement of a lattice on the \n image of the created bone defect .", "the intersection lines localized on bone were \n marked to calculate the new bone area whereas intersections lines localized on bg \n particles were not marked and therefore were not included for this purpose .", "the measurements were performed using a microscope ( zeiss axioskop 2 , carl zeiss \n instruments , gottingen , lower saxony , germany ) with a 1.25/.035 objective associated \n with a video camera / computer / software ( image pro plus version 3.0 , media cybernetics , \n silver spring , md , usa ) .", "the mean values for all \n groups were determined using the individual means from the 9 dogs .", "the hypothesis \n that there were no differences among the groups was tested by one - way anova .", "if a statistical \n difference was detected , tukey s pairwise multiple comparison test was used ." ], [ "four 5-ml \n tubes containing 0.5 ml of 3.2% sodium citrate solution ( an anticoagulant ) were drawn \n from each dog .", "the blood was thus separated into 3 basic parts : red blood cells ( at the \n bottom of the tube ) , platelet enriched plasma ( a discrete grey line in the middle of \n the tube ) and platelet poor plasma ( at the top of the tube ) .", "the portion \n corresponding to the platelet poor plasma was discarded from each tube and the \n remaining content was centrifuged again at 1,200 rpm for 15 min .", "four hundred \n microliters of the middle portion , corresponding to the platelet enriched plasma , \n were pipetted from each tube . in order to obtain the gel ,", "30 l of 10% calcium \n chloride was added to prp and heated in a water bath at 37c .", "the gel was achieved \n after a period of 10 to 15 min . a tube with a sample of whole blood \n as well as a sample of prp , retrieved before adding calcium chloride of 10% ," ], [ "in the moment of the defect treatment , an apical reference notch was made with \n curettes on the root surface at the remaining bone level . each defect in each animal \n", "( a total of 4 defects/ animal ) was randomly assigned to one of the following \n treatments ( the treatments with prp were located at the same side that was randomly \n determined ) : control : the defect was naturally filled with coagulum ; \n bg ( perioglas , us biomaterials , alachua , fl , usa ) : bg particles \n were applied , filling the defect ; prp : after obtaining the prp gel , \n it was immediately applied on the defect ; prp+bg : bg was \n incorporated to the prp ( 1:1 ) and this assemblage was immediately taken to a water \n bath at 37c for formation of a mixture of the gel and the bg graft .", "primary , tension - free wound closure was accomplished following defect treatment , with \n the gingival flaps positioned and sutured with interrupted sutures ( vicryl , ethicon \n inc , so jos dos campos , sp , brazil ) at their presurgical position . \n", "immediately after the surgeries , an intramuscular injection of penicillin ( 1.5 ml \n 150,000 iu ) was given to the animal .", "postoperative plaque control was performed by irrigation with a solution of 1% \n chlorhexidine gluconate ( every other day ) .", "after 90 days , under general anesthesia , \n the animals were sacrificed with a perfusion of 10% neutral formalin solution .", "the \n jaws were dissected and the blocks containing the experimental specimens were \n removed , and decalcified in a solution of equal parts of 50% formic acid and 20% \n sodium citrate for 5 months .", "the decalcified specimens were washed in running water , \n dehydrated and embedded in paraffin .", "five sections representing the midbuccal portion of each defect were selected for the \n histometric procedures .", "the \n following distances were measured : total epithelium ( sulcular and junctional epithelium ) : from the \n gingival margin to the apical border of the junctional epithelium . connective tissue adaptation ( without cementum ) : from the apical \n border of the junctional epithelium to the coronal end of new cementum .", "new cementum : from the apical notch to the most coronal part of new \n cementum . new bone ( bone position ) : from the apical notch to the most coronal \n extent of new bone .", "negative values would be assigned if the bony crest was located \n apically to the apical notch .", "new bone area : area of newly formed bone filling the defect area \n ( limited by the distance between the two notches in an apico - coronal direction and 4 \n mm away from the root surface in a lateral direction ) .", "the area of new bone was \n measured with an image analysis system that allowed the placement of a lattice on the \n image of the created bone defect .", "the intersection lines localized on bone were \n marked to calculate the new bone area whereas intersections lines localized on bg \n particles were not marked and therefore were not included for this purpose .", "the measurements were performed using a microscope ( zeiss axioskop 2 , carl zeiss \n instruments , gottingen , lower saxony , germany ) with a 1.25/.035 objective associated \n with a video camera / computer / software ( image pro plus version 3.0 , media cybernetics , \n silver spring , md , usa ) ." ], [ "mean values for each parameter were obtained per defect . the mean values for all \n groups", "the hypothesis \n that there were no differences among the groups was tested by one - way anova .", "if a statistical \n difference was detected , tukey s pairwise multiple comparison test was used ." ], [ "all \n groups presented a similar healing pattern characterized mainly by the presence of \n new cementum covering most of the root surface associated with the defect and new \n bone , up to the top of the defect , with a small amount of connective tissue and \n epithelium ( figure 3 ) .", "the periodontal ligament \n was characterized by the presence of collagen fibers obliquely oriented to the root \n surface , extending between the new cementum and the newly formed bone .", "photomicrographs of the treated 3-wall intrabony defect showing new bone and \n cementum formation extending coronally to the apical notch ( h&e , original \n magnification x20 ) .", "a ) control ; b ) prp ; c ) bg ; d ) bg+prp .", "note the remaining \n particles of bg surrounded by new bone and connective tissue ( c and d ) .", "arrows \n indicate areas with new bone in contact with bg particles in the defects treated with bg , it was possible to observe that part of the material \n seemed to be resorbed and replaced by bone , while some of the remaining bg particles \n showed dissolution of their central part with newly formed bone inside of them . some \n defects presented bone in direct contact and/or surrounding the bg particles ( figure 4 ) .", "almost all defects that received bg \n presented a dense connective tissue surrounding the remaining particles and areas \n suggesting the formation of new bone .", "new bone formation observed in the 3-wall intrabony defect treated with bg \n ( h&e , original magnification x100 ) .", "arrows indicate bone in direct contact \n with bg particles the histometric results are shown in table 1 . \n", "a superior area of new bone was observed in the sites treated with prp+bg and bg \n ( 13.802.32 mm and 15.632.64 mm , \n respectively ) . in the other groups ,", "the new bone area was 8.191.46 \n mm ( control group ) and 8.811.47 mm ( prp ) . although \n the difference in the new bone area , no significant difference was found in the \n linear parameter new bone , e.g. bone position : from the apical notch to the most \n coronal extent of new bone ( 4.370.44 mm , 4.240.68 mm , \n 4.370.46 mm and 4.440.42 mm , for control group , bg , prp and prp+bg , \n respectively ; p=0.85 ) . mean and standard deviation ( sd ) for the parameters evaluated after all the \n treatments ( control , bg , prp and bg+prp ) .", "means followed by different letters \n are statistically different at 5% data analysis showed no significant differences among the groups regarding the \n initial defect extension ( 4.550.17 mm , 4.480.20 mm , 4.480.28 \n mm and 4.570.32 mm , for control group , bg , prp and prp+bg , respectively ; \n p=0.74 ) .", "in addition , intergroup analysis demonstrated that no significant difference \n was observed among the groups in the following parameters : total epithelium extension \n ( 2.240.58 mm , 1.940.37 mm , 1.970.37 mm and 1.810.61 , \n for control group , bg , prp and prp+bg , respectively ; p=0.45 ) , connective tissue \n extension ( 0.900.28 mm , 0.840.41 mm , 1.070.27 mm and \n 1.150.32 mm , for control group , bg , prp and prp+bg , respectively ; p=0.59 ) and \n new cementum extension ( 2.630.70 mm , 2.560.36 mm , 2.370.38 mm \n and 3.100.47 mm , for control group , bg , prp and prp+bg , respectively ; \n p=0.85 ) .", "the results from the histometric measurements , i.e. , epithelial \n length ( the portion of epithelium located on the root ) , connective tissue adaptation \n and new cementum formation , expressed as the percentage of the distance between the \n coronal and the apical notch are presented in figure \n 5 ." ], [ "" ], [ "no undesirable events like inflammation and foreign body reactions were observed . all \n groups presented a similar healing pattern characterized mainly by the presence of \n new cementum covering most of the root surface associated with the defect and new \n bone , up to the top of the defect , with a small amount of connective tissue and \n epithelium ( figure 3 ) .", "the periodontal ligament \n was characterized by the presence of collagen fibers obliquely oriented to the root \n surface , extending between the new cementum and the newly formed bone .", "photomicrographs of the treated 3-wall intrabony defect showing new bone and \n cementum formation extending coronally to the apical notch ( h&e , original \n magnification x20 ) .", "a ) control ; b ) prp ; c ) bg ; d ) bg+prp .", "note the remaining \n particles of bg surrounded by new bone and connective tissue ( c and d ) .", "arrows \n indicate areas with new bone in contact with bg particles in the defects treated with bg , it was possible to observe that part of the material \n seemed to be resorbed and replaced by bone , while some of the remaining bg particles \n showed dissolution of their central part with newly formed bone inside of them . some \n defects presented bone in direct contact and/or surrounding the bg particles ( figure 4 ) .", "almost all defects that received bg \n presented a dense connective tissue surrounding the remaining particles and areas \n suggesting the formation of new bone .", "new bone formation observed in the 3-wall intrabony defect treated with bg \n ( h&e , original magnification x100 ) ." ], [ "a superior area of new bone was observed in the sites treated with prp+bg and bg \n ( 13.802.32 mm and 15.632.64 mm , \n respectively ) . in the other groups , the new bone area was 8.191.46 \n mm ( control group ) and 8.811.47 mm ( prp ) . although \n the difference in the new bone area , no significant difference was found in the \n linear parameter new bone , e.g. bone position : from the apical notch to the most \n coronal extent of new bone ( 4.370.44 mm , 4.240.68 mm , \n 4.370.46 mm and 4.440.42 mm , for control group , bg , prp and prp+bg , \n respectively ; p=0.85 ) . mean and standard deviation ( sd ) for the parameters evaluated after all the \n treatments ( control , bg , prp and bg+prp ) .", "means followed by different letters \n are statistically different at 5% data analysis showed no significant differences among the groups regarding the \n initial defect extension ( 4.550.17 mm , 4.480.20 mm , 4.480.28 \n mm and 4.570.32 mm , for control group , bg , prp and prp+bg , respectively ; \n p=0.74 ) .", "in addition , intergroup analysis demonstrated that no significant difference \n was observed among the groups in the following parameters : total epithelium extension \n ( 2.240.58 mm , 1.940.37 mm , 1.970.37 mm and 1.810.61 , \n for control group , bg , prp and prp+bg , respectively ; p=0.45 ) , connective tissue \n extension ( 0.900.28 mm , 0.840.41 mm , 1.070.27 mm and \n 1.150.32 mm , for control group , bg , prp and prp+bg , respectively ; p=0.59 ) and \n new cementum extension ( 2.630.70 mm , 2.560.36 mm , 2.370.38 mm \n and 3.100.47 mm , for control group , bg , prp and prp+bg , respectively ; \n p=0.85 ) .", "the results from the histometric measurements , i.e. , epithelial \n length ( the portion of epithelium located on the root ) , connective tissue adaptation \n and new cementum formation , expressed as the percentage of the distance between the \n coronal and the apical notch are presented in figure \n 5 ." ], [ "the use of different regenerative approaches in the treatment of periodontal defects has \n demonstrated a variable clinical result .", "some techniques have been used in association , aiming to increase \n their effect on periodontal regeneration .", "the present study was \n designed to evaluate , histomorphometrically , the healing process of 3-wall intrabony \n defects treated with bg , prp and their association .", "prp has become a focus of current studies due to its potential to accelerate wound \n healing .", "it has been previously shown that prp stimulated pdl \n cells and fibroblastic cell proliferationbut suppressed \n epithelial cell division in vitro .", "consequently , by ordering these cellular responses into a series \n of related events , prp may facilitate wound - healing and tissue regeneration .", "the claimed \n ability to suppress epithelial cell proliferation seems advantageous for periodontal \n regeneration by favoring the formation of a new connective tissue attachment and new \n cementum on the root surface .", "the \n results of the present study failed to demonstrate that the use of prp can promote an \n additional effect in terms of cementum formation when treating 3-wall intrabony defects . \n", "cementum formation for bg and prp followed the same trend observed for the control group \n ( bg=2.5 mm/57% , prp=2.4 mm/53% , control=58% ) while the prp+bg showed a slightly greater \n cementum formation ( prp+bg=3.10 mm/68% ) , but the differences among the groups were not \n statistically significant . in this respect", ", it should be recognized that a non - contained \n osseous defect might have favored the discrimination of quantitative differences among \n the treatments . in a previous histological study", ", kim , et al ( 2004 ) evaluated the healing patterns following flap surgery in \n experimentally induced 1- , 2- and 3-wall intrabony defects . according to the authors , \n one- and 3-wall intrabony defects", "can be considered pre - clinical models that are \n relatively easy to manage and from which homogeneous healing outcomes may be expected . \n", "they suggested that these defects appear to be reproducible models to evaluate candidate \n technologies for periodontal regeneration .", "regarding the 3-wall intrabony defect , they reported a mean \n cementum formation of approximately 70% of the initial defect extension . in the present \n study ,", "a period of 1 month of plaque accumulation was allowed as a way to obtain some \n chronification of the defect .", "the control group showed 2.6 mm ( 58% ) of the original \n defect covered with new cementum .", "this result confirms the high regenerative potential \n of this type of defect that provides favorable anatomic characteristics for clot \n stabilization and protection .", "it is well accepted that healing of intrabony defects is positively correlated to the \n number of bone walls limiting the defect .", "therefore , the 3-wall intrabony defect used in \n the present study provides ideal extent and location of tissue resources , cells and \n vascularity circumscribing the defect , which might have favored bone formation .", "the \n linear measurement of the new bone height revealed an impressive coronal bone formation \n for all the groups , with no significant differences among the treatments .", "the claimed osteoinductive properties of prp could not be supported by the results of the present study .", "it \n has been suggested that prp could be used to increase the bone deposition rate and the \n quality of bone regeneration when augmenting sites prior to or in conjunction with \n dental implant placement .", "nevertheless , more recent animal studies could not find \n increased bone regeneration when prp was used .", "recently , bioactive glass has been used as a way to achieve bone and periodontal \n regeneration based on its osteoconductive properties . the need for an \n alternative to autologous and allogenic bone grafts", "have encouraged the search of a \n material that could be safe , surgically convenient and predictably promote regeneration \n with a reduced morbidity . in this study ,", "the use of bg was not associated with any \n foreign body reaction , showing biocompatibility with periodontal tissues .", "histological observations after treatment of periodontal defects with bg in nonhuman \n primates showed a significant increase of new cementum and inhibition of downgrowth of \n junctional epithelium .", "when bg is \n implanted in vivo , the ph of the site increases , a layer rich in silica \n gel is formed on the surface of the particles and subsequently an amorphous calcium \n phosphate layer is formed by the interaction between calcium and phosphate from the bg \n and the surrounding medium .", "its \n superficial bioactivity may stimulate a rapid formation of a connective tissue seal that \n is supposed to have the ability to block the epithelium migration and allow for the \n repopulation of the previously contaminated area by periodontal ligament cells . in the present study ,", "almost all defects \n that received bg presented a dense connective tissue surrounding the remaining particles \n in accordance with observations of previous studies . in the groups", "treated with bg , the area of new bone was 13.802.32 mm \n ( prp+bg ) and 15.632.64 mm ( bg ) , respectively . in the groups that did \n", "not receive this material , the new bone area was 8.191.46 mm \n ( control group ) and 8.811.47 mm ( prp ) . based on the significant \n differences observed in the area of newly formed bone when groups with and without bg \n were compared the present study corroborates the hypothesis that this material could act \n as an osteoconductive graft .", "in addition , it was observed that some remaining bg \n granules showed dissolution of their core with newly formed bone , as described in the \n literature . aiming to increase the periodontal and bone regeneration", ", some studies evaluated the \n additional effect of prp , combined with different types of grafting materials , on the \n treatment of intrabony defects . in spite of some clinical results showing a greater \n reduction of pocket depth and", "gain in clinical attachment level , other clinical studies \n failed to demonstrate an additional effect . in 3-wall periimplant defects in dogs \n treated with prp associated to xenogenic bone graft ,", "the same \n authors also reported no significant effect with the addition of prp to xenogenic bone \n grafts on bone mineral density or graft maturity .", "these observations are in agreement with the results of the \n present study , which could not demonstrate an additional effect of prp in periodontal \n and bone regeneration of 3 wall intrabony defects .", "the histomorphometric findings \n demonstrated that the sites treated with prp+bg showed a similar amount of new bone \n ( 13.802.32 mm ) when compared with the sites that only received bg \n ( 15.632.64 mm ) .", "the ideal platelet and growth factor concentration to promote periodontal regeneration \n has not yet been established . to the date , there is insufficient information about \n growth factor interactions and how they influence the activations of gene expression and \n protein production . in the present study ,", "the achieved percentage mean of platelet \n concentration was 227.02% ( mean of 463.015 platelets / ml ) in relation to blood platelets \n count .", "( 2003 ) \n reported that growth factors may act at specific times and at appropriate concentrations \n and this may be other explanation for the different results obtained in studies \n evaluating the use of prp .", "the great variability in the outcomes of the studies with prp \n may also be explained by the diversity of methods to obtain it .", "the technique to produce \n prp in the present study was used in other animal studies by our research \n group .", "this technique enables the production of a platelet \n concentration generally four times greater than the whole blood . according to landsberg , \n et al .", "( 1998 ) , thrombin promotes the \n release of antibodies to coagulation factors v and vi , which could be a potentially \n life - threatening event .", "these antibodies cross - react with human factor v thereby , \n causing a factor v deficiency that can be sufficiently severe to induce excessive \n bleeding and even death . considering that , the use of thrombin in this type of study is \n not allowed in our country . in the present study , instead of thrombin", ", 10% calcium \n chloride was added to prp and heated in a water bath at 37c .", "( 2003 ) measured the concentrations of \n bfgf , vegf , pdgf - bb and tgf-1 released from platelet concentrate and whole \n blood , before and after the addition of calcium alone , thrombin alone and various \n concentrations of calcium and thrombin .", "those authors observed that calcium chloride , \n regardless of the use of thrombin , released platelet growth factors . in this \n study ,", "the concentration of \n growth factors that was released was sufficient to promote endothelial cell \n proliferation in vitro .", "recently , the tissue engineering science has provided a new possibility of cell therapy \n as a future alternative for the use of conventional regenerative techniques .", "this strategy that exploits the \n regenerative capacity of stem cells grown in a three - dimensional scaffold and \n subsequently implanted into the defect , in the presence of molecule modulators , may help \n overcoming several limitations of current regeneration modalities .", "in an in vitro study , \n the application of growth factors found in prp ( tgf-1 and igf - i ) in cultures of \n periodontal ligament cells , increased their mitotic activity and their capacity for \n adhesion on the root fragments surface . the use of platelet \n growth factors associated with cell therapy could be an alternative favoring the process \n of periodontal regeneration ." ], [ "within the limits of this animal model study , it may be concluded that the association \n of prp with bg did not exert an additional effect to periodontal regeneration of 3-wall \n intrabony defects in dogs .", "further studies are necessary to investigate the advantages \n and limitations of these materials in the treatment of periodontal defects ." ] ]

[ "the prevalence of diabetes is increasing globally , associated with an increase in obesity and in sedentary lifestyle [ 13 ] .", "this is associated with morbidity and mortality due to the effects of hyperglycemia related complication and by its association with atherosclerosis - heart disease and stroke , as the leading causes for mortality in diabetics .", "hospitalization for acute internal and coronary disorders is therefore high among patients with diabetes ; estimates from several studies show that the percentage of patients with diabetes admitted is up to three times the percent of diabetes in the relevant population . ", "the increase in the prevalence diabetes and obesity is , however , not distributed evenly between different racial and ethnic groups , with some groups showing higher susceptibility to environmental changes brought by immigration or rapid socioeconomic changes . ", "the arab population in israel has recently been observed to have an alarming high prevalence of diabetes , reaching 50% in women above 50 years old .", "this high prevalence seems to be higher than observed in arab countries or in arab immigrants to the usa or western europe .", "the unique situation of this population calls for both studies and intervention on a national level to promote health and prevent the consequences of the metabolic perturbation . considering the high prevalence of diabetes in the arab population in israel", ", we hypothesized that we will find substantial number of patients in the internal and the intensive coronary unit ( icu ) wards diagnosed and undiagnosed diabetic patients .", "seeking such a high risk population will enable the characterization of demographic , clinical state and diabetes related parameters .", "this data will serve to form intervention strategy in order to enhance health related outcomes .", "the study was performed at the internal medicine wards and icu of the holy family ( hfh ) hospital and the nazareth hospital in nazareth .", "the hfh hospital ( established 1882 ) has 32 beds in the internal ward and icu .", "the nazareth hospital was established by the edinburgh medical mission society in 1861 in which 40 beds are in the internal ward and the icu .", "the rural population has shifted from mainly an agrarian society toward an urban like society .", "both hospitals serve mainly the arab population in northern israel with an estimated population of 253,000 . ", "diabetes was defined as either known or unknown diabetes . known diabetes : pervious diagnosis and/or treatment with diabetes related medication . ", "unknown diabetes : two measurements of blood glucose > 200 mg% and/or hba1c > 6.5% .", "dwelling was defined as urban residing in the areas of major cities nazareth and acre ; rural - residing in the areas of smaller towns and villages . ", "data on all patients hospitalized in the internal medicine and icu units in hfh and nazareth hospitals between 1.1.2009 and 31.12.2009 were reviewed .", "charts of all diabetics were probed for data concerning demographics and previous diagnosis , and data pertaining to the hospitalization cause and the outcomes .", "all patients who were hospitalized during the months : january , april , july , and october served as a control group .", "all measured variables and derived parameters were tabulated by descriptive statistics . for descriptive statistics summary tables", "were provided giving sample size , absolute and relative frequency for categorical variables and sample size , arithmetic mean , standard deviation , median , minimum , and maximum for continuous variables .", "the following statistical tests were used in the analysis of the data presented in this study . ", "chi - square test was applied for testing the statistical significance of the differences in frequency of categorical variables between the study groups . ", "the two - sample t - test was applied for testing the statistical significance of the differences of continuous variables between the study groups . ", "analysis of covariance ( anova ) was applied for comparing the differences of continuous variables between the outcomes of patients . ", "logistic regression was applied using patient 's death as outcome and glucose on day 1 as predictor variable . ", "multiple regression was applied using days of hospitalization as outcome and glucose on day 1 as predictor variable .", "chi - square test was applied for testing the statistical significance of the differences in frequency of categorical variables between the study groups .", "the two - sample t - test was applied for testing the statistical significance of the differences of continuous variables between the study groups .", "analysis of covariance ( anova ) was applied for comparing the differences of continuous variables between the outcomes of patients .", "logistic regression was applied using patient 's death as outcome and glucose on day 1 as predictor variable .", "multiple regression was applied using days of hospitalization as outcome and glucose on day 1 as predictor variable .", "all tests applied were two - tailed , and p value of 5% or less was considered statistically significant .", "the data was analyzed using the sas version 9.1 ( sas institute , cary , nc ) .", "the number of all patients hospitalized one or more during the study year was 3784 .", "thus , the proportion of diabetic patients hospitalized for one or more times during the study year was 39% . ", "patient demographics show the preponderance of the arab patients admitted : 92.8% of diabetic and 90.7% in the control group ( p = nonsignificant ( ns ) ) .", "the minority were jews ( 3.45 , 5.1% , resp . ) or other ethnic groups ( p = ns ) .", "more patients with diabetes reported residence in rural versus urban areas than in the control group ( table 1 ) . ", "clinical characteristics at admission differed between patients with diabetes and controls ( table 2 ) .", "diabetic patients admitted were significantly older than control 66.53 12.72 years and 54.26 21.53 years , respectively ( p < 0.0001 ) .", "of the diabetic patients admitted , 52.9% were women in comparison to 45.0% of the control group ( p = 0.0003 ) . ", "weight did not differ between groups 82.39 49.12 versus 85.08 19.08 ( p = 0.47 ) .", "the indications for hospitalization were different between diabetes and controls and between genders : the main difference was in the higher occurrence of hospitalization due to atherosclerotic related diseases both cardiovascular disease and strokes in the diabetic group versus controls ( 37% versus 27% , resp .", "significant differences in the indications for hospitalization were maintained within the gender in each group .", "while only 18.4% of control women were hospitalized for cardiac indications , 32.9% of diabetic women were hospitalized for cardiac reasons a similar rate as in the nondiabetic men ( 34.2% ) . in comorbidities", ", we also detected significant difference between groups with a noticeable higher renal disease in the diabetic group ( 16.6% versus 7.9% ) ( table 3 ) .", "hospitalization : duration of hospitalization was significantly longer for the diabetic patients than for control patients : 3.71 2.99 days versus 3.27 2.97 days , respectively ( p = 0.0008 ) ( table 2 ) .", "no significant differences in discharge status were noted between the two groups with the majority of patients being home discharged and with a low rate of in - hospital death2.6% control and 2.3% diabetic ( table 4 ) .", "one - year outcome was poorer in the diabetic group but without reaching statistical significance with a major difference in readmission rates in the diabetic group between the two hospitals .", "mortality during the first year following discharge was 11.5% in the control versus 13.2% in the diabetic group ( p = 0.48 ) . a high readmission rate for both groups 19.9% and 20.3% control and diabetic group respectively , but there was an inequality between the two hospitals with one hospital demonstrating a significant difference between the two groups in the rate of readmission being significantly higher in the diabetic group during the first year after discharge in comparison to the control readmission rate . ", "average daily glucose levels during hospitalization as described in figure 2 demonstrate a decline in values from day one 211.4 ( 118.1 ) mg / dl to less than 200 mg / dl and thereafter stable levels while glucose levels were stable in the control group ( figure 2 ) .", "the glucose level at admission was a significant and an independent predictor for longer hospitalization and death in the nondiabetic but not in the diabetic group ( data not shown ) . ", "as a group , indications for hospitalization differed between patients with diabetes and controls . for both groups ,", "the leading indication for hospitalization was cardiovascular disease , but cardiovascular disease and urinary tract infection were more prevalent as an indication for hospitalization in the diabetic group ( 36.8% versus 27.1% and 7.7% versus 6.9% , resp . ) while chest infections were significantly less prevalent in the diabetic group versus control group ( 12.5% versus 16.9% , resp . ) . ", "the glucose lowering treatment for the patients with diabetes , prior to their hospitalization , was diet alone in 13.1% , oral therapy in 60% , insulin in 19% , and combination therapy in 7% of the patients . at discharge", ", there was no significant shift in the treatment paradigm in the total group ( table 5 ) .", " this study sheds light on the characteristics of hospitalized patients with diabetes in hospitals serving a high risk for diabetes population .", "we found high percentage of diabetes among the patients hospitalized in the internal medicine wards and cardiac intensive units , reflecting the high prevalence of diabetes in the population .", "39.3% of total patient admitted to the internal medical ward and intensive care unit were diabetic .", "there was a female preponderance among patients admitted with diabetes 52.9% while only 45.0% of patients without diabetes , hospitalized , were women ( p = 0.0003 ) .", "all this reflects the alarming prevalence of obesity and diabetes among adult arab women in northern israel .", "it has been recently reported that the prevalence of overweigh and obesity among arab woman over 40 years of age who attend primary health clinic is 74% [ 79 ] with diabetes near 50% .", "this novel observation is seminal by bringing attention to this specific group of women with high morbidity risk and now observing high rate of hospitalization . interestingly ,", "diabetic women tend to have similar prevalence of coronary heart disease as main indication for admission , as for nondiabetic men ( 32.9% versus 34.2% , resp . ) . as cardiovascular disease and urinary tract infection were more prevalent as an indication for hospitalization in the diabetic group , prevention of cardiovascular disease and urinary tract infection", "should therefore be reinforced particularly in the group of arab diabetic women within diabetic population . ", "patients with diabetes had a significant greater length of stay than hospitalized patients without diabetes , similar to data described in previous publications [ 1012 ] .", "the reasons might be multifactorial , and it is important to note that interventions with chronic disease management programs for outpatients and diabetes team consultation during hospitalization have been proved to be an effective means to reduce the likelihood and duration of hospitalizations for individuals with diabetes . implementing similar strategies in this high risk group and evaluating the impact of this intervention on outcomes of rate and recurrence of hospitalization and hospital length of stay is therefore of paramount importance .", "in this study , we can additionally identify areas for intervention ; as is evident in table 5 , the stay in the hospital did not affect the patients treatment regimens , although glycemic levels at discharge were above the target ( mean glucose levels at discharge > 190 mg / dl ) .", "furthermore , in only a minority of patients , an hba1c measurement was performed during hospitalization .", "these issues reflect the current practice and constraint on hospitalization length and on the performance of blood tests , pushing toward shorter hospital stay and cutting back on performing blood tests not directly relevant to the immediate hospital care .", "it will be of interest to examine whether interventions as described above will cause changes in treatment regimens with improvement in long - term outcomes and readmissions . ", "the glycemic control goals in nonsurgical hospitalized patients continue to be debated , but a consensus defining upper limits below 180 m / dl in noncritical patients is well based on outcomes studies and is therefore recommended by recent position papers .", "average glucose levels during hospitalization were above 180 mg% throughout the hospital stay ( figure 2 ) ; therefore , as noted there is a need for team consultation during hospitalization tailored to hospitals that provide medical care to populations in high risk for diabetes . ", "results of this study show the impact of the high prevalence of diabetes on the hospitalization of arab patients in northern israel , with specific emphasis on the burden of diabetes of the arab women .", "it calls attention to the need of in - hospital consultation team and postdischarge plan .", "information from the cohort described within this study might be applicable to other medical centers providing large population of arab descent [ 1517 ] ." ]

[ "<S> \n aims . to seek high risk population for diabetes and to improve their health care by investigating the characteristics and outcome of hospitalization in hospitals with predominant arab patients in northern israel </S>", "<S> . methods . </S>", "<S> retrospective analysis of the prevalence of diabetes and the outcome of diabetic in comparison to nondiabetic patients hospitalized in the internal medicine and intensive cardiac units in two major hospitals with one - year postdischarge data between 1.1.2009 and 31.12.2009 . </S>", "<S> results . </S>", "<S> thirty - nine percent of the patients were diagnosed with diabetes . </S>", "<S> the preponderance of women in the diabetes group was noted . </S>", "<S> diabetic patients had an increase in the duration of hospitalization ( p = 0.0008 ) , with one hospital having a high readmission rate for the diabetic patients . </S>", "<S> the average glycemia during hospitalization exceeded the recommended threshold of 180 mg% without major changes in the therapeutic regimens in comparison to preadmission regimens . </S>", "<S> conclusions . </S>", "<S> arab populations , women in particular , in westernizing societies are at high risk for diabetes which exemplifies as high rate of patients with diabetes among hospitalized patients . </S>", "<S> areas for intervention during hospitalization and at predischarge have been identified to improve health outcomes and prevent readmissions . </S>" ]

[ "1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion" ]

[ [ "the prevalence of diabetes is increasing globally , associated with an increase in obesity and in sedentary lifestyle [ 13 ] .", "this is associated with morbidity and mortality due to the effects of hyperglycemia related complication and by its association with atherosclerosis - heart disease and stroke , as the leading causes for mortality in diabetics .", "hospitalization for acute internal and coronary disorders is therefore high among patients with diabetes ; estimates from several studies show that the percentage of patients with diabetes admitted is up to three times the percent of diabetes in the relevant population . ", "the increase in the prevalence diabetes and obesity is , however , not distributed evenly between different racial and ethnic groups , with some groups showing higher susceptibility to environmental changes brought by immigration or rapid socioeconomic changes . ", "the arab population in israel has recently been observed to have an alarming high prevalence of diabetes , reaching 50% in women above 50 years old .", "this high prevalence seems to be higher than observed in arab countries or in arab immigrants to the usa or western europe .", "the unique situation of this population calls for both studies and intervention on a national level to promote health and prevent the consequences of the metabolic perturbation . considering the high prevalence of diabetes in the arab population in israel", ", we hypothesized that we will find substantial number of patients in the internal and the intensive coronary unit ( icu ) wards diagnosed and undiagnosed diabetic patients .", "seeking such a high risk population will enable the characterization of demographic , clinical state and diabetes related parameters .", "this data will serve to form intervention strategy in order to enhance health related outcomes ." ], [ "the study was performed at the internal medicine wards and icu of the holy family ( hfh ) hospital and the nazareth hospital in nazareth .", "the hfh hospital ( established 1882 ) has 32 beds in the internal ward and icu .", "the nazareth hospital was established by the edinburgh medical mission society in 1861 in which 40 beds are in the internal ward and the icu .", "the rural population has shifted from mainly an agrarian society toward an urban like society .", "both hospitals serve mainly the arab population in northern israel with an estimated population of 253,000 . ", "diabetes was defined as either known or unknown diabetes . known diabetes : pervious diagnosis and/or treatment with diabetes related medication . ", "unknown diabetes : two measurements of blood glucose > 200 mg% and/or hba1c > 6.5% .", "dwelling was defined as urban residing in the areas of major cities nazareth and acre ; rural - residing in the areas of smaller towns and villages . ", "data on all patients hospitalized in the internal medicine and icu units in hfh and nazareth hospitals between 1.1.2009 and 31.12.2009 were reviewed .", "charts of all diabetics were probed for data concerning demographics and previous diagnosis , and data pertaining to the hospitalization cause and the outcomes .", "all patients who were hospitalized during the months : january , april , july , and october served as a control group .", "all measured variables and derived parameters were tabulated by descriptive statistics . for descriptive statistics summary tables", "were provided giving sample size , absolute and relative frequency for categorical variables and sample size , arithmetic mean , standard deviation , median , minimum , and maximum for continuous variables .", "the following statistical tests were used in the analysis of the data presented in this study . ", "chi - square test was applied for testing the statistical significance of the differences in frequency of categorical variables between the study groups . ", "the two - sample t - test was applied for testing the statistical significance of the differences of continuous variables between the study groups . ", "analysis of covariance ( anova ) was applied for comparing the differences of continuous variables between the outcomes of patients . ", "logistic regression was applied using patient 's death as outcome and glucose on day 1 as predictor variable . ", "multiple regression was applied using days of hospitalization as outcome and glucose on day 1 as predictor variable .", "chi - square test was applied for testing the statistical significance of the differences in frequency of categorical variables between the study groups .", "the two - sample t - test was applied for testing the statistical significance of the differences of continuous variables between the study groups .", "analysis of covariance ( anova ) was applied for comparing the differences of continuous variables between the outcomes of patients .", "logistic regression was applied using patient 's death as outcome and glucose on day 1 as predictor variable .", "multiple regression was applied using days of hospitalization as outcome and glucose on day 1 as predictor variable .", "all tests applied were two - tailed , and p value of 5% or less was considered statistically significant .", "the data was analyzed using the sas version 9.1 ( sas institute , cary , nc ) ." ], [ "the number of all patients hospitalized one or more during the study year was 3784 .", "thus , the proportion of diabetic patients hospitalized for one or more times during the study year was 39% . ", "patient demographics show the preponderance of the arab patients admitted : 92.8% of diabetic and 90.7% in the control group ( p = nonsignificant ( ns ) ) .", "the minority were jews ( 3.45 , 5.1% , resp . ) or other ethnic groups ( p = ns ) .", "more patients with diabetes reported residence in rural versus urban areas than in the control group ( table 1 ) . ", "clinical characteristics at admission differed between patients with diabetes and controls ( table 2 ) .", "diabetic patients admitted were significantly older than control 66.53 12.72 years and 54.26 21.53 years , respectively ( p < 0.0001 ) .", "of the diabetic patients admitted , 52.9% were women in comparison to 45.0% of the control group ( p = 0.0003 ) . ", "weight did not differ between groups 82.39 49.12 versus 85.08 19.08 ( p = 0.47 ) .", "the indications for hospitalization were different between diabetes and controls and between genders : the main difference was in the higher occurrence of hospitalization due to atherosclerotic related diseases both cardiovascular disease and strokes in the diabetic group versus controls ( 37% versus 27% , resp .", "significant differences in the indications for hospitalization were maintained within the gender in each group .", "while only 18.4% of control women were hospitalized for cardiac indications , 32.9% of diabetic women were hospitalized for cardiac reasons a similar rate as in the nondiabetic men ( 34.2% ) . in comorbidities", ", we also detected significant difference between groups with a noticeable higher renal disease in the diabetic group ( 16.6% versus 7.9% ) ( table 3 ) .", "hospitalization : duration of hospitalization was significantly longer for the diabetic patients than for control patients : 3.71 2.99 days versus 3.27 2.97 days , respectively ( p = 0.0008 ) ( table 2 ) .", "no significant differences in discharge status were noted between the two groups with the majority of patients being home discharged and with a low rate of in - hospital death2.6% control and 2.3% diabetic ( table 4 ) .", "one - year outcome was poorer in the diabetic group but without reaching statistical significance with a major difference in readmission rates in the diabetic group between the two hospitals .", "mortality during the first year following discharge was 11.5% in the control versus 13.2% in the diabetic group ( p = 0.48 ) . a high readmission rate for both groups 19.9% and 20.3% control and diabetic group respectively , but there was an inequality between the two hospitals with one hospital demonstrating a significant difference between the two groups in the rate of readmission being significantly higher in the diabetic group during the first year after discharge in comparison to the control readmission rate . ", "average daily glucose levels during hospitalization as described in figure 2 demonstrate a decline in values from day one 211.4 ( 118.1 ) mg / dl to less than 200 mg / dl and thereafter stable levels while glucose levels were stable in the control group ( figure 2 ) .", "the glucose level at admission was a significant and an independent predictor for longer hospitalization and death in the nondiabetic but not in the diabetic group ( data not shown ) . ", "as a group , indications for hospitalization differed between patients with diabetes and controls . for both groups ,", "the leading indication for hospitalization was cardiovascular disease , but cardiovascular disease and urinary tract infection were more prevalent as an indication for hospitalization in the diabetic group ( 36.8% versus 27.1% and 7.7% versus 6.9% , resp . ) while chest infections were significantly less prevalent in the diabetic group versus control group ( 12.5% versus 16.9% , resp . ) . ", "the glucose lowering treatment for the patients with diabetes , prior to their hospitalization , was diet alone in 13.1% , oral therapy in 60% , insulin in 19% , and combination therapy in 7% of the patients . at discharge", ", there was no significant shift in the treatment paradigm in the total group ( table 5 ) ." ], [ " this study sheds light on the characteristics of hospitalized patients with diabetes in hospitals serving a high risk for diabetes population .", "we found high percentage of diabetes among the patients hospitalized in the internal medicine wards and cardiac intensive units , reflecting the high prevalence of diabetes in the population .", "39.3% of total patient admitted to the internal medical ward and intensive care unit were diabetic .", "there was a female preponderance among patients admitted with diabetes 52.9% while only 45.0% of patients without diabetes , hospitalized , were women ( p = 0.0003 ) .", "all this reflects the alarming prevalence of obesity and diabetes among adult arab women in northern israel .", "it has been recently reported that the prevalence of overweigh and obesity among arab woman over 40 years of age who attend primary health clinic is 74% [ 79 ] with diabetes near 50% .", "this novel observation is seminal by bringing attention to this specific group of women with high morbidity risk and now observing high rate of hospitalization . interestingly ,", "diabetic women tend to have similar prevalence of coronary heart disease as main indication for admission , as for nondiabetic men ( 32.9% versus 34.2% , resp . ) . as cardiovascular disease and urinary tract infection were more prevalent as an indication for hospitalization in the diabetic group , prevention of cardiovascular disease and urinary tract infection", "should therefore be reinforced particularly in the group of arab diabetic women within diabetic population . ", "patients with diabetes had a significant greater length of stay than hospitalized patients without diabetes , similar to data described in previous publications [ 1012 ] .", "the reasons might be multifactorial , and it is important to note that interventions with chronic disease management programs for outpatients and diabetes team consultation during hospitalization have been proved to be an effective means to reduce the likelihood and duration of hospitalizations for individuals with diabetes . implementing similar strategies in this high risk group and evaluating the impact of this intervention on outcomes of rate and recurrence of hospitalization and hospital length of stay is therefore of paramount importance .", "in this study , we can additionally identify areas for intervention ; as is evident in table 5 , the stay in the hospital did not affect the patients treatment regimens , although glycemic levels at discharge were above the target ( mean glucose levels at discharge > 190 mg / dl ) .", "furthermore , in only a minority of patients , an hba1c measurement was performed during hospitalization .", "these issues reflect the current practice and constraint on hospitalization length and on the performance of blood tests , pushing toward shorter hospital stay and cutting back on performing blood tests not directly relevant to the immediate hospital care .", "it will be of interest to examine whether interventions as described above will cause changes in treatment regimens with improvement in long - term outcomes and readmissions . ", "the glycemic control goals in nonsurgical hospitalized patients continue to be debated , but a consensus defining upper limits below 180 m / dl in noncritical patients is well based on outcomes studies and is therefore recommended by recent position papers .", "average glucose levels during hospitalization were above 180 mg% throughout the hospital stay ( figure 2 ) ; therefore , as noted there is a need for team consultation during hospitalization tailored to hospitals that provide medical care to populations in high risk for diabetes . ", "results of this study show the impact of the high prevalence of diabetes on the hospitalization of arab patients in northern israel , with specific emphasis on the burden of diabetes of the arab women .", "it calls attention to the need of in - hospital consultation team and postdischarge plan .", "information from the cohort described within this study might be applicable to other medical centers providing large population of arab descent [ 1517 ] ." ] ]

[ "the ivy sign might be suggestive of diffuse leptomeningeal collaterals found on post - contrast t1-weighted mr images in patients with moyamoya disease11,19 ) .", "it has also been defined as a continuous linear leptomeningeal high - signal intensity along the cortical sulci and subarachnoid spaces on mr fluid - attenuated inversion recovery ( flair ) images13 ) .", "its proliferation is suggestive of a retrograde slow vascular flow that is highly correlated with the existence of a decreased cerebrovascular reserve ( cvr)2,4,5,10,18 ) .", "accordingly , recent studies have examined the usefulness of mr flair images as a follow - up study after superficial temporal artery ( sta)-middle cerebral artery ( mca ) anastomosis in patients with moyamoya disease6,9 ) .", "kawashima et al.9 ) reported that the ivy sign on mr flair images was a useful indicator in detecting brain hemodynamic changes at a follow - up in patients with moyamoya disease who underwent sta - mca anastomosis .", "but , most of these studies have been conducted in younger patients with a mean age of 20 - 29 years .", "considering the age distribution , the higher incidence of moyamoya disease was observed in adults 45 to 49 years of age and the second in children 5 to 9 years of age , their reliability would be questionable on aspect of age1 ) . given the above background , we conducted this study to examine the usefulness of the ivy sign on mr flair images as compared with single photon emission computerized tomography ( spect ) and mr perfusion studies in detecting brain hemodynamic changes between preoperatively and postoperatively in adult patients ( above 20 years ) with moyamoya disease3,12,17 ) .", "from sep 2010 to dec 2012 , twelve adult patients with moyamoya disease visited our medical institution . in these patients ( average age , 45 years ; range , 23 - 64 years ) , moyamoya disease", "was incidentally found on a regular check - up without notable symptoms or it was detected on examinations for transient ischemic attack ( tia ) or cerebral infarction .", "the suzuki grade of moyamoya disease was analyzed and then compared with the mean ivy score .", "twelve patients and thirteen cases ( one having surgery at both hemispheres ) underwent sta - mca anastomosis .", "our patients had a mean age of 45 years and they underwent sta - mca anastomosis on preoperative and postoperative mr flair images . in each patient , such imaging studies as a mri , a ct angiography , and a spect were performed both preoperatively and postoperatively .", "a board - certified specialist in radiology reviewed the images and rated the ivy sign scores .", "mr perfusion imaging represents a form of functional imaging that assesses alterations in blood flow with additional information on metabolism and regional measures of a specific tracer .", "this technique can be used to find salvageable ischemic brain tissue which represents the \" schemic penumbra \" .", "patients identified with a larger penumbra are more likely to benefit from recanalization therapies making it crucial to properly recognize them23 ) .", "mr perfusion image was performed on 1.5 and 3 t machine ( magnetom skyra , siemens , germany ) with spin echo - planar imaging sequences by using the following parameters : repetition time ( tr)/echo time ( te ) , 1950/30 ms ; flip angle , 90 ; matrix , 128128 ; field of view 220220 ; section thickness 5 mm ; and intersection gap , 1 mm in all patients .", "sixty sections and 25 images per section in all patients were obtained before , during and after the administration of contrast agent .", "contrast agent 0.2 mmol gadopentetate dimeglumine ( multihense , patheon , italy ) per kilogram of body weight at a rate of 3 ml / sec was injected by using an mr imaging - compatible power injector ( spectrics ; medrad , pittsburgh , pa , usa ) .", "this was followed by a 20 ml bolus of saline administered at the same injection rate .", "perfusion maps of cbv were generated after eliminating the effect of contrast agent recirculation by using gamma - variate curve fitting .", "we utilized software program ( numaris/4 , version syngo mr d13 ) for evaluation of cbv .", "mr flair was also performed on a 3 t machine ( magnetom skyra , siemens , germany ) with spin echo - planar imaging sequences .", "protocol imaging included axial flair sequence ( tr : 9000 ms ; te : 95 ms ; field of view 199220 ; section thickness 5 mm ; flip angle , 90 ; matrix size : 512278 ) . in studies of cerebral vascular reserve by spect with the use of n - isopropyl-4-iodoamphetamine ( 123i - imp ) ,", "it is necessary to measure cerebral blood flow at rest and after acetazolamide ( acz ) challenge .", "the spect scanner e - cam ( siemens , germany ) and image processor e - soft ( siemens ) were used .", "the collimator used was a low - energy , ultra - high resolution , fan - beam collimator ( 83 cps / mbq ) .", "conditions for acquisition were as follows : 5-min acquisition ( 7.5 s / step , 3/step , continuous ) , matrix of 128128 ( 3.56 mm / pixel ) , and radius of rotation of 13 cm . a filtered backprojection method using a ramp filter was utilized to reconstruct images from 5-min data .", "as a 3d post - filter , a low - pass filter ( cutoff 0.4 cycles / cm ; order 5.0 ) was used .", "the corticosubcortical area of cerebral hemisphere was divided into the following four regions on each hemisphere : the anterior cerebral artery ( aca ) , the anterior half of the middle cerebral artery ( ant - mca ) , the posterior half of the mca ( post - mca ) , and the posterior cerebral artery ( pca)16 ) ( fig .", "1 ) . the central sulcus is a landmark that separates between the ant - mca regions and the post - mca ones .", "we compared both flair images obtained preoperatively and postoperatively based on the ivy sign scores , for which we used a 3-grade system : grade 0 ( negative ; no ivy sign ) , grade 1 ( minimal ; the ivy sign is defined as minimal or equivocal high signal intensity ) and grade 2 ( positive ; the ivy sign is obviously linear and punctate high signal intensity ) .", "the spect results were demonstrated using 20 levels of color ranging from black to white .", "we give the score from 0 to 100 points , it means that each color was given 5 points .", "this scoring system was possible because the 20 levels of color spectrum is exactly specified in the spect image and this result was confirmed the radiologist .", "when there was a sufficient cerebral perfusion , it was shown as red colors with an intensity of 60 to 80 .", "we applied cvr which results of post - administration of acetazolamide to this study because that it is significantly associated with an increased risk of stroke recurrence in patients with symptomatic cerebral infarction8 ) .", "we analyzed the relationship between the ivy sign on mr flair images and cvr on spect ones and then compared them between prior to and following the sta - mca anastomosis .", "the statistical analysis was performed with the student t - test for correlations between patients with an ivy sign on mr flair and cvr on spect .", "the difference was considered to be significant if the p value was less than 5% ( p<0.05 ) .", "from sep 2010 to dec 2012 , twelve adult patients with moyamoya disease visited our medical institution . in these patients ( average age , 45 years ; range , 23 - 64 years ) , moyamoya disease", "was incidentally found on a regular check - up without notable symptoms or it was detected on examinations for transient ischemic attack ( tia ) or cerebral infarction .", "the suzuki grade of moyamoya disease was analyzed and then compared with the mean ivy score .", "twelve patients and thirteen cases ( one having surgery at both hemispheres ) underwent sta - mca anastomosis .", "our patients had a mean age of 45 years and they underwent sta - mca anastomosis on preoperative and postoperative mr flair images .", "in each patient , such imaging studies as a mri , a ct angiography , and a spect were performed both preoperatively and postoperatively .", "a board - certified specialist in radiology reviewed the images and rated the ivy sign scores .", "mr perfusion imaging represents a form of functional imaging that assesses alterations in blood flow with additional information on metabolism and regional measures of a specific tracer .", "this technique can be used to find salvageable ischemic brain tissue which represents the \" schemic penumbra \" .", "patients identified with a larger penumbra are more likely to benefit from recanalization therapies making it crucial to properly recognize them23 ) .", "mr perfusion image was performed on 1.5 and 3 t machine ( magnetom skyra , siemens , germany ) with spin echo - planar imaging sequences by using the following parameters : repetition time ( tr)/echo time ( te ) , 1950/30 ms ; flip angle , 90 ; matrix , 128128 ; field of view 220220 ; section thickness 5 mm ; and intersection gap , 1 mm in all patients .", "sixty sections and 25 images per section in all patients were obtained before , during and after the administration of contrast agent .", "contrast agent 0.2 mmol gadopentetate dimeglumine ( multihense , patheon , italy ) per kilogram of body weight at a rate of 3 ml / sec was injected by using an mr imaging - compatible power injector ( spectrics ; medrad , pittsburgh , pa , usa ) . this was followed by a 20 ml bolus of saline administered at the same injection rate .", "perfusion maps of cbv were generated after eliminating the effect of contrast agent recirculation by using gamma - variate curve fitting .", "we utilized software program ( numaris/4 , version syngo mr d13 ) for evaluation of cbv .", "mr flair was also performed on a 3 t machine ( magnetom skyra , siemens , germany ) with spin echo - planar imaging sequences .", "protocol imaging included axial flair sequence ( tr : 9000 ms ; te : 95 ms ; field of view 199220 ; section thickness 5 mm ; flip angle , 90 ; matrix size : 512278 ) . in studies of cerebral vascular reserve by spect with the use of n - isopropyl-4-iodoamphetamine ( 123i - imp ) , it is necessary to measure cerebral blood flow at rest and after acetazolamide ( acz ) challenge .", "the spect scanner e - cam ( siemens , germany ) and image processor e - soft ( siemens ) were used .", "the collimator used was a low - energy , ultra - high resolution , fan - beam collimator ( 83 cps / mbq ) .", "conditions for acquisition were as follows : 5-min acquisition ( 7.5 s / step , 3/step , continuous ) , matrix of 128128 ( 3.56 mm / pixel ) , and radius of rotation of 13 cm .", "a filtered backprojection method using a ramp filter was utilized to reconstruct images from 5-min data .", "as a 3d post - filter , a low - pass filter ( cutoff 0.4 cycles / cm ; order 5.0 ) was used .", "the corticosubcortical area of cerebral hemisphere was divided into the following four regions on each hemisphere : the anterior cerebral artery ( aca ) , the anterior half of the middle cerebral artery ( ant - mca ) , the posterior half of the mca ( post - mca ) , and the posterior cerebral artery ( pca)16 ) ( fig .", "1 ) . the central sulcus is a landmark that separates between the ant - mca regions and the post - mca ones .", "we compared both flair images obtained preoperatively and postoperatively based on the ivy sign scores , for which we used a 3-grade system : grade 0 ( negative ; no ivy sign ) , grade 1 ( minimal ; the ivy sign is defined as minimal or equivocal high signal intensity ) and grade 2 ( positive ; the ivy sign is obviously linear and punctate high signal intensity ) .", "the spect results were demonstrated using 20 levels of color ranging from black to white .", "we give the score from 0 to 100 points , it means that each color was given 5 points .", "this scoring system was possible because the 20 levels of color spectrum is exactly specified in the spect image and this result was confirmed the radiologist .", "when there was a sufficient cerebral perfusion , it was shown as red colors with an intensity of 60 to 80 .", "we applied cvr which results of post - administration of acetazolamide to this study because that it is significantly associated with an increased risk of stroke recurrence in patients with symptomatic cerebral infarction8 ) .", "we analyzed the relationship between the ivy sign on mr flair images and cvr on spect ones and then compared them between prior to and following the sta - mca anastomosis .", "the statistical analysis was performed with the student t - test for correlations between patients with an ivy sign on mr flair and cvr on spect .", "the difference was considered to be significant if the p value was less than 5% ( p<0.05 ) .", "thirteen cases of twelve patients , one patient to undergo surgery , bilateral hemispheres , were diagnosed with moyamoya disease on angiography and they underwent sta - mca anastomosis ( table 1 ) .", "with that as a result of the sum of cvr in all patients , post - contrast image , on the spect images was 3035 preoperatively and 3480 postoperatively ( p<0.05 ) , the surgery was successful in all patients . among these patients ,", "the cerebral hemisphere was divided into four regions : the aca , ant - mca , post - mca , and pca .", "thus , 4 regions of 13 cases , a total of 52 regions were selected .", "we compared changes in the sum of cvr between preoperatively and postoperatively in each 4 regions .", "we aimed to proof of the ivy sign could be another marker of hemodynamic change , so the analysis of preoperative and postoperative change of ivy sign is the most important topic in this reports . but ivy sign was not observed on whole region of hemisphere , so we selected the slice of mr flair images which demonstrated the typical ivy signs and compared that with cvr on the same slice of spect images .", "this showed that cvr was increased from 745 to 815 in the aca region ( p<0.05 ) , from 720 to 885 ( p<0.05 ) in the ant - mca region , and from 710 to 895 ( p<0.05 ) in the post - mca region .", "these results indicate that there was a significant increase in the sum of cvr in all of these three regions . in the pca region , however , it was increased from 860 to 885 but this difference did not reach a statistical significance .", "to evaluate the usefulness of ivy sign at a follow - up , we compared ivy sign changes on mr flair images between preoperatively and postoperatively with cvr changes on spect images between prior to and following the sta - mca anastomosis7,19 ) .", "all of the patients who showed the minimal or positive ivy sign were identified in 18 of 24 hemispheres ( 75% ) .", "preoperative and postoperative mr flair images were taken at 46.7 days on average ( range , 8 - 99 days ) and at 196.6 days ( range , 33 - 368 days ) , respectively .", "minimal or positive ivy signs were found in 8 aca regions , 13 ant - mca regions , 19 post - mca regions and 3 pca regions . of these regions ,", "the ivy signs were predominantly recorded from the mca territory ; their prevalence was the highest in the post - mca region . by the corticosubcortical area of cerebral hemisphere after sta - mca anastomosis , ivy score was 8 and 4 in the aca regions , 13 and 4 in the ant - mca regions and 19 and 9 in the post - mca regions .", "all of these differences reached a statistical significance ( p<0.05 ) . in the pca regions ,", "however , there was no significant difference in the sum of ivy score between preoperatively ( 3 ) and postoperatively ( 3 ) .", "in particular , the sum of ivy score was significantly higher in the post - mca region as compared with other regions and the degree of the reduction in the sum of ivy score was also the highest in the post - mca region .", "we compared these results with cvr scores between preoperatively and postoperatively on spect images and checked these correlation outcomes reached a statistical significance ( p<0.05 ) ( table 2 ) .", "we compared ivy sign changes on mr flair images with mr perfusion images between prior to and following the sta - mca anastomosis .", "we selected the same number of the slice of mr perfusion images with the slice of mr flair image , same methods as compared mr flair with spect , which demonstrated the typical ivy signs .", "we compared on mr perfusion images , the degree of change of mean transit time ( mtt ) was higher in the regions with positive or minimal ivy sign as compared with those with negative one .", "after sta - mca anastomosis , it was also demonstrated that the hemodynamic profile was improved in the regions both spect ( fig .", "4 ) and perfusion mri images where the ivy sign was missing or decreased ( fig .", "we divided into three groups on patients clinical states ; asymptomatic groups , tia and complete stroke group and checked the change of ivy score by clinical symptoms .", "the mean preoperative ivy score was 2 points in the asymptomatic group , 3 points in the tia group and 5.2 points in the complete stroke group , thus indicating that higher ivy score was noticed on the patients with more severe clinical symptoms .", "to date , numerous studies have evaluated brain hemodynamic changes . since brain hemodynamic changes were first measured based on co2 reactivity in a published study , the spect has been regarded as a standard diagnostic modality where the cvr is measured based on the iatrogenic metabolic effect by using an intravenous administration of acetazolamide20,21,23 ) . in addition , the results of mtt on mr perfusion are also useful to determine the hemodynamic change in moyamoya patients12,13 ) .", "ohta et al.19 ) first reported that the ivy sign is a diffuse leptomeningeal enhancement found on t1-weighted mr images of patients with moyamoya disease in childhood .", "based on published studies supporting the applicability of ivy sign on t1-weighted mr images in measuring brain hemodynamic changes have been conducted up to present11 ) .", "but , there are some reports that indicated the side effects occur during the spect or mr perfusion , t1-weighted mri including discomfort to patients . according to settakis et al.22 ) , side effects such as headache and discomfort may occur as a result of the activation of acz . in patients with a low hemodynamic state", ", there is also a possibility that the cbf might be further reduced with the administration of acz because of the vascular steal phenomenon in the adjacent area where the cvr is relatively preserved .", "special attention should therefore be paid to the administration of acz in patients with severe ischemic symptoms .", "mr perfusion and t1-weighted mri also requires administration of contrast agent which eventually require the patient should be evaluated via an intravenous route in a fasting state , so patients should keep the empty stomach for several hours . according to recent studies , the ivy sign on mr flair images showed a new diagnostic value in detecting brain hemodynamic changes10,13 ) .", "several studies had shown that the ivy sign is also seen on mr falir images and it is suggestive of brain hemodynamic status in patients with moyamoya disease .", "we assume that mr flair would be more useful in performing a follow - up evaluation as compared with spect or perfusion mri , t1-weighted mri study because it causes less discomfort to patients with moyamoya disease15,22 ) .", "the mr flair study is advantageous in that it can be easily performed in a clinical setting .", "as compared with spect or mr perfusion , t1-weighted mri study , the mr flair imaging is more advantageous in that it takes less than ten minutes , causes a less side effects , do not need to feel hunger and is a non - invasive modality .", "based on the published studies about the usefulness of the ivy sign on mr flair images in evaluating brain hemodynamic changes , further studies have been conducted to examine whether it is also a useful indicator at a follow - up in patients who underwent sta - mca anastomosis . in 2010 , kawashima et al.9 ) used the ivy sign on mr flair images for a follow - up study of patients with moyamoya disease who underwent sta - mca anastomosis , thus reporting that the ivy sign was a useful indicator in evaluating the preoperative and postoperative hemodynamic status of brain .", "ideguchi et al.6 ) reported that the ivy sign is a non - invasive indicator in detecting brain hemodynamic changes through a comparison between spect and mr flair study .", "however , it should be noted that the above studies enrolled relatively younger patients ; kawashima et al . enrolled patients with a mean age of 25 years and ideguchi et al . did those with a mean age of 24 years .", "the detection rate of moyamoya disease per year was 0.94 patients per 100000 people , and prevalence was 10.5 patients per 100000 people .", "the incidence of ischemia concerned with the disease was 0.53 patients per 100000 people - years and hemorrhage was 0.2 patients per 100000 people - years . and , considering the age distribution ,", "the highest incidence of moyamoya disease was observed in adults 45 to 49 years of age and the second in children 5 to 9 years of age , their reliability would be questionable on aspect of age , the above two studies have the limitation of subject selection in appropriate age1 ) .", "we performed mr flair study to evaluate the diagnostic value of the ivy sign in adult patients ( average age , 45 years ; range , 23 - 64 years ) .", "thus , our results support that it can be considered as a useful indicator for adult patients with moyamoya disease .", "we underwent sta - mca anastomosis in thirteen cases of twelve patients ( one patient having surgery on bilateral hemispheres ) who were diagnosed with moyamoya disease on angiography .", "all of the surgery was successful as a result of the sum of cvr on the spect images was 3035 preoperatively and 3480 postoperatively ( p<0.05 ) . based on the reports by ideguchi et al.6 ) , we compared the ivy signs between preoperatively and postoperatively in each region .", "our results showed that it was no significant different in hemodynamic state evaluation between mr flair images and spect ones , and based on these results , we conclude that the ivy sign on mr flair images is a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively as much as cvr on spect ones . to assess the accuracy of mr flair , we separated the results by four regions . of four areas (", "aca , ant - mca , post - mca , pca ) , results were significant changes in the ivy sign on mr flair images and the cvr on spect ones postoperatively as compared with preoperatively in both the aca and mca territories and the degree of changes in both indicators was the highest in the mca territory ( p<0.05 ) .", "but , there were no significant differences in two indicators between preoperatively and postoperatively in the pca territory ( table 2 ) .", "these findings can be explained based on the relative preservation of posterior circulation by pca in patients with moyamoya disease , as reported by fujiwara et al.23 ) .", "compared to other reports , although it is not absolute comparison , our reports showed that 75% of regions of cerebral hemispheres were positive for the ivy sign ; it was higher than 64.7% reported by kawashima et al .", "our report was consistent the report by mori et al.14,22 ) , ivy sign was a significant correlation with the severity of symptoms .", "the sum of the ivy scores was 5.2 points in the complete stroke group , 3 points in the tia group and 2 points in the asymptomatic group .", "these results are different from other studies indicating that the ivy sign on mr flair is also a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively in adult patients ( above 20 years ) with moyamoya disease .", "also , mr flair was not performed at constant tesla , some patients have taken mri at 1.5 tesla and others at 3 tesla .", "further studies are warranted to compare the ivy signs between preoperatively and postoperatively in patients presenting with clinical symptoms .", "if there is a significant correlation between the ivy sign and the symptoms , this would be useful to evaluate their prognosis .", "the ivy sign on flair images was present in the regions where both the cerebral perfusion and the cvr were decreased .", "after sta - mca anastomosis , the ivy sign was disappeared or its intensity was decreased in the hemisphere .", "as compared with conventional diagnostic modalities such as spect or mr perfusion , the ivy sign is considered as a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively .", "our results also show that it is also a useful indicator in detecting brain hemodynamic changes in adult patients .", "furthermore , the degree of ivy sign was higher in patients with a greater severity of symptoms .", "further studies are warranted to examine whether the ivy sign is also useful in identifying the improvement of hemodynamic status and assessing the effectiveness of sta - mca anastomosis in adult patients with moyamoya disease ." ]

[ "<S> objectivemr perfusion and single photon emission computerized tomography ( spect ) are well known imaging studies to evaluate hemodynamic change between prior to and following superficial temporal artery ( sta)-middle cerebral artery ( mca ) anastomosis in moyamoya disease . </S>", "<S> but their side effects and invasiveness make discomfort to patients . </S>", "<S> we evaluated the ivy sign on mr fluid attenuated inversion recovery ( flair ) images in adult patients with moyamoya disease and compared it with result of spect and mr perfusion images.methodswe enrolled twelve patients ( thirteen cases ) who were diagnosed with moyamoya disease and underwent sta - mca anastomosis at our medical institution during a period ranging from september of 2010 to december of 2012 . </S>", "<S> the presence of the ivy sign on mr flair images was classified as negative ( 0 ) , minimal ( 1 ) , and positive ( 2 ) . </S>", "<S> regions were classified into four territories : the anterior cerebral artery ( aca ) , the anterior mca , the posterior mca and the posterior cerebral artery.resultsivy signs on preoperative and postoperative mr flair were improved ( 8 and 4 in the aca regions , 13 and 4 in the anterior mca regions and 19 and 9 in the posterior mca regions ) . like this result , the cerebrovascular reserve ( cvr ) on spect was significantly increased in the sum of cvr in same regions after sta - mca anastomosis.conclusionafter sta - mca anastomosis , ivy signs were decreased in the cerebral hemisphere . as compared with conventional diagnostic modalities such as spect and mr perfusion images , the ivy sign on mr flair </S>", "<S> is considered as a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively in adult moyamoya patients . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "Patients", "Imaging studies", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSION" ]

[ [ "the ivy sign might be suggestive of diffuse leptomeningeal collaterals found on post - contrast t1-weighted mr images in patients with moyamoya disease11,19 ) .", "it has also been defined as a continuous linear leptomeningeal high - signal intensity along the cortical sulci and subarachnoid spaces on mr fluid - attenuated inversion recovery ( flair ) images13 ) .", "its proliferation is suggestive of a retrograde slow vascular flow that is highly correlated with the existence of a decreased cerebrovascular reserve ( cvr)2,4,5,10,18 ) .", "accordingly , recent studies have examined the usefulness of mr flair images as a follow - up study after superficial temporal artery ( sta)-middle cerebral artery ( mca ) anastomosis in patients with moyamoya disease6,9 ) .", "kawashima et al.9 ) reported that the ivy sign on mr flair images was a useful indicator in detecting brain hemodynamic changes at a follow - up in patients with moyamoya disease who underwent sta - mca anastomosis .", "but , most of these studies have been conducted in younger patients with a mean age of 20 - 29 years .", "considering the age distribution , the higher incidence of moyamoya disease was observed in adults 45 to 49 years of age and the second in children 5 to 9 years of age , their reliability would be questionable on aspect of age1 ) . given the above background , we conducted this study to examine the usefulness of the ivy sign on mr flair images as compared with single photon emission computerized tomography ( spect ) and mr perfusion studies in detecting brain hemodynamic changes between preoperatively and postoperatively in adult patients ( above 20 years ) with moyamoya disease3,12,17 ) ." ], [ "from sep 2010 to dec 2012 , twelve adult patients with moyamoya disease visited our medical institution . in these patients ( average age , 45 years ; range , 23 - 64 years ) , moyamoya disease", "was incidentally found on a regular check - up without notable symptoms or it was detected on examinations for transient ischemic attack ( tia ) or cerebral infarction .", "the suzuki grade of moyamoya disease was analyzed and then compared with the mean ivy score .", "twelve patients and thirteen cases ( one having surgery at both hemispheres ) underwent sta - mca anastomosis .", "our patients had a mean age of 45 years and they underwent sta - mca anastomosis on preoperative and postoperative mr flair images . in each patient , such imaging studies as a mri , a ct angiography , and a spect were performed both preoperatively and postoperatively .", "a board - certified specialist in radiology reviewed the images and rated the ivy sign scores .", "mr perfusion imaging represents a form of functional imaging that assesses alterations in blood flow with additional information on metabolism and regional measures of a specific tracer .", "this technique can be used to find salvageable ischemic brain tissue which represents the \" schemic penumbra \" .", "patients identified with a larger penumbra are more likely to benefit from recanalization therapies making it crucial to properly recognize them23 ) .", "mr perfusion image was performed on 1.5 and 3 t machine ( magnetom skyra , siemens , germany ) with spin echo - planar imaging sequences by using the following parameters : repetition time ( tr)/echo time ( te ) , 1950/30 ms ; flip angle , 90 ; matrix , 128128 ; field of view 220220 ; section thickness 5 mm ; and intersection gap , 1 mm in all patients .", "sixty sections and 25 images per section in all patients were obtained before , during and after the administration of contrast agent .", "contrast agent 0.2 mmol gadopentetate dimeglumine ( multihense , patheon , italy ) per kilogram of body weight at a rate of 3 ml / sec was injected by using an mr imaging - compatible power injector ( spectrics ; medrad , pittsburgh , pa , usa ) .", "this was followed by a 20 ml bolus of saline administered at the same injection rate .", "perfusion maps of cbv were generated after eliminating the effect of contrast agent recirculation by using gamma - variate curve fitting .", "we utilized software program ( numaris/4 , version syngo mr d13 ) for evaluation of cbv .", "mr flair was also performed on a 3 t machine ( magnetom skyra , siemens , germany ) with spin echo - planar imaging sequences .", "protocol imaging included axial flair sequence ( tr : 9000 ms ; te : 95 ms ; field of view 199220 ; section thickness 5 mm ; flip angle , 90 ; matrix size : 512278 ) . in studies of cerebral vascular reserve by spect with the use of n - isopropyl-4-iodoamphetamine ( 123i - imp ) ,", "it is necessary to measure cerebral blood flow at rest and after acetazolamide ( acz ) challenge .", "the spect scanner e - cam ( siemens , germany ) and image processor e - soft ( siemens ) were used .", "the collimator used was a low - energy , ultra - high resolution , fan - beam collimator ( 83 cps / mbq ) .", "conditions for acquisition were as follows : 5-min acquisition ( 7.5 s / step , 3/step , continuous ) , matrix of 128128 ( 3.56 mm / pixel ) , and radius of rotation of 13 cm . a filtered backprojection method using a ramp filter was utilized to reconstruct images from 5-min data .", "as a 3d post - filter , a low - pass filter ( cutoff 0.4 cycles / cm ; order 5.0 ) was used .", "the corticosubcortical area of cerebral hemisphere was divided into the following four regions on each hemisphere : the anterior cerebral artery ( aca ) , the anterior half of the middle cerebral artery ( ant - mca ) , the posterior half of the mca ( post - mca ) , and the posterior cerebral artery ( pca)16 ) ( fig .", "1 ) . the central sulcus is a landmark that separates between the ant - mca regions and the post - mca ones .", "we compared both flair images obtained preoperatively and postoperatively based on the ivy sign scores , for which we used a 3-grade system : grade 0 ( negative ; no ivy sign ) , grade 1 ( minimal ; the ivy sign is defined as minimal or equivocal high signal intensity ) and grade 2 ( positive ; the ivy sign is obviously linear and punctate high signal intensity ) .", "the spect results were demonstrated using 20 levels of color ranging from black to white .", "we give the score from 0 to 100 points , it means that each color was given 5 points .", "this scoring system was possible because the 20 levels of color spectrum is exactly specified in the spect image and this result was confirmed the radiologist .", "when there was a sufficient cerebral perfusion , it was shown as red colors with an intensity of 60 to 80 .", "we applied cvr which results of post - administration of acetazolamide to this study because that it is significantly associated with an increased risk of stroke recurrence in patients with symptomatic cerebral infarction8 ) .", "we analyzed the relationship between the ivy sign on mr flair images and cvr on spect ones and then compared them between prior to and following the sta - mca anastomosis .", "the statistical analysis was performed with the student t - test for correlations between patients with an ivy sign on mr flair and cvr on spect .", "the difference was considered to be significant if the p value was less than 5% ( p<0.05 ) ." ], [ "from sep 2010 to dec 2012 , twelve adult patients with moyamoya disease visited our medical institution . in these patients ( average age , 45 years ; range , 23 - 64 years ) , moyamoya disease", "was incidentally found on a regular check - up without notable symptoms or it was detected on examinations for transient ischemic attack ( tia ) or cerebral infarction .", "the suzuki grade of moyamoya disease was analyzed and then compared with the mean ivy score .", "twelve patients and thirteen cases ( one having surgery at both hemispheres ) underwent sta - mca anastomosis .", "our patients had a mean age of 45 years and they underwent sta - mca anastomosis on preoperative and postoperative mr flair images ." ], [ "in each patient , such imaging studies as a mri , a ct angiography , and a spect were performed both preoperatively and postoperatively .", "a board - certified specialist in radiology reviewed the images and rated the ivy sign scores .", "mr perfusion imaging represents a form of functional imaging that assesses alterations in blood flow with additional information on metabolism and regional measures of a specific tracer .", "this technique can be used to find salvageable ischemic brain tissue which represents the \" schemic penumbra \" .", "patients identified with a larger penumbra are more likely to benefit from recanalization therapies making it crucial to properly recognize them23 ) .", "mr perfusion image was performed on 1.5 and 3 t machine ( magnetom skyra , siemens , germany ) with spin echo - planar imaging sequences by using the following parameters : repetition time ( tr)/echo time ( te ) , 1950/30 ms ; flip angle , 90 ; matrix , 128128 ; field of view 220220 ; section thickness 5 mm ; and intersection gap , 1 mm in all patients .", "sixty sections and 25 images per section in all patients were obtained before , during and after the administration of contrast agent .", "contrast agent 0.2 mmol gadopentetate dimeglumine ( multihense , patheon , italy ) per kilogram of body weight at a rate of 3 ml / sec was injected by using an mr imaging - compatible power injector ( spectrics ; medrad , pittsburgh , pa , usa ) . this was followed by a 20 ml bolus of saline administered at the same injection rate .", "perfusion maps of cbv were generated after eliminating the effect of contrast agent recirculation by using gamma - variate curve fitting .", "we utilized software program ( numaris/4 , version syngo mr d13 ) for evaluation of cbv .", "mr flair was also performed on a 3 t machine ( magnetom skyra , siemens , germany ) with spin echo - planar imaging sequences .", "protocol imaging included axial flair sequence ( tr : 9000 ms ; te : 95 ms ; field of view 199220 ; section thickness 5 mm ; flip angle , 90 ; matrix size : 512278 ) . in studies of cerebral vascular reserve by spect with the use of n - isopropyl-4-iodoamphetamine ( 123i - imp ) , it is necessary to measure cerebral blood flow at rest and after acetazolamide ( acz ) challenge .", "the spect scanner e - cam ( siemens , germany ) and image processor e - soft ( siemens ) were used .", "the collimator used was a low - energy , ultra - high resolution , fan - beam collimator ( 83 cps / mbq ) .", "conditions for acquisition were as follows : 5-min acquisition ( 7.5 s / step , 3/step , continuous ) , matrix of 128128 ( 3.56 mm / pixel ) , and radius of rotation of 13 cm .", "a filtered backprojection method using a ramp filter was utilized to reconstruct images from 5-min data .", "as a 3d post - filter , a low - pass filter ( cutoff 0.4 cycles / cm ; order 5.0 ) was used .", "the corticosubcortical area of cerebral hemisphere was divided into the following four regions on each hemisphere : the anterior cerebral artery ( aca ) , the anterior half of the middle cerebral artery ( ant - mca ) , the posterior half of the mca ( post - mca ) , and the posterior cerebral artery ( pca)16 ) ( fig .", "1 ) . the central sulcus is a landmark that separates between the ant - mca regions and the post - mca ones .", "we compared both flair images obtained preoperatively and postoperatively based on the ivy sign scores , for which we used a 3-grade system : grade 0 ( negative ; no ivy sign ) , grade 1 ( minimal ; the ivy sign is defined as minimal or equivocal high signal intensity ) and grade 2 ( positive ; the ivy sign is obviously linear and punctate high signal intensity ) .", "the spect results were demonstrated using 20 levels of color ranging from black to white .", "we give the score from 0 to 100 points , it means that each color was given 5 points .", "this scoring system was possible because the 20 levels of color spectrum is exactly specified in the spect image and this result was confirmed the radiologist .", "when there was a sufficient cerebral perfusion , it was shown as red colors with an intensity of 60 to 80 .", "we applied cvr which results of post - administration of acetazolamide to this study because that it is significantly associated with an increased risk of stroke recurrence in patients with symptomatic cerebral infarction8 ) .", "we analyzed the relationship between the ivy sign on mr flair images and cvr on spect ones and then compared them between prior to and following the sta - mca anastomosis ." ], [ "the statistical analysis was performed with the student t - test for correlations between patients with an ivy sign on mr flair and cvr on spect .", "the difference was considered to be significant if the p value was less than 5% ( p<0.05 ) ." ], [ "thirteen cases of twelve patients , one patient to undergo surgery , bilateral hemispheres , were diagnosed with moyamoya disease on angiography and they underwent sta - mca anastomosis ( table 1 ) .", "with that as a result of the sum of cvr in all patients , post - contrast image , on the spect images was 3035 preoperatively and 3480 postoperatively ( p<0.05 ) , the surgery was successful in all patients . among these patients ,", "the cerebral hemisphere was divided into four regions : the aca , ant - mca , post - mca , and pca .", "thus , 4 regions of 13 cases , a total of 52 regions were selected .", "we compared changes in the sum of cvr between preoperatively and postoperatively in each 4 regions .", "we aimed to proof of the ivy sign could be another marker of hemodynamic change , so the analysis of preoperative and postoperative change of ivy sign is the most important topic in this reports . but ivy sign was not observed on whole region of hemisphere , so we selected the slice of mr flair images which demonstrated the typical ivy signs and compared that with cvr on the same slice of spect images .", "this showed that cvr was increased from 745 to 815 in the aca region ( p<0.05 ) , from 720 to 885 ( p<0.05 ) in the ant - mca region , and from 710 to 895 ( p<0.05 ) in the post - mca region .", "these results indicate that there was a significant increase in the sum of cvr in all of these three regions . in the pca region , however , it was increased from 860 to 885 but this difference did not reach a statistical significance .", "to evaluate the usefulness of ivy sign at a follow - up , we compared ivy sign changes on mr flair images between preoperatively and postoperatively with cvr changes on spect images between prior to and following the sta - mca anastomosis7,19 ) .", "all of the patients who showed the minimal or positive ivy sign were identified in 18 of 24 hemispheres ( 75% ) .", "preoperative and postoperative mr flair images were taken at 46.7 days on average ( range , 8 - 99 days ) and at 196.6 days ( range , 33 - 368 days ) , respectively .", "minimal or positive ivy signs were found in 8 aca regions , 13 ant - mca regions , 19 post - mca regions and 3 pca regions . of these regions ,", "the ivy signs were predominantly recorded from the mca territory ; their prevalence was the highest in the post - mca region . by the corticosubcortical area of cerebral hemisphere after sta - mca anastomosis , ivy score was 8 and 4 in the aca regions , 13 and 4 in the ant - mca regions and 19 and 9 in the post - mca regions .", "all of these differences reached a statistical significance ( p<0.05 ) . in the pca regions ,", "however , there was no significant difference in the sum of ivy score between preoperatively ( 3 ) and postoperatively ( 3 ) .", "in particular , the sum of ivy score was significantly higher in the post - mca region as compared with other regions and the degree of the reduction in the sum of ivy score was also the highest in the post - mca region .", "we compared these results with cvr scores between preoperatively and postoperatively on spect images and checked these correlation outcomes reached a statistical significance ( p<0.05 ) ( table 2 ) .", "we compared ivy sign changes on mr flair images with mr perfusion images between prior to and following the sta - mca anastomosis .", "we selected the same number of the slice of mr perfusion images with the slice of mr flair image , same methods as compared mr flair with spect , which demonstrated the typical ivy signs .", "we compared on mr perfusion images , the degree of change of mean transit time ( mtt ) was higher in the regions with positive or minimal ivy sign as compared with those with negative one .", "after sta - mca anastomosis , it was also demonstrated that the hemodynamic profile was improved in the regions both spect ( fig .", "4 ) and perfusion mri images where the ivy sign was missing or decreased ( fig .", "we divided into three groups on patients clinical states ; asymptomatic groups , tia and complete stroke group and checked the change of ivy score by clinical symptoms .", "the mean preoperative ivy score was 2 points in the asymptomatic group , 3 points in the tia group and 5.2 points in the complete stroke group , thus indicating that higher ivy score was noticed on the patients with more severe clinical symptoms ." ], [ "to date , numerous studies have evaluated brain hemodynamic changes . since brain hemodynamic changes were first measured based on co2 reactivity in a published study , the spect has been regarded as a standard diagnostic modality where the cvr is measured based on the iatrogenic metabolic effect by using an intravenous administration of acetazolamide20,21,23 ) . in addition , the results of mtt on mr perfusion are also useful to determine the hemodynamic change in moyamoya patients12,13 ) .", "ohta et al.19 ) first reported that the ivy sign is a diffuse leptomeningeal enhancement found on t1-weighted mr images of patients with moyamoya disease in childhood .", "based on published studies supporting the applicability of ivy sign on t1-weighted mr images in measuring brain hemodynamic changes have been conducted up to present11 ) .", "but , there are some reports that indicated the side effects occur during the spect or mr perfusion , t1-weighted mri including discomfort to patients . according to settakis et al.22 ) , side effects such as headache and discomfort may occur as a result of the activation of acz . in patients with a low hemodynamic state", ", there is also a possibility that the cbf might be further reduced with the administration of acz because of the vascular steal phenomenon in the adjacent area where the cvr is relatively preserved .", "special attention should therefore be paid to the administration of acz in patients with severe ischemic symptoms .", "mr perfusion and t1-weighted mri also requires administration of contrast agent which eventually require the patient should be evaluated via an intravenous route in a fasting state , so patients should keep the empty stomach for several hours . according to recent studies , the ivy sign on mr flair images showed a new diagnostic value in detecting brain hemodynamic changes10,13 ) .", "several studies had shown that the ivy sign is also seen on mr falir images and it is suggestive of brain hemodynamic status in patients with moyamoya disease .", "we assume that mr flair would be more useful in performing a follow - up evaluation as compared with spect or perfusion mri , t1-weighted mri study because it causes less discomfort to patients with moyamoya disease15,22 ) .", "the mr flair study is advantageous in that it can be easily performed in a clinical setting .", "as compared with spect or mr perfusion , t1-weighted mri study , the mr flair imaging is more advantageous in that it takes less than ten minutes , causes a less side effects , do not need to feel hunger and is a non - invasive modality .", "based on the published studies about the usefulness of the ivy sign on mr flair images in evaluating brain hemodynamic changes , further studies have been conducted to examine whether it is also a useful indicator at a follow - up in patients who underwent sta - mca anastomosis . in 2010 , kawashima et al.9 ) used the ivy sign on mr flair images for a follow - up study of patients with moyamoya disease who underwent sta - mca anastomosis , thus reporting that the ivy sign was a useful indicator in evaluating the preoperative and postoperative hemodynamic status of brain .", "ideguchi et al.6 ) reported that the ivy sign is a non - invasive indicator in detecting brain hemodynamic changes through a comparison between spect and mr flair study .", "however , it should be noted that the above studies enrolled relatively younger patients ; kawashima et al . enrolled patients with a mean age of 25 years and ideguchi et al . did those with a mean age of 24 years .", "the detection rate of moyamoya disease per year was 0.94 patients per 100000 people , and prevalence was 10.5 patients per 100000 people .", "the incidence of ischemia concerned with the disease was 0.53 patients per 100000 people - years and hemorrhage was 0.2 patients per 100000 people - years . and , considering the age distribution ,", "the highest incidence of moyamoya disease was observed in adults 45 to 49 years of age and the second in children 5 to 9 years of age , their reliability would be questionable on aspect of age , the above two studies have the limitation of subject selection in appropriate age1 ) .", "we performed mr flair study to evaluate the diagnostic value of the ivy sign in adult patients ( average age , 45 years ; range , 23 - 64 years ) .", "thus , our results support that it can be considered as a useful indicator for adult patients with moyamoya disease .", "we underwent sta - mca anastomosis in thirteen cases of twelve patients ( one patient having surgery on bilateral hemispheres ) who were diagnosed with moyamoya disease on angiography .", "all of the surgery was successful as a result of the sum of cvr on the spect images was 3035 preoperatively and 3480 postoperatively ( p<0.05 ) . based on the reports by ideguchi et al.6 ) , we compared the ivy signs between preoperatively and postoperatively in each region .", "our results showed that it was no significant different in hemodynamic state evaluation between mr flair images and spect ones , and based on these results , we conclude that the ivy sign on mr flair images is a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively as much as cvr on spect ones . to assess the accuracy of mr flair , we separated the results by four regions . of four areas (", "aca , ant - mca , post - mca , pca ) , results were significant changes in the ivy sign on mr flair images and the cvr on spect ones postoperatively as compared with preoperatively in both the aca and mca territories and the degree of changes in both indicators was the highest in the mca territory ( p<0.05 ) .", "but , there were no significant differences in two indicators between preoperatively and postoperatively in the pca territory ( table 2 ) .", "these findings can be explained based on the relative preservation of posterior circulation by pca in patients with moyamoya disease , as reported by fujiwara et al.23 ) .", "compared to other reports , although it is not absolute comparison , our reports showed that 75% of regions of cerebral hemispheres were positive for the ivy sign ; it was higher than 64.7% reported by kawashima et al .", "our report was consistent the report by mori et al.14,22 ) , ivy sign was a significant correlation with the severity of symptoms .", "the sum of the ivy scores was 5.2 points in the complete stroke group , 3 points in the tia group and 2 points in the asymptomatic group .", "these results are different from other studies indicating that the ivy sign on mr flair is also a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively in adult patients ( above 20 years ) with moyamoya disease .", "also , mr flair was not performed at constant tesla , some patients have taken mri at 1.5 tesla and others at 3 tesla .", "further studies are warranted to compare the ivy signs between preoperatively and postoperatively in patients presenting with clinical symptoms .", "if there is a significant correlation between the ivy sign and the symptoms , this would be useful to evaluate their prognosis ." ], [ "the ivy sign on flair images was present in the regions where both the cerebral perfusion and the cvr were decreased .", "after sta - mca anastomosis , the ivy sign was disappeared or its intensity was decreased in the hemisphere .", "as compared with conventional diagnostic modalities such as spect or mr perfusion , the ivy sign is considered as a useful indicator in detecting brain hemodynamic changes between preoperatively and postoperatively .", "our results also show that it is also a useful indicator in detecting brain hemodynamic changes in adult patients .", "furthermore , the degree of ivy sign was higher in patients with a greater severity of symptoms .", "further studies are warranted to examine whether the ivy sign is also useful in identifying the improvement of hemodynamic status and assessing the effectiveness of sta - mca anastomosis in adult patients with moyamoya disease ." ] ]

[ "although alzheimer 's disease ( ad ) and its main brain histopathology , that is , senile plaques and \n neurofibrillary tangles ( nfts ) , were described a century ago , \n significant research advances in the disease began only a few \n decades ago .", "the discoveries of the major protein components of \n senile plaques as amyloid -peptide [ 1 , 2 ] and of nfts \n as abnormally hyperphosphorylated tau [ 3 , 4 ] in the 1980s \n initiated a new era of ad research .", "since then , much research has \n focused on the molecular mechanisms of initiation and formation of \n the senile plaques and nfts and their roles in the pathogenesis of \n ad .", "evidence accumulated in the last two decades indicates that \n malprocessing of both tau and -amyloid precursor protein , \n which produces -peptide , is pivotal , if not central , to the \n molecular mechanism of ad .", "the severity of dementia symptoms in ad \n strongly correlates to the number of nfts , but not of senile \n plaques , in ad brains [ 59 ] , suggesting that tau \n pathology might be associated with the disease mechanism more \n directly .", "abnormal hyperphosphorylation of tau and its deposits in \n the brain is also seen in several other neurodegenerative diseases \n that are collectively named tauopathies ( for review , see \n [ 10 , 11 ] ) .", "the discovery of tau mutations that cause \n hereditary frontotemporal dementia and parkinsonism linked to \n chromosome 17 ( ftdp-17 ) [ 1214 ] further indicates that \n tau abnormality alone is sufficient to produce dementia . \n", "therefore , for developing rational therapeutic \n treatment of ad , it is essential to understand the molecular \n mechanism by which tau abnormalities lead to neurofibrillary \n degeneration . because tau aggregated in the brain of ad and all other \n tauopathies", "is always abnormally hyperphosphorylated , numerous \n studies have focused on the roles of the abnormal \n hyperphosphorylation and the mechanism leading to tau \n hyperphosphorylation .", "recent studies demonstrate that it is the \n abnormal hyperphosphorylation that makes tau lose its normal \n function to stimulate microtubule assembly , gain toxic activity , \n and aggregate into nfts [ 1523 ] . in \n addition to tau ,", "several other brain proteins such as \n neurofilaments , microtubule - associated protein ( map ) 1 b , \n -tubulin , and -catenin are also found to be \n hyperphosphorylated [ 2427 ] , suggesting that the \n protein phosphorylation / dephosphorylation system might be \n dysregulated in ad brain .", "because abnormally \n hyperphosphorylated tau is pivotal to ad and has been extensively \n studied , this review focuses on tau hyperphosphorylation . \n", "prevention and reversal of abnormal hyperphosphorylation of tau as \n a potential promising therapeutic strategy is also discussed .", "tau was first discovered by weingarten et al as a microtubule - associated protein that stimulates \n microtubule assembly .", "there was not much research \n interest in tau protein until a decade later , when it was found to \n make up the paired helical filaments ( phfs ) that form nfts in ad \n brain [ 3 , 4 , 29 ] .", "human tau gene was found on the long arm of \n chromosome 17 ( position 17q21 ) and was found to contain 16 exons \n .", "this single tau gene encodes six tau isoforms in adult \n human brain as a result of alternative splicing of its mrna \n .", "the six isoforms of tau differ from each other by the \n presence or absence of one or two inserts ( 29 or 58 amino acids ) \n in the n - terminal part and by the presence of either three or \n four repeats in the c - terminal half .", "the repeats in the c - terminal half of tau are \n the domains that bind to microtubules [ 3234 ] .", "the region \n upstream of the microtubule - binding domains contains many proline \n residues and , hence , is called the proline - rich region .", "the best - known biological functions of tau are to stimulate \n microtubule assembly and to stabilize microtubule structure .", "tau \n binds to microtubules via its microtubule - binding domains located \n at the c - terminal half of the molecule [ 3234 ] .", "the \n n - terminal part projects from the microtubule surface , where it \n may interact with other cytoskeletal elements and the plasma \n membrane [ 35 , 36 ] .", "each of the six tau isoforms possibly has \n its particular physiological roles and differential biological \n activities , because they are differentially expressed during \n development and have different activities to stimulate microtubule \n assembly [ 37 , 38 ] .", "only the shortest isoform of tau is \n expressed in fetal brain , whereas all six isoforms are seen in \n adult brain [ 39 , 40 ] .", "in addition to stimulating microtubule \n assembly , several studies have suggested that tau may have other \n physiological functions .", "it appears to interfere with binding of \n kinesin and kinesin - like motors to microtubules , leading to a \n preferential inhibition of plus - end - directed axonal transport \n .", "overexpression of tau inhibits kinesin - dependent \n trafficking of vesicles , mitochondria , and endoplasmic reticulum \n .", "this may explain the symptoms of amyotrophic lateral \n sclerosis with neurofilament accumulation in motor neurons of \n several transgenic models of tau overexpression \n [ 4346 ] .", "tau has been found to interact with \n mitochondria , plasma membrane , and nucleic \n acids [ 48 , 49 ] , suggesting that it may act as a mediator \n between microtubules and these organelles .", "tau also appears to \n interact with src - family nonreceptor tyrosine kinases such as fyn \n [ 50 , 51 ] and phospholipase c- [ 52 , 53 ] via \n its proline - rich region .", "these data suggest that tau may also play \n a role in the signal transduction pathways involving src - family \n tyrosine kinases and phospholipase c-. however , the physiological significance of these interactions remains to be \n elucidated . as early as 1977", ", tau was found to be a phosphoprotein . \n in 1984 , it was demonstrated that phosphorylation of tau \n negatively regulates its activity in promoting microtubule \n assembly . because tau is abnormally hyperphosphorylated \n in ad and other tauopathies , tau phosphorylation has been studied \n extensively .", "normal brain tau contains 2 or 3 moles of phosphates \n per mole of tau [ 5658 ] .", "studies on human brain biopsy \n tissue indicated that several serine and threonine residues of tau \n are normally phosphorylated at substoichiometrical levels \n [ 59 , 60 ]", ". a normal level of phosphorylation appears to be \n required for tau 's optimal function , whereas the \n hyperphosphorylated tau loses its biological activity [ 15 , \n 16 , 6169 ] .", "the discovery that tau aggregated in ad brain is abnormally \n hyperphosphorylated has stimulated many studies on the extent and \n sites of tau hyperphosphorylation and their role in the \n pathogenesis of ad .", "the phosphorylation level of tau isolated from \n autopsied ad brains is 3- to 4-fold higher than that from normal \n human brains [ 5658 ] .", "in addition , the hyperphosphorylated \n tau is accumulated in both brains [ 70 , 71 ] and cerebral \n spinal fluid [ 7280 ] of individuals with \n ad .", "all six isoforms of tau are aggregated into phfs in the \n abnormally hyperphosphorylated forms in ad brains [ 3 , 4 , 31 , 81 ] . to date , at least 37 serine and threonine residues have been \n found to be phosphorylated in phf - tau ( for review , see ) . \n", "these residues include thr39 , ser46 , thr69 , thr123 , ser137 , \n thr153 , thr175 , thr181 , ser198 , ser199 , ser202 , thr205 , ser208 , \n ser210 , thr212 , ser214 , thr217 , thr231 , ser235 , ser237 , ser238 , \n ser241 , ser262 , ser285 , ser305 , ser324 , ser352 , ser356 , ser396 , \n ser400 , thr403 , ser404 , ser409 , ser412 , ser413 , ser416 , and \n ser422 .", "many of these residues are also phosphorylated in normal \n human brains without nfts at smaller extents , but they are rapidly \n dephosphorylated during postmortem delay and tissue processing \n [ 59 , 60 ] . however , the phosphate groups at these sites are \n not readily dephosphorylated during the postmortem period and \n tissue processing of ad brain , probably because of the deficient \n protein phosphatase activities [ 8389 ] .", "some of the phosphorylation sites seen in phf - tau are not phosphorylated at all in normal brains .", "because all of the previously identified phosphorylation sites of \n normal tau and phf - tau are at either serine or threonine residues , \n it was thought that tau was phosphorylated only at serine and \n threonine residues .", "however , recent studies suggest that tau in \n developing brain and in ad brain is also phosphorylated at \n tyrosine residues .", "the src - family nonreceptor tyrosine kinase fyn \n can bind to and phosphorylate tau in vitro and in transfected \n cells [ 50 , 51 , 93 ] .", "tyrosine phosphorylated tau at this position is \n also seen immunohistochemically in the brain of transgenic mice \n that express mutated human taup301l . \n", "williamson et al demonstrated that in primary human and rat brain cortical cultures tau is phosphorylated at tyr 29 upon \n treatment with a. the tyrosine phosphorylation of tau \n appears rapid and transient .", "interestingly , antibodies specific to \n tyrosine phosphorylated tau labeled purified phf - tau , but not \n normal tau , suggesting that phf - tau is phosphorylated at the \n tyrosine residues [ 93 , 94 ] .", "in addition , tyr394 was also \n found to be phosphorylated in phf - tau and in tau from fetal \n brains , and the phosphorylation at this site is catalyzed by \n another nonreceptor tyrosine kinase c - abl .", "it is not \n clear if the phosphorylation at any of the above tyrosine residues \n is stoichiometrically significant .", "therefore , whether the tyrosine \n phosphorylation of tau has any pathophysiological relevance \n remains to be elucidated .", "numerous studies have demonstrated the important role of abnormal \n hyperphosphorylation of tau in its aggregation into nfts and in \n alzheimer 's neurofibrillary degeneration . in cultured cells , \n", "hyperphosphorylation of tau after treatment with phosphatase \n inhibitors impairs its activity to bind to microtubules and \n induces filamentous aggregation of tau . \n", "pseudohyperphosphorylated tau that simulates abnormally \n hyperphosphorylated tau by mutation of serine or threonine \n residues into glutamate at selected ad - related sites exerts a \n cytotoxic effect , whereas wild - type tau is neutral . in \n contrast , neurons from tau - knockout mice are resistant to a-induced neurotoxicity .", "overexpression of human tau in \n combination with phosphorylation by drosophila gsk-3 homologue shaggy , but not tau overexpression alone , exacerbates \n tau - induced neurodegeneration and results in the formation of \n nft - like filamentous tau aggregates .", "this study shows a \n causal relationship between tau hyperphosphorylation and \n neurofibrillary degeneration in vivo .", "a study in disabled-1 ( an \n adapter protein ) knockout mice further demonstrates that tau \n hyperphosphorylation causes early death of the animals . \n", "most importantly , tau in inclusions of all tauopathies in human \n and animal models is always hyperphosphorylated ( for reviews , see \n [ 11 , 98 ] ) .", "abnormal hyperphosphorylation of tau appears to \n precede its aggregation into nfts in ad brain [ 57 , 99101 ] .", "taken together , these studies suggest that the \n abnormal hyperphosphorylation of tau is crucial to neurofibrillary \n degeneration in ad and other tauopathies", ". \n the largest isoform of human brain tau ( 441 amino acids ) contains \n 80 serine and threonine residues and five tyrosine residues \n .", "phosphorylation at nearly half of these residues has \n been reported in phf - tau ( see for review ) .", "many studies \n have demonstrated that phosphorylation of tau at different sites \n has different impacts on its biological function and on its \n pathogenic role .", "for instance , a quantitative in vitro study \n demonstrated that phosphorylation of tau at ser262 , thr231 , and \n ser235 inhibits its binding to microtubules by 35% , \n 25% , and 10% , respectively .", "in cultured \n cells , phosphorylation of tau at ser214 and ser262 decreases its \n binding to microtubules and appears to inhibit its assembly to \n filaments . in vitro kinetic studies of", "the binding \n between hyperphosphorylated tau and normal tau suggest that \n phosphorylation of tau at ser199/ser202/thr205 , thr212 , \n thr231/ser235 , ser262/ser356 , and ser422 are among the critical \n phosphorylation sites that convert tau to a toxic molecule to \n sequester normal maps from microtubules .", "further \n phosphorylation at thr231 , ser396 , and ser422 promotes \n self - assembly of tau into filaments .", "similarly , tau mutated at \n ser396 and ser404 ( changing ser into glu ) to mimic phosphoserine \n is more fibrillogenic than wild - type tau , and a tau \n construct in which ser422 is mutated to glu shows a significantly \n increased propensity to aggregate .", "consistent with \n these observations is that mutation of ser422 to ala prevents \n a-induced tau aggregation .", "these results \n suggest that phosphorylation of ser422 may play a key role in tau \n filament formation in vivo . \n ", "an important question is , by what mechanism is the tau abnormality \n involved in the pathological cascades that lead to \n neurodegeneration in ad and other tauopathies . does a \n hyperphosphorylation - induced defect in its activity to stimulate \n microtubule - assembly contribute to cell dysfunction ?", "although \n tau loses its activity to stimulate microtubules , lack of overt \n phenotype of tau knockout transgenic mice suggests that \n it is very unlikely that tau abnormality contributes to \n neurodegeneration via loss of normal function due to its \n hyperphosphorylation . by a series of studies", ", we have found that \n both the abnormally hyperphosphorylated tau isolated from ad brain \n and in vitro hyperphosphorylation tau gain a toxic activity to \n sequester normal tau and other maps , such as map1 and map2 , and \n cause microtubule disassembly [ 16 , 18 , 66 , 108 ] . upon \n dephosphorylation", "polymerization \n of the hyperphosphorylated tau into phfs also abolishes this toxic \n activity ( alonso a et al , unpublished observation ) .", "hence , we \n speculate that the abnormal hyperphosphorylation of tau causes \n neurodegeneration by gain of toxic activity rather than by loss of \n normal activity that can be compensated for by other maps and that \n formation of phfs / nfts from the hyperphosphorylated tau in neurons \n is a defense mechanism by which neurons aim to reduce the toxic \n activity of the abnormally hyperphosphorylated tau .", "conditional \n overexpression of gsk-3 in the transgenic mouse brains \n induces tau hyperphosphorylation and neurodegeneration , but no tau \n aggregation .", "in contrast , there are nfts but no memory \n loss in several lines of tau transgenic mice ( for review , see \n ) .", "this phenomenon is probably common to other diseases \n characterized by abnormal protein aggregates such as huntington \n disease and cardiomyopathy , in which the abnormal , nonfibrillar \n protein oligomers , rather than the aggregates themselves , appear \n to be pathogenic [ 111 , 112 ] .", "to understand the mechanism leading to abnormal \n hyperphosphorylation of tau in ad , protein kinases and \n phosphatases that regulate tau phosphorylation level must be \n identified first . in the last two decades , numerous studies aimed \n to the identification of tau kinases and phosphatases have been \n carried out .", "it was found that in vitro , dozens of \n phosphoseryl / phosphothroenyl protein kinases and most of the major \n protein phosphatases could act on tau protein at various \n phosphorylation sites ( for reviews in detail , see [ 82 , 113 , 114 ] ) .", "tau appears to be a universal substrate for protein kinases \n and phosphatases in vitro .", "this may not be surprising , because \n nearly 20% of the amino acid residues of tau molecule are serines \n and threonines , and nearly 50% of these residues are \n phosphorylated to certain degrees in ad brain ( see for \n review ) .", "however , it is unlikely that all these enzymes that act \n on tau in vitro catalyze tau phosphorylation / dephosphorylation in \n vivo .", "immunohistochemical studies also have shown a colocalization \n of more than a dozen protein kinases and several protein \n phosphatases with nfts of ad brain . as we now know that nfts are \n very sticky structures that can be stained \n immunohistochemically by antibodies to numerous antigens , \n immunohistochemical colocalization with nfts can only support \n other data that indicate a role of the specific protein or enzyme \n in the formation of nfts , but itself can not indicate such a role .", "further studies in cultured cells , in situ , and especially in vivo \n suggest that a few protein kinases and phosphatases may be \n involved in regulation of tau phosphorylation in the brain .", "the \n kinases that most likely play a role in phosphorylation of tau in \n the brain include glycogen synthase kanase-3 ( gsk-3 ) , cyclin - dependent kinase 5 ( cdk5 ) , camp - dependent \n protein kinase ( pka ) , stress - activated protein kinases , and \n calcium / calmodulin - dependent kinase ii ( camk - ii ) .", "the \n sites of tau phosphorylation by these kinases , except \n stress - activity protein kinases , have been summarized in our \n recent review . among protein phosphatases", ", pp2a has been \n shown to be the major tau phosphatase in the brain [ 69 , \n 116120 ] . in a recent study , we compared the catalytic \n kinetics of tau dephosphorylation by various major brain protein \n phosphatases and determined the relative contributions of these \n phosphatases to the regulation of tau phosphorylation \n quantitatively .", "we found that pp2a accounts for 70% of \n the total tau phosphatase activity , whereas pp1 , pp2b , and pp5 \n each accounts for only 10% of the total tau phosphatase \n activity . because pp2b activity is upregulated rather \n than downregulated in ad brain , it is unlikely that it regulates \n tau phosphorylation in vivo .", "accumulated evidence indicates that tau phosphorylation is \n regulated by several protein kinases and that more than one kinase \n might be involved in abnormal hyperphosphorylation of tau in ad \n brain .", "interestingly , gsk-3 phosphorylates tau at both \n prime sites ( ie , tau needs to be primed by phosphorylation with \n other kinases at other sites ) and unprimed sites \n [ 122126 ] . in a cotransfection study", ", \n cho and johnson found that a gsk-3 \n mutant ( gsk-3-r96a ) that only phosphorylates unprimed sites has no \n negative impact on tau 's ability to bind to microtubules , in \n contrast to wild - type gsk-3 , which significantly impairs \n tau 's ability to bind to microtubules .", "further studies demonstrate \n that primed phosphorylation of tau at thr231 by gsk-3 plays a critical role in decreasing tau 's ability to both bind to \n and stabilize microtubules . in rat brains ,", "activation of pka not only induces primed phosphorylation of tau by \n gsk-3 , but also impairs the spatial memory of rats \n [ 124 , 127 ] .", "gsk-3 appears to be regulated by both \n phosphoinositol-3 kinase and protein kinase c \n pathways \n [ 128131 ] .", "an obvious approach to understanding how tau becomes abnormally \n hyperphosphorylated in ad is to study whether tau kinase(s ) or tau \n phosphatase(s ) are dysregulated in ad brain .", "several studies have \n focused on whether the activities and expression of these enzymes \n are altered in ad brain . among protein kinases ,", "cdk5 was reported \n to be upregulated in ad brain by one laboratory , but \n this result was challenged by others [ 133136 ] .", "on \n the other hand , both the activity and the expression of pp2a as \n well as the activities of pp1 and pp5 are decreased in the \n selected areas of ad brain [ 8389 ] .", "consistent with this finding , several other neuronal proteins such as neurofilaments , map1b , -tubulin , and -catenin are \n also hyperphosphorylated in ad brain [ 2427 ] .", "hence , it \n appears that downregulation of the phosphatases , especially of \n pp2a , might underlie the abnormal hyperphosphorylation of tau and \n other proteins in ad brain .", "studies of metabolically active rat \n brain slices and transgenic mice suggest that the downregulation \n of pp2a may produce hyperphosphorylation of tau , not only by the \n deficient dephosphorylation of tau , but also through the \n activation of several pp2a - regulated protein kinases , including \n pka , camk - ii , map kinases , and \n stress - activated protein kinases [ 139141 ] .", "nevertheless , \n inhibition of pp2a activity in animal brain could only induce \n hyperphosphorylation of tau at some of the hyperphosphorylation \n sites seen in phf - tau , but does not result in nfts .", "attempts to \n produce massive tangles of phfs in animal models merely via \n alteration of tau phosphatase and/or kinase activities have not \n yet been successful .", "these observations suggest that the \n downregulation of tau phosphatases in ad brain may be only \n partially responsible for the abnormal hyperphosphorylation of \n tau .", "the causes leading to decreased pp2a activity in ad brain are not \n well understood .", "downregulation of pp2a expression and \n upregulation of pp2a endogenous inhibitor proteins \n i1pp2a and i2pp2a in ad \n brain may both contribute to the downregulation of pp2a activity . \n", "because the activities of pp1 [ 83 , 88 ] and pp5 [ 88 , 89 ] , \n which contribute to regulation of tau phosphorylation to a much \n smaller extent than pp2a , are also decreased in ad \n brain , there might be a common factor that downregulates the \n activities of the major brain protein phosphatases in ad brain .", "in addition to tau kinases and phosphatases , alterations of tau \n itself , the substrate of these enzymes , may also play an important \n role in its abnormal hyperphosphorylation and conversion into \n phfs .", "tau is also modified post - translationally by \n -n - acetylglucosamine ( glcnac ) via a glycosidic bond at \n the hydroxyl groups of serine and/or threonine residues , and this \n modification is called o - glcnacylation [ 143145 ] . \n because o - glcnac could modify the same serine or threonine \n residues of tau as phosphate does and a reciprocal relationship \n between o - glcnacylation and phosphorylation has been seen in \n many proteins ( for review , see ) , o - glcnacylation \n could affect phosphorylation of tau .", "recent studies in various \n systems found that tau phosphorylation is indeed regulated by \n o - glcnacylation inversely [ 144 , 145 , 147 ] .", "most \n interestingly , fasting of mice induces downregulation of tau \n o - glcnacylation , which relies on glucose metabolism to supply \n udp - glcnac as a donor for protein o - glcnacylation , and in turn \n leads to hyperphosphorylation of tau .", "these findings \n led to the novel hypothesis that impaired glucose \n uptake / metabolism in ad brain , which was well established decades \n ago , contributes to the disease pathogenesis via downregulation of \n tau o - glcnacylation and , consequently , upregulation of tau \n phosphorylation that leads to neurofibrillary degeneration \n .", "classical n - linked glycosylation is a modification of protein at \n asparagine residues by oligosaccharides , which normally modifies \n only membrane proteins and secreted proteins .", "tau in ad brain , but \n not in normal human brain , was found to be modified by \n n - glycosylation [ 68 , 149 , 150 ] , and this aberrant tau modification appears to precede and facilitate abnormal \n hyperphosphorylation of tau [ 150152 ] .", "there is no doubt that the abnormality of tau plays a central role \n in neurofibrillary degeneration in ad and other tauopathies .", "a \n critical review of the literature accumulated in the last two \n decades sheds light onto the probable mechanism of neurofibrillary \n degeneration of ad ( figure 1 ) .", "tau is the major microtubule - associated protein of mature neurons \n where it stimulates microtubule assembly and stabilizes \n microtubule structure .", "the phosphorylation level \n of tau is regulated by tau kinases and tau phosphatases , as well \n as by the alteration of tau itself . in ad and", "probably also in \n other tauopathies , metabolic and genetic abnormalities lead to \n dysregulation of signal transduction pathways , which in turn \n causes an imbalance of the phosphorylation / dephosphorylation \n system , that is , downregulation of pp2a in the brain .", "this \n imbalance results in increased phosphorylation ( ie , \n hyperphosphorylation ) of tau .", "the impaired brain glucose \n uptake / metabolism that precedes ad also facilitates \n hyperphosphorylation of tau via downregulation of tau \n o - glcnacylation .", "aberrant n - glycosylation of tau in \n ad brain also makes tau a more favorable substrate for major tau \n kinases and less favorable for tau phosphatases [ 151 , 152 ] , \n thereby facilitating tau hyperphosphorylation . \n ", "the abnormally hyperphosphorylated tau resulting from any of the \n above causes not only loses its biological activity to stimulate \n microtubule assembly , but also becomes a toxic molecule , \n sequesters normal tau , map1 , and map2 , and causes disassembly of \n microtubules .", "the breakdown of the microtubule network in the \n affected neurons compromises axonal transport and leads to \n retrograde degeneration , which in turn results in neuronal death \n and dementia . on the other hand , the abnormally \n hyperphosphorylated tau detached from microtubules is not only \n easier to polymerize into phfs as a result of \n hyperphosphorylation , but it also causes increased intraneuronal \n soluble tau concentration due to sequestration of normal tau from \n microtubules , which further facilitates tau aggregation into phfs . \n", "the polymerized abnormal tau is further modified by \n ubiquitination , glycation , polyamination , nitration , and \n truncation ( for review , see ) , and forms mature \n phfs / nfts . unlike the unpolymerized hyperphosphorylated tau that \n is toxic , phfs / nfts appears to be inert ( alonso a et al unpublished observation ) , but these lesions grow in size \n with disease progression and eventually might physically choke the \n affected neuron to death .", "because neurofibrillary degeneration plays a central role in the \n pathogenesis of ad , one of the most attractive therapeutic targets \n of ad is to inhibit neurofibrillary degeneration .", "as outlined in \n figure 1 , the most promising approaches to achieve \n this goal are to inhibit the abnormal hyperphosphorylation of tau \n and to inhibit its sequestration of normal maps .", "the former \n approach is more effective since it should both rescue the \n disruption of microtubule and axoplasmic flow and prevent further \n deposition of nfts .", "several academic groups and pharmaceutical \n companies have been investigating this approach by restoring pp2a \n activity or inhibiting tau kinase activity in the brain . \n", "memantine , a low - to - moderate - affinity antagonist of nmda receptor , \n which improves mental function and the quality of daily life of \n individuals with moderate to severe ad [ 153 , 154 ] , reverses \n the okadaic - acid - induced inhibition of pp2a activity and prevents \n tau hyperphosphorylation in hippocampal slice cultures from adult \n rats .", "the restoration of pp2a activity to normal level \n by memantine also leads to restoration of the expression of map2 \n in the neuropil and a reversal of hyperphosphorylation and \n accumulation of neurofilaments .", "wang 's group has demonstrated that \n treatment of brain slices and rats with melatonin can restore pp2a \n activity that is inhibited by okadaic acid or calyculin a and \n reverse hyperphosphorylation of tau and neurofilament proteins as \n well as cytotoxicities [ 156158 ] .", "melatonin also prevents \n tau hyperphosphorylation and aggregation induced by overactivation \n of gsk-3 or pka [ 131 , 159 ] .", "these are examples showing that \n inhibition of dysregulation of protein \n phosphorylation / dephosphorylation is a promising target to treat \n ad .", "further investigation of new compounds that can inhibit \n abnormal hyperphosphorylation of tau will likely provide new \n treatments for ad .", "maps , microtubule - associated proteins ; mts , microtubules ; phfs , paired helical filaments ; nfts , neurofibrillary tangles ." ]

[ "<S> studies during the last two decades have provided new insights \n into the molecular mechanism of alzheimer 's disease ( ad ) . </S>", "<S> one of \n the milestone findings in ad research was the demonstration that \n neurofibrillary degeneration characterized by tau pathology is \n central to the pathogenesis of ad and other tauopathies and that \n abnormal hyperphosphorylation of tau is pivotal to neurofibrillary \n degeneration . </S>", "<S> this article reviews the recent research advances in \n tau pathology and the underlying dysregulation of the protein \n phosphorylation / dephosphorylation system . </S>", "<S> an updated model of the \n mechanism of neurofibrillary degeneration is also presented , and a \n promising therapeutic target to treat ad by correcting \n dysregulation of protein phosphorylation / dephosphorylation is \n discussed . </S>" ]

[ "INTRODUCTION", "TAU PROTEIN", "ABNORMAL HYPERPHOSPHORYLATION OF TAU IN AD BRAIN", "IMBALANCE OF PHOSPHORYLATION/DEPHOSPHORYLATION IN AD BRAIN", "MECHANISM OF NEUROFIBRILLARY DEGENERATION", "THERAPEUTIC TARGET TO TREAT AD BY CORRECTING DYSREGULATION OF PROTEIN PHOSPHORYLATION/DEPHOSPHORYLATION", "Figures and Tables" ]

[ [ "although alzheimer 's disease ( ad ) and its main brain histopathology , that is , senile plaques and \n neurofibrillary tangles ( nfts ) , were described a century ago , \n significant research advances in the disease began only a few \n decades ago .", "the discoveries of the major protein components of \n senile plaques as amyloid -peptide [ 1 , 2 ] and of nfts \n as abnormally hyperphosphorylated tau [ 3 , 4 ] in the 1980s \n initiated a new era of ad research .", "since then , much research has \n focused on the molecular mechanisms of initiation and formation of \n the senile plaques and nfts and their roles in the pathogenesis of \n ad .", "evidence accumulated in the last two decades indicates that \n malprocessing of both tau and -amyloid precursor protein , \n which produces -peptide , is pivotal , if not central , to the \n molecular mechanism of ad .", "the severity of dementia symptoms in ad \n strongly correlates to the number of nfts , but not of senile \n plaques , in ad brains [ 59 ] , suggesting that tau \n pathology might be associated with the disease mechanism more \n directly .", "abnormal hyperphosphorylation of tau and its deposits in \n the brain is also seen in several other neurodegenerative diseases \n that are collectively named tauopathies ( for review , see \n [ 10 , 11 ] ) .", "the discovery of tau mutations that cause \n hereditary frontotemporal dementia and parkinsonism linked to \n chromosome 17 ( ftdp-17 ) [ 1214 ] further indicates that \n tau abnormality alone is sufficient to produce dementia . \n", "therefore , for developing rational therapeutic \n treatment of ad , it is essential to understand the molecular \n mechanism by which tau abnormalities lead to neurofibrillary \n degeneration . because tau aggregated in the brain of ad and all other \n tauopathies", "is always abnormally hyperphosphorylated , numerous \n studies have focused on the roles of the abnormal \n hyperphosphorylation and the mechanism leading to tau \n hyperphosphorylation .", "recent studies demonstrate that it is the \n abnormal hyperphosphorylation that makes tau lose its normal \n function to stimulate microtubule assembly , gain toxic activity , \n and aggregate into nfts [ 1523 ] . in \n addition to tau ,", "several other brain proteins such as \n neurofilaments , microtubule - associated protein ( map ) 1 b , \n -tubulin , and -catenin are also found to be \n hyperphosphorylated [ 2427 ] , suggesting that the \n protein phosphorylation / dephosphorylation system might be \n dysregulated in ad brain .", "because abnormally \n hyperphosphorylated tau is pivotal to ad and has been extensively \n studied , this review focuses on tau hyperphosphorylation . \n", "prevention and reversal of abnormal hyperphosphorylation of tau as \n a potential promising therapeutic strategy is also discussed ." ], [ "tau was first discovered by weingarten et al as a microtubule - associated protein that stimulates \n microtubule assembly .", "there was not much research \n interest in tau protein until a decade later , when it was found to \n make up the paired helical filaments ( phfs ) that form nfts in ad \n brain [ 3 , 4 , 29 ] .", "human tau gene was found on the long arm of \n chromosome 17 ( position 17q21 ) and was found to contain 16 exons \n .", "this single tau gene encodes six tau isoforms in adult \n human brain as a result of alternative splicing of its mrna \n .", "the six isoforms of tau differ from each other by the \n presence or absence of one or two inserts ( 29 or 58 amino acids ) \n in the n - terminal part and by the presence of either three or \n four repeats in the c - terminal half .", "the repeats in the c - terminal half of tau are \n the domains that bind to microtubules [ 3234 ] .", "the region \n upstream of the microtubule - binding domains contains many proline \n residues and , hence , is called the proline - rich region .", "the best - known biological functions of tau are to stimulate \n microtubule assembly and to stabilize microtubule structure .", "tau \n binds to microtubules via its microtubule - binding domains located \n at the c - terminal half of the molecule [ 3234 ] .", "the \n n - terminal part projects from the microtubule surface , where it \n may interact with other cytoskeletal elements and the plasma \n membrane [ 35 , 36 ] .", "each of the six tau isoforms possibly has \n its particular physiological roles and differential biological \n activities , because they are differentially expressed during \n development and have different activities to stimulate microtubule \n assembly [ 37 , 38 ] .", "only the shortest isoform of tau is \n expressed in fetal brain , whereas all six isoforms are seen in \n adult brain [ 39 , 40 ] .", "in addition to stimulating microtubule \n assembly , several studies have suggested that tau may have other \n physiological functions .", "it appears to interfere with binding of \n kinesin and kinesin - like motors to microtubules , leading to a \n preferential inhibition of plus - end - directed axonal transport \n .", "overexpression of tau inhibits kinesin - dependent \n trafficking of vesicles , mitochondria , and endoplasmic reticulum \n .", "this may explain the symptoms of amyotrophic lateral \n sclerosis with neurofilament accumulation in motor neurons of \n several transgenic models of tau overexpression \n [ 4346 ] .", "tau has been found to interact with \n mitochondria , plasma membrane , and nucleic \n acids [ 48 , 49 ] , suggesting that it may act as a mediator \n between microtubules and these organelles .", "tau also appears to \n interact with src - family nonreceptor tyrosine kinases such as fyn \n [ 50 , 51 ] and phospholipase c- [ 52 , 53 ] via \n its proline - rich region .", "these data suggest that tau may also play \n a role in the signal transduction pathways involving src - family \n tyrosine kinases and phospholipase c-. however , the physiological significance of these interactions remains to be \n elucidated . as early as 1977", ", tau was found to be a phosphoprotein . \n in 1984 , it was demonstrated that phosphorylation of tau \n negatively regulates its activity in promoting microtubule \n assembly . because tau is abnormally hyperphosphorylated \n in ad and other tauopathies , tau phosphorylation has been studied \n extensively .", "normal brain tau contains 2 or 3 moles of phosphates \n per mole of tau [ 5658 ] .", "studies on human brain biopsy \n tissue indicated that several serine and threonine residues of tau \n are normally phosphorylated at substoichiometrical levels \n [ 59 , 60 ]", ". a normal level of phosphorylation appears to be \n required for tau 's optimal function , whereas the \n hyperphosphorylated tau loses its biological activity [ 15 , \n 16 , 6169 ] ." ], [ "the discovery that tau aggregated in ad brain is abnormally \n hyperphosphorylated has stimulated many studies on the extent and \n sites of tau hyperphosphorylation and their role in the \n pathogenesis of ad .", "the phosphorylation level of tau isolated from \n autopsied ad brains is 3- to 4-fold higher than that from normal \n human brains [ 5658 ] .", "in addition , the hyperphosphorylated \n tau is accumulated in both brains [ 70 , 71 ] and cerebral \n spinal fluid [ 7280 ] of individuals with \n ad .", "all six isoforms of tau are aggregated into phfs in the \n abnormally hyperphosphorylated forms in ad brains [ 3 , 4 , 31 , 81 ] . to date , at least 37 serine and threonine residues have been \n found to be phosphorylated in phf - tau ( for review , see ) . \n", "these residues include thr39 , ser46 , thr69 , thr123 , ser137 , \n thr153 , thr175 , thr181 , ser198 , ser199 , ser202 , thr205 , ser208 , \n ser210 , thr212 , ser214 , thr217 , thr231 , ser235 , ser237 , ser238 , \n ser241 , ser262 , ser285 , ser305 , ser324 , ser352 , ser356 , ser396 , \n ser400 , thr403 , ser404 , ser409 , ser412 , ser413 , ser416 , and \n ser422 .", "many of these residues are also phosphorylated in normal \n human brains without nfts at smaller extents , but they are rapidly \n dephosphorylated during postmortem delay and tissue processing \n [ 59 , 60 ] . however , the phosphate groups at these sites are \n not readily dephosphorylated during the postmortem period and \n tissue processing of ad brain , probably because of the deficient \n protein phosphatase activities [ 8389 ] .", "some of the phosphorylation sites seen in phf - tau are not phosphorylated at all in normal brains .", "because all of the previously identified phosphorylation sites of \n normal tau and phf - tau are at either serine or threonine residues , \n it was thought that tau was phosphorylated only at serine and \n threonine residues .", "however , recent studies suggest that tau in \n developing brain and in ad brain is also phosphorylated at \n tyrosine residues .", "the src - family nonreceptor tyrosine kinase fyn \n can bind to and phosphorylate tau in vitro and in transfected \n cells [ 50 , 51 , 93 ] .", "tyrosine phosphorylated tau at this position is \n also seen immunohistochemically in the brain of transgenic mice \n that express mutated human taup301l . \n", "williamson et al demonstrated that in primary human and rat brain cortical cultures tau is phosphorylated at tyr 29 upon \n treatment with a. the tyrosine phosphorylation of tau \n appears rapid and transient .", "interestingly , antibodies specific to \n tyrosine phosphorylated tau labeled purified phf - tau , but not \n normal tau , suggesting that phf - tau is phosphorylated at the \n tyrosine residues [ 93 , 94 ] .", "in addition , tyr394 was also \n found to be phosphorylated in phf - tau and in tau from fetal \n brains , and the phosphorylation at this site is catalyzed by \n another nonreceptor tyrosine kinase c - abl .", "it is not \n clear if the phosphorylation at any of the above tyrosine residues \n is stoichiometrically significant .", "therefore , whether the tyrosine \n phosphorylation of tau has any pathophysiological relevance \n remains to be elucidated .", "numerous studies have demonstrated the important role of abnormal \n hyperphosphorylation of tau in its aggregation into nfts and in \n alzheimer 's neurofibrillary degeneration . in cultured cells , \n", "hyperphosphorylation of tau after treatment with phosphatase \n inhibitors impairs its activity to bind to microtubules and \n induces filamentous aggregation of tau . \n", "pseudohyperphosphorylated tau that simulates abnormally \n hyperphosphorylated tau by mutation of serine or threonine \n residues into glutamate at selected ad - related sites exerts a \n cytotoxic effect , whereas wild - type tau is neutral . in \n contrast , neurons from tau - knockout mice are resistant to a-induced neurotoxicity .", "overexpression of human tau in \n combination with phosphorylation by drosophila gsk-3 homologue shaggy , but not tau overexpression alone , exacerbates \n tau - induced neurodegeneration and results in the formation of \n nft - like filamentous tau aggregates .", "this study shows a \n causal relationship between tau hyperphosphorylation and \n neurofibrillary degeneration in vivo .", "a study in disabled-1 ( an \n adapter protein ) knockout mice further demonstrates that tau \n hyperphosphorylation causes early death of the animals . \n", "most importantly , tau in inclusions of all tauopathies in human \n and animal models is always hyperphosphorylated ( for reviews , see \n [ 11 , 98 ] ) .", "abnormal hyperphosphorylation of tau appears to \n precede its aggregation into nfts in ad brain [ 57 , 99101 ] .", "taken together , these studies suggest that the \n abnormal hyperphosphorylation of tau is crucial to neurofibrillary \n degeneration in ad and other tauopathies", ". \n the largest isoform of human brain tau ( 441 amino acids ) contains \n 80 serine and threonine residues and five tyrosine residues \n .", "phosphorylation at nearly half of these residues has \n been reported in phf - tau ( see for review ) .", "many studies \n have demonstrated that phosphorylation of tau at different sites \n has different impacts on its biological function and on its \n pathogenic role .", "for instance , a quantitative in vitro study \n demonstrated that phosphorylation of tau at ser262 , thr231 , and \n ser235 inhibits its binding to microtubules by 35% , \n 25% , and 10% , respectively .", "in cultured \n cells , phosphorylation of tau at ser214 and ser262 decreases its \n binding to microtubules and appears to inhibit its assembly to \n filaments . in vitro kinetic studies of", "the binding \n between hyperphosphorylated tau and normal tau suggest that \n phosphorylation of tau at ser199/ser202/thr205 , thr212 , \n thr231/ser235 , ser262/ser356 , and ser422 are among the critical \n phosphorylation sites that convert tau to a toxic molecule to \n sequester normal maps from microtubules .", "further \n phosphorylation at thr231 , ser396 , and ser422 promotes \n self - assembly of tau into filaments .", "similarly , tau mutated at \n ser396 and ser404 ( changing ser into glu ) to mimic phosphoserine \n is more fibrillogenic than wild - type tau , and a tau \n construct in which ser422 is mutated to glu shows a significantly \n increased propensity to aggregate .", "consistent with \n these observations is that mutation of ser422 to ala prevents \n a-induced tau aggregation .", "these results \n suggest that phosphorylation of ser422 may play a key role in tau \n filament formation in vivo . \n ", "an important question is , by what mechanism is the tau abnormality \n involved in the pathological cascades that lead to \n neurodegeneration in ad and other tauopathies . does a \n hyperphosphorylation - induced defect in its activity to stimulate \n microtubule - assembly contribute to cell dysfunction ?", "although \n tau loses its activity to stimulate microtubules , lack of overt \n phenotype of tau knockout transgenic mice suggests that \n it is very unlikely that tau abnormality contributes to \n neurodegeneration via loss of normal function due to its \n hyperphosphorylation . by a series of studies", ", we have found that \n both the abnormally hyperphosphorylated tau isolated from ad brain \n and in vitro hyperphosphorylation tau gain a toxic activity to \n sequester normal tau and other maps , such as map1 and map2 , and \n cause microtubule disassembly [ 16 , 18 , 66 , 108 ] . upon \n dephosphorylation", "polymerization \n of the hyperphosphorylated tau into phfs also abolishes this toxic \n activity ( alonso a et al , unpublished observation ) .", "hence , we \n speculate that the abnormal hyperphosphorylation of tau causes \n neurodegeneration by gain of toxic activity rather than by loss of \n normal activity that can be compensated for by other maps and that \n formation of phfs / nfts from the hyperphosphorylated tau in neurons \n is a defense mechanism by which neurons aim to reduce the toxic \n activity of the abnormally hyperphosphorylated tau .", "conditional \n overexpression of gsk-3 in the transgenic mouse brains \n induces tau hyperphosphorylation and neurodegeneration , but no tau \n aggregation .", "in contrast , there are nfts but no memory \n loss in several lines of tau transgenic mice ( for review , see \n ) .", "this phenomenon is probably common to other diseases \n characterized by abnormal protein aggregates such as huntington \n disease and cardiomyopathy , in which the abnormal , nonfibrillar \n protein oligomers , rather than the aggregates themselves , appear \n to be pathogenic [ 111 , 112 ] ." ], [ "to understand the mechanism leading to abnormal \n hyperphosphorylation of tau in ad , protein kinases and \n phosphatases that regulate tau phosphorylation level must be \n identified first . in the last two decades , numerous studies aimed \n to the identification of tau kinases and phosphatases have been \n carried out .", "it was found that in vitro , dozens of \n phosphoseryl / phosphothroenyl protein kinases and most of the major \n protein phosphatases could act on tau protein at various \n phosphorylation sites ( for reviews in detail , see [ 82 , 113 , 114 ] ) .", "tau appears to be a universal substrate for protein kinases \n and phosphatases in vitro .", "this may not be surprising , because \n nearly 20% of the amino acid residues of tau molecule are serines \n and threonines , and nearly 50% of these residues are \n phosphorylated to certain degrees in ad brain ( see for \n review ) .", "however , it is unlikely that all these enzymes that act \n on tau in vitro catalyze tau phosphorylation / dephosphorylation in \n vivo .", "immunohistochemical studies also have shown a colocalization \n of more than a dozen protein kinases and several protein \n phosphatases with nfts of ad brain . as we now know that nfts are \n very sticky structures that can be stained \n immunohistochemically by antibodies to numerous antigens , \n immunohistochemical colocalization with nfts can only support \n other data that indicate a role of the specific protein or enzyme \n in the formation of nfts , but itself can not indicate such a role .", "further studies in cultured cells , in situ , and especially in vivo \n suggest that a few protein kinases and phosphatases may be \n involved in regulation of tau phosphorylation in the brain .", "the \n kinases that most likely play a role in phosphorylation of tau in \n the brain include glycogen synthase kanase-3 ( gsk-3 ) , cyclin - dependent kinase 5 ( cdk5 ) , camp - dependent \n protein kinase ( pka ) , stress - activated protein kinases , and \n calcium / calmodulin - dependent kinase ii ( camk - ii ) .", "the \n sites of tau phosphorylation by these kinases , except \n stress - activity protein kinases , have been summarized in our \n recent review . among protein phosphatases", ", pp2a has been \n shown to be the major tau phosphatase in the brain [ 69 , \n 116120 ] . in a recent study , we compared the catalytic \n kinetics of tau dephosphorylation by various major brain protein \n phosphatases and determined the relative contributions of these \n phosphatases to the regulation of tau phosphorylation \n quantitatively .", "we found that pp2a accounts for 70% of \n the total tau phosphatase activity , whereas pp1 , pp2b , and pp5 \n each accounts for only 10% of the total tau phosphatase \n activity . because pp2b activity is upregulated rather \n than downregulated in ad brain , it is unlikely that it regulates \n tau phosphorylation in vivo .", "accumulated evidence indicates that tau phosphorylation is \n regulated by several protein kinases and that more than one kinase \n might be involved in abnormal hyperphosphorylation of tau in ad \n brain .", "interestingly , gsk-3 phosphorylates tau at both \n prime sites ( ie , tau needs to be primed by phosphorylation with \n other kinases at other sites ) and unprimed sites \n [ 122126 ] . in a cotransfection study", ", \n cho and johnson found that a gsk-3 \n mutant ( gsk-3-r96a ) that only phosphorylates unprimed sites has no \n negative impact on tau 's ability to bind to microtubules , in \n contrast to wild - type gsk-3 , which significantly impairs \n tau 's ability to bind to microtubules .", "further studies demonstrate \n that primed phosphorylation of tau at thr231 by gsk-3 plays a critical role in decreasing tau 's ability to both bind to \n and stabilize microtubules . in rat brains ,", "activation of pka not only induces primed phosphorylation of tau by \n gsk-3 , but also impairs the spatial memory of rats \n [ 124 , 127 ] .", "gsk-3 appears to be regulated by both \n phosphoinositol-3 kinase and protein kinase c \n pathways \n [ 128131 ] .", "an obvious approach to understanding how tau becomes abnormally \n hyperphosphorylated in ad is to study whether tau kinase(s ) or tau \n phosphatase(s ) are dysregulated in ad brain .", "several studies have \n focused on whether the activities and expression of these enzymes \n are altered in ad brain . among protein kinases ,", "cdk5 was reported \n to be upregulated in ad brain by one laboratory , but \n this result was challenged by others [ 133136 ] .", "on \n the other hand , both the activity and the expression of pp2a as \n well as the activities of pp1 and pp5 are decreased in the \n selected areas of ad brain [ 8389 ] .", "consistent with this finding , several other neuronal proteins such as neurofilaments , map1b , -tubulin , and -catenin are \n also hyperphosphorylated in ad brain [ 2427 ] .", "hence , it \n appears that downregulation of the phosphatases , especially of \n pp2a , might underlie the abnormal hyperphosphorylation of tau and \n other proteins in ad brain .", "studies of metabolically active rat \n brain slices and transgenic mice suggest that the downregulation \n of pp2a may produce hyperphosphorylation of tau , not only by the \n deficient dephosphorylation of tau , but also through the \n activation of several pp2a - regulated protein kinases , including \n pka , camk - ii , map kinases , and \n stress - activated protein kinases [ 139141 ] .", "nevertheless , \n inhibition of pp2a activity in animal brain could only induce \n hyperphosphorylation of tau at some of the hyperphosphorylation \n sites seen in phf - tau , but does not result in nfts .", "attempts to \n produce massive tangles of phfs in animal models merely via \n alteration of tau phosphatase and/or kinase activities have not \n yet been successful .", "these observations suggest that the \n downregulation of tau phosphatases in ad brain may be only \n partially responsible for the abnormal hyperphosphorylation of \n tau .", "the causes leading to decreased pp2a activity in ad brain are not \n well understood .", "downregulation of pp2a expression and \n upregulation of pp2a endogenous inhibitor proteins \n i1pp2a and i2pp2a in ad \n brain may both contribute to the downregulation of pp2a activity . \n", "because the activities of pp1 [ 83 , 88 ] and pp5 [ 88 , 89 ] , \n which contribute to regulation of tau phosphorylation to a much \n smaller extent than pp2a , are also decreased in ad \n brain , there might be a common factor that downregulates the \n activities of the major brain protein phosphatases in ad brain .", "in addition to tau kinases and phosphatases , alterations of tau \n itself , the substrate of these enzymes , may also play an important \n role in its abnormal hyperphosphorylation and conversion into \n phfs .", "tau is also modified post - translationally by \n -n - acetylglucosamine ( glcnac ) via a glycosidic bond at \n the hydroxyl groups of serine and/or threonine residues , and this \n modification is called o - glcnacylation [ 143145 ] . \n because o - glcnac could modify the same serine or threonine \n residues of tau as phosphate does and a reciprocal relationship \n between o - glcnacylation and phosphorylation has been seen in \n many proteins ( for review , see ) , o - glcnacylation \n could affect phosphorylation of tau .", "recent studies in various \n systems found that tau phosphorylation is indeed regulated by \n o - glcnacylation inversely [ 144 , 145 , 147 ] .", "most \n interestingly , fasting of mice induces downregulation of tau \n o - glcnacylation , which relies on glucose metabolism to supply \n udp - glcnac as a donor for protein o - glcnacylation , and in turn \n leads to hyperphosphorylation of tau .", "these findings \n led to the novel hypothesis that impaired glucose \n uptake / metabolism in ad brain , which was well established decades \n ago , contributes to the disease pathogenesis via downregulation of \n tau o - glcnacylation and , consequently , upregulation of tau \n phosphorylation that leads to neurofibrillary degeneration \n .", "classical n - linked glycosylation is a modification of protein at \n asparagine residues by oligosaccharides , which normally modifies \n only membrane proteins and secreted proteins .", "tau in ad brain , but \n not in normal human brain , was found to be modified by \n n - glycosylation [ 68 , 149 , 150 ] , and this aberrant tau modification appears to precede and facilitate abnormal \n hyperphosphorylation of tau [ 150152 ] ." ], [ "there is no doubt that the abnormality of tau plays a central role \n in neurofibrillary degeneration in ad and other tauopathies .", "a \n critical review of the literature accumulated in the last two \n decades sheds light onto the probable mechanism of neurofibrillary \n degeneration of ad ( figure 1 ) .", "tau is the major microtubule - associated protein of mature neurons \n where it stimulates microtubule assembly and stabilizes \n microtubule structure .", "the phosphorylation level \n of tau is regulated by tau kinases and tau phosphatases , as well \n as by the alteration of tau itself . in ad and", "probably also in \n other tauopathies , metabolic and genetic abnormalities lead to \n dysregulation of signal transduction pathways , which in turn \n causes an imbalance of the phosphorylation / dephosphorylation \n system , that is , downregulation of pp2a in the brain .", "this \n imbalance results in increased phosphorylation ( ie , \n hyperphosphorylation ) of tau .", "the impaired brain glucose \n uptake / metabolism that precedes ad also facilitates \n hyperphosphorylation of tau via downregulation of tau \n o - glcnacylation .", "aberrant n - glycosylation of tau in \n ad brain also makes tau a more favorable substrate for major tau \n kinases and less favorable for tau phosphatases [ 151 , 152 ] , \n thereby facilitating tau hyperphosphorylation . \n ", "the abnormally hyperphosphorylated tau resulting from any of the \n above causes not only loses its biological activity to stimulate \n microtubule assembly , but also becomes a toxic molecule , \n sequesters normal tau , map1 , and map2 , and causes disassembly of \n microtubules .", "the breakdown of the microtubule network in the \n affected neurons compromises axonal transport and leads to \n retrograde degeneration , which in turn results in neuronal death \n and dementia . on the other hand , the abnormally \n hyperphosphorylated tau detached from microtubules is not only \n easier to polymerize into phfs as a result of \n hyperphosphorylation , but it also causes increased intraneuronal \n soluble tau concentration due to sequestration of normal tau from \n microtubules , which further facilitates tau aggregation into phfs . \n", "the polymerized abnormal tau is further modified by \n ubiquitination , glycation , polyamination , nitration , and \n truncation ( for review , see ) , and forms mature \n phfs / nfts . unlike the unpolymerized hyperphosphorylated tau that \n is toxic , phfs / nfts appears to be inert ( alonso a et al unpublished observation ) , but these lesions grow in size \n with disease progression and eventually might physically choke the \n affected neuron to death ." ], [ "because neurofibrillary degeneration plays a central role in the \n pathogenesis of ad , one of the most attractive therapeutic targets \n of ad is to inhibit neurofibrillary degeneration .", "as outlined in \n figure 1 , the most promising approaches to achieve \n this goal are to inhibit the abnormal hyperphosphorylation of tau \n and to inhibit its sequestration of normal maps .", "the former \n approach is more effective since it should both rescue the \n disruption of microtubule and axoplasmic flow and prevent further \n deposition of nfts .", "several academic groups and pharmaceutical \n companies have been investigating this approach by restoring pp2a \n activity or inhibiting tau kinase activity in the brain . \n", "memantine , a low - to - moderate - affinity antagonist of nmda receptor , \n which improves mental function and the quality of daily life of \n individuals with moderate to severe ad [ 153 , 154 ] , reverses \n the okadaic - acid - induced inhibition of pp2a activity and prevents \n tau hyperphosphorylation in hippocampal slice cultures from adult \n rats .", "the restoration of pp2a activity to normal level \n by memantine also leads to restoration of the expression of map2 \n in the neuropil and a reversal of hyperphosphorylation and \n accumulation of neurofilaments .", "wang 's group has demonstrated that \n treatment of brain slices and rats with melatonin can restore pp2a \n activity that is inhibited by okadaic acid or calyculin a and \n reverse hyperphosphorylation of tau and neurofilament proteins as \n well as cytotoxicities [ 156158 ] .", "melatonin also prevents \n tau hyperphosphorylation and aggregation induced by overactivation \n of gsk-3 or pka [ 131 , 159 ] .", "these are examples showing that \n inhibition of dysregulation of protein \n phosphorylation / dephosphorylation is a promising target to treat \n ad .", "further investigation of new compounds that can inhibit \n abnormal hyperphosphorylation of tau will likely provide new \n treatments for ad ." ], [ "maps , microtubule - associated proteins ; mts , microtubules ; phfs , paired helical filaments ; nfts , neurofibrillary tangles ." ] ]

[ "noninfectious rhinitis ( nir ) affects 40% of the adult population , has a significant impact on health - related quality of life , and results in substantial health ", "economic costs to the society.13 nir is characterized by symptoms of upper airway inflammation , such as nasal obstruction , secretion , itching and sneezing , and represents several pathological entities , including allergic rhinitis , vasomotor rhinitis , and chronic rhinosinusitis.4 the disease has previously been linked to asthma , smoking , occupational exposure to gas , fumes , or dust , and also to gastroesophageal reflux disease , but for many patients with nir , the pathophysiology remains unknown.1,3,5,6 nir has been reported in individuals with chronic obstructive pulmonary disease ( copd ) , but epidemiological evidence is lacking.7 copd is an inflammatory , progressive airway disease with persistent airflow limitation .", "the airflow limitation in copd is caused by a combination of small airway disease ( obstructive bronchiolitis ) and the destruction of lung parenchyma ( emphysema ) .", "exposure to noxious particles and gases , particularly tobacco smoking , but also occupational exposure to gas , fumes , or dust , is related to the development of copd.8,9 interestingly , the same risk factors have also been associated with nir.1,6 copd occurs predominantly in patients aged > 40 years as a direct result of the slow initial development of the disease.1012 furthermore , new research has shown paranasal sinus opacification to be associated with copd and self - reported respiratory symptoms.13 the estimated prevalence of copd in the nordic countries , defined according to the global initiative for chronic obstructive lung disease stage i and higher , varies from 9.4% to 18.8%.1418 to date , only a few studies have evaluated a possible link between nir and copd .", "this is surprising , as both nir and copd are common inflammatory conditions in the respiratory tract and could potentially be linked through a generalized airway inflammation in accordance with the united airways ", "concept introduced to explain the link between upper and lower inflammatory airway disease in asthma and nir , even though other inflammatory mechanisms are likely to be involved.7 the diagnosis and classification of copd is based on a clinical assessment and spirometry is mandatory.8 to the authors knowledge , there are no large population - based studies of the relationship between rhinitis and copd based on spirometry data . in the present study ,", "data on spirometry and respiratory symptoms were combined from a large longitudinal population - based cohort , followed for 5 years .", "the main hypothesis of the study was that copd is related to the development of nir .", "this study is a part of the longitudinal population - based study called adonix ( adult onset asthma and nitric oxide ) .", "adonix was approved by the regional ethical review board of gothenburg ( dnr 092 - 01 ) and has previously been described in detail.5 the adonix study was designed to evaluate risk factors for airway disease such as nir , copd , and asthma .", "a total of 6,665 subjects , randomly selected , aged 2575 years , from the municipal register of gothenburg , sweden , were included at baseline between 2001 and 2003 .", "the subjects performed spirometry and answered a questionnaire with questions on upper and lower respiratory symptoms and diseases .", "the subjects also underwent a measurement of fractional exhaled nitric oxide ( feno ) and a laboratory analysis of specific immunoglobulin e antibodies for atopy status .", "after 5 years , the included subjects were asked to answer a follow - up questionnaire .", "this study evaluates the risk of developing new - onset nir in relation to having copd during a 5-year observation period .", "the questions used for new - onset nir and associated risk factors , such as smoking , educational status , and occupational exposure to gas , fumes , or dust , are shown in table 1 .", "the questions on occupational exposure to gas , fumes , or dust , and educational status were added in 2005 and were therefore not answered by all the participants at baseline . for this reason", "subjects who reported nir at baseline were excluded from the study population , along with subjects who did not answer the nir question at follow - up . to avoid misclassification between asthma and copd , all the subjects who reported asthma during the past 12 months at baseline were also excluded . as a result , 3,612 subjects were included in the analyses .", "spirometry was performed with a dry wedge spirometer ( vitalograph , buckingham , uk ) . according to the guidelines available at the time of the study,19 the best of a minimum of three similar maneuvers", "the ratio of the forced expiratory volume in 1 second ( fev1 ) divided by forced vital capacity ( fvc ) was calculated and a cut - off value for the fev1/fvc ratio of < 0.7 defined copd in accordance with the global initiative for chronic obstructive lung disease guidelines,8 apart from the fact that no reversibility test was performed .", "feno was measured before spirometry using a chemiluminescence analyzer and has been extensively described elsewhere.20 a cut - off value of 50 ppb or more in the exhaled no concentration was used to indicate a high likelihood of asthma in patients with copd.21 atopy was defined as a positive phadiatop test ( pharmacia , uppsala , sweden ) .", "class 0 was regarded as negative and class 1 as positive ( atopic).22 data on age , gender , and body mass index ( bmi ) were also collected ( table 2 ) .", "analyses were performed with the sas statistical software version 9.4 ( sas institute inc . , cary , nc , usa ) .", "univariate analyses included using t - tests and chi - square tests for continuous and categorical variables respectively .", "a multivariate logistic regression model was used to calculate odds ratios and 95% confidence intervals .", "this study is a part of the longitudinal population - based study called adonix ( adult onset asthma and nitric oxide ) .", "adonix was approved by the regional ethical review board of gothenburg ( dnr 092 - 01 ) and has previously been described in detail.5 the adonix study was designed to evaluate risk factors for airway disease such as nir , copd , and asthma .", "a total of 6,665 subjects , randomly selected , aged 2575 years , from the municipal register of gothenburg , sweden , were included at baseline between 2001 and 2003 .", "the subjects performed spirometry and answered a questionnaire with questions on upper and lower respiratory symptoms and diseases .", "the subjects also underwent a measurement of fractional exhaled nitric oxide ( feno ) and a laboratory analysis of specific immunoglobulin e antibodies for atopy status .", "after 5 years , the included subjects were asked to answer a follow - up questionnaire .", "this study evaluates the risk of developing new - onset nir in relation to having copd during a 5-year observation period .", "the questions used for new - onset nir and associated risk factors , such as smoking , educational status , and occupational exposure to gas , fumes , or dust , are shown in table 1 .", "the questions on occupational exposure to gas , fumes , or dust , and educational status were added in 2005 and were therefore not answered by all the participants at baseline . for this reason ,", "subjects who reported nir at baseline were excluded from the study population , along with subjects who did not answer the nir question at follow - up . to avoid misclassification between asthma and copd , all the subjects who reported asthma during the past 12 months at baseline were also excluded . as a result ,", "spirometry was performed with a dry wedge spirometer ( vitalograph , buckingham , uk ) . according to the guidelines available at the time of the study,19 the best of a minimum of three similar maneuvers", "the ratio of the forced expiratory volume in 1 second ( fev1 ) divided by forced vital capacity ( fvc ) was calculated and a cut - off value for the fev1/fvc ratio of < 0.7 defined copd in accordance with the global initiative for chronic obstructive lung disease guidelines,8 apart from the fact that no reversibility test was performed .", "feno was measured before spirometry using a chemiluminescence analyzer and has been extensively described elsewhere.20 a cut - off value of 50 ppb or more in the exhaled no concentration was used to indicate a high likelihood of asthma in patients with copd.21 atopy was defined as a positive phadiatop test ( pharmacia , uppsala , sweden ) .", "class 0 was regarded as negative and class 1 as positive ( atopic).22 data on age , gender , and body mass index ( bmi ) were also collected ( table 2 ) .", "analyses were performed with the sas statistical software version 9.4 ( sas institute inc . , cary , nc , usa ) .", "univariate analyses included using t - tests and chi - square tests for continuous and categorical variables respectively .", "a multivariate logistic regression model was used to calculate odds ratios and 95% confidence intervals .", "the comparison between subjects with new - onset nir and no nir at follow - up is presented in table 2 .", "as shown , there was no significant difference in mean age , gender , or bmi .", "the prevalence of copd was 8% in the total study population and 9.2% for subjects aged over 40 years .", "new - onset nir was significantly associated with copd ( 10.8 vs 7.4 p=0.005 ) .", "new - onset nir was also related to smoking ( p=0.004 ) and atopy ( p<0.001 ) in the univariate analyses . in the logistic regression analysis adjusted for age , gender , bmi , copd , smoking , and atopy , copd , smoking , and atopy remained independently associated with new - onset nir ( figure 2 ) .", "the interaction between copd and smoking in relation to new - onset nir was calculated and was found to be nonsignificant ( data not shown ) .", "there was no association between atopy and copd , indicating a low risk of asthma misclassification ( 19.1% vs 19.2% , p=1 ) .", "as shown in figure 3 , the vast majority of the subjects with copd were aged > 40 years , as expected .", "only 4.3% ( 11 of 257 subjects with copd ) displayed a feno value > 50 ppb , ie , values that indicate the presence of atopy and asthma .", "the sub - analysis of occupational exposure to gas , fumes , or dust ( n=2,257 ) , and educational status ( n=2,256 ) introduced in 2005 revealed a significant association with new - onset nir for gas , fumes , or dust ( p=0.006 ) but not for educational status ( p=0.75 ) ( data not shown ) .", "in this longitudinal population - based study of a large cohort , the risk of developing nir , over a 5-year period increased in individuals with copd . smoking and atopy were also risk factors for nir .", "the results indicate that there is a link present between upper and lower respiratory inflammation in nir and copd , and it is therefore important to assess nir in patients with copd .", "nir is identified by the presence of nasal symptoms , but identifying individuals with copd requires spirometry .", "the authors are not aware of any previous studies of nir in copd , including spirometry data from a large general population sample . in this study ,", "this could have resulted in misclassification between copd and asthma . in an attempt to minimize the risk of misclassification to eosinophilic inflammation and asthma ,", "all subjects reporting asthma within 12 months at baseline were excluded from the analyses . also , atopy in subjects with copd was analyzed and no significant association with copd was identified .", "furthermore , only 4.3% of the subjects in the copd group had feno > 50 ppb , which indicates a low prevalence of eosinophilic inflammation in this group .", "therefore , the subjects in this study with an fev1/fvc of < 0.7 are believed to be a representative of copd .", "the prevalence of copd in this general population sample was 8.8% , which is consistent with previous estimates ranging between 9.4% and 18.8%.1418 the prevalence was also higher in subjects aged 40 and above , which is in accordance with other studies of copd.11 the main finding in this study was that the 5-year cumulative incidence of nir was higher in the subjects with copd , both in the univariate analyses and the logistic regression analyses .", "smoking and atopy also remained independent risk factors for new - onset nir in the logistic regression analyses .", "although a previous study based on questionnaire data alone has indicated a relationship between rhinitis and copd in a large population sample , this study adds vital information on lung function that is needed for a copd diagnosis and confirms that there is a relationship between the two diseases.7 although the study shows a link between upper and lower respiratory inflammation in copd and nir , it is important to note that the findings do not imply a causal relationship between copd and nir . in spite of this , both diseases share common features that could be of importance for disease development in the airway at different levels .", "a similar inflammatory response can be elicited in both the upper and lower airways following exposure to inhaled irritants and sensitizers in the common airstream .", "gas , fumes , or dust are regarded as risk factors for copd,9 as well as for nir.6 individual vulnerability in the airway mucosa to irritants or deficits in systemic inflammatory responses could also explain why copd and nir can more commonly be found in the same subjects .", "the humoral spread of inflammatory mediators via the blood stream to both the upper and lower airways as seen in rhinitis and asthma is another possibility.23 tobacco smoke is an airborne irritant that is significantly associated not only with the development of copd but also with nir.1,11,12,24 in terms of the temporal relationship between the onset of copd and nir , it is important to recognize that the airway inflammation in copd and nir is likely to develop over time . in the absence of histological specimens from the airway , it is not possible to determine when and where inflammation starts and symptoms may begin later than signs of airway inflammation .", "differences in the concentrations to which the upper and lower airways are exposed may also explain different patterns of symptom debut .", "tobacco smoke is mainly inhaled through the mouth directly to the lungs and the tobacco load on the nasal mucosa is likely to be lower . to be able to establish plausible common pathophysiological mechanisms", "two possible risk factors for new - onset nir were analyzed separately , due to their late introduction in the questionnaire that missed a part of the study population , ie , educational status and occupational exposure to gas , fumes , or dust .", "educational status was not associated with new - onset nir , despite having previously been considered to be a risk factor for copd.25 occupational exposure to gas , fumes , or dust was not included in the logistic regression due to missing data , but in the sub - analyses it showed an association with new - onset nir . in developing countries , this is a major risk factor for copd due to cooking over open fires and it may also be important for nir , but the exposure in developed countries , such as sweden , is expected to be low .", "the main strength of the present study is its longitudinal population - based design with a large general population , including the availability of spirometry data for 93% of the subjects .", "nir includes several phenotypes of rhinitis , both allergic and nonallergic , as well as chronic rhinosinusitis . as a result", ", the nir question does not enable us to link copd to a specific phenotype of rhinitis .", "the interval of 5 years in the follow - up questionnaire may have introduced recall bias when answering the nir question .", "furthermore , this epidemiological study did not include a clinical assessment of copd by a doctor and therefore have no information on the severity of the disease .", "the copd definition that was used may also have introduced misclassification to other lower airway diseases showing a similar spirometry pattern , but , as previously discussed , the risk of including asthmatics in this study was minimized .", "there is an established inflammatory link between asthma and rhinitis , which many regard as the concept of generalized airway inflammation the united airways , helping to define and understand the disease.3 this study provides new evidence linking nir to copd , supported by the fact that both diseases involve inflammation of the airway mucosa and that they share common risk factors , such as exposure to tobacco smoke and cooking fumes .", "in contrast to the link between allergic rhinitis and asthma , the authors are aware of no common inflammatory pathway in copd and nir , such as the allergic inflammation that could explain the coexistence of the two diseases . to be able to confirm a pathophysiological relationship , further studies are needed , but the present study shows that copd should be regarded as a co - factor in the development of nir . the link between", "nir and copd also suggest that the diagnosis and treatment of the two diseases in the same patient could be of importance for patient outcome in terms of early diagnosis , disease control , and patient satisfaction , which has to be addressed further .", "this longitudinal population - based study of a large cohort shows that copd is a risk factor for developing nir .", "the results indicate that there is a link present between upper and lower respiratory inflammation in nir and copd ." ]

[ "<S> backgroundthe aim of this population - based study was to investigate the risk of developing noninfectious rhinitis ( nir ) in subjects with chronic obstructive pulmonary disease ( copd).materials and methodsthis is a longitudinal population - based study comprising 3,612 randomly selected subjects from gothenburg , sweden , aged 2575 years . </S>", "<S> lung function was measured at baseline with spirometry and the included subjects answered a questionnaire on respiratory symptoms . at follow - up , the subjects answered a questionnaire with a response rate of 87% . </S>", "<S> nir was defined as symptoms of nasal obstruction , nasal secretion , and/or sneezing attacks without having a cold , during the last 5 years . </S>", "<S> copd was defined as a spirometry ratio of forced expiratory volume in 1 second divided by forced vital capacity ( fev1/fvc ) < 0.7 . subjects who reported asthma and nir at baseline were excluded from the study . </S>", "<S> the odds ratios for developing nir ( ie , new - onset nir ) in relation to age , gender , body mass index , copd , smoking , and atopy were calculated.resultsin subjects with copd , the 5-year incidence of nir was significantly increased ( 10.8% vs 7.4% , p=0.005 ) and was higher among subjects aged > 40 years . </S>", "<S> smoking , atopy , and occupational exposure to gas , fumes , or dust were also associated with new - onset nir . </S>", "<S> copd , smoking , and atopy remained individual risk factors for new - onset nir in the logistic regression analysis.conclusionsthis longitudinal population - based study of a large cohort showed that copd is a risk factor for developing nir . </S>", "<S> smoking and atopy are also risk factors for nir . </S>", "<S> the results indicate that there is a link present between upper and lower respiratory inflammation in nir and copd . </S>" ]

[ "Introduction", "Material and methods", "The adult onset asthma and nitric oxide cohort", "Study design", "Statistics", "Results", "Discussion", "Conclusion" ]

[ [ "noninfectious rhinitis ( nir ) affects 40% of the adult population , has a significant impact on health - related quality of life , and results in substantial health ", "economic costs to the society.13 nir is characterized by symptoms of upper airway inflammation , such as nasal obstruction , secretion , itching and sneezing , and represents several pathological entities , including allergic rhinitis , vasomotor rhinitis , and chronic rhinosinusitis.4 the disease has previously been linked to asthma , smoking , occupational exposure to gas , fumes , or dust , and also to gastroesophageal reflux disease , but for many patients with nir , the pathophysiology remains unknown.1,3,5,6 nir has been reported in individuals with chronic obstructive pulmonary disease ( copd ) , but epidemiological evidence is lacking.7 copd is an inflammatory , progressive airway disease with persistent airflow limitation .", "the airflow limitation in copd is caused by a combination of small airway disease ( obstructive bronchiolitis ) and the destruction of lung parenchyma ( emphysema ) .", "exposure to noxious particles and gases , particularly tobacco smoking , but also occupational exposure to gas , fumes , or dust , is related to the development of copd.8,9 interestingly , the same risk factors have also been associated with nir.1,6 copd occurs predominantly in patients aged > 40 years as a direct result of the slow initial development of the disease.1012 furthermore , new research has shown paranasal sinus opacification to be associated with copd and self - reported respiratory symptoms.13 the estimated prevalence of copd in the nordic countries , defined according to the global initiative for chronic obstructive lung disease stage i and higher , varies from 9.4% to 18.8%.1418 to date , only a few studies have evaluated a possible link between nir and copd .", "this is surprising , as both nir and copd are common inflammatory conditions in the respiratory tract and could potentially be linked through a generalized airway inflammation in accordance with the united airways ", "concept introduced to explain the link between upper and lower inflammatory airway disease in asthma and nir , even though other inflammatory mechanisms are likely to be involved.7 the diagnosis and classification of copd is based on a clinical assessment and spirometry is mandatory.8 to the authors knowledge , there are no large population - based studies of the relationship between rhinitis and copd based on spirometry data . in the present study ,", "data on spirometry and respiratory symptoms were combined from a large longitudinal population - based cohort , followed for 5 years .", "the main hypothesis of the study was that copd is related to the development of nir ." ], [ "this study is a part of the longitudinal population - based study called adonix ( adult onset asthma and nitric oxide ) .", "adonix was approved by the regional ethical review board of gothenburg ( dnr 092 - 01 ) and has previously been described in detail.5 the adonix study was designed to evaluate risk factors for airway disease such as nir , copd , and asthma .", "a total of 6,665 subjects , randomly selected , aged 2575 years , from the municipal register of gothenburg , sweden , were included at baseline between 2001 and 2003 .", "the subjects performed spirometry and answered a questionnaire with questions on upper and lower respiratory symptoms and diseases .", "the subjects also underwent a measurement of fractional exhaled nitric oxide ( feno ) and a laboratory analysis of specific immunoglobulin e antibodies for atopy status .", "after 5 years , the included subjects were asked to answer a follow - up questionnaire .", "this study evaluates the risk of developing new - onset nir in relation to having copd during a 5-year observation period .", "the questions used for new - onset nir and associated risk factors , such as smoking , educational status , and occupational exposure to gas , fumes , or dust , are shown in table 1 .", "the questions on occupational exposure to gas , fumes , or dust , and educational status were added in 2005 and were therefore not answered by all the participants at baseline . for this reason", "subjects who reported nir at baseline were excluded from the study population , along with subjects who did not answer the nir question at follow - up . to avoid misclassification between asthma and copd , all the subjects who reported asthma during the past 12 months at baseline were also excluded . as a result , 3,612 subjects were included in the analyses .", "spirometry was performed with a dry wedge spirometer ( vitalograph , buckingham , uk ) . according to the guidelines available at the time of the study,19 the best of a minimum of three similar maneuvers", "the ratio of the forced expiratory volume in 1 second ( fev1 ) divided by forced vital capacity ( fvc ) was calculated and a cut - off value for the fev1/fvc ratio of < 0.7 defined copd in accordance with the global initiative for chronic obstructive lung disease guidelines,8 apart from the fact that no reversibility test was performed .", "feno was measured before spirometry using a chemiluminescence analyzer and has been extensively described elsewhere.20 a cut - off value of 50 ppb or more in the exhaled no concentration was used to indicate a high likelihood of asthma in patients with copd.21 atopy was defined as a positive phadiatop test ( pharmacia , uppsala , sweden ) .", "class 0 was regarded as negative and class 1 as positive ( atopic).22 data on age , gender , and body mass index ( bmi ) were also collected ( table 2 ) .", "analyses were performed with the sas statistical software version 9.4 ( sas institute inc . , cary , nc , usa ) .", "univariate analyses included using t - tests and chi - square tests for continuous and categorical variables respectively .", "a multivariate logistic regression model was used to calculate odds ratios and 95% confidence intervals ." ], [ "this study is a part of the longitudinal population - based study called adonix ( adult onset asthma and nitric oxide ) .", "adonix was approved by the regional ethical review board of gothenburg ( dnr 092 - 01 ) and has previously been described in detail.5 the adonix study was designed to evaluate risk factors for airway disease such as nir , copd , and asthma .", "a total of 6,665 subjects , randomly selected , aged 2575 years , from the municipal register of gothenburg , sweden , were included at baseline between 2001 and 2003 .", "the subjects performed spirometry and answered a questionnaire with questions on upper and lower respiratory symptoms and diseases .", "the subjects also underwent a measurement of fractional exhaled nitric oxide ( feno ) and a laboratory analysis of specific immunoglobulin e antibodies for atopy status .", "after 5 years , the included subjects were asked to answer a follow - up questionnaire ." ], [ "this study evaluates the risk of developing new - onset nir in relation to having copd during a 5-year observation period .", "the questions used for new - onset nir and associated risk factors , such as smoking , educational status , and occupational exposure to gas , fumes , or dust , are shown in table 1 .", "the questions on occupational exposure to gas , fumes , or dust , and educational status were added in 2005 and were therefore not answered by all the participants at baseline . for this reason ,", "subjects who reported nir at baseline were excluded from the study population , along with subjects who did not answer the nir question at follow - up . to avoid misclassification between asthma and copd , all the subjects who reported asthma during the past 12 months at baseline were also excluded . as a result ,", "spirometry was performed with a dry wedge spirometer ( vitalograph , buckingham , uk ) . according to the guidelines available at the time of the study,19 the best of a minimum of three similar maneuvers", "the ratio of the forced expiratory volume in 1 second ( fev1 ) divided by forced vital capacity ( fvc ) was calculated and a cut - off value for the fev1/fvc ratio of < 0.7 defined copd in accordance with the global initiative for chronic obstructive lung disease guidelines,8 apart from the fact that no reversibility test was performed .", "feno was measured before spirometry using a chemiluminescence analyzer and has been extensively described elsewhere.20 a cut - off value of 50 ppb or more in the exhaled no concentration was used to indicate a high likelihood of asthma in patients with copd.21 atopy was defined as a positive phadiatop test ( pharmacia , uppsala , sweden ) .", "class 0 was regarded as negative and class 1 as positive ( atopic).22 data on age , gender , and body mass index ( bmi ) were also collected ( table 2 ) ." ], [ "analyses were performed with the sas statistical software version 9.4 ( sas institute inc . , cary , nc , usa ) .", "univariate analyses included using t - tests and chi - square tests for continuous and categorical variables respectively .", "a multivariate logistic regression model was used to calculate odds ratios and 95% confidence intervals ." ], [ "the comparison between subjects with new - onset nir and no nir at follow - up is presented in table 2 .", "as shown , there was no significant difference in mean age , gender , or bmi .", "the prevalence of copd was 8% in the total study population and 9.2% for subjects aged over 40 years .", "new - onset nir was significantly associated with copd ( 10.8 vs 7.4 p=0.005 ) .", "new - onset nir was also related to smoking ( p=0.004 ) and atopy ( p<0.001 ) in the univariate analyses . in the logistic regression analysis adjusted for age , gender , bmi , copd , smoking , and atopy , copd , smoking , and atopy remained independently associated with new - onset nir ( figure 2 ) .", "the interaction between copd and smoking in relation to new - onset nir was calculated and was found to be nonsignificant ( data not shown ) .", "there was no association between atopy and copd , indicating a low risk of asthma misclassification ( 19.1% vs 19.2% , p=1 ) .", "as shown in figure 3 , the vast majority of the subjects with copd were aged > 40 years , as expected .", "only 4.3% ( 11 of 257 subjects with copd ) displayed a feno value > 50 ppb , ie , values that indicate the presence of atopy and asthma .", "the sub - analysis of occupational exposure to gas , fumes , or dust ( n=2,257 ) , and educational status ( n=2,256 ) introduced in 2005 revealed a significant association with new - onset nir for gas , fumes , or dust ( p=0.006 ) but not for educational status ( p=0.75 ) ( data not shown ) ." ], [ "in this longitudinal population - based study of a large cohort , the risk of developing nir , over a 5-year period increased in individuals with copd . smoking and atopy were also risk factors for nir .", "the results indicate that there is a link present between upper and lower respiratory inflammation in nir and copd , and it is therefore important to assess nir in patients with copd .", "nir is identified by the presence of nasal symptoms , but identifying individuals with copd requires spirometry .", "the authors are not aware of any previous studies of nir in copd , including spirometry data from a large general population sample . in this study ,", "this could have resulted in misclassification between copd and asthma . in an attempt to minimize the risk of misclassification to eosinophilic inflammation and asthma ,", "all subjects reporting asthma within 12 months at baseline were excluded from the analyses . also , atopy in subjects with copd was analyzed and no significant association with copd was identified .", "furthermore , only 4.3% of the subjects in the copd group had feno > 50 ppb , which indicates a low prevalence of eosinophilic inflammation in this group .", "therefore , the subjects in this study with an fev1/fvc of < 0.7 are believed to be a representative of copd .", "the prevalence of copd in this general population sample was 8.8% , which is consistent with previous estimates ranging between 9.4% and 18.8%.1418 the prevalence was also higher in subjects aged 40 and above , which is in accordance with other studies of copd.11 the main finding in this study was that the 5-year cumulative incidence of nir was higher in the subjects with copd , both in the univariate analyses and the logistic regression analyses .", "smoking and atopy also remained independent risk factors for new - onset nir in the logistic regression analyses .", "although a previous study based on questionnaire data alone has indicated a relationship between rhinitis and copd in a large population sample , this study adds vital information on lung function that is needed for a copd diagnosis and confirms that there is a relationship between the two diseases.7 although the study shows a link between upper and lower respiratory inflammation in copd and nir , it is important to note that the findings do not imply a causal relationship between copd and nir . in spite of this , both diseases share common features that could be of importance for disease development in the airway at different levels .", "a similar inflammatory response can be elicited in both the upper and lower airways following exposure to inhaled irritants and sensitizers in the common airstream .", "gas , fumes , or dust are regarded as risk factors for copd,9 as well as for nir.6 individual vulnerability in the airway mucosa to irritants or deficits in systemic inflammatory responses could also explain why copd and nir can more commonly be found in the same subjects .", "the humoral spread of inflammatory mediators via the blood stream to both the upper and lower airways as seen in rhinitis and asthma is another possibility.23 tobacco smoke is an airborne irritant that is significantly associated not only with the development of copd but also with nir.1,11,12,24 in terms of the temporal relationship between the onset of copd and nir , it is important to recognize that the airway inflammation in copd and nir is likely to develop over time . in the absence of histological specimens from the airway , it is not possible to determine when and where inflammation starts and symptoms may begin later than signs of airway inflammation .", "differences in the concentrations to which the upper and lower airways are exposed may also explain different patterns of symptom debut .", "tobacco smoke is mainly inhaled through the mouth directly to the lungs and the tobacco load on the nasal mucosa is likely to be lower . to be able to establish plausible common pathophysiological mechanisms", "two possible risk factors for new - onset nir were analyzed separately , due to their late introduction in the questionnaire that missed a part of the study population , ie , educational status and occupational exposure to gas , fumes , or dust .", "educational status was not associated with new - onset nir , despite having previously been considered to be a risk factor for copd.25 occupational exposure to gas , fumes , or dust was not included in the logistic regression due to missing data , but in the sub - analyses it showed an association with new - onset nir . in developing countries , this is a major risk factor for copd due to cooking over open fires and it may also be important for nir , but the exposure in developed countries , such as sweden , is expected to be low .", "the main strength of the present study is its longitudinal population - based design with a large general population , including the availability of spirometry data for 93% of the subjects .", "nir includes several phenotypes of rhinitis , both allergic and nonallergic , as well as chronic rhinosinusitis . as a result", ", the nir question does not enable us to link copd to a specific phenotype of rhinitis .", "the interval of 5 years in the follow - up questionnaire may have introduced recall bias when answering the nir question .", "furthermore , this epidemiological study did not include a clinical assessment of copd by a doctor and therefore have no information on the severity of the disease .", "the copd definition that was used may also have introduced misclassification to other lower airway diseases showing a similar spirometry pattern , but , as previously discussed , the risk of including asthmatics in this study was minimized .", "there is an established inflammatory link between asthma and rhinitis , which many regard as the concept of generalized airway inflammation the united airways , helping to define and understand the disease.3 this study provides new evidence linking nir to copd , supported by the fact that both diseases involve inflammation of the airway mucosa and that they share common risk factors , such as exposure to tobacco smoke and cooking fumes .", "in contrast to the link between allergic rhinitis and asthma , the authors are aware of no common inflammatory pathway in copd and nir , such as the allergic inflammation that could explain the coexistence of the two diseases . to be able to confirm a pathophysiological relationship , further studies are needed , but the present study shows that copd should be regarded as a co - factor in the development of nir . the link between", "nir and copd also suggest that the diagnosis and treatment of the two diseases in the same patient could be of importance for patient outcome in terms of early diagnosis , disease control , and patient satisfaction , which has to be addressed further ." ], [ "this longitudinal population - based study of a large cohort shows that copd is a risk factor for developing nir .", "the results indicate that there is a link present between upper and lower respiratory inflammation in nir and copd ." ] ]

[ "immunocompromised patients might bear a variety of presentations such as rhino - orbito - cerebral , pulmonary , gastrointestinal , cutaneous , central nervous system and disseminated form .", "rhino - orbito - cerebral and pulmonary mucormycosis are the most common forms ; the latter progresses rapidly to devastating diseases .", "presentation of pulmonary mucormycosis may be confused with several infectious and non infectious diseases ; therefore its diagnosis is difficult .", "chest ct scan of a patient with pulmonary mucormycosis might demonstrate such presentations as air crescent sign , ct halo sign , solitary or multiple pulmonary nodules or masses , bronchopulmonary fistula and pulmonary artery pseudo aneurysm .", "the occurrence of pneumothorax and its subsequent rhs along with sinusitis , supported by radiology and pathology in a 33 years old patient , is considered as a rare case of pulmonary mucormycosis .", "following is the treatment history of a man , 33 years old in july 2014 ( day + 14 ) , when he died in our hospital as a result of multiorgan failure .", "he was a farmer , married , with no history of alcohol consumption , illicit drugs use and extramarital relationships . on day 330 : because of edema he visited a medical center in another city , where he was admitted and membranous glomerulonephritis was diagnosed .", "consequently the followings administered ; prednisolone and cyclosporine for 2 months , cellcept for 1 month , prednisolone and cyclophosphomide for 7 months . on day 75 : because of progressive generalized edema he was admitted again .", "he was discharged under treatment with prednisolone ( 50 mg / daily ) and tacrolymus ( 1 cap / bd ) . during the next 2 months he was feeling fine . on day 28 , he travelled to village for a few days and he had close contact with sheep . on day", "14 the patient reported fever , cough and dyspnea with mild erythema and pain in right eye .", "anorexia , weakness , dyspnea and conjunctivitis developed and persisted despite of outpatient antibiotic treatment . in the emergency department (", "day 0 ) : the patient suffered from dyspnea , weakness and right eye pain . on examination , he was agitated but completely conscious with diaphoretic skin .", "the vital signs have been as follows ; temperature , 36.7 c ; blood pressure , 100/60 mmgh ; pulse rate , 99 beats per minute ; respiratory rate , 22 beats per minute and oxygen saturation in air room , 99% .", "in right eye , there was moderate conjunctivitis with eyelid erythema ; its movement was normal .", "the first day chest ct scan revealed large pleural effusion with pneumothorax in the left side and a shift of mediastinum to the right side .", "1 ) . consequently drainage with chest tube was administered by the surgeon immediately . because of the immunocompromised state of the patient , parenteral trimethoprim- sulfamethaxazol ( 160 mg / iv / tds ) and meropenem ( 1 g / iv / bd ) were administered for pneumocystis jiroveci and multi drug resistant organisms .", "we also started fluconazole for oral lesions . on day + 3 , right eye erythema was extended with small size necrosis in right nasal ala without bleeding ( fig .", "with doubt of rhino - orbital mucormycosis , biopsies of tongue and nasal septa were carried out and amphotericin b deoxycholate ( 1 mg / kg / daily ) was administered . on day + 4", ", paranasal sinuses ( pns ) ct scan revealed soft tissue density lesion in left ethmoid sinus and nasal passage with extension to left orbit ( fig .", ", he was transferred to intensive care unit ( icu ) because of respiratory distress and was intubated after 48 h ; his conscious level was 14+t/15 .", "smear of sputum for acid fast bacilli , stain and culture of sputum for pneumocystis jiroveci were negative .", "hiv antibody test , d - dimer enzyme -linked immunosorbent assay ( elisa ) and collagen vascular diseases screening tests were negative ( table 1 ) .", "pleural fluid cytologic examination and polymerase chain reaction ( pcr ) for fungi were negative . on day + 6 , laboratory results revealed pancytopenia ( table 1 ) , however in peripheral blood smear there was no schistocyte or immature cells .", "bone marrow biopsy which could help in finding the cause of pancytopenia was nevertheless postponed due to the patient 's poor condition and low platelet count .", "meanwhile his blood pressure was decreased and we started inotropic drugs.hemodynamic instability of the patient prohibited further attempts such as bronchoscopy and ct guided lung biopsy to identify the causative agent . on day + 9 ,", "section of tounge biopsy showed ulcerated squamous epithelial mucosa with subepithelial fibro fatty tissue and skeletal muscle fibers of tongue that is on site of ulceration .", "the inflammatory granulation tissue and inflammatory multinucleated giant cells were also seen in gimsa staining , but no cyst and trophozoite of pneumocystis jiroveci were identified .", "pulmonary mucormycosis is a devastating infection when it affects immunocompromised patients such as diabetes mellitus , hematologic malignancies , chronic renal failure , post transplantaion and immunosuppressed persons . herein , we present a rhino - orbital mucormycosis with pulmonary involvement .", "reticulation inside the rhs , presence of pleural effusion , multiple nodules and concomitant rhino - orbital mucormycosis as well as documented pathology , collectively were considered as indicating that pulmonary involvement has been due to mucormycosis .", "the pathological finding is achieved by tongue tissue ; lung biopsy has not been possible due to poor condition of the patient .", "our case was an immunocompromised patient with documented rhino - orbital mucormycosis ( figs . 3 , 5 and 6 ) .", "chest ct scan showed bilateral pleural effusion , multiple nodules and rhs with thin reticulation ( figs . 1 and 4 ) .", "although histopathology findings are usually needed to establish the diagnosis of pulmonary mucormycosis , using ct imaging and clinical presentation we can avoid the invasive procedures such as lung biopsy", ". there are few case reports of pneumothorax in pulmonary mucormycosis , some of which are iatrogenic during mechanical ventilation and bronchoscopic procedure [ 911 ] .", "combined mucormycosis and methicillin - resistant staphylococcus aureus ( mrsa ) were found to be responsible for life threatening pneumothorax in an immunocompromised patient .", "mucormycosis and aspergillus have been recognized as leading to pneumonia and pneunothorax in a dialysis patient .", "however , spontaneous pneumothorax in pulmonary mucormycosis that is followed by rhs seems to be a unique case . based on patient 's background and clinical signs , amphotericin b deoxycholate ( 1 mg", "the lower dose of antifungal drug has been due to the patient 's renal insufficiency .", "ambisome would have certainly been a better choice than amphotericin b ; however , the former has not been available in iran .", "however the lack of bone erosion in sinus ct scan , delay in pathologic result and patient 's poor clinical condition , lead into the reluctance of surgeon for extensive debridement that had negative impact on patient 's prognosis .", "pneumothorax which is due to mucormycosis can be fatal particularly in immunocompromised persons , so clinicians should take immediate measures ( follow up imaging and pathology ) to confirm or reject the presence of invasive fungal infection . if confirmed , antifungal therapy is necessary as soon as possible ." ]

[ "<S> mucormycosis , an invasive fungus with a variety of clinical presentation , is a devastating infection in immunocompromised host . here </S>", "<S> an unusual case of pulmonary mucormycosis is introduced in an immunodeficient patient in which pneumothorax was followed by reversed halo sign ( rhs ) . </S>", "<S> the clinicians , who visit immunocompromised persons with pneumothorax , should be considerate to take immediate imaging and pathologic measures to confirm or reject mucormycosis . </S>" ]

[ "Introduction", "Case", "Discussion", "Conflict of interest", "Ethical form" ]

[ [ "immunocompromised patients might bear a variety of presentations such as rhino - orbito - cerebral , pulmonary , gastrointestinal , cutaneous , central nervous system and disseminated form .", "rhino - orbito - cerebral and pulmonary mucormycosis are the most common forms ; the latter progresses rapidly to devastating diseases .", "presentation of pulmonary mucormycosis may be confused with several infectious and non infectious diseases ; therefore its diagnosis is difficult .", "chest ct scan of a patient with pulmonary mucormycosis might demonstrate such presentations as air crescent sign , ct halo sign , solitary or multiple pulmonary nodules or masses , bronchopulmonary fistula and pulmonary artery pseudo aneurysm .", "the occurrence of pneumothorax and its subsequent rhs along with sinusitis , supported by radiology and pathology in a 33 years old patient , is considered as a rare case of pulmonary mucormycosis ." ], [ "following is the treatment history of a man , 33 years old in july 2014 ( day + 14 ) , when he died in our hospital as a result of multiorgan failure .", "he was a farmer , married , with no history of alcohol consumption , illicit drugs use and extramarital relationships . on day 330 : because of edema he visited a medical center in another city , where he was admitted and membranous glomerulonephritis was diagnosed .", "consequently the followings administered ; prednisolone and cyclosporine for 2 months , cellcept for 1 month , prednisolone and cyclophosphomide for 7 months . on day 75 : because of progressive generalized edema he was admitted again .", "he was discharged under treatment with prednisolone ( 50 mg / daily ) and tacrolymus ( 1 cap / bd ) . during the next 2 months he was feeling fine . on day 28 , he travelled to village for a few days and he had close contact with sheep . on day", "14 the patient reported fever , cough and dyspnea with mild erythema and pain in right eye .", "anorexia , weakness , dyspnea and conjunctivitis developed and persisted despite of outpatient antibiotic treatment . in the emergency department (", "day 0 ) : the patient suffered from dyspnea , weakness and right eye pain . on examination , he was agitated but completely conscious with diaphoretic skin .", "the vital signs have been as follows ; temperature , 36.7 c ; blood pressure , 100/60 mmgh ; pulse rate , 99 beats per minute ; respiratory rate , 22 beats per minute and oxygen saturation in air room , 99% .", "in right eye , there was moderate conjunctivitis with eyelid erythema ; its movement was normal .", "the first day chest ct scan revealed large pleural effusion with pneumothorax in the left side and a shift of mediastinum to the right side .", "1 ) . consequently drainage with chest tube was administered by the surgeon immediately . because of the immunocompromised state of the patient , parenteral trimethoprim- sulfamethaxazol ( 160 mg / iv / tds ) and meropenem ( 1 g / iv / bd ) were administered for pneumocystis jiroveci and multi drug resistant organisms .", "we also started fluconazole for oral lesions . on day + 3 , right eye erythema was extended with small size necrosis in right nasal ala without bleeding ( fig .", "with doubt of rhino - orbital mucormycosis , biopsies of tongue and nasal septa were carried out and amphotericin b deoxycholate ( 1 mg / kg / daily ) was administered . on day + 4", ", paranasal sinuses ( pns ) ct scan revealed soft tissue density lesion in left ethmoid sinus and nasal passage with extension to left orbit ( fig .", ", he was transferred to intensive care unit ( icu ) because of respiratory distress and was intubated after 48 h ; his conscious level was 14+t/15 .", "smear of sputum for acid fast bacilli , stain and culture of sputum for pneumocystis jiroveci were negative .", "hiv antibody test , d - dimer enzyme -linked immunosorbent assay ( elisa ) and collagen vascular diseases screening tests were negative ( table 1 ) .", "pleural fluid cytologic examination and polymerase chain reaction ( pcr ) for fungi were negative . on day + 6 , laboratory results revealed pancytopenia ( table 1 ) , however in peripheral blood smear there was no schistocyte or immature cells .", "bone marrow biopsy which could help in finding the cause of pancytopenia was nevertheless postponed due to the patient 's poor condition and low platelet count .", "meanwhile his blood pressure was decreased and we started inotropic drugs.hemodynamic instability of the patient prohibited further attempts such as bronchoscopy and ct guided lung biopsy to identify the causative agent . on day + 9 ,", "section of tounge biopsy showed ulcerated squamous epithelial mucosa with subepithelial fibro fatty tissue and skeletal muscle fibers of tongue that is on site of ulceration .", "the inflammatory granulation tissue and inflammatory multinucleated giant cells were also seen in gimsa staining , but no cyst and trophozoite of pneumocystis jiroveci were identified ." ], [ "pulmonary mucormycosis is a devastating infection when it affects immunocompromised patients such as diabetes mellitus , hematologic malignancies , chronic renal failure , post transplantaion and immunosuppressed persons . herein , we present a rhino - orbital mucormycosis with pulmonary involvement .", "reticulation inside the rhs , presence of pleural effusion , multiple nodules and concomitant rhino - orbital mucormycosis as well as documented pathology , collectively were considered as indicating that pulmonary involvement has been due to mucormycosis .", "the pathological finding is achieved by tongue tissue ; lung biopsy has not been possible due to poor condition of the patient .", "our case was an immunocompromised patient with documented rhino - orbital mucormycosis ( figs . 3 , 5 and 6 ) .", "chest ct scan showed bilateral pleural effusion , multiple nodules and rhs with thin reticulation ( figs . 1 and 4 ) .", "although histopathology findings are usually needed to establish the diagnosis of pulmonary mucormycosis , using ct imaging and clinical presentation we can avoid the invasive procedures such as lung biopsy", ". there are few case reports of pneumothorax in pulmonary mucormycosis , some of which are iatrogenic during mechanical ventilation and bronchoscopic procedure [ 911 ] .", "combined mucormycosis and methicillin - resistant staphylococcus aureus ( mrsa ) were found to be responsible for life threatening pneumothorax in an immunocompromised patient .", "mucormycosis and aspergillus have been recognized as leading to pneumonia and pneunothorax in a dialysis patient .", "however , spontaneous pneumothorax in pulmonary mucormycosis that is followed by rhs seems to be a unique case . based on patient 's background and clinical signs , amphotericin b deoxycholate ( 1 mg", "the lower dose of antifungal drug has been due to the patient 's renal insufficiency .", "ambisome would have certainly been a better choice than amphotericin b ; however , the former has not been available in iran .", "however the lack of bone erosion in sinus ct scan , delay in pathologic result and patient 's poor clinical condition , lead into the reluctance of surgeon for extensive debridement that had negative impact on patient 's prognosis .", "pneumothorax which is due to mucormycosis can be fatal particularly in immunocompromised persons , so clinicians should take immediate measures ( follow up imaging and pathology ) to confirm or reject the presence of invasive fungal infection . if confirmed , antifungal therapy is necessary as soon as possible ." ], [ "" ], [ "" ] ]

[ "ovarian follicular fate ( i.e. development / ovulation versus atresia ) in the mammals is determined by the \n fate of the cells within the follicles and is regulated by complex interactions of endocrine \n ( gonadotropin and steroid hormones ) and locally produced ( growth factors and cytokines ) factors ( hirshfield , 1991 ) .", "although follicular atresia can occur at all the \n stages of follicular development , a high incidence of granulosa cell apoptosis , a well recognized \n cellular basis for this degenerative process , is predominantly observed at the early antral stage of \n follicular development during the ovarian cycle ( kim et al . , \n 1998 ) .", "fas antigen ( fas ) , a member of the tnf receptor family , and its ligand fas ligand ( fasl ) , have been \n identified as cell death mediator and factor , respectively in a variety of cell types ( watanabefukunaga et al . , 1992 ; suda \n et al . , 1993 ) .", "their involvement in the induction of granulosa and luteal cell apoptosis has \n been demonstrated in several mammalian species ( quirk et al . , \n 1995 ; hakuno et al . , 1996 ; kondo et al .", "fas \n and fasl expression is up - regulated in granulosa cells undergoing apoptosis during early stage of \n follicular development ( kim et al . , 1998 ) and", "p53-mediated \n granulosa cell apoptosis is associated with the activation of fas / fasl system during follicular atresia \n ( kim et al . , 1999 ) .", "in addition , follicle stimulating hormone \n ( fsh ) is an important negative regulator of the fas / fasl system and a key cell survival factor during \n the follicular development and the role of the fas / fasl system in granulosa cell apoptosis appears more \n prominent at early than at late stages of follicular maturation .", "interferon gamma ( ifn- ) is a member of a family of heterogeneous regulatory proteins having antiviral , \n immunomodulatory , antiproliferative , and cytodifferentiation regulatory activities ( pestka et al . , 1987 ) .", "it increases adrenal steroidogenesis ( blalock and harp , 1981 ) , increases iodide uptake by thyroid cells \n ( friedman et al . , 1982 ) , and suppresses hcg - induced testosterone production by leydig cells ( orava et al . , 1989 ) .", "at physiological concentrations , ifn- \n inhibits granulosa cell differentiation and steroidogenesis ( gorospe et \n al . , 1988 ; xiao and findlay , 1992 ) .", "in addition , ifn- \n increases fas mrna expression in mouse lymphoma cells and bam3 cells ( watanabefukunaga et al . , 1992 ) and", "fas content in human granulosa / luteal cells ( quirk et al . , 1995 ) .", "apoptosis in ovarian surface epithelial cells \n is mediated by increased fas expression induced by ifn- ( quirk et al . , \n 1997 ) .", "moreover , ifn- induces fas - mediated apoptosis in differrentiated mouse granulosa \n cells which could be potentiated by the presence of tnf ( quirk et al . , \n 1998 ) .", "although the importance of ifn- in undifferentiated or poorly differentiated \n granulosa cells is well established , the presence of this cytokine in the ovary throughout follicular \n development has not been investigated .", "in addition , the regulation of fas system in undifferentiated \n granulosa cells by ifn- , subsequent fas - mediated apoptotic response as well as their modulation by \n gonadotropin in vivo remain unclear .", "the purpose of the present study was to examine the role of ifn- in regulation of fas expression and \n apoptosis in granulosa cells at different stages of follicular development and granulosa cell \n differentiation in vitro .", "in addition , we assessed the presence of ifn- in the ovary \n during follicular maturation and studied the relationship between the presence of ifn- , fas / fasl system \n and apoptosis in vivo .", "[ p]-ddatp and dntps were obtained from amersham pharmacia biotech ( piscataway , nj ) . \n", "horse radish peroxidase ( hrp)-conjugated goat anti - rabbit secondary antibody was purchased from \n bio - rad laboratories ( hercule , ca ) .", "rabbit anti - rat interferon- ( ifn- ) was obtained from biosource \n international ( camarillo , ca ) .", "biotinylated 16-dutp , in situ cell death detection \n ( fluorescein ) kit , proteinase - k and terminal deoxynucleotidyl transferase ( tdt ) were purchased from \n boehringer - mannheim ( indianapolis , in ) .", "recombinant rat ifn- was from genzyme ( cambridge , ma ) . \n fetal bovine serum ( fbs ) , minimal essential medium ( mem ) , non - essential amino acids ( naa ) , \n penicillin and streptomycin were from gibco / brl ( burlington , on , canada ) .", "fitc - conjugated mouse \n anti - hamster igg , hamster anti - mouse fas monoclonal antibody ( clone jo2 ) and hamster igg were \n obtained from pharmingen ( san diego , ca ) .", "avian myeloblastosis virus ( amv ) reverse transcriptase , \n oligo(dt)15 primer , rrnasin ribonucleotide inhibitor and taq dna polymerase were from \n promega ( madison , wi ) .", "rabbit igg , rabbit peroxidase kits , rabbit polyclonal anti - mouse fas ( sc-716 ) \n and anti - rat fasl ( sc-834 ) antibodies , and neutralization peptides ( fas ; sc-716p , fasl ; sc-834p ) \n were from santa cruz biotechnology ( santa cruz , ca ) .", "agarose , bovine serum albumin ( bsa ; fraction \n v ) , diethylstilbesterol ( des ) , equine chorionic gonadotropin ( ecg ) , methyl green , normal goat serum , \n phenylmethylsulfonyl fluoride ( pmsf ) and veronal acetate were purchased from sigma chemical co. ( st \n louis , mo ) . to obtain ovaries consisting mainly of follicles synchronized at the preantral / early antral and \n medium / large antral stages in immature ( 21 - 23 days old ) sprague - dawley rats ( charles river canada ; \n quebec , canada or samtako bio - korea ; osan , korea ) , des ( 1mg / day , sc , for 3 consecutive days and \n sacrificed 24 hr after last injection ) and ecg (", "15 iu , ip and sacrificed 48 hr thereafter ) were \n administered , respectively .", "a 14 h light:10 h dark photo - cycle was maintained with \n lights - on at 0600 h. all procedures were performed in accordance with protocols approved by the \n dong - a university animal care and use committee . following removal of connective tissues ,", "the \n ovaries were washed in pbs ( ph 7.4 ) to remove excess blood and were either immediately fixed in 10% \n neutral buffered formalin ( ph 7.4 ) for histological processing or used for granulosa cell isolation \n by follicle puncture ( rao et al . ,", "granulosa cells ( 2.510 ) from ovaries of des- and ecg - primed rats were plated for 6 hr \n ( 5% co2 , 37 ) in 60 mm falcon plastic culture dishes ( becton dickins , lincoln park , nj ) \n in mem supplemented with 2% fbs , 1x naa , penicillin ( 100 u / ml ) and streptomycin ( 100 g / ml ) .", "the \n medium was then replaced with mem supplemented with 0.1% bsa , 1x naa , penicillin ( 100 u / ml ) and \n streptomycin ( 100 g / ml ) in subsequent culture .", "non - viable ( floating ) cells were removed at this \n medium - replacement step .", "for induction of fas mrna and protein expression , granulosa cells were cultured in the presence of \n recombinant rat ifn- ( 0 - 1,000 u / ml of the medium ) for 18 hr . floating and attached cells harvested \n by centrifugation and trypsinization respectively , were combined and washed with ice - cold pbs , \n pending protein and rna extraction , and flowcytometric analysis . for immunocytochemistry and tunel , \n sterilized cover glasses ( 2222 mm ) were added onto the bottom of the culture dishes prior to cell \n plating . at the end of the culture period ,", "the cover glasses were removed , washed briefly with \n ice - cold pbs and processed for immunocytochemistry . to assess the influence of ifn- on fas expression and subsequent granulosa cell apoptotic response , \n cells pretreated for 18 hr with / without the cytokine were incubated for an additional 6 h with \n either agonistic fas monoclonal antibody ( hamster anti - mouse fas mab , igg ; 1 g / ml ) or hamster igg \n ( control ; 1 g / ml ) .", "the effect of the treatment was assessed by phase - contrast microscopy , tunel and \n dna fragmentation analysis . ", "paraffin embedded whole ovarian sections ( 4 - 5 m ) were used for ifn- , fas and fasl \n immunohistochemistry .", "the deparaffinized and hydrated sections were quenched in \n h2o2 ( 0.3% ; 30 min ) and rinsed thoroughly with pbs ( 315 min ) .", "the \n sections were blocked with normal goat serum in pbs [ 1.5% ; room temperature ( rt ) , 1 hr ] , and \n then incubated ( rt , 45 min ) with rabbit polyclonal anti - rat ifn- ( 0.8 g / ml ) or rabbit \n polyclonal anti - mouse fas ( 0.3 g / ml ) , or rabbit polyclonal anti - rat fasl antibodies ( 0.3 g / ml ) \n in pbs containing normal goat serum ( 1.5% ) .", "the sections were incubated at rt with \n biotin - conjugated goat anti - rabbit igg ( 1:200 ; 1 hr ) , avidin - biotin - peroxidase complex ( santa \n cruz rabbit peroxidase kit ; 1 hr ) and dab solution ( 1 - 5 min ) , with nuclei lightly counterstained \n with hematoxylin . for negative controls , rabbit igg ( 1 g / ml ) instead of the primary antibodies \n was added to the reaction . in this experiment ,", "antibody specificity was confirmed by antibody \n preabsorption test , using recombinant rat ifn- ( 2 - 3 g ) and fas and fasl neutralizing peptides \n ( 1 g ) in the respective primary antibody reaction . for localization of fas protein in cultured granulosa cells ,", "the cells were fixed for 10 min in \n ice - cold paraformaldehyde in pbs ( 2% ; ph 7.4 ) . after several washes with pbs , the cells were \n incubated ( 20 min ) in pbs containing normal goat serum ( 10% ) to suppress nonspecific igg \n binding , washed with pbs ( 35 min ) and incubated for 1h with rabbit anti - mouse antibody [ 0.1 \n g / ml in pbs containing normal goat serum ( 1.5% ) ] . following a pbs wash ,", "the cells were \n incubated ( 30 min in the dark ) with fluorescein - conjugated goat anti - rabbit antibody [ 1 g / ml \n pbs with normal goat serum ( 1.5% ) ] , mounted with 90% glycerol in pbs and observed under \n fluorescence microscope .", "( 1992 ) was used to localize apoptotic cells in paraffin whole ovarian sections \n mounted on positively charged slides ( probeon plus ; fisher scientific ltd , ottawa , on ) .", "briefly , \n the sections were deparaffinized , hydrated , treated with proteinase - k ( 10 g / ml . , 30 min . ; 37 ) . \n", "endogenous peroxidase activity was removed by immersing the sections in methanol containing \n h2o2 ( 0.3% , 30 min . ,", "the sections were soaked in the tdt buffer \n [ tris - hcl ( 25 mm ) , sodium cacodylate ( 200 mm ) , cobalt chloride ( 5 mm ) , bsa ( 250 g / ml ) , ph 6.6 ; \n 15 min ) , incubated in 50 l of tdt buffer containing tdt ( 10 u ) and biotinylated 16-dutp ( 1 \n nmol ) [ 60 min . , 37 ] , reacted with avidin - biotin - peroxidase complex ( santa cruz rabbit \n peroxidase kit ; 30 min ) and subsequently with dab solution ( 1 - 5 min ) , as per manufacturer s \n instruction .", "if required , the nuclei were counterstained with methyl green ( 5% ; in 0.1 m veronal \n acetate , ph 4.0 ) .", "tdt enzyme or biotinylated 16-dutp in the labelling reaction were omitted in \n the negative control slides . for in situ detection of apoptosis in cultured \n cells , cells were fixed ( 10 min , 0 ) in fresh paraformaldehyde ( 4% in pbs ) and an in \n situ cell death detection ( fluorescein ) kit ( boehringer mannheim ; quebec , canada ) \n was used , as per manufacturer s instructions . total dna was extracted from cultured granulosa cells ( floating cells + trypsinized attached \n cells ) according to the modified procedure of gross - bellard \n et al .", "( 1973 ) , as previously described ( li et al . , 1998 ) . for biochemical assessment of dna fragmentation", ", dna was \n radiolabeled at the 3-ends according to an established method ( tilly et al . , 1993 ) .", "briefly , one microgram of dna was incubated ( 37 ; 60 min ) with \n 50 l of the labelling buffer [ tris - hcl ( 25 mm ) , sodium cacodylate ( 200 mm ) , cobalt chloride ( 5 \n mm ) , bsa ( 250 g / ml ) , ph 6.6 ] containing terminal deoxynucleotidyl transferase ( tdt ; 25 u ) and \n [ p]-ddatp ( 5 mci , 3,000 ci / mmol ) . the labeled dna were resolved on 1.8% \n agarose gel ( 3.5 h , 60 v ) in tae buffer .", "the gel was dried and subsequently exposed to x - ray \n film at -70. total rna was isolated from cultured granulosa cells using the qiagen rneasy mini rna isolation \n kit ( valencia , ca ) .", "briefly , the cells attached to the culture dishes were lyzed with lysis \n solution ( buffer rlt ) provided by the manufacturer , and the lysate was passed through the qiagen \n qia shredder ( valencia , ca ) . to ensure the extracted rna for pcr was free of dna ,", "two micrograms of total rna was reverse - transcribed ( 42 , 15 min ) with oligo ( dt)15 \n primers ( 0.5 g/g rna ) and avian myeloblastosis virus ( amv ) reverse transcriptase ( 15 u/g rna ) \n in a 20 l reaction mixture containing 10x reverse transcription buffer [ tris - hcl ( 100 mm ) ( ph \n 8.8 ) , kcl ( 500 mm ) , triton x-100 ( 1% ) ] , mgcl2 ( 25 mm ) , dntp mixture ( 10 mm ) and \n rrnasin ribonucleotide inhibitor ( 2 u ) . the reaction was terminated by inactivating the reverse \n transcriptase ( heating at 99 , 5 min ) . ", "gene - specific oligonucleotide primers ( 46.1% gc content ) were designed based on published rat fas \n cdna sequence [ 22 ; genbank accession no .", "d26112 ] and synthesized using a beckman oligo 1000 m dna \n synthesizer ( fullerton , ca ) .", "primers for rat -actin ( nudel et \n al . , 1983 ) were obtained from clontech ( palo alto , ca ) .", "the primers used for pcr \n amplification for fas were 5'-tgcacctcgtgtggacttgaag-3'[479 - 500 ; forward primer ] and \n 5'-ccagtcttcccgtgagattgatac-3 ' [ 888 - 865 ; reverse primer ] .", "polymerase chain reactions were \n carried out with a pct-100 programmable thermal controller ( mj research , inc . , watertown , ma ) . \n", "the pcr reaction mixture included the oligonucleotide primers ( 5 pmol each ) , and taq dna \n polymerase ( 0.5 u ) for each reaction .", "the total volume was brought up to 50 l with 1x pcr \n buffer [ tris - hcl ( 50 mm ) , kcl ( 20 mm ) , mgcl2 ( 3 mm ) , dmso ( 0.5% ) ] and dntp ( 20 m ) . \n", "amplification was carried out for 35 cycles under the annealing temperature of 60 ( 45 sec ) , \n primer extention at 72 ( 1 min ) and denaturation at 94 ( 30 sec ) .", "the amplified dna was \n electrophoresed on 2% agarose gel , and the gel was stained with ethidium bromide for \n visualization under uv light . at initial stage of this experiment ,", "the amplified fas product was \n confirmed by southern hybridization ( sambrook et al . , \n 1989 ) , using the rat fas cdna probe .", "the intensities of the bands were \n densitometrically scanned and each fas amplified product was normalized by -actin . harvested granulosa cells ( 210 cells ) were resuspend in ice - cold wash buffer \n [ nan3 ( 0.1% ) , fbs ( 0.5% ) in pbs ] and centrifuged ( 500 g ; 5 min , 4 ) .", "the cells \n were immediately incubated in primary antibody solution [ hamster anti - mouse fas ( 0.5 g / ml ) in \n wash buffer ) ] at 4 for 30 min . , washed ( 500 g , 25 min in wash buffer ) , and incubated ( 30 \n min . , 4 in dark ) , with fitc - conjugated mouse anti - hamster igg ( 1.0 g / ml ) .", "the cells were then \n washed twice with the wash buffer , filtered through a 35 m nylon mesh to remove cell clumps and \n aliquoted for appropriate cell concentration ( final concentration , 110 cells in 500 \n l ) for flow cytometric analysis ( coulter epics profile ii , hialeah , fl ) .", "as negative controls , nonspecific binding in \n flowcytogram was determined in the absence of primary antibody . ", "pcr and flowcytometry data presented herein were expressed as the meansem of three separate \n experiments .", "the presence and relative abundance of ifn- protein in the follicles during development were \n screened by immunohistochemistry , using a rat specific ifn- antibody ( fig .", "1a ) exhibit immunoreactivity for ifn- , however , the intense and aggregated \n signals were detected in granulosa cells ( arrows ) as well as oocyte ( including its nucleus ) . in \n contrast to its intense immunoreactivity in the cytoplasm ,", "the nuclei of oocytes of secondary and \n tertiary preantral follicles showed considerably lower immunostaining .", "1b ) , granulosa cells in preantral follicles exhibited \n immunopositivity for ifn- , although with lesser intensity and aggregated characteristics than those \n in primordial and early stage of preantral follicles .", "1d ) antral follicles was much less than that seen in the \n preantral follicles . generally , the immunereactivity in theca cells was minimal in the antral \n follicles . a , secondary ( preantral ) follicles ; b , preantral follicle ; c , medium antral follicle ; d , \n large antral follicle .", "gc , granulosa cells ; tc , theca cells ; it , interstitial cells ; cc , \n cumulus cells ; o , oocyte ; n nucleus ; at , antrum .", "arrows indicate intense and aggregated \n immunoreactivity for ifn-. magnification : 400. scare bar : 50 m . based upon the immunohistochemical evidence for the presence of inf- at different stage of \n follicular development in vivo ( fig .", "1 ) , the \n potential role of ifn- in the regulation of fas mrna and protein in relatively undifferentiated \n ( des - primed ) and differentiated ( ecg - primed ) granulosa cells was investigated in \n vitro , using rt - pcr ( fig .", "3 ) . in cultured des - primed granulosa \n cells , ifn- up - regulated fas mrna in a concentration - dependent manner ( fig .", "high concentration ( > 100 u / ml ) of ifn- ( 1,000 u / ml ) resulted in the \n loss of effectiveness of the cytokine in inducing fas mrna and protein expression ( fig .", "2 ) . a , analysis of fas mrna expression by rt - pcr ; b , densitometric quantification of fas mrna \n expression obtained from panel a. * , p<0.05 ; * * * , p < \n 0.001 ; # # # , p<0.001 vs. control group ( 0 u / ml ) .", "insert in each flowcytogram shows representative fluorescence microphotograph of fas \n immunocytochemistry ( arrows and arrowheads indicate immunoreactivity in cytoplasm and \n membrane , respectively .", "numbers 1 and 2 in each flowcytogram \n indicate nonspecific ( partially overlapped with low intensity of fas - positive portion ) and \n specific fas - positive ( high fluorescence intensity ) portion , respectively .", "although cultured ecg - primed granulosa cells also expressed fas mrna , its abundance was considerably \n lower than that in des - primed cells .", "in addition , while fas mrna also increased in a \n concentration - dependent manner in the presence of ifn- , significantly higher concentrations of the \n cytokine ( 100 - 1,000 u / ml , p<0.01 ) were required to elicit a significant increase \n in fas expression than those in des - primed cells ( fig .", "the \n fas protein was localized in the des- and ecg - primed granulosa cells after ifn- treatment and the \n expression levels were subjected to fas antibody - conjugated immuno - flowcytometric analysis ( fig .", "3 ) . as shown in the inserts within each representative \n flowcytogram ( fig .", "3 ) , fas protein was localized in both the \n cellular membrane ( arrow head ) and the cytoplasm [ peri - nuclear region ( arrow ) ] . in addition , intense \n and aggregated immuno - fluorescence was observed in both des- ( fig .", "3 ) granulosa cells \n after ifn- exposure . whereas a significant population of the control group in the des - primed \n granulosa cells exhibited the high intensity of immunofluorescence for endogenous fas \n immunoreactivity [ fig .", "3a , portion 2 ( 184.3% ) ] , the ecg - primed \n control granulosa cells showed relatively smaller population with high immunofluorescence intensity \n for fas [ fig .", "ifn- treatment ( 100 \n u / ml ) of des - primed granulosa cells significantly increased high intensity ( 337.5% ) portion of fas \n immunofluorescene ( fig .", "however , treatment with \n high concentration of ifn- ( 1,000 u / ml ) resulted in a lower number of cells exhibiting \n immunofluorescence positivity for fas in high intensity ( 252.3% ) portion than those with 100 u / ml \n but higher than that of the control group . in ecg - primed cells ,", "however , only small increases in \n immunofluorescence were detected with ifn- at concentrations of 100 and 1,000 u / ml when compared to \n controls ( fig . 3e and 3f ) .", "the functional role of ifn--induced fas expression in induction of apoptosis were tested in \n undifferentiated ( des - primed ) and differentiated ( ecg - primed ) granulosa cells in vitro \n was determined by assessing their the ability to undergo apoptosis following a challenge \n with an agonistic effect of fas mab . while typical cellular morphology and apoptosis in des - primed \n granulosa cells are illustrated in fig .", "4 , differences in these \n features between des- and ecg - primed groups are summarized in table \n 1 . in des - primed ( undifferentiated ) granulosa cells , ifn- had minimal effects on \n cellular morphology ( fig .", "however , addition of fas mab to the cell \n cultures resulted in the appearance of apoptotic cellular morphology and characteristics ( fig .", "4c & 4 g ) , which could be enhanced with pretreatment with \n ifn- ( fig .", "4d & 4h ) . in ecg - primed ( differentiated ) \n granulosa cells , however , ifn- pretreatment failed to potentiate the pro - apoptotic effects of \n agonistic fas mab , which were observed prominently in des - primed cells ( table 1 ) . left and right panels illustrate cellular morphology ( phase - contrast microscopy ) and \n in situ apoptotic cell detection ( tunel ) , respectively .", "a and e , \n control group [ ifn- vehicle ( medium ) ( 24 hr ) + igg ( 6 hr ) ] ; b and f , ifn- group [ ifn- ( 24 \n hr ) + igg ( 6 hr ) ] ; c and g , fas mab group [ ifn- vehicle ( medium ) ( 24 hr ) + fas mab ( 6 hr ) ] ; \n d and h , ifn- and fas mab group [ ifn- ( 24 hr ) + fas mab ( 6 hr ) ] .", "arrowheads in left and right panels indicate floating cells and \n tunel - positive cells , respecttively .", "magnification : 400. inserts in right panels show \n representative apoptotic dna fragmentation pattern in each treatment group .", "- : rare , + : occasional , + + : frequent , + + + : extensive , + + + + : very extensive to further examine the colocalization of ifn- , fas and fasl expression and occurrence of \n apoptosis in vivo , follicles at very early stage of antral formation ( estimated \n between preantral and early antral stages ) were analyzed serially by immunohistochemistry and \n in situ tunel method ( fig . 5 ) . \n", "immunoreactivity for ifn- was distributed in the granulosa but not the thecal layers ( fig .", "likewise , the most intense and aggregated form of fas \n immuno - positive signals were detected in the loosely attached granulosa cells ( fig .", "although fasl immunoreactivity could be colocalized in fas - positive \n cells , he most aggregated and intense ( arrows ) immunoreactivity was present in granulosa cells \n mainly lining the antrum ( fig .", "5c ) . similarly , apoptotic \n ( tunel - positive ) granulosa cells were also detected in antral granulosa cells ( fig .", "a , ifn- ; b , fas ; c , fasl ; d , tunel .", "gc , tc and it represent granulosa cells , theca cells and \n interstitial cells , respectively .", "in the present study , we have localized rat ifn- in the rat ovary during the follicular development and \n tested its role on granulosa cell death via induction of the fas / fasl system in granulosa cells at \n different stage of differentiation .", "we have found that the responsiveness of granulosa cells to ifn- in \n terms of fas receptor expression was significantly compromised with follicular maturation .", "the \n immunoreactivity of ifn- was most intense in early stages of folliculogenesis , such as in preantral \n follicles .", "up - regulation of fas by ifn- and the subsequent increased responsiveness to an apoptotic \n death signal ( e.g. fas activation by agonistic fas mab ) were more evident in granulosa cells from the \n preantral follicles than those from antral follicles previously exposed to gonadotropins .", "the importance of intraovarian interaction between immune cells ( lymphocytes and macrophages ) and \n granulosa cells in ovarian physiology via the synthesis and actions of cytokines ( e.g.tnf and il ) has \n been studied extensively ( adashi , 1992 ; terranova et al . , 1993 ) . although the modulatory roles of ifn- in granulosa cell \n differentiation and steroidogenesis have been demonstrated in vitro ( gorospe et al . , 1998 ; xiao and \n findlay , 1992 ) ,", "ifn- is \n detectable in the follicular fluids ( grasso et al . , 1988 ) and \n lymphocytes present in the ovary have been suggested to be an important contributor to the follicular \n ifn- ( adashi , 1992 ) .", "immune cells ( lymphocytes / macrophages ) are \n believed to be mainly present in atretic antral follicles ( best et al . , \n 1996 ) ,", ", 1994a ; brannstrom et al . , 1994b ) \n where they remove damaged and dead cells by secreting cytotoxic cytokines and phagocytosis .", "the presence \n of immune cells during early stage of follicular development has not been reported and the possibility \n that immune cells are the only the source of ifn- in preantral follicles can not be excluded . in this \n study , ifn- was localized predominantly in granulosa cells and oocytes throughout the follicular \n development but more abundantly in those at the early follicular ( preantral ) stage .", "it has been \n demonstrated that tnf- and fas ligand are expressed in granulosa cells and oocytes ( chen et al .", "although we have not studied ifn- gene expression in ovarian cells in the current \n study , it is possible that oocytes and granulosa cells in the preantral follicles may be an important \n source of ifn- in the ovary and that this cytokine plays an autocrine and/or paracrine role in \n regulating the expression and actions of other cytokines involved in cell removal process in the \n degrading follicles .", "ifn- is an important modulator of granulosa cell differentiation during the early stage of follicular \n development ( gorospe et al . , 1988 ; xiao and findlay , 1992 ) ,", "although its functional role in differentiated granulosa \n cells in late follicular development ( i.e. , preovulatory follicles ) remains to be further established . \n", "granulosa cell apoptosis is frequently observed in early antral ( penultimate ) stage of follicular \n development , it is only assumed that up - regulation of fas receptor by ifn- in granulosa cells of \n preantral follicles is prerequisite for fasl ligation and that the latter could be activated by \n intra - ovarian factor(s ) following antrum formation .", "it has been suggested that ifn- facilitate \n apoptosis in luteinized granulosa cells by decreasing endogenous igf activity via stimulation of igfbps \n rather than of fas activation ( cataldo et al . , 1998 ) .", "however , the \n concentrations of ifn- used in above studies could have been pharmacological and cytotoxic to the \n cells .", "quirk et al ( 1995 ) demonstrated fas - mediated apoptosis in \n human granulosa - luteal cells and suggested that ifn- up - regulates fas mrna and protein expression and \n that pretreatment of cells with ifn- alone or in combination with hcg facilitates fas mab - induced \n cytotoxicity .", "therefore , it is unlikely that the physiological concentration of ifn- could induce fas \n expression in fully differentiated luteinized ( or luteinizing ) granulosa cells .", "recently , quirk et al ( 1998 ) showed that combined ifn- and tnf treatment \n was more effective in inducing fas expression and potentiating fas mab - induced apoptosis in cultured \n granulosa cells ( mostly from large preovulatory follicles in ecg - primed mice ) than by either ifn- or \n tnf alone .", "the results suggest that the mechanism(s ) of induction of apoptosis and responsiveness \n against apoptotic insult ( e.g. ifn- ) in differentiated granulosa cells might be much more complex and \n resistant than those of undifferentiated granulsoa cells , as also seen in the present study .", "the possible mediatory role of the fas / fasl system in granulosa and luteal cell apoptosis during \n follicular atresia and luteal regression , respectively have been investigated in several mammalian \n species ( quirk et al . , 1995 ;", "previous reports have demonstrated that increased granulosa cell fas and fasl protein \n content is associated with apoptosis at the penultimate ( early ) stage of follicular development \n in vivo ( kim et al . , 1998 ) , and that \n p53-mediated granulosa cell apoptosis involves the activation of the fas / fasl system ( kim et al . , 1999 ) .", "these studies also suggested that fsh is a key \n inhibitory factor on granulosa cell death as well as the involvement of the fas / fasl system in \n follicular atresia .", "the nature of the regulation of fas receptor expression throughout the follicular \n development , however , remains unclear .", "although the regulation of fas receptor expression by ifn- has \n been demonstrated in human granulosa - luteal cells in vitro ( quirk et al . , 1995 ) , the potential regulatory role of ifn- in fas mrna and protein \n expression in undifferentiated granulosa cells during early stages of folliculogenesis has not been \n thoroughly investigated . in the present studies ,", "we have studied the possible role of ifn- in the \n regulation of granulsoa cell fas expression during follicular development . while increased fas \n expression in granulsoa cells from preantral or early antral follicles could be induced with low \n concentrations of ifn- ( 10 - 100 u / ml ) , higher concentrations ( 1,000 u / ml ) were required in ecg - primed \n cells .", "our present immuno - flowcytometric analysis also confirmed that undifferentiated ( des - primed ) \n granulsoa cells were more responsive to ifn- in the induction of fas receptor than cells pretreated \n with gonadotropin .", "these findings are not only consistent with the studies by quirk et al ( 1995 ) which showed increased expression of fas protein in \n gonadotropin - primed human granulosa cells following exposure to very high concentration ( > 10,000 \n u / ml ) of ifn- in vitro , but extended their observations to suggest a role for the \n cytokine in the ovary at physiological concentrations .", "it is well established that fsh is an important cell survival factor in the mammalian ovarian follicle \n ( chun et al .", "whereas stimulation of immature rats with ecg resulted in suppressed basal \n granulosa cell fas / fasl levels and apoptosis , decreased atresia and enhanced follicular growth , \n withdrawal of gonadotropic support with the injection of a neutralizing antibody in \n vivo during early antral development led to increased granulosa cell fas / fasl expression \n and apoptotic cell death , and eventual follicular demise ( kim et al . , \n 1998 ) . in the present studies , we have demonstrated that while ifn- effectively up - regulated \n fas expression in granulosa cells from preantral and early antral follicles and sensitized them to the \n pro - apoptotic action of agonistic fas antibody , cells isolated from large antral follicles previously \n exposed to the gonadotropin in vivo were more differentiated but less responsive to \n ifn-. these findings suggest that the anti - apoptotic role of fsh in the granulosa cells may in part \n involve its modulatory influence on ifn- action , and are consistent with the concept that ovarian \n follicles beyond the penultimate stage of development are protected physiologically from pro - apoptotic \n insult and destined to ovulate ( chun et al . , 1996 ; boone et al . , 1997 ) .", "the mechanism(s ) by which the gonadotropin \n modulates ifn--induced fas expression granulosa cells is unclear .", "it has been demonstrated that fas \n expression is regulated by the newly identified fas - regulatory gene tdag51 , the expression of which is \n pkc - dependent ( wang et al . , 1998 ) . since ifn- , like tnf ( sancho tello and terranova , 1991 ) and il-3 ( farrar et al . , 1985 ) , appears capable of activating pkc ( ostrowski et al . , 1988 ) , it is possible that the reduced responsiveness of \n differentiated granulosa cells to ifn- might be due to pka activation by fsh .", "in addition , activation \n of pka pathway is known to inhibit pkc - dependent mechanism(s ) in a variety of biological systems \n including the granulosa cell ( shinohara et al . , 1985 ) .", "in this \n context , differential responsiveness of granulosa cells at different cytodifferentiation to ifn- may be \n related to possible interactions between signal transduction pathways of ifn- and gonadotropin .", "it is \n possible that the efficient fas gene expression in undifferentiated granulosa cells may involve \n activation of pkc pathway by ifn-. fas receptor signaling is a complex process and continues to capture considerable attention and research \n efforts .", "fas activation initiated by ligation and oligomerrization results in recruitment of fadd \n ( fasassociated death domain - containing protein ) ( chinnaiyan et al . , \n 1995 ) .", "fadd then activates procaspase8 ( muzio et al . , \n 1996 ) , which appears to be the first step of a proteolytic cascade that activates other \n caspases such as caspases3 , 6 , and 7 ( hirata et al . , 1998 )", "ifn- significantly increased the population of granulosa cells exhibiting high \n fluorescence intensity of fas immunoreactivity . while this increase might merely represent an increase \n in the cell population with enhanced fas expression , it is tempting to speculate that the high intensity \n of fluorescence reflects the oligomerization of fas receptor proteins , and that fas oligomerization in \n response to ifn- in undifferentiated granulsoa cells may be more efficient and functional than in \n differentiated granulosa cells .", "whether this indeed is the case requires further investigation and \n confirmation . in this context , intense and aggregated immunoreactive fas protein has consistently been \n detected in cytoplasm as well as cellular membrane of cultured granulosa cells of the present study . \n", "these results support our previous observation suggesting the presence of aggregated fas immunostaining \n in the granulosa cells of atretic early antral follicles in the ovary ( kim et al . , 1998 ) .", "moreover , recent studies have indicated that fas is also localized in the \n peri - nuclear region of the cytoplasm and that fas - trafficking from golgi apparatus to membrane is \n p53-dependent and required for fas - mediated apoptosis ( bennett et al . , \n 1998 ) .", "in light of our recent demonstration of the role of p53 in the regulation of fas \n expression in rat granulosa cells ( kim et al . , 1999 ) and", "their \n intense fas immunostaining following ifn- challenge , investigations into whether this cytokine plays a \n role in fas trafficking in the granulosa cell would provide important clues on the cellular mechanism \n involved in the control of fas - mediated granulsoa cell apoptosis during early folliculogenesis . in summary , we have demonstrated that ifn- plays a pivotal role in regulating granulosa cell \n differentiation and apoptosis by potentiating fas function , a process modulated by gonadotropin ( i.e. \n fsh ) .", "the role of ifn- is most prominent in undifferentiated granulosa cells during early stage of \n follicular development ." ]

[ "<S> abstractfas ligand ( fasl ) and its receptor fas have been implicated in granulosa cell apoptosis during \n follicular atresia . </S>", "<S> although interferon - gamma ( ifn- ) is believed to be involved in the regulation \n fas expression in differentiated granulosa or granulosa - luteal cells , the expression of this \n cytokine and its role in the regulation of the granulosa cell fas / fasl system and apoptosis during \n follicular maturation have not been thoroughly investigated . in the present study , we have examined \n the presence of ifn- in ovarian follicles at different stage of development by immunohistochemistry \n and related their relative intensities with follicular expression of fas and fasl , and with \n differences in granulosa cell sensitivity to fas activation by exogenous agonistic anti - fas \n monoclonal antibody ( fas mab ) . although ifn- immunostaining was detectable in oocyte and granulosa \n cells in antral follicles , most intense immunoreactivity for the cytokine was observed in these \n cells of preantral follicles . </S>", "<S> intense immunoreactivity for ifn- was most evident in granulosa cells \n of atretic early antral follicles where increased fas and fasl expression and apoptosis were also \n observed . whereas low concentrations of ifn- ( 10 - 100 u / ml ) significantly increased fas expression \n in undifferentiated granulosa cells ( from preantral or very early antral follicles ) in \n vitro , very higher concentrations ( 1,000 u / ml ) </S>", "<S> were required to up - regulate of fas in \n differentiated cells isolated from ecg - primed ( antral ) follicles . </S>", "<S> addition of agonistic fas mab to \n cultures of granulosa cells at the two stages of differentiation and pretreated with ifn- ( 100 \n u / ml ) elicited morphological and biochemical apoptotic features which were more prominent in cells \n not previously exposed to the gonadotropin in vivo . </S>", "<S> these findings suggested that \n ifn- is an important physiologic intra - ovarian regulator of follicular atresia and plays a pivotal \n role in regulation of expression of fas receptor and subsequent apoptotic response in \n undifferentiated ( or poorly differentiated ) granulosa cells at an early ( penultimate ) stage of \n follicular development . </S>" ]

[ "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "ovarian follicular fate ( i.e. development / ovulation versus atresia ) in the mammals is determined by the \n fate of the cells within the follicles and is regulated by complex interactions of endocrine \n ( gonadotropin and steroid hormones ) and locally produced ( growth factors and cytokines ) factors ( hirshfield , 1991 ) .", "although follicular atresia can occur at all the \n stages of follicular development , a high incidence of granulosa cell apoptosis , a well recognized \n cellular basis for this degenerative process , is predominantly observed at the early antral stage of \n follicular development during the ovarian cycle ( kim et al . , \n 1998 ) .", "fas antigen ( fas ) , a member of the tnf receptor family , and its ligand fas ligand ( fasl ) , have been \n identified as cell death mediator and factor , respectively in a variety of cell types ( watanabefukunaga et al . , 1992 ; suda \n et al . , 1993 ) .", "their involvement in the induction of granulosa and luteal cell apoptosis has \n been demonstrated in several mammalian species ( quirk et al . , \n 1995 ; hakuno et al . , 1996 ; kondo et al .", "fas \n and fasl expression is up - regulated in granulosa cells undergoing apoptosis during early stage of \n follicular development ( kim et al . , 1998 ) and", "p53-mediated \n granulosa cell apoptosis is associated with the activation of fas / fasl system during follicular atresia \n ( kim et al . , 1999 ) .", "in addition , follicle stimulating hormone \n ( fsh ) is an important negative regulator of the fas / fasl system and a key cell survival factor during \n the follicular development and the role of the fas / fasl system in granulosa cell apoptosis appears more \n prominent at early than at late stages of follicular maturation .", "interferon gamma ( ifn- ) is a member of a family of heterogeneous regulatory proteins having antiviral , \n immunomodulatory , antiproliferative , and cytodifferentiation regulatory activities ( pestka et al . , 1987 ) .", "it increases adrenal steroidogenesis ( blalock and harp , 1981 ) , increases iodide uptake by thyroid cells \n ( friedman et al . , 1982 ) , and suppresses hcg - induced testosterone production by leydig cells ( orava et al . , 1989 ) .", "at physiological concentrations , ifn- \n inhibits granulosa cell differentiation and steroidogenesis ( gorospe et \n al . , 1988 ; xiao and findlay , 1992 ) .", "in addition , ifn- \n increases fas mrna expression in mouse lymphoma cells and bam3 cells ( watanabefukunaga et al . , 1992 ) and", "fas content in human granulosa / luteal cells ( quirk et al . , 1995 ) .", "apoptosis in ovarian surface epithelial cells \n is mediated by increased fas expression induced by ifn- ( quirk et al . , \n 1997 ) .", "moreover , ifn- induces fas - mediated apoptosis in differrentiated mouse granulosa \n cells which could be potentiated by the presence of tnf ( quirk et al . , \n 1998 ) .", "although the importance of ifn- in undifferentiated or poorly differentiated \n granulosa cells is well established , the presence of this cytokine in the ovary throughout follicular \n development has not been investigated .", "in addition , the regulation of fas system in undifferentiated \n granulosa cells by ifn- , subsequent fas - mediated apoptotic response as well as their modulation by \n gonadotropin in vivo remain unclear .", "the purpose of the present study was to examine the role of ifn- in regulation of fas expression and \n apoptosis in granulosa cells at different stages of follicular development and granulosa cell \n differentiation in vitro .", "in addition , we assessed the presence of ifn- in the ovary \n during follicular maturation and studied the relationship between the presence of ifn- , fas / fasl system \n and apoptosis in vivo ." ], [ "[ p]-ddatp and dntps were obtained from amersham pharmacia biotech ( piscataway , nj ) . \n", "horse radish peroxidase ( hrp)-conjugated goat anti - rabbit secondary antibody was purchased from \n bio - rad laboratories ( hercule , ca ) .", "rabbit anti - rat interferon- ( ifn- ) was obtained from biosource \n international ( camarillo , ca ) .", "biotinylated 16-dutp , in situ cell death detection \n ( fluorescein ) kit , proteinase - k and terminal deoxynucleotidyl transferase ( tdt ) were purchased from \n boehringer - mannheim ( indianapolis , in ) .", "recombinant rat ifn- was from genzyme ( cambridge , ma ) . \n fetal bovine serum ( fbs ) , minimal essential medium ( mem ) , non - essential amino acids ( naa ) , \n penicillin and streptomycin were from gibco / brl ( burlington , on , canada ) .", "fitc - conjugated mouse \n anti - hamster igg , hamster anti - mouse fas monoclonal antibody ( clone jo2 ) and hamster igg were \n obtained from pharmingen ( san diego , ca ) .", "avian myeloblastosis virus ( amv ) reverse transcriptase , \n oligo(dt)15 primer , rrnasin ribonucleotide inhibitor and taq dna polymerase were from \n promega ( madison , wi ) .", "rabbit igg , rabbit peroxidase kits , rabbit polyclonal anti - mouse fas ( sc-716 ) \n and anti - rat fasl ( sc-834 ) antibodies , and neutralization peptides ( fas ; sc-716p , fasl ; sc-834p ) \n were from santa cruz biotechnology ( santa cruz , ca ) .", "agarose , bovine serum albumin ( bsa ; fraction \n v ) , diethylstilbesterol ( des ) , equine chorionic gonadotropin ( ecg ) , methyl green , normal goat serum , \n phenylmethylsulfonyl fluoride ( pmsf ) and veronal acetate were purchased from sigma chemical co. ( st \n louis , mo ) . to obtain ovaries consisting mainly of follicles synchronized at the preantral / early antral and \n medium / large antral stages in immature ( 21 - 23 days old ) sprague - dawley rats ( charles river canada ; \n quebec , canada or samtako bio - korea ; osan , korea ) , des ( 1mg / day , sc , for 3 consecutive days and \n sacrificed 24 hr after last injection ) and ecg (", "15 iu , ip and sacrificed 48 hr thereafter ) were \n administered , respectively .", "a 14 h light:10 h dark photo - cycle was maintained with \n lights - on at 0600 h. all procedures were performed in accordance with protocols approved by the \n dong - a university animal care and use committee . following removal of connective tissues ,", "the \n ovaries were washed in pbs ( ph 7.4 ) to remove excess blood and were either immediately fixed in 10% \n neutral buffered formalin ( ph 7.4 ) for histological processing or used for granulosa cell isolation \n by follicle puncture ( rao et al . ,", "granulosa cells ( 2.510 ) from ovaries of des- and ecg - primed rats were plated for 6 hr \n ( 5% co2 , 37 ) in 60 mm falcon plastic culture dishes ( becton dickins , lincoln park , nj ) \n in mem supplemented with 2% fbs , 1x naa , penicillin ( 100 u / ml ) and streptomycin ( 100 g / ml ) .", "the \n medium was then replaced with mem supplemented with 0.1% bsa , 1x naa , penicillin ( 100 u / ml ) and \n streptomycin ( 100 g / ml ) in subsequent culture .", "non - viable ( floating ) cells were removed at this \n medium - replacement step .", "for induction of fas mrna and protein expression , granulosa cells were cultured in the presence of \n recombinant rat ifn- ( 0 - 1,000 u / ml of the medium ) for 18 hr . floating and attached cells harvested \n by centrifugation and trypsinization respectively , were combined and washed with ice - cold pbs , \n pending protein and rna extraction , and flowcytometric analysis . for immunocytochemistry and tunel , \n sterilized cover glasses ( 2222 mm ) were added onto the bottom of the culture dishes prior to cell \n plating . at the end of the culture period ,", "the cover glasses were removed , washed briefly with \n ice - cold pbs and processed for immunocytochemistry . to assess the influence of ifn- on fas expression and subsequent granulosa cell apoptotic response , \n cells pretreated for 18 hr with / without the cytokine were incubated for an additional 6 h with \n either agonistic fas monoclonal antibody ( hamster anti - mouse fas mab , igg ; 1 g / ml ) or hamster igg \n ( control ; 1 g / ml ) .", "the effect of the treatment was assessed by phase - contrast microscopy , tunel and \n dna fragmentation analysis . ", "paraffin embedded whole ovarian sections ( 4 - 5 m ) were used for ifn- , fas and fasl \n immunohistochemistry .", "the deparaffinized and hydrated sections were quenched in \n h2o2 ( 0.3% ; 30 min ) and rinsed thoroughly with pbs ( 315 min ) .", "the \n sections were blocked with normal goat serum in pbs [ 1.5% ; room temperature ( rt ) , 1 hr ] , and \n then incubated ( rt , 45 min ) with rabbit polyclonal anti - rat ifn- ( 0.8 g / ml ) or rabbit \n polyclonal anti - mouse fas ( 0.3 g / ml ) , or rabbit polyclonal anti - rat fasl antibodies ( 0.3 g / ml ) \n in pbs containing normal goat serum ( 1.5% ) .", "the sections were incubated at rt with \n biotin - conjugated goat anti - rabbit igg ( 1:200 ; 1 hr ) , avidin - biotin - peroxidase complex ( santa \n cruz rabbit peroxidase kit ; 1 hr ) and dab solution ( 1 - 5 min ) , with nuclei lightly counterstained \n with hematoxylin . for negative controls , rabbit igg ( 1 g / ml ) instead of the primary antibodies \n was added to the reaction . in this experiment ,", "antibody specificity was confirmed by antibody \n preabsorption test , using recombinant rat ifn- ( 2 - 3 g ) and fas and fasl neutralizing peptides \n ( 1 g ) in the respective primary antibody reaction . for localization of fas protein in cultured granulosa cells ,", "the cells were fixed for 10 min in \n ice - cold paraformaldehyde in pbs ( 2% ; ph 7.4 ) . after several washes with pbs , the cells were \n incubated ( 20 min ) in pbs containing normal goat serum ( 10% ) to suppress nonspecific igg \n binding , washed with pbs ( 35 min ) and incubated for 1h with rabbit anti - mouse antibody [ 0.1 \n g / ml in pbs containing normal goat serum ( 1.5% ) ] . following a pbs wash ,", "the cells were \n incubated ( 30 min in the dark ) with fluorescein - conjugated goat anti - rabbit antibody [ 1 g / ml \n pbs with normal goat serum ( 1.5% ) ] , mounted with 90% glycerol in pbs and observed under \n fluorescence microscope .", "( 1992 ) was used to localize apoptotic cells in paraffin whole ovarian sections \n mounted on positively charged slides ( probeon plus ; fisher scientific ltd , ottawa , on ) .", "briefly , \n the sections were deparaffinized , hydrated , treated with proteinase - k ( 10 g / ml . , 30 min . ; 37 ) . \n", "endogenous peroxidase activity was removed by immersing the sections in methanol containing \n h2o2 ( 0.3% , 30 min . ,", "the sections were soaked in the tdt buffer \n [ tris - hcl ( 25 mm ) , sodium cacodylate ( 200 mm ) , cobalt chloride ( 5 mm ) , bsa ( 250 g / ml ) , ph 6.6 ; \n 15 min ) , incubated in 50 l of tdt buffer containing tdt ( 10 u ) and biotinylated 16-dutp ( 1 \n nmol ) [ 60 min . , 37 ] , reacted with avidin - biotin - peroxidase complex ( santa cruz rabbit \n peroxidase kit ; 30 min ) and subsequently with dab solution ( 1 - 5 min ) , as per manufacturer s \n instruction .", "if required , the nuclei were counterstained with methyl green ( 5% ; in 0.1 m veronal \n acetate , ph 4.0 ) .", "tdt enzyme or biotinylated 16-dutp in the labelling reaction were omitted in \n the negative control slides . for in situ detection of apoptosis in cultured \n cells , cells were fixed ( 10 min , 0 ) in fresh paraformaldehyde ( 4% in pbs ) and an in \n situ cell death detection ( fluorescein ) kit ( boehringer mannheim ; quebec , canada ) \n was used , as per manufacturer s instructions . total dna was extracted from cultured granulosa cells ( floating cells + trypsinized attached \n cells ) according to the modified procedure of gross - bellard \n et al .", "( 1973 ) , as previously described ( li et al . , 1998 ) . for biochemical assessment of dna fragmentation", ", dna was \n radiolabeled at the 3-ends according to an established method ( tilly et al . , 1993 ) .", "briefly , one microgram of dna was incubated ( 37 ; 60 min ) with \n 50 l of the labelling buffer [ tris - hcl ( 25 mm ) , sodium cacodylate ( 200 mm ) , cobalt chloride ( 5 \n mm ) , bsa ( 250 g / ml ) , ph 6.6 ] containing terminal deoxynucleotidyl transferase ( tdt ; 25 u ) and \n [ p]-ddatp ( 5 mci , 3,000 ci / mmol ) . the labeled dna were resolved on 1.8% \n agarose gel ( 3.5 h , 60 v ) in tae buffer .", "the gel was dried and subsequently exposed to x - ray \n film at -70. total rna was isolated from cultured granulosa cells using the qiagen rneasy mini rna isolation \n kit ( valencia , ca ) .", "briefly , the cells attached to the culture dishes were lyzed with lysis \n solution ( buffer rlt ) provided by the manufacturer , and the lysate was passed through the qiagen \n qia shredder ( valencia , ca ) . to ensure the extracted rna for pcr was free of dna ,", "two micrograms of total rna was reverse - transcribed ( 42 , 15 min ) with oligo ( dt)15 \n primers ( 0.5 g/g rna ) and avian myeloblastosis virus ( amv ) reverse transcriptase ( 15 u/g rna ) \n in a 20 l reaction mixture containing 10x reverse transcription buffer [ tris - hcl ( 100 mm ) ( ph \n 8.8 ) , kcl ( 500 mm ) , triton x-100 ( 1% ) ] , mgcl2 ( 25 mm ) , dntp mixture ( 10 mm ) and \n rrnasin ribonucleotide inhibitor ( 2 u ) . the reaction was terminated by inactivating the reverse \n transcriptase ( heating at 99 , 5 min ) . ", "gene - specific oligonucleotide primers ( 46.1% gc content ) were designed based on published rat fas \n cdna sequence [ 22 ; genbank accession no .", "d26112 ] and synthesized using a beckman oligo 1000 m dna \n synthesizer ( fullerton , ca ) .", "primers for rat -actin ( nudel et \n al . , 1983 ) were obtained from clontech ( palo alto , ca ) .", "the primers used for pcr \n amplification for fas were 5'-tgcacctcgtgtggacttgaag-3'[479 - 500 ; forward primer ] and \n 5'-ccagtcttcccgtgagattgatac-3 ' [ 888 - 865 ; reverse primer ] .", "polymerase chain reactions were \n carried out with a pct-100 programmable thermal controller ( mj research , inc . , watertown , ma ) . \n", "the pcr reaction mixture included the oligonucleotide primers ( 5 pmol each ) , and taq dna \n polymerase ( 0.5 u ) for each reaction .", "the total volume was brought up to 50 l with 1x pcr \n buffer [ tris - hcl ( 50 mm ) , kcl ( 20 mm ) , mgcl2 ( 3 mm ) , dmso ( 0.5% ) ] and dntp ( 20 m ) . \n", "amplification was carried out for 35 cycles under the annealing temperature of 60 ( 45 sec ) , \n primer extention at 72 ( 1 min ) and denaturation at 94 ( 30 sec ) .", "the amplified dna was \n electrophoresed on 2% agarose gel , and the gel was stained with ethidium bromide for \n visualization under uv light . at initial stage of this experiment ,", "the amplified fas product was \n confirmed by southern hybridization ( sambrook et al . , \n 1989 ) , using the rat fas cdna probe .", "the intensities of the bands were \n densitometrically scanned and each fas amplified product was normalized by -actin . harvested granulosa cells ( 210 cells ) were resuspend in ice - cold wash buffer \n [ nan3 ( 0.1% ) , fbs ( 0.5% ) in pbs ] and centrifuged ( 500 g ; 5 min , 4 ) .", "the cells \n were immediately incubated in primary antibody solution [ hamster anti - mouse fas ( 0.5 g / ml ) in \n wash buffer ) ] at 4 for 30 min . , washed ( 500 g , 25 min in wash buffer ) , and incubated ( 30 \n min . , 4 in dark ) , with fitc - conjugated mouse anti - hamster igg ( 1.0 g / ml ) .", "the cells were then \n washed twice with the wash buffer , filtered through a 35 m nylon mesh to remove cell clumps and \n aliquoted for appropriate cell concentration ( final concentration , 110 cells in 500 \n l ) for flow cytometric analysis ( coulter epics profile ii , hialeah , fl ) .", "as negative controls , nonspecific binding in \n flowcytogram was determined in the absence of primary antibody . ", "pcr and flowcytometry data presented herein were expressed as the meansem of three separate \n experiments ." ], [ "the presence and relative abundance of ifn- protein in the follicles during development were \n screened by immunohistochemistry , using a rat specific ifn- antibody ( fig .", "1a ) exhibit immunoreactivity for ifn- , however , the intense and aggregated \n signals were detected in granulosa cells ( arrows ) as well as oocyte ( including its nucleus ) . in \n contrast to its intense immunoreactivity in the cytoplasm ,", "the nuclei of oocytes of secondary and \n tertiary preantral follicles showed considerably lower immunostaining .", "1b ) , granulosa cells in preantral follicles exhibited \n immunopositivity for ifn- , although with lesser intensity and aggregated characteristics than those \n in primordial and early stage of preantral follicles .", "1d ) antral follicles was much less than that seen in the \n preantral follicles . generally , the immunereactivity in theca cells was minimal in the antral \n follicles . a , secondary ( preantral ) follicles ; b , preantral follicle ; c , medium antral follicle ; d , \n large antral follicle .", "gc , granulosa cells ; tc , theca cells ; it , interstitial cells ; cc , \n cumulus cells ; o , oocyte ; n nucleus ; at , antrum .", "arrows indicate intense and aggregated \n immunoreactivity for ifn-. magnification : 400. scare bar : 50 m . based upon the immunohistochemical evidence for the presence of inf- at different stage of \n follicular development in vivo ( fig .", "1 ) , the \n potential role of ifn- in the regulation of fas mrna and protein in relatively undifferentiated \n ( des - primed ) and differentiated ( ecg - primed ) granulosa cells was investigated in \n vitro , using rt - pcr ( fig .", "3 ) . in cultured des - primed granulosa \n cells , ifn- up - regulated fas mrna in a concentration - dependent manner ( fig .", "high concentration ( > 100 u / ml ) of ifn- ( 1,000 u / ml ) resulted in the \n loss of effectiveness of the cytokine in inducing fas mrna and protein expression ( fig .", "2 ) . a , analysis of fas mrna expression by rt - pcr ; b , densitometric quantification of fas mrna \n expression obtained from panel a. * , p<0.05 ; * * * , p < \n 0.001 ; # # # , p<0.001 vs. control group ( 0 u / ml ) .", "insert in each flowcytogram shows representative fluorescence microphotograph of fas \n immunocytochemistry ( arrows and arrowheads indicate immunoreactivity in cytoplasm and \n membrane , respectively .", "numbers 1 and 2 in each flowcytogram \n indicate nonspecific ( partially overlapped with low intensity of fas - positive portion ) and \n specific fas - positive ( high fluorescence intensity ) portion , respectively .", "although cultured ecg - primed granulosa cells also expressed fas mrna , its abundance was considerably \n lower than that in des - primed cells .", "in addition , while fas mrna also increased in a \n concentration - dependent manner in the presence of ifn- , significantly higher concentrations of the \n cytokine ( 100 - 1,000 u / ml , p<0.01 ) were required to elicit a significant increase \n in fas expression than those in des - primed cells ( fig .", "the \n fas protein was localized in the des- and ecg - primed granulosa cells after ifn- treatment and the \n expression levels were subjected to fas antibody - conjugated immuno - flowcytometric analysis ( fig .", "3 ) . as shown in the inserts within each representative \n flowcytogram ( fig .", "3 ) , fas protein was localized in both the \n cellular membrane ( arrow head ) and the cytoplasm [ peri - nuclear region ( arrow ) ] . in addition , intense \n and aggregated immuno - fluorescence was observed in both des- ( fig .", "3 ) granulosa cells \n after ifn- exposure . whereas a significant population of the control group in the des - primed \n granulosa cells exhibited the high intensity of immunofluorescence for endogenous fas \n immunoreactivity [ fig .", "3a , portion 2 ( 184.3% ) ] , the ecg - primed \n control granulosa cells showed relatively smaller population with high immunofluorescence intensity \n for fas [ fig .", "ifn- treatment ( 100 \n u / ml ) of des - primed granulosa cells significantly increased high intensity ( 337.5% ) portion of fas \n immunofluorescene ( fig .", "however , treatment with \n high concentration of ifn- ( 1,000 u / ml ) resulted in a lower number of cells exhibiting \n immunofluorescence positivity for fas in high intensity ( 252.3% ) portion than those with 100 u / ml \n but higher than that of the control group . in ecg - primed cells ,", "however , only small increases in \n immunofluorescence were detected with ifn- at concentrations of 100 and 1,000 u / ml when compared to \n controls ( fig . 3e and 3f ) .", "the functional role of ifn--induced fas expression in induction of apoptosis were tested in \n undifferentiated ( des - primed ) and differentiated ( ecg - primed ) granulosa cells in vitro \n was determined by assessing their the ability to undergo apoptosis following a challenge \n with an agonistic effect of fas mab . while typical cellular morphology and apoptosis in des - primed \n granulosa cells are illustrated in fig .", "4 , differences in these \n features between des- and ecg - primed groups are summarized in table \n 1 . in des - primed ( undifferentiated ) granulosa cells , ifn- had minimal effects on \n cellular morphology ( fig .", "however , addition of fas mab to the cell \n cultures resulted in the appearance of apoptotic cellular morphology and characteristics ( fig .", "4c & 4 g ) , which could be enhanced with pretreatment with \n ifn- ( fig .", "4d & 4h ) . in ecg - primed ( differentiated ) \n granulosa cells , however , ifn- pretreatment failed to potentiate the pro - apoptotic effects of \n agonistic fas mab , which were observed prominently in des - primed cells ( table 1 ) . left and right panels illustrate cellular morphology ( phase - contrast microscopy ) and \n in situ apoptotic cell detection ( tunel ) , respectively .", "a and e , \n control group [ ifn- vehicle ( medium ) ( 24 hr ) + igg ( 6 hr ) ] ; b and f , ifn- group [ ifn- ( 24 \n hr ) + igg ( 6 hr ) ] ; c and g , fas mab group [ ifn- vehicle ( medium ) ( 24 hr ) + fas mab ( 6 hr ) ] ; \n d and h , ifn- and fas mab group [ ifn- ( 24 hr ) + fas mab ( 6 hr ) ] .", "arrowheads in left and right panels indicate floating cells and \n tunel - positive cells , respecttively .", "magnification : 400. inserts in right panels show \n representative apoptotic dna fragmentation pattern in each treatment group .", "- : rare , + : occasional , + + : frequent , + + + : extensive , + + + + : very extensive to further examine the colocalization of ifn- , fas and fasl expression and occurrence of \n apoptosis in vivo , follicles at very early stage of antral formation ( estimated \n between preantral and early antral stages ) were analyzed serially by immunohistochemistry and \n in situ tunel method ( fig . 5 ) . \n", "immunoreactivity for ifn- was distributed in the granulosa but not the thecal layers ( fig .", "likewise , the most intense and aggregated form of fas \n immuno - positive signals were detected in the loosely attached granulosa cells ( fig .", "although fasl immunoreactivity could be colocalized in fas - positive \n cells , he most aggregated and intense ( arrows ) immunoreactivity was present in granulosa cells \n mainly lining the antrum ( fig .", "5c ) . similarly , apoptotic \n ( tunel - positive ) granulosa cells were also detected in antral granulosa cells ( fig .", "a , ifn- ; b , fas ; c , fasl ; d , tunel .", "gc , tc and it represent granulosa cells , theca cells and \n interstitial cells , respectively ." ], [ "in the present study , we have localized rat ifn- in the rat ovary during the follicular development and \n tested its role on granulosa cell death via induction of the fas / fasl system in granulosa cells at \n different stage of differentiation .", "we have found that the responsiveness of granulosa cells to ifn- in \n terms of fas receptor expression was significantly compromised with follicular maturation .", "the \n immunoreactivity of ifn- was most intense in early stages of folliculogenesis , such as in preantral \n follicles .", "up - regulation of fas by ifn- and the subsequent increased responsiveness to an apoptotic \n death signal ( e.g. fas activation by agonistic fas mab ) were more evident in granulosa cells from the \n preantral follicles than those from antral follicles previously exposed to gonadotropins .", "the importance of intraovarian interaction between immune cells ( lymphocytes and macrophages ) and \n granulosa cells in ovarian physiology via the synthesis and actions of cytokines ( e.g.tnf and il ) has \n been studied extensively ( adashi , 1992 ; terranova et al . , 1993 ) . although the modulatory roles of ifn- in granulosa cell \n differentiation and steroidogenesis have been demonstrated in vitro ( gorospe et al . , 1998 ; xiao and \n findlay , 1992 ) ,", "ifn- is \n detectable in the follicular fluids ( grasso et al . , 1988 ) and \n lymphocytes present in the ovary have been suggested to be an important contributor to the follicular \n ifn- ( adashi , 1992 ) .", "immune cells ( lymphocytes / macrophages ) are \n believed to be mainly present in atretic antral follicles ( best et al . , \n 1996 ) ,", ", 1994a ; brannstrom et al . , 1994b ) \n where they remove damaged and dead cells by secreting cytotoxic cytokines and phagocytosis .", "the presence \n of immune cells during early stage of follicular development has not been reported and the possibility \n that immune cells are the only the source of ifn- in preantral follicles can not be excluded . in this \n study , ifn- was localized predominantly in granulosa cells and oocytes throughout the follicular \n development but more abundantly in those at the early follicular ( preantral ) stage .", "it has been \n demonstrated that tnf- and fas ligand are expressed in granulosa cells and oocytes ( chen et al .", "although we have not studied ifn- gene expression in ovarian cells in the current \n study , it is possible that oocytes and granulosa cells in the preantral follicles may be an important \n source of ifn- in the ovary and that this cytokine plays an autocrine and/or paracrine role in \n regulating the expression and actions of other cytokines involved in cell removal process in the \n degrading follicles .", "ifn- is an important modulator of granulosa cell differentiation during the early stage of follicular \n development ( gorospe et al . , 1988 ; xiao and findlay , 1992 ) ,", "although its functional role in differentiated granulosa \n cells in late follicular development ( i.e. , preovulatory follicles ) remains to be further established . \n", "granulosa cell apoptosis is frequently observed in early antral ( penultimate ) stage of follicular \n development , it is only assumed that up - regulation of fas receptor by ifn- in granulosa cells of \n preantral follicles is prerequisite for fasl ligation and that the latter could be activated by \n intra - ovarian factor(s ) following antrum formation .", "it has been suggested that ifn- facilitate \n apoptosis in luteinized granulosa cells by decreasing endogenous igf activity via stimulation of igfbps \n rather than of fas activation ( cataldo et al . , 1998 ) .", "however , the \n concentrations of ifn- used in above studies could have been pharmacological and cytotoxic to the \n cells .", "quirk et al ( 1995 ) demonstrated fas - mediated apoptosis in \n human granulosa - luteal cells and suggested that ifn- up - regulates fas mrna and protein expression and \n that pretreatment of cells with ifn- alone or in combination with hcg facilitates fas mab - induced \n cytotoxicity .", "therefore , it is unlikely that the physiological concentration of ifn- could induce fas \n expression in fully differentiated luteinized ( or luteinizing ) granulosa cells .", "recently , quirk et al ( 1998 ) showed that combined ifn- and tnf treatment \n was more effective in inducing fas expression and potentiating fas mab - induced apoptosis in cultured \n granulosa cells ( mostly from large preovulatory follicles in ecg - primed mice ) than by either ifn- or \n tnf alone .", "the results suggest that the mechanism(s ) of induction of apoptosis and responsiveness \n against apoptotic insult ( e.g. ifn- ) in differentiated granulosa cells might be much more complex and \n resistant than those of undifferentiated granulsoa cells , as also seen in the present study .", "the possible mediatory role of the fas / fasl system in granulosa and luteal cell apoptosis during \n follicular atresia and luteal regression , respectively have been investigated in several mammalian \n species ( quirk et al . , 1995 ;", "previous reports have demonstrated that increased granulosa cell fas and fasl protein \n content is associated with apoptosis at the penultimate ( early ) stage of follicular development \n in vivo ( kim et al . , 1998 ) , and that \n p53-mediated granulosa cell apoptosis involves the activation of the fas / fasl system ( kim et al . , 1999 ) .", "these studies also suggested that fsh is a key \n inhibitory factor on granulosa cell death as well as the involvement of the fas / fasl system in \n follicular atresia .", "the nature of the regulation of fas receptor expression throughout the follicular \n development , however , remains unclear .", "although the regulation of fas receptor expression by ifn- has \n been demonstrated in human granulosa - luteal cells in vitro ( quirk et al . , 1995 ) , the potential regulatory role of ifn- in fas mrna and protein \n expression in undifferentiated granulosa cells during early stages of folliculogenesis has not been \n thoroughly investigated . in the present studies ,", "we have studied the possible role of ifn- in the \n regulation of granulsoa cell fas expression during follicular development . while increased fas \n expression in granulsoa cells from preantral or early antral follicles could be induced with low \n concentrations of ifn- ( 10 - 100 u / ml ) , higher concentrations ( 1,000 u / ml ) were required in ecg - primed \n cells .", "our present immuno - flowcytometric analysis also confirmed that undifferentiated ( des - primed ) \n granulsoa cells were more responsive to ifn- in the induction of fas receptor than cells pretreated \n with gonadotropin .", "these findings are not only consistent with the studies by quirk et al ( 1995 ) which showed increased expression of fas protein in \n gonadotropin - primed human granulosa cells following exposure to very high concentration ( > 10,000 \n u / ml ) of ifn- in vitro , but extended their observations to suggest a role for the \n cytokine in the ovary at physiological concentrations .", "it is well established that fsh is an important cell survival factor in the mammalian ovarian follicle \n ( chun et al .", "whereas stimulation of immature rats with ecg resulted in suppressed basal \n granulosa cell fas / fasl levels and apoptosis , decreased atresia and enhanced follicular growth , \n withdrawal of gonadotropic support with the injection of a neutralizing antibody in \n vivo during early antral development led to increased granulosa cell fas / fasl expression \n and apoptotic cell death , and eventual follicular demise ( kim et al . , \n 1998 ) . in the present studies , we have demonstrated that while ifn- effectively up - regulated \n fas expression in granulosa cells from preantral and early antral follicles and sensitized them to the \n pro - apoptotic action of agonistic fas antibody , cells isolated from large antral follicles previously \n exposed to the gonadotropin in vivo were more differentiated but less responsive to \n ifn-. these findings suggest that the anti - apoptotic role of fsh in the granulosa cells may in part \n involve its modulatory influence on ifn- action , and are consistent with the concept that ovarian \n follicles beyond the penultimate stage of development are protected physiologically from pro - apoptotic \n insult and destined to ovulate ( chun et al . , 1996 ; boone et al . , 1997 ) .", "the mechanism(s ) by which the gonadotropin \n modulates ifn--induced fas expression granulosa cells is unclear .", "it has been demonstrated that fas \n expression is regulated by the newly identified fas - regulatory gene tdag51 , the expression of which is \n pkc - dependent ( wang et al . , 1998 ) . since ifn- , like tnf ( sancho tello and terranova , 1991 ) and il-3 ( farrar et al . , 1985 ) , appears capable of activating pkc ( ostrowski et al . , 1988 ) , it is possible that the reduced responsiveness of \n differentiated granulosa cells to ifn- might be due to pka activation by fsh .", "in addition , activation \n of pka pathway is known to inhibit pkc - dependent mechanism(s ) in a variety of biological systems \n including the granulosa cell ( shinohara et al . , 1985 ) .", "in this \n context , differential responsiveness of granulosa cells at different cytodifferentiation to ifn- may be \n related to possible interactions between signal transduction pathways of ifn- and gonadotropin .", "it is \n possible that the efficient fas gene expression in undifferentiated granulosa cells may involve \n activation of pkc pathway by ifn-. fas receptor signaling is a complex process and continues to capture considerable attention and research \n efforts .", "fas activation initiated by ligation and oligomerrization results in recruitment of fadd \n ( fasassociated death domain - containing protein ) ( chinnaiyan et al . , \n 1995 ) .", "fadd then activates procaspase8 ( muzio et al . , \n 1996 ) , which appears to be the first step of a proteolytic cascade that activates other \n caspases such as caspases3 , 6 , and 7 ( hirata et al . , 1998 )", "ifn- significantly increased the population of granulosa cells exhibiting high \n fluorescence intensity of fas immunoreactivity . while this increase might merely represent an increase \n in the cell population with enhanced fas expression , it is tempting to speculate that the high intensity \n of fluorescence reflects the oligomerization of fas receptor proteins , and that fas oligomerization in \n response to ifn- in undifferentiated granulsoa cells may be more efficient and functional than in \n differentiated granulosa cells .", "whether this indeed is the case requires further investigation and \n confirmation . in this context , intense and aggregated immunoreactive fas protein has consistently been \n detected in cytoplasm as well as cellular membrane of cultured granulosa cells of the present study . \n", "these results support our previous observation suggesting the presence of aggregated fas immunostaining \n in the granulosa cells of atretic early antral follicles in the ovary ( kim et al . , 1998 ) .", "moreover , recent studies have indicated that fas is also localized in the \n peri - nuclear region of the cytoplasm and that fas - trafficking from golgi apparatus to membrane is \n p53-dependent and required for fas - mediated apoptosis ( bennett et al . , \n 1998 ) .", "in light of our recent demonstration of the role of p53 in the regulation of fas \n expression in rat granulosa cells ( kim et al . , 1999 ) and", "their \n intense fas immunostaining following ifn- challenge , investigations into whether this cytokine plays a \n role in fas trafficking in the granulosa cell would provide important clues on the cellular mechanism \n involved in the control of fas - mediated granulsoa cell apoptosis during early folliculogenesis . in summary , we have demonstrated that ifn- plays a pivotal role in regulating granulosa cell \n differentiation and apoptosis by potentiating fas function , a process modulated by gonadotropin ( i.e. \n fsh ) .", "the role of ifn- is most prominent in undifferentiated granulosa cells during early stage of \n follicular development ." ] ]

[ "colorectal cancer is the third most common incident cancer among men and the second most common cancer among women worldwide .", "incidence rates vary 10-fold in both sexes worldwide , and the highest rates are estimated in more developed regions , such as north america and western europe , whereas the lowest rates are estimated in africa ( except southern africa ) and south - central asia . in asia , colorectal cancer is the fourth most common incident cancer among men and the fifth most common cancer among women . although incidence rates are relatively low in asian countries , those for east asian countries are relatively high . in korea ,", "annual percentage changes in age - standardized incidence rates were 6.2% in men and 6.8% in women between 1999 and 2009 using the world standard population as a standard population . in 2009 , 24,986 new colorectal cancer cases ( 15,068 men and 9,918 women ) were diagnosed , accounting for 13.0% of all cancer occurrences .", "colorectal cancer is the second most common cancer after stomach cancer among men and the third most common cancer after cancers of the thyroid and breast among women .", "there have been several reports on differences in patterns of colorectal incidence trends according to age group , sex , and anatomical location [ 3 - 5 ] .", "however , it has not been properly investigated whether colorectal cancers of a specific age group , sex , or anatomical location show more rapid increases in the korean population .", "the aim of the current study was to demonstrate changing trends of colorectal cancer incidence according to age group , sex , and anatomical location .", "the korea central cancer registry ( kccr ) , a nation - wide , hospital - based cancer registry , was initiated by the ministry of health and welfare , korea in 1980 .", "the registry collected information on approximately 80 - 90% of cancer cases from more than 150 training hospitals across the country , and , in 1999 , the kccr expanded cancer registration to cover the entire korean population under the population - based regional cancer registry program .", "age ( five - year intervals ) and sex - specific incidence rates and the number of cases of colorectal cancer between 1999 and 2009 were obtained from the korea national cancer incidence database .", "anatomical subsites were defined based on the tenth version of the international statistical classification of diseases and related health problems , 10th revision ( icd-10 ) .", "we defined the proximal colon as the cecum ( c18.0 ) , appendix ( c18.1 ) , ascending colon ( c18.2 ) , hepatic flexure ( c18.3 ) , transverse colon ( c18.4 ) , and splenic flexure of the colon ( c18.5 ) ; the distal colon was defined as the descending colon ( c18.6 ) and sigmoid colon ( c18.7 ) .", "overlapping lesions of the colon ( c18.8 ) and colon not otherwise specified ( c18.9 ) were not included in the subsite analysis .", "age - standardized rates ( asrs ) were calculated using the middle - year population of 2,000 as the standard population .", "annual percent changes ( apcs ) for the incidence rates were calculated using a linear model , according to the following formula ; ( exp(b)-1)100 , where b is the slope of the regression of the natural logarithm of the asr in a calendar year .", "the 95% confidence intervals ( cis ) were obtained with a standard error from the fit of the regression and the t - distribution function .", "all analyses were conducted by sex , subsite , and age group ( 10-year intervals ) .", "male - to - female incidence rate ratios ( irr ) were calculated using the numbers of cancer patients for each site and the sex - specific population structure for each year using stata / se 10.0 for windows ( stata corp lp , college station , tx ) . statistical significance in difference between irrs", "the asrs and apcs overall and for each subsite of colorectal cancer by sex are shown in tables 1 and 2 and fig .", "1 . among men , the asr for colorectal cancer was 27 per 100,000 in 1999 and increased to 50.2 in 2009 ( apc , 6.6% ) .", "the asr for colorectal cancer among women was 17.2 in 1999 and 26.9 in 2009 ( apc , 5.1% ) .", "the rectum was the most common cancer site among both men and women in 1999 and 2009 .", "cancer of the distal colon showed the highest apc ( 10.8% among men and 8.4% among women ) , followed by the proximal colon ( 7.9% among men and 6.6% among women ) and rectum ( 5.2% among men and 2.4% among women ) . as a result", ", the proportion of rectal cancer decreased from 51.5% in 1999 to 47.1% in 2009 among men , and from 50.5% in 1999 to 42.8% in 2009 among women , whereas the proportion of proximal and distal colon cancers increased among both men and women ( appendix 2 ) . in all subsites ,", "significantly higher apcs were observed among men , compared with women . as a result ,", "the male - to - female irrs showed a significant increase between 1999 and 2009 for overall colorectal cancer ( 1.20 in 1999 to 1.51 in 2009 ) , distal colon cancer ( 1.32 to 1.60 ) , and rectal cancer ( 1.22 to 1.67 ) , whereas the irr for proximal colon cancer did not show a significant change ( 1.09 to 1.16 ) ( table 3 ) .", "the asrs and apcs for colorectal cancer subsites by age group are shown in tables 4 and 5 . for overall colorectal cancer ,", "men who were in their 60s showed the highest increase , whereas , for women , those in their 80s showed the highest increase .", "distal colon cancer consistently showed the highest apc in most age groups among both men and women , except for women in their 80s , who showed the highest apc for proximal colon cancer . among women , for almost every subsite , the most notable increases in incidence were observed for the oldest age group .", "proximal colon cancer in men also showed the highest apc in the oldest age groups , whereas apcs were significantly higher only in the 60s for distal colon cancer and 40s and 60s for rectal cancer .", "however , the apcs showed narrow ranges of between 10.1 and 11.7 for distal colon cancer and between 4.7 and 5.8 for rectal cancer among men over 40 years old .", "the highest male - to - female irrs for overall colorectal cancer were observed for the oldest age groups , whereas the lowest irrs were observed for younger age groups ( table 6 ) .", "however , significant increases in male - to - female irrs were observed in their 50s and 60s between 1999 and 2009 . in the subsite - specific analyses ,", "the changes in male - to - female irrs between 1999 and 2009 showed a statistically significantly increase for rectal cancer among men and women in their 50s , and 60s and for distal colon cancer among those in their 60s .", "incidence of colorectal cancer has increased in most countries , except for the united states and some areas of japan .", "the most significant increases have been observed in eastern european countries and most asian countries .", "although the korean population has experienced a rapid increase in colorectal cancer incidence , it has not been properly investigated whether the increasing pattern differs according to age group , sex , or anatomical location .", "there have been suggestions that colorectal cancer incidence by subsite differs according to race , sex , age group , and time period . in our study ,", "rectal cancer accounted for the highest proportion among all subsites and ranged from 41% to 51% of all colorectal cancers .", "this result is consistent in part with a report from the us , which showed that the rectum ( c19.9 and c20.9 ) was the most common subsite for male asians and pacific islanders living in the us ( 35% ) , whereas the proximal colon was the most common site for whites and blacks among both men and women , as well as female asians and pacific islanders .", "however , in the current study , the apc for rectal cancer was the lowest among the subsites included , thus , it appears that the proportion of rectal cancer has decreased among both men and women in korea .", "instead , colon cancer , particularly distal colon cancer , has shown a rapid increase .", "we expect that the proportion of rectal cancer will continue to decrease in the future , and the proportion will eventually reach a level similar to that of western countries .", "one study suggested that incidence of colorectal cancer has increased among younger age groups in the us population , although overall incidence of colorectal cancer has declined .", "the increases observed in the younger population were mainly attributed to rectal cancer . in our study , we did not find clear evidence for a more rapid increase in overall incidence of colorectal cancer or rectal cancer in younger age groups , compared to older age groups in women . in men , however , it is worthy of mention that , unlike proximal colon cancer and distal colon cancer , where the apcs were lowest in their 30s and 40s , the apcs of rectal cancer in men in their 30s and 40s were statistically higher than those for men in their 70s and 80s .", "it is notable that although the incidence was higher among men than among women , the apcs were also higher among men than among women for most subsites . as a result ,", "the male - to - female irr for overall colorectal cancer increased from 1.20 in 1999 to 1.51 in 2009 .", "a high male - to - female irr was observed for most subsites across ethnic groups in the us population .", "our study also showed that the lowest male - to - female irr was observed for proximal colon cancer , indicating that the proportion of proximal colon cancer was higher among women than men .", "this result is consistent with results reported from the us , germany , and japan .", "traditionally , differences in the distribution of colorectal cancer subsites between men and women have been explained by the role of female hormonal factors .", "rapid increases in colorectal cancer incidence and the stabilization of colorectal cancer mortality in the korean population could be explained by introduction of colorectal cancer screening .", "colorectal cancer screening programs were introduced in 2004 as a part of the national cancer screening program for medical aid recipients and national health insurance beneficiaries in the lower income bracket .", "the fecal occult blood test ( fobt ) is provided free of charge as a primary modality for men and women aged 50 years or older .", "fobt - positive individuals were provided follow - up by either colonoscopy or double - contrast barium enema .", "the participation rate was only 10.5% in 2004 , however , it increased to 21.1% in 2008 . according to the korea national cancer screening survey , which covers organized and opportunistic cancer screening programs , lifetime screening rates for colorectal cancer were 25.3% in 2004 and 54.2% in 2010 , whereas screening rates by recommended guidelines were 19.9% in 2004 and 35.5% in 2010 . in the early phase of screening ,", "however , the introduction of colorectal cancer screening can not completely explain the differential increase in colorectal cancer incidence by subsite .", "fobt usually has a higher sensitivity for advanced neoplasia , including colorectal cancer and advanced colorectal adenomas , in the left vs. right colon .", "this explains in part the increase in the rate of distal colon cancer , but not the low increase in rectal cancer .", "in addition , the national cancer screening program guidelines recommend colono - scopy or double - contrast barium enema as a follow - up modality for fobt positive patients .", "sigmoidoscopy , which is used as a screening tool for an average - risk population in the us , is not considered as a follow - up modality in korea .", "therefore , there is little possibility that screening - detected cancers are more likely to be distal colon and rectal cancer .", "a transition in risk factors may explain the differential increase in colorectal cancer according to incidence according to subsite . in a large insurance", "database - based study , frequent alcohol consumption and high consumption amount were more strongly associated with risk of distal colon cancer among men and risk of rectal cancer risk among women . high body mass index ( bmi25 kg / m ) was associated with increased risk for distal colon cancer among men and for proximal colon cancer among women .", "in addition , frequent meat intake was associated with proximal colon cancer risk among men and risk of proximal colon and rectal cancer among women .", "daily alcohol consumption in korea increased from 10.6 g in 1999 to 17.3 g in 2007 among men and from 1.4 g in 1999 to 2.9 g in 2007 among women .", "the proportion of adult men with a bmi of 25 kg / m and was 25.1% in 1998 and increased to 35.3% in 2008 .", "daily per capita meat consumption was 6.6 g in 1969 and increased to 95.1 g in 2005 .", "in contrast , the cigarette smoking rate in the adult population decreased from 75.1% in 1992 to 43.1% in 2009 among men and from 5.1% in 1992 to 3.9% in 2009 among women .", "cigarette smoking has shown a stronger association with risk of rectal cancer than colon cancer . in our subsite analysis ,", "we excluded overlapping lesions of the colon ( c18.8 ) and colon not otherwise specified ( c18.9 ) .", "the proportions of c18.8 did not change between 1999 and 2009 ( 1.0% for men and 0.8% for women ) , however , the proportions of c18.9 during the same period decreased from 9.8% to 5.5% among men and from 11.1% to 5.9% among women . improved accuracy in topology classification", "may explain in part the increase in the incidence of distal colon cancer , however , the changes exceeded the portion that might be explained by improved accuracy in topology coding .", "the rapid increase in colorectal cancer incidence in korea between 1999 and 2009 is mainly attributed to increases in colon cancer , especially distal colon cancer .", "increases in the proportion of colon cancer may be explained by a transition in risk factors for subsites and the effect of colorectal cancer screening programs .", "the male - to - female irrs were higher for distal colon and rectal cancer and increased between 1999 and 2009 due to more rapid changes in male colorectal cancer incidence ." ]

[ "<S> purposethis study was conducted in order to demonstrate changing trends in colorectal cancer incidence according to sex , age group , and anatomical location in the korean population.materials and methodsdata from the korea central cancer registry between 1999 and 2009 were analyzed . </S>", "<S> annual percent changes ( apcs ) of sex- and age - specific incidence rates for cancer of the proximal colon ( international statistical classification of diseases and related health problems , 10th revision [ icd-10 ] code c18.0 - 18.5 ) , distal colon ( c18.6 - 18.7 ) , and rectum ( c19 - 20 ) , and male - to - female incidence rate ratios ( irr ) were calculated.resultsthe age - standardized incidence rate ( asr ) of colorectal cancer was 27 ( per 100,000 ) in 1999 and increased to 50.2 in 2009 among men ( apc , 6.6% ) . </S>", "<S> the asr for women was 17.2 in 1999 and 26.9 in 2009 ( apc , 5.1% ) . </S>", "<S> the rectum was the most common site of cancer among both men and women during 1999 and 2009 . </S>", "<S> however , the distal colon had the highest apc ( 10.8% among men and 8.4% among women ) , followed by the proximal colon ( 7.9% among men and 6.6% among women ) , and rectum ( 5.2% among men and 2.4% among women ) . </S>", "<S> the proportion of rectal cancer decreased from 51.5% in 1999 to 47.1% in 2009 among men , and from 50.5% to 42.8% among women . </S>", "<S> an increase in the male - to - female irr was observed for distal colon cancer and rectal cancer , whereas the irr for proximal colon cancer was stable.conclusionthe rapid increase in colorectal cancer incidence is mainly attributed to the increase in colon cancer , especially distal colon cancer , and may be explained by a transition of risk factors for subsites or by the effect of colorectal cancer screening . </S>" ]

[ "Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion", "Mid-year population in 2000, Korea", "The number of colorectal cancer cases by subsites, the Korea Central Cancer Registry, 1999 and 2009" ]

[ [ "colorectal cancer is the third most common incident cancer among men and the second most common cancer among women worldwide .", "incidence rates vary 10-fold in both sexes worldwide , and the highest rates are estimated in more developed regions , such as north america and western europe , whereas the lowest rates are estimated in africa ( except southern africa ) and south - central asia . in asia , colorectal cancer is the fourth most common incident cancer among men and the fifth most common cancer among women . although incidence rates are relatively low in asian countries , those for east asian countries are relatively high . in korea ,", "annual percentage changes in age - standardized incidence rates were 6.2% in men and 6.8% in women between 1999 and 2009 using the world standard population as a standard population . in 2009 , 24,986 new colorectal cancer cases ( 15,068 men and 9,918 women ) were diagnosed , accounting for 13.0% of all cancer occurrences .", "colorectal cancer is the second most common cancer after stomach cancer among men and the third most common cancer after cancers of the thyroid and breast among women .", "there have been several reports on differences in patterns of colorectal incidence trends according to age group , sex , and anatomical location [ 3 - 5 ] .", "however , it has not been properly investigated whether colorectal cancers of a specific age group , sex , or anatomical location show more rapid increases in the korean population .", "the aim of the current study was to demonstrate changing trends of colorectal cancer incidence according to age group , sex , and anatomical location ." ], [ "the korea central cancer registry ( kccr ) , a nation - wide , hospital - based cancer registry , was initiated by the ministry of health and welfare , korea in 1980 .", "the registry collected information on approximately 80 - 90% of cancer cases from more than 150 training hospitals across the country , and , in 1999 , the kccr expanded cancer registration to cover the entire korean population under the population - based regional cancer registry program .", "age ( five - year intervals ) and sex - specific incidence rates and the number of cases of colorectal cancer between 1999 and 2009 were obtained from the korea national cancer incidence database .", "anatomical subsites were defined based on the tenth version of the international statistical classification of diseases and related health problems , 10th revision ( icd-10 ) .", "we defined the proximal colon as the cecum ( c18.0 ) , appendix ( c18.1 ) , ascending colon ( c18.2 ) , hepatic flexure ( c18.3 ) , transverse colon ( c18.4 ) , and splenic flexure of the colon ( c18.5 ) ; the distal colon was defined as the descending colon ( c18.6 ) and sigmoid colon ( c18.7 ) .", "overlapping lesions of the colon ( c18.8 ) and colon not otherwise specified ( c18.9 ) were not included in the subsite analysis .", "age - standardized rates ( asrs ) were calculated using the middle - year population of 2,000 as the standard population .", "annual percent changes ( apcs ) for the incidence rates were calculated using a linear model , according to the following formula ; ( exp(b)-1)100 , where b is the slope of the regression of the natural logarithm of the asr in a calendar year .", "the 95% confidence intervals ( cis ) were obtained with a standard error from the fit of the regression and the t - distribution function .", "all analyses were conducted by sex , subsite , and age group ( 10-year intervals ) .", "male - to - female incidence rate ratios ( irr ) were calculated using the numbers of cancer patients for each site and the sex - specific population structure for each year using stata / se 10.0 for windows ( stata corp lp , college station , tx ) . statistical significance in difference between irrs" ], [ "the asrs and apcs overall and for each subsite of colorectal cancer by sex are shown in tables 1 and 2 and fig .", "1 . among men , the asr for colorectal cancer was 27 per 100,000 in 1999 and increased to 50.2 in 2009 ( apc , 6.6% ) .", "the asr for colorectal cancer among women was 17.2 in 1999 and 26.9 in 2009 ( apc , 5.1% ) .", "the rectum was the most common cancer site among both men and women in 1999 and 2009 .", "cancer of the distal colon showed the highest apc ( 10.8% among men and 8.4% among women ) , followed by the proximal colon ( 7.9% among men and 6.6% among women ) and rectum ( 5.2% among men and 2.4% among women ) . as a result", ", the proportion of rectal cancer decreased from 51.5% in 1999 to 47.1% in 2009 among men , and from 50.5% in 1999 to 42.8% in 2009 among women , whereas the proportion of proximal and distal colon cancers increased among both men and women ( appendix 2 ) . in all subsites ,", "significantly higher apcs were observed among men , compared with women . as a result ,", "the male - to - female irrs showed a significant increase between 1999 and 2009 for overall colorectal cancer ( 1.20 in 1999 to 1.51 in 2009 ) , distal colon cancer ( 1.32 to 1.60 ) , and rectal cancer ( 1.22 to 1.67 ) , whereas the irr for proximal colon cancer did not show a significant change ( 1.09 to 1.16 ) ( table 3 ) .", "the asrs and apcs for colorectal cancer subsites by age group are shown in tables 4 and 5 . for overall colorectal cancer ,", "men who were in their 60s showed the highest increase , whereas , for women , those in their 80s showed the highest increase .", "distal colon cancer consistently showed the highest apc in most age groups among both men and women , except for women in their 80s , who showed the highest apc for proximal colon cancer . among women , for almost every subsite , the most notable increases in incidence were observed for the oldest age group .", "proximal colon cancer in men also showed the highest apc in the oldest age groups , whereas apcs were significantly higher only in the 60s for distal colon cancer and 40s and 60s for rectal cancer .", "however , the apcs showed narrow ranges of between 10.1 and 11.7 for distal colon cancer and between 4.7 and 5.8 for rectal cancer among men over 40 years old .", "the highest male - to - female irrs for overall colorectal cancer were observed for the oldest age groups , whereas the lowest irrs were observed for younger age groups ( table 6 ) .", "however , significant increases in male - to - female irrs were observed in their 50s and 60s between 1999 and 2009 . in the subsite - specific analyses ,", "the changes in male - to - female irrs between 1999 and 2009 showed a statistically significantly increase for rectal cancer among men and women in their 50s , and 60s and for distal colon cancer among those in their 60s ." ], [ "incidence of colorectal cancer has increased in most countries , except for the united states and some areas of japan .", "the most significant increases have been observed in eastern european countries and most asian countries .", "although the korean population has experienced a rapid increase in colorectal cancer incidence , it has not been properly investigated whether the increasing pattern differs according to age group , sex , or anatomical location .", "there have been suggestions that colorectal cancer incidence by subsite differs according to race , sex , age group , and time period . in our study ,", "rectal cancer accounted for the highest proportion among all subsites and ranged from 41% to 51% of all colorectal cancers .", "this result is consistent in part with a report from the us , which showed that the rectum ( c19.9 and c20.9 ) was the most common subsite for male asians and pacific islanders living in the us ( 35% ) , whereas the proximal colon was the most common site for whites and blacks among both men and women , as well as female asians and pacific islanders .", "however , in the current study , the apc for rectal cancer was the lowest among the subsites included , thus , it appears that the proportion of rectal cancer has decreased among both men and women in korea .", "instead , colon cancer , particularly distal colon cancer , has shown a rapid increase .", "we expect that the proportion of rectal cancer will continue to decrease in the future , and the proportion will eventually reach a level similar to that of western countries .", "one study suggested that incidence of colorectal cancer has increased among younger age groups in the us population , although overall incidence of colorectal cancer has declined .", "the increases observed in the younger population were mainly attributed to rectal cancer . in our study , we did not find clear evidence for a more rapid increase in overall incidence of colorectal cancer or rectal cancer in younger age groups , compared to older age groups in women . in men , however , it is worthy of mention that , unlike proximal colon cancer and distal colon cancer , where the apcs were lowest in their 30s and 40s , the apcs of rectal cancer in men in their 30s and 40s were statistically higher than those for men in their 70s and 80s .", "it is notable that although the incidence was higher among men than among women , the apcs were also higher among men than among women for most subsites . as a result ,", "the male - to - female irr for overall colorectal cancer increased from 1.20 in 1999 to 1.51 in 2009 .", "a high male - to - female irr was observed for most subsites across ethnic groups in the us population .", "our study also showed that the lowest male - to - female irr was observed for proximal colon cancer , indicating that the proportion of proximal colon cancer was higher among women than men .", "this result is consistent with results reported from the us , germany , and japan .", "traditionally , differences in the distribution of colorectal cancer subsites between men and women have been explained by the role of female hormonal factors .", "rapid increases in colorectal cancer incidence and the stabilization of colorectal cancer mortality in the korean population could be explained by introduction of colorectal cancer screening .", "colorectal cancer screening programs were introduced in 2004 as a part of the national cancer screening program for medical aid recipients and national health insurance beneficiaries in the lower income bracket .", "the fecal occult blood test ( fobt ) is provided free of charge as a primary modality for men and women aged 50 years or older .", "fobt - positive individuals were provided follow - up by either colonoscopy or double - contrast barium enema .", "the participation rate was only 10.5% in 2004 , however , it increased to 21.1% in 2008 . according to the korea national cancer screening survey , which covers organized and opportunistic cancer screening programs , lifetime screening rates for colorectal cancer were 25.3% in 2004 and 54.2% in 2010 , whereas screening rates by recommended guidelines were 19.9% in 2004 and 35.5% in 2010 . in the early phase of screening ,", "however , the introduction of colorectal cancer screening can not completely explain the differential increase in colorectal cancer incidence by subsite .", "fobt usually has a higher sensitivity for advanced neoplasia , including colorectal cancer and advanced colorectal adenomas , in the left vs. right colon .", "this explains in part the increase in the rate of distal colon cancer , but not the low increase in rectal cancer .", "in addition , the national cancer screening program guidelines recommend colono - scopy or double - contrast barium enema as a follow - up modality for fobt positive patients .", "sigmoidoscopy , which is used as a screening tool for an average - risk population in the us , is not considered as a follow - up modality in korea .", "therefore , there is little possibility that screening - detected cancers are more likely to be distal colon and rectal cancer .", "a transition in risk factors may explain the differential increase in colorectal cancer according to incidence according to subsite . in a large insurance", "database - based study , frequent alcohol consumption and high consumption amount were more strongly associated with risk of distal colon cancer among men and risk of rectal cancer risk among women . high body mass index ( bmi25 kg / m ) was associated with increased risk for distal colon cancer among men and for proximal colon cancer among women .", "in addition , frequent meat intake was associated with proximal colon cancer risk among men and risk of proximal colon and rectal cancer among women .", "daily alcohol consumption in korea increased from 10.6 g in 1999 to 17.3 g in 2007 among men and from 1.4 g in 1999 to 2.9 g in 2007 among women .", "the proportion of adult men with a bmi of 25 kg / m and was 25.1% in 1998 and increased to 35.3% in 2008 .", "daily per capita meat consumption was 6.6 g in 1969 and increased to 95.1 g in 2005 .", "in contrast , the cigarette smoking rate in the adult population decreased from 75.1% in 1992 to 43.1% in 2009 among men and from 5.1% in 1992 to 3.9% in 2009 among women .", "cigarette smoking has shown a stronger association with risk of rectal cancer than colon cancer . in our subsite analysis ,", "we excluded overlapping lesions of the colon ( c18.8 ) and colon not otherwise specified ( c18.9 ) .", "the proportions of c18.8 did not change between 1999 and 2009 ( 1.0% for men and 0.8% for women ) , however , the proportions of c18.9 during the same period decreased from 9.8% to 5.5% among men and from 11.1% to 5.9% among women . improved accuracy in topology classification", "may explain in part the increase in the incidence of distal colon cancer , however , the changes exceeded the portion that might be explained by improved accuracy in topology coding ." ], [ "the rapid increase in colorectal cancer incidence in korea between 1999 and 2009 is mainly attributed to increases in colon cancer , especially distal colon cancer .", "increases in the proportion of colon cancer may be explained by a transition in risk factors for subsites and the effect of colorectal cancer screening programs .", "the male - to - female irrs were higher for distal colon and rectal cancer and increased between 1999 and 2009 due to more rapid changes in male colorectal cancer incidence ." ], [ "" ], [ "" ] ]

[ "they may be the first manifestation of metastatic spread of an internal malignancy or herald cancer recurrence long after treatment of a primary tumor .", "breast cancer , colorectal cancer , and melanoma frequently metastasize to the skin in women . in men ; melanoma , lung cancer , and colorectal cancer are the most common sources of cutaneous metastases .", "however , such patterns may differ geographically as a study in taiwan found internal malignancies metastasize to the skin with different frequencies and at rates differing from primarily caucasian populations .", "an extensive literature review was conducted using pubmed from may 26 , 2011-july 16 , 2013 relating cutaneous metastases .", "articles chosen for reference were queried with the following prompts : cutaneous metastases , clinical presentation , histological features , and", "further searches included treatment and management options for metastatic breast , metastatic colorectal , metastatic melanoma , metastatic lung , and hematologic cancers .", "we review the literature on clinical presentation , diagnosis , and advances in the management .", "cutaneous metastases may be asymptomatic or be associated with pain and tenderness [ figure 1a ] .", "they frequently present as a rapidly growing painless dermal or subcutaneous nodules with intact overlying epidermis [ figures 1b d ] , but can also mimic an inflammatory dermatosis .", "metastases may also present as macules , infiltrated or indurated plaques , discoid lesions , tumor nodules with telangiectasias , bullous or papulosquamous lesions , scarred plaques , or pigmented tumors . while there is no single diagnostic characteristic which predominates ; a few patterns have been recognized .", "( a ) gingival metastases from a primary chondrosarcoma.(b ) firm nodule at the base of the ear from a primary lung cancer .", "( c ) two subcutaneous firm nodules in the clavicular fossa from metastatic lung cancer .", "( d ) isolated firm nodular lesion from metastatic colon cancer metastases originating from breast cancer tend to appear in the anterior chest wall , either from direct extension of underlying tumor or by lymphatic spread [ figures 2a and 3a ] .", "extensive cutaneous involvement of metastatic breast cancer can simulate cellulitis ( carcinoma erysipeloides ) or a breast - plate of armor ( \" en cuirasse \" pattern ) .", "interestingly , a subset of breast cancer patients has superior prognosis , even among breast cancer patients with stage iv disease .", "( a ) cellulitis - like appearance of metastatic breast cancer.(b ) plaque - like area with multiple nodules from a primary breast cancer .", "( c ) massive recurrence of breast cancer on the chest wall and abdomen of the en cuirasse type .", "( d ) breast cancer , metastatic to the thoracic spine with extension to the skin , and lceration secondary to radiation therapy ( a ) cellulitic - type appearance with multiple nodules in metastatic breast cancer of the shoulder and upper back .", "a cam 5.2 stain reveals perinuclear staining ( original magnification , 400).(c ) metastatic squamous cell carcinoma of the skin reveals neoplastic cells in the dermis ( cytokeratin 5/6 stain ; original magnification , 100 ) .", "a her2/neu stain is positive and decorates the neoplastic cells in the dermis indicating possible metastasis from breast carcinoma ( original magnification , 200 ) while most cancers present as solitary nodules , melanoma and carcinomas with an unknown primary frequently present as multiple nodules .", "locoregional melanocytic metastases may be explained by angiotropism , an extravascular migratory phenotype , and an important marker and prognostic factor of metastasis in melanoma .", "the face and scalp are most commonly affected by metastases , suggesting that blood vessels and patterns of innervation may influence the spread of metastases .", "these patterns can also been associated with cancers of gastric , pulmonary , prostatic , ovarian , laryngeal , palatine - tonsillar , pancreatic , colorectal , parotid , thyroid , or uterine origin and may suggest local treatments that work for melanoma may also work for other metastatic cutaneous malignancies .", "although metastases may be comprised of cells which are more undifferentiated than the primary tumor , careful examination usually reveals important clues .", "for example , melanoma is associated with pigment in at least some of the neoplastic cells in most cases .", "squamous cell carcinoma is associated with keratin pearl formation , and adenocarcinomas usually exhibit gland formation .", "thyroid cancer may exhibit colloid bodies , colon cancer may be associated with mucinous cells , and other tumors may exhibit telltale clues as to their origin . when confronted with a possible metastasis ,", "comparison with the histologic appearance of any prior malignant neoplasms is usually one of the first steps that may help identify the source .", "additionally , studies have indicated the majority of metastatic cancers to the skin are adenocarcinomas .", "when tumors are poorly differentiated or anaplastic , screening immunohistochemical studies can be very helpful .", "when neoplastic cells stain with cytokeratin markers such as ae1/ae3 and cam 5.2 , a carcinoma is likely [ figure 3b ] .", "staining with cd45/leukocyte common antigen ( lca ) indicates that neoplastic cells are of lymphoid origin , while s-100 , melan - a , mart-1 , hmb-45 , and sox-10 decorate most melanomas .", "more detailed immunohistochemical studies [ table 2 ] can be performed when screening studies and clinical correlation have not been fruitful in identifying a tumor of origin .", "although advances in immunohistochemistry are very promising , there is still much work that needs to be done to characterize metastases .", "a recent letter to the editor highlights the pitfalls that can occur when differentiating primary cutaneous apocrine carcinoma from a metastasis .", "although cytokeratin 5/6 is associated with cutaneous apocrine carcinoma , this staining pattern has also been noted with metastatic squamous cell carcinoma [ figure 3c ] .", "similarly , cytokeratin 7 usually stains metastases diffusely , but is associated with only focal staining in primary adnexal carcinoma , but this pattern is not absolute .", "mammaglobin is of interest as it is associated with more widespread staining of metastases than primary apocrine carcinoma , but the results are still preliminary .", "cyclooxygenase 2 is associated with tumors of the mid - gut ( ileum and appendix ) , gut , colorectal adenocarcinoma as well as gastroesophageal adenocarcinoma .", "staining of adenocarcinoma of the lung and endometrioid adenocarcinoma can also occur , highlighting the fact that all immunochemical marker studies must be interpreted in the context of other findings .", "when an epithelial carcinoma is identified , cytokeratin 7 and 20 stains can provide important useful characterization of tumors .", "additional testing can include thyroid transcription factor-1 ( ttf-1 ) and other markers as needed . when clinical appearance , clinical history , and the histologic findings on biopsy are all taken into account", "unfortunately , there are still many cases that elude precise diagnosis regarding whether a tumor is primary or metastatic .", "recent reports have proposed a diagnostic algorithm for metastatic tumors of unknown primary . until better methods are developed", "we recommend excising metastases when surgically feasible and when this will result in a significant decrease in total tumor burden , improve quality of life , or result in increased functionality .", "novel treatments have had a profound effect on prolonging the survival and improving quality of life with metastatic cancer .", "oftentimes , treatment of the primary cancer with a successful regimen will cause cutaneous lesions to lessen .", "interestingly , the abscopal response represents an unusual response to radiation therapy in which radiation of a tumor causes untreated distant skin lesions to dissipate without recurrence .", "the pathophysiology and mechanism of the abscopal effect have not been well - defined , but one study used monoclonal antibodies to analyze infiltrating lymphocytes and suggested that the abscopal effect was caused by activated cellular immunity .", "the abscopal response has been reported with treatment of melanoma , merkel cell carcinoma , hematologic , and solid tumors .", "the study of effective local treatments for melanoma may serve as a model for other malignancies that disseminate via lymphatics and the blood stream to the skin . for melanomas ,", "we typically excise isolated metastases with a 1 cm margin of normal skin , understanding that there are no evidence based studies looking at the utility of margins for metastatic disease . in areas of high cosmetic importance or when therapy is limited to palliation", ", we sometimes perform excisions with very limited margins . therapy for patients with widespread cutaneous and subcutaneous metastases of advanced melanoma that are nonresectable is limited to other types of palliative therapy .", "options include radiotherapy , systemic chemotherapy , polychemotherapy , isolated limb perfusion , interferon alpha injections , cryotherapy , laser ablation , or radiofrequency ablation .", "electrochemotherapy is a novel therapeutic option based on enhanced drug delivery into cancer cells using externally applied electrical fields that increase cell membrane permeability . using targeted electric pulses on tumor - specific areas , cytotoxicity of cisplatin", "increased 80 times and bleomycin 8,000 times with a large reduction in systemic side effects .", "a study comparing tumor nodules treated with electrochemotherapy with cisplatin versus cisplatin alone found at a 124-week follow - up , 77% of control rate of tumor nodules treated with electrochemotherapy was observed compared to 19% for those treated with cisplatin only .", "electrochemotherapy is well - tolerated by patients , highly effective with low doses of cisplatin or bleomycin , has a short duration of treatment sessions , insignificant side effects , and is more cost - effective than isolated limb perfusion , radiotherapy , interferon - alpha , and hyperthermia associated with radiotherapy and chemotherapy .", "oncogene - targeted therapy for metastatic melanoma using vemurafenib ( plx4032 ) for melanoma associated with a mutation of the braf gene v600 mutation results in complete or partial tumor regression in the majority of patients with an estimated median progression - free survival of more than 7 months .", "side effects include arthralgia , rash , nausea , photosensitivity , fatigue , cutaneous squamous - cell carcinoma , pruritus , and palmoplantar dysesthesia .", "imiquimod 5% cream has also been used successfully in the treatment of metastatic melanoma that is difficult to remove by surgery . in one report ,", "a 58-year - old woman with a superficial spreading melanoma with a breslow thickness 0.75 mm was treated with wide local excision followed by flap reconstruction .", "melanoma reemerged 14 months later that was treated with wide local excision followed by split - thickness graft repair .", "the patient was enrolled in a melanoma peptide vaccine trial 5 months later without improvement .", "twenty - one months later , the patient was treated with imiquimod 5% cream and histopathologic evaluation revealed regression of the widespread cutaneous metastases after 6 months of imiquimod .", "any change in the skin should raise the suspicion of metastases in the correct clinical context .", "biopsy is mandatory in patients with a history of cancer or those with at high risk of cancer .", "immunohistochemical screening methods continue to improve and assist in diagnosing the primary tumors of origin .", "immunohistochemical findings can also help direct treatment options . with advances in novel therapies that prolong survival ," ]

[ "<S> skin metastases are rare in the routine clinical practice of dermatology , but are of major clinical significance because they usually indicate advanced disease . </S>", "<S> we reviewed the literature on skin metastasis regarding recent trends in clinical presentation and diagnosis of the most common cutaneous lesions . </S>", "<S> an extensive literature review was conducted using pubmed from may 26 , 2011 to july 16 , 2013 relating cutaneous metastases . </S>", "<S> articles chosen for reference were queried with the following prompts : cutaneous metastases , clinical presentation , histological features , and </S>", "<S> immunohistochemistry . </S>", "<S> further searches included treatment and management options for metastatic breast , metastatic colorectal , metastatic melanoma , metastatic lung , and hematologic cancers . </S>", "<S> we also reviewed the literature on the current management of melanoma as a model for all cutaneous metastatic disease . </S>", "<S> our own clinical findings are presented and compared to the literature . </S>", "<S> additionally , we highlight the most useful immunohistochemical studies that aid in diagnoses . </S>", "<S> several novel therapies and combination therapies such as electrochemotherapy , vemurafenib , and imiquimod will be discussed for palliative treatment of cancers that have been found to improve cutaneous lesions . </S>", "<S> we review these notable findings and developments regarding skin metastases for the general dermatologist . </S>" ]

[ "Introduction", "Clinical Presentation", "Diagnosis and Immunohistochemistry", "Current Advances in Prognosis and the Management", "Conclusion" ]

[ [ "they may be the first manifestation of metastatic spread of an internal malignancy or herald cancer recurrence long after treatment of a primary tumor .", "breast cancer , colorectal cancer , and melanoma frequently metastasize to the skin in women . in men ; melanoma , lung cancer , and colorectal cancer are the most common sources of cutaneous metastases .", "however , such patterns may differ geographically as a study in taiwan found internal malignancies metastasize to the skin with different frequencies and at rates differing from primarily caucasian populations .", "an extensive literature review was conducted using pubmed from may 26 , 2011-july 16 , 2013 relating cutaneous metastases .", "articles chosen for reference were queried with the following prompts : cutaneous metastases , clinical presentation , histological features , and", "further searches included treatment and management options for metastatic breast , metastatic colorectal , metastatic melanoma , metastatic lung , and hematologic cancers .", "we review the literature on clinical presentation , diagnosis , and advances in the management ." ], [ "cutaneous metastases may be asymptomatic or be associated with pain and tenderness [ figure 1a ] .", "they frequently present as a rapidly growing painless dermal or subcutaneous nodules with intact overlying epidermis [ figures 1b d ] , but can also mimic an inflammatory dermatosis .", "metastases may also present as macules , infiltrated or indurated plaques , discoid lesions , tumor nodules with telangiectasias , bullous or papulosquamous lesions , scarred plaques , or pigmented tumors . while there is no single diagnostic characteristic which predominates ; a few patterns have been recognized .", "( a ) gingival metastases from a primary chondrosarcoma.(b ) firm nodule at the base of the ear from a primary lung cancer .", "( c ) two subcutaneous firm nodules in the clavicular fossa from metastatic lung cancer .", "( d ) isolated firm nodular lesion from metastatic colon cancer metastases originating from breast cancer tend to appear in the anterior chest wall , either from direct extension of underlying tumor or by lymphatic spread [ figures 2a and 3a ] .", "extensive cutaneous involvement of metastatic breast cancer can simulate cellulitis ( carcinoma erysipeloides ) or a breast - plate of armor ( \" en cuirasse \" pattern ) .", "interestingly , a subset of breast cancer patients has superior prognosis , even among breast cancer patients with stage iv disease .", "( a ) cellulitis - like appearance of metastatic breast cancer.(b ) plaque - like area with multiple nodules from a primary breast cancer .", "( c ) massive recurrence of breast cancer on the chest wall and abdomen of the en cuirasse type .", "( d ) breast cancer , metastatic to the thoracic spine with extension to the skin , and lceration secondary to radiation therapy ( a ) cellulitic - type appearance with multiple nodules in metastatic breast cancer of the shoulder and upper back .", "a cam 5.2 stain reveals perinuclear staining ( original magnification , 400).(c ) metastatic squamous cell carcinoma of the skin reveals neoplastic cells in the dermis ( cytokeratin 5/6 stain ; original magnification , 100 ) .", "a her2/neu stain is positive and decorates the neoplastic cells in the dermis indicating possible metastasis from breast carcinoma ( original magnification , 200 ) while most cancers present as solitary nodules , melanoma and carcinomas with an unknown primary frequently present as multiple nodules .", "locoregional melanocytic metastases may be explained by angiotropism , an extravascular migratory phenotype , and an important marker and prognostic factor of metastasis in melanoma .", "the face and scalp are most commonly affected by metastases , suggesting that blood vessels and patterns of innervation may influence the spread of metastases .", "these patterns can also been associated with cancers of gastric , pulmonary , prostatic , ovarian , laryngeal , palatine - tonsillar , pancreatic , colorectal , parotid , thyroid , or uterine origin and may suggest local treatments that work for melanoma may also work for other metastatic cutaneous malignancies ." ], [ "although metastases may be comprised of cells which are more undifferentiated than the primary tumor , careful examination usually reveals important clues .", "for example , melanoma is associated with pigment in at least some of the neoplastic cells in most cases .", "squamous cell carcinoma is associated with keratin pearl formation , and adenocarcinomas usually exhibit gland formation .", "thyroid cancer may exhibit colloid bodies , colon cancer may be associated with mucinous cells , and other tumors may exhibit telltale clues as to their origin . when confronted with a possible metastasis ,", "comparison with the histologic appearance of any prior malignant neoplasms is usually one of the first steps that may help identify the source .", "additionally , studies have indicated the majority of metastatic cancers to the skin are adenocarcinomas .", "when tumors are poorly differentiated or anaplastic , screening immunohistochemical studies can be very helpful .", "when neoplastic cells stain with cytokeratin markers such as ae1/ae3 and cam 5.2 , a carcinoma is likely [ figure 3b ] .", "staining with cd45/leukocyte common antigen ( lca ) indicates that neoplastic cells are of lymphoid origin , while s-100 , melan - a , mart-1 , hmb-45 , and sox-10 decorate most melanomas .", "more detailed immunohistochemical studies [ table 2 ] can be performed when screening studies and clinical correlation have not been fruitful in identifying a tumor of origin .", "although advances in immunohistochemistry are very promising , there is still much work that needs to be done to characterize metastases .", "a recent letter to the editor highlights the pitfalls that can occur when differentiating primary cutaneous apocrine carcinoma from a metastasis .", "although cytokeratin 5/6 is associated with cutaneous apocrine carcinoma , this staining pattern has also been noted with metastatic squamous cell carcinoma [ figure 3c ] .", "similarly , cytokeratin 7 usually stains metastases diffusely , but is associated with only focal staining in primary adnexal carcinoma , but this pattern is not absolute .", "mammaglobin is of interest as it is associated with more widespread staining of metastases than primary apocrine carcinoma , but the results are still preliminary .", "cyclooxygenase 2 is associated with tumors of the mid - gut ( ileum and appendix ) , gut , colorectal adenocarcinoma as well as gastroesophageal adenocarcinoma .", "staining of adenocarcinoma of the lung and endometrioid adenocarcinoma can also occur , highlighting the fact that all immunochemical marker studies must be interpreted in the context of other findings .", "when an epithelial carcinoma is identified , cytokeratin 7 and 20 stains can provide important useful characterization of tumors .", "additional testing can include thyroid transcription factor-1 ( ttf-1 ) and other markers as needed . when clinical appearance , clinical history , and the histologic findings on biopsy are all taken into account", "unfortunately , there are still many cases that elude precise diagnosis regarding whether a tumor is primary or metastatic .", "recent reports have proposed a diagnostic algorithm for metastatic tumors of unknown primary . until better methods are developed" ], [ "we recommend excising metastases when surgically feasible and when this will result in a significant decrease in total tumor burden , improve quality of life , or result in increased functionality .", "novel treatments have had a profound effect on prolonging the survival and improving quality of life with metastatic cancer .", "oftentimes , treatment of the primary cancer with a successful regimen will cause cutaneous lesions to lessen .", "interestingly , the abscopal response represents an unusual response to radiation therapy in which radiation of a tumor causes untreated distant skin lesions to dissipate without recurrence .", "the pathophysiology and mechanism of the abscopal effect have not been well - defined , but one study used monoclonal antibodies to analyze infiltrating lymphocytes and suggested that the abscopal effect was caused by activated cellular immunity .", "the abscopal response has been reported with treatment of melanoma , merkel cell carcinoma , hematologic , and solid tumors .", "the study of effective local treatments for melanoma may serve as a model for other malignancies that disseminate via lymphatics and the blood stream to the skin . for melanomas ,", "we typically excise isolated metastases with a 1 cm margin of normal skin , understanding that there are no evidence based studies looking at the utility of margins for metastatic disease . in areas of high cosmetic importance or when therapy is limited to palliation", ", we sometimes perform excisions with very limited margins . therapy for patients with widespread cutaneous and subcutaneous metastases of advanced melanoma that are nonresectable is limited to other types of palliative therapy .", "options include radiotherapy , systemic chemotherapy , polychemotherapy , isolated limb perfusion , interferon alpha injections , cryotherapy , laser ablation , or radiofrequency ablation .", "electrochemotherapy is a novel therapeutic option based on enhanced drug delivery into cancer cells using externally applied electrical fields that increase cell membrane permeability . using targeted electric pulses on tumor - specific areas , cytotoxicity of cisplatin", "increased 80 times and bleomycin 8,000 times with a large reduction in systemic side effects .", "a study comparing tumor nodules treated with electrochemotherapy with cisplatin versus cisplatin alone found at a 124-week follow - up , 77% of control rate of tumor nodules treated with electrochemotherapy was observed compared to 19% for those treated with cisplatin only .", "electrochemotherapy is well - tolerated by patients , highly effective with low doses of cisplatin or bleomycin , has a short duration of treatment sessions , insignificant side effects , and is more cost - effective than isolated limb perfusion , radiotherapy , interferon - alpha , and hyperthermia associated with radiotherapy and chemotherapy .", "oncogene - targeted therapy for metastatic melanoma using vemurafenib ( plx4032 ) for melanoma associated with a mutation of the braf gene v600 mutation results in complete or partial tumor regression in the majority of patients with an estimated median progression - free survival of more than 7 months .", "side effects include arthralgia , rash , nausea , photosensitivity , fatigue , cutaneous squamous - cell carcinoma , pruritus , and palmoplantar dysesthesia .", "imiquimod 5% cream has also been used successfully in the treatment of metastatic melanoma that is difficult to remove by surgery . in one report ,", "a 58-year - old woman with a superficial spreading melanoma with a breslow thickness 0.75 mm was treated with wide local excision followed by flap reconstruction .", "melanoma reemerged 14 months later that was treated with wide local excision followed by split - thickness graft repair .", "the patient was enrolled in a melanoma peptide vaccine trial 5 months later without improvement .", "twenty - one months later , the patient was treated with imiquimod 5% cream and histopathologic evaluation revealed regression of the widespread cutaneous metastases after 6 months of imiquimod ." ], [ "any change in the skin should raise the suspicion of metastases in the correct clinical context .", "biopsy is mandatory in patients with a history of cancer or those with at high risk of cancer .", "immunohistochemical screening methods continue to improve and assist in diagnosing the primary tumors of origin .", "immunohistochemical findings can also help direct treatment options . with advances in novel therapies that prolong survival ," ] ]

[ "ampullary adenomas arise from the ampulla of vater , the confluence of the pancreatic and common bile ducts , located at the major duodenal papilla in the second part of the duodenum . based upon autopsy studies , ampullary adenomas", "however , the vast majority of these patients will remain asymptomatic [ 1 , 2 , 3 , 4 ] . as with colonic adenocarcinoma", ", ampullary adenomas also follow the adenoma - carcinoma sequence and may thus evolve from localised premalignant lesions into widespread carcinoma .", "such lesions may be removed endoscopically or via surgical excision , with either a local resection or an extended procedure , for example , a pancreaticoduodenectomy .", "this may occur as an idiopathic process , as is more common in the paediatric population , or it may be due to a structural pathology , such as colonic malignancy . as other gastrointestinal adenomas , ampullary adenomas assume tubular , tubulovillous or villous forms .", "they can occur either sporadically or in patients with a genetic predisposition , such as familial adenomatous polyposis or gardner 's syndrome , and such lesions may therefore be identified via endoscopic surveillance programmes .", "indeed , previous studies have suggested a further genetic link to other disorders , such as adult polycystic kidney disease , whilst no specific environmental factors such as diet have been identified in their development .", "clinically , they are usually asymptomatic and found incidentally at endoscopy ; however , some patients may present with symptoms caused by obstruction of the main gastrointestinal tract and/or the distal biliary / pancreatic ducts .", "the most common presenting symptoms are gastric outlet obstruction or gastrointestinal bleeding [ 8 , 9 ] .", "however , rarer presenting complaints may include acute pancreatitis , biliary obstruction or , as in this case , intussusception .", "indications for the excision of such lesions are essentially two - fold : to attempt resolution of immediate symptoms and to prevent malignant progression", ". the management of such lesions may be endoscopic surveillance , endoscopic excision or surgical resection .", "surgical resection has the advantage of lower recurrence rates as opposed to endoscopic resection ; however , with the obvious caveat of a much higher risk of morbidity and mortality . in this article , we present a case where a female patient developed recurrent chest infections and gastric outlet obstruction secondary to an intussuscepting ampullary adenoma . due to the size and location of the mass ,", "it was surgically removed by pancreaticoduodenectomy . to the best of our knowledge , this is the first such presentation worldwide , and we hope that it may be of educational value to both physicians and surgeons alike .", "a 49-year - old nigerian female presented to her local hospital with a 1-year history of recurrent chest infections .", "she had no relevant past family history of any genetic disorders , in addition to no recent foreign travel or contact exposure to tuberculosis .", "when she initially presented , plain chest radiographs were performed and demonstrated multiple lung lesions suggestive of an infective / inflammatory process .", "she grew pseudomonas on sputum culture , but despite treatment for atypical pneumonia with multiple courses of antibiotics , she failed to adequately improve .", "chest ct was consequently performed and demonstrated multiple cavitating lung lesions ( fig 1 ) . during this time", ", she had intermittent vomiting with an associated 15-kg weight loss over a 12-month period .", "these symptoms were initially attributed to her recurrent chest infections and were not further investigated .", "a working diagnosis of aspergillus infection was established , and the patient remained under the care of the respiratory physicians . at the time of chest ct ,", "however , an incidental finding was noted in the second to the fourth part of the duodenum .", "concurrently , she developed subacute bowel obstruction with persistent vomiting and was unable to tolerate oral intake .", "a dedicated ct scan of the abdomen and pelvis was therefore performed , demonstrating an obstructing polypoid mass at d2 ( fig 2 ) .", "this lesion was confirmed on upper gastrointestinal endoscopy , and a biopsy demonstrated a tubulovillous adenoma with high - grade dysplasia .", "nasojejunal tube insertion was attempted at her local hospital ; however , this failed to bypass the obstructing lesion .", "her case was discussed at the regional hepatopancreaticobiliary multidisciplinary team meeting at the royal london hospital . as her biopsy demonstrated high - grade dysplasia , in addition to her symptomatic presentation , it was felt that a resection was required . based upon her cross - sectional and endoscopic imaging", ", it was deemed that this would not be amenable to an endoscopic excision , and she was likely to require surgical resection and should be transferred to the hpb unit at the royal london hospital .", "interestingly , despite this large lesion , she never became jaundiced and maintained a normal bilirubin level .", "on arrival at the hospital , she was admitted to the surgical ward , and although she had persistent vomiting and was cachectic , she was haemodynamically stable .", "g / l ( normal range 3550 ) due to the failed nasojejunal tube insertion .", "consequently , a picc line was inserted by interventional radiology , and in an attempt to optimise her for major abdominal surgery , total parenteral nutrition was administered . however , due to these ongoing nutritional problems and a failure to adequately improve , she underwent a semi - urgent conventional pancreaticoduodenectomy .", "intraoperatively , a large mass at d2 with a duodeno - duodenal intussusception and distal duodenal ischaemia was identified ( fig 3 ) .", "histological analysis reported a polypoid tumour at the ampulla of vater , 75 28 30 mm in size , with a reduced intussusception and an ischaemic distal duodenum ( fig 3 ) .", "these findings were in keeping with a duodenal ampullary low - grade tubular adenoma , with intussusception and complete resection margins .", "there was no evidence of any high - grade dysplasia , in contrast to her previous diagnostic biopsy , or invasive malignancy .", "the patient was admitted to the high - dependency unit postoperatively and was stepped down to the surgical ward on postoperative day 2 .", "she progressed well with early mobilisation , and her nutritional status greatly improved during this time , with due care not to induce a refeeding syndrome .", "she did not develop any exocrine pancreatic insufficiency or any derangement from her baseline glycaemic control .", "to date , the patient has not suffered any long - term complications , and remains clinically well on follow - up .", "intussusception in adults is rare and is responsible for approximately 15% of all adult intestinal obstructions", ". these can be classified by location into enteroenteric , colocolonic , ileocolic and ileocaecal , respectively .", "the presenting symptoms are usually non - specific and chronic , with pain and symptoms of intermittent intestinal obstruction , such as vomiting , bloating and/or diarrhoea or constipation being the major complaints .", "the classic paediatric triad of cramping abdominal pain , bloody diarrhoea and a tender abdominal mass is rare in adults .", "as opposed to the paediatric population , where the aetiology is normally idiopathic , in adults , the cause is usually a structural pathology , with two - thirds of colonic cases being a malignant neoplasm , as opposed to one - third of those occurring in the small intestine [ 11 , 12 , 13 ] .", "abdominal ct is now regarded as the modality of choice for investigating such lesions . due to the preponderance for a structural aetiology ,", "the management of such cases is almost always via laparotomy , with no role for the use of air insufflation as utilised in the paediatric population .", "intraoperative reduction of the intussuscepted bowel is possible ; however , there are associated risks such as tumour seeding and anastomotic complications due to the pathological bowel tissue .", "it may , however , be carried out if the lesion has been confirmed as benign in the preoperative period , and can be performed by milking the lesion in a distal to proximal direction , followed by a limited resection [ 15 , 16 ] .", "a literature search revealed 13 cases of duodenal / ampullary adenomas causing duodeno - duodenal intussusception , and to the best of our knowledge , no case of an ampullary adenoma causing symptomatic intussusception with recurrent chest infections was previously reported .", "incidental duodenal polyps are reported in the literature at a rate of 0.34.6% and have a variety of pathological origins , including adenomas , submucosal tumours and hamartomas amongst others .", "ampullary adenomas are thought to be precursors to adenocarcinomas , as such tissue is present in 90% of the resected ampullary adenocarcinoma specimens , whilst based on autopsy studies , they may occur at a rate of 0.040.12% in the general population [ 1 , 2 , 3 ] . due to their location", ", ampullary adenomas may clinically manifest via obstruction of the main gastrointestinal tract or the biliary and/or pancreatic ducts .", "however , they are usually asymptomatic and found incidentally at endoscopy , whilst some patients may present with symptoms , most commonly gastric outlet obstruction or gastrointestinal bleeding [ 8 , 9 ] .", "rarer presenting complaints may include acute pancreatitis , biliary obstruction or , as in this case , intussusception .", "depending on the clinical urgency of definitive treatment , such lesions may be further investigated using a wide range of modalities , including ultrasonography , ct , endoscopic ultrasonography ( eus ) , magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography .", "eus has proven to be a vital technique in the local assessment of periampullary tumours and has a higher sensitivity to both us and ct when detecting such lesions and assessing muscular ( suggesting invasion , and therefore malignancy ) and vascular involvement ( to assess resectability ) . however , ercp is also extremely useful and has a similar sensitivity to eus in detecting ampullary tumours .", "there are three broad management strategies for ampullary adenomas : surveillance , endoscopic resection and surgery .", "endoscopic resection may be possible in adenomas with low - grade dysplasia , which are < 1 cm in size and demonstrate benign characteristics at endoscopy .", "however , chini and draganov suggest that they believe endoscopic resection to be an appropriate course of action even in the presence of high - grade dysplasia if the two above criteria are met . the presence of an ampullary adenocarcinoma warrants surgical resection if the candidate is fit .", "however , there have been sporadic case reports where such malignancies have been removed endoscopically [ 22 , 23 , 24 , 25 , 26 ] .", "however , debate surrounding their management exists , as some groups speculate that the progression from adenoma to carcinoma is a low percentage , whilst others argue that the quality of forceps biopsy via endoscopy may miss occult foci of adenocarcinoma within the adenoma .", "asymptomatic patients may be treated with endoscopic management with endoscopic surveillance as follow up ; however , there is no current consensus as to the ideal surveillance post endoscopic resection .", "clinicians must ensure that patients who are previously well and present recurrently with chest infections are fully investigated and that they consider the possibility of a lesion causing gastric outlet obstruction .", "clinicians should consider dedicated abdominal imaging , and if a mass is identified , undergo endoscopy to further characterise the lesion and develop an on - going management plan with appropriate specialist input .", "the index of suspicion should of course be higher if the patient goes on to further develop signs of an obstructing lesion , such as persistent vomiting ." ]

[ "<S> ampullary adenomas are a rare clinical entity , occurring at a rate of 0.040.12% in the general population . </S>", "<S> they are premalignant lesions which have the capability to progress to malignancy , and they should be excised if they are causing immediate symptoms and/or are likely to degenerate to carcinoma . </S>", "<S> intestinal intussusception in adults is rare and , unlike in children , is often due to a structural pathology . </S>", "<S> intussuscepting duodenal / ampullary adenomas have been reported in the literature on 13 previous occasions , however never before with this presentation . </S>", "<S> we report the case of a woman who presented with a 1-year history of recurrent chest infections . </S>", "<S> she was treated with numerous antibiotics , whilst intermittent symptoms of recurrent vomiting and weight loss were initially attributed to her lung infections . </S>", "<S> a chest ct demonstrated multiple cavitating lung lesions , whilst an obstructing polypoid mass was noted at d2 on dedicated abdominal imaging . </S>", "<S> due to ongoing nutritional problems , she had a semi - urgent pancreaticoduodenectomy . </S>", "<S> intraoperative findings demonstrated a large mass at d2 with a duodeno - duodenal intussusception . </S>", "<S> histological analysis reported a duodenal , ampullary , low - grade tubular adenoma , 75 28 30 mm in size , with intussusception and complete resection margins . </S>", "<S> the patient recovered well and was discharged on postoperative day 10 , with no complications to date . </S>", "<S> ampullary adenomas may present with obstruction of the main gastrointestinal tract and/or biliary / pancreatic ducts . </S>", "<S> common presentations include gastric outlet obstruction , gastrointestinal bleeding or acute pancreatitis . </S>", "<S> this unique presentation should remind clinicians of the need to investigate recurrent chest infections for a possible gastrointestinal cause . </S>" ]

[ "Introduction", "Case Presentation", "Discussion", "Conclusion", "Statement of Ethics", "Disclosure Statement" ]

[ [ "ampullary adenomas arise from the ampulla of vater , the confluence of the pancreatic and common bile ducts , located at the major duodenal papilla in the second part of the duodenum . based upon autopsy studies , ampullary adenomas", "however , the vast majority of these patients will remain asymptomatic [ 1 , 2 , 3 , 4 ] . as with colonic adenocarcinoma", ", ampullary adenomas also follow the adenoma - carcinoma sequence and may thus evolve from localised premalignant lesions into widespread carcinoma .", "such lesions may be removed endoscopically or via surgical excision , with either a local resection or an extended procedure , for example , a pancreaticoduodenectomy .", "this may occur as an idiopathic process , as is more common in the paediatric population , or it may be due to a structural pathology , such as colonic malignancy . as other gastrointestinal adenomas , ampullary adenomas assume tubular , tubulovillous or villous forms .", "they can occur either sporadically or in patients with a genetic predisposition , such as familial adenomatous polyposis or gardner 's syndrome , and such lesions may therefore be identified via endoscopic surveillance programmes .", "indeed , previous studies have suggested a further genetic link to other disorders , such as adult polycystic kidney disease , whilst no specific environmental factors such as diet have been identified in their development .", "clinically , they are usually asymptomatic and found incidentally at endoscopy ; however , some patients may present with symptoms caused by obstruction of the main gastrointestinal tract and/or the distal biliary / pancreatic ducts .", "the most common presenting symptoms are gastric outlet obstruction or gastrointestinal bleeding [ 8 , 9 ] .", "however , rarer presenting complaints may include acute pancreatitis , biliary obstruction or , as in this case , intussusception .", "indications for the excision of such lesions are essentially two - fold : to attempt resolution of immediate symptoms and to prevent malignant progression", ". the management of such lesions may be endoscopic surveillance , endoscopic excision or surgical resection .", "surgical resection has the advantage of lower recurrence rates as opposed to endoscopic resection ; however , with the obvious caveat of a much higher risk of morbidity and mortality . in this article , we present a case where a female patient developed recurrent chest infections and gastric outlet obstruction secondary to an intussuscepting ampullary adenoma . due to the size and location of the mass ,", "it was surgically removed by pancreaticoduodenectomy . to the best of our knowledge , this is the first such presentation worldwide , and we hope that it may be of educational value to both physicians and surgeons alike ." ], [ "a 49-year - old nigerian female presented to her local hospital with a 1-year history of recurrent chest infections .", "she had no relevant past family history of any genetic disorders , in addition to no recent foreign travel or contact exposure to tuberculosis .", "when she initially presented , plain chest radiographs were performed and demonstrated multiple lung lesions suggestive of an infective / inflammatory process .", "she grew pseudomonas on sputum culture , but despite treatment for atypical pneumonia with multiple courses of antibiotics , she failed to adequately improve .", "chest ct was consequently performed and demonstrated multiple cavitating lung lesions ( fig 1 ) . during this time", ", she had intermittent vomiting with an associated 15-kg weight loss over a 12-month period .", "these symptoms were initially attributed to her recurrent chest infections and were not further investigated .", "a working diagnosis of aspergillus infection was established , and the patient remained under the care of the respiratory physicians . at the time of chest ct ,", "however , an incidental finding was noted in the second to the fourth part of the duodenum .", "concurrently , she developed subacute bowel obstruction with persistent vomiting and was unable to tolerate oral intake .", "a dedicated ct scan of the abdomen and pelvis was therefore performed , demonstrating an obstructing polypoid mass at d2 ( fig 2 ) .", "this lesion was confirmed on upper gastrointestinal endoscopy , and a biopsy demonstrated a tubulovillous adenoma with high - grade dysplasia .", "nasojejunal tube insertion was attempted at her local hospital ; however , this failed to bypass the obstructing lesion .", "her case was discussed at the regional hepatopancreaticobiliary multidisciplinary team meeting at the royal london hospital . as her biopsy demonstrated high - grade dysplasia , in addition to her symptomatic presentation , it was felt that a resection was required . based upon her cross - sectional and endoscopic imaging", ", it was deemed that this would not be amenable to an endoscopic excision , and she was likely to require surgical resection and should be transferred to the hpb unit at the royal london hospital .", "interestingly , despite this large lesion , she never became jaundiced and maintained a normal bilirubin level .", "on arrival at the hospital , she was admitted to the surgical ward , and although she had persistent vomiting and was cachectic , she was haemodynamically stable .", "g / l ( normal range 3550 ) due to the failed nasojejunal tube insertion .", "consequently , a picc line was inserted by interventional radiology , and in an attempt to optimise her for major abdominal surgery , total parenteral nutrition was administered . however , due to these ongoing nutritional problems and a failure to adequately improve , she underwent a semi - urgent conventional pancreaticoduodenectomy .", "intraoperatively , a large mass at d2 with a duodeno - duodenal intussusception and distal duodenal ischaemia was identified ( fig 3 ) .", "histological analysis reported a polypoid tumour at the ampulla of vater , 75 28 30 mm in size , with a reduced intussusception and an ischaemic distal duodenum ( fig 3 ) .", "these findings were in keeping with a duodenal ampullary low - grade tubular adenoma , with intussusception and complete resection margins .", "there was no evidence of any high - grade dysplasia , in contrast to her previous diagnostic biopsy , or invasive malignancy .", "the patient was admitted to the high - dependency unit postoperatively and was stepped down to the surgical ward on postoperative day 2 .", "she progressed well with early mobilisation , and her nutritional status greatly improved during this time , with due care not to induce a refeeding syndrome .", "she did not develop any exocrine pancreatic insufficiency or any derangement from her baseline glycaemic control .", "to date , the patient has not suffered any long - term complications , and remains clinically well on follow - up ." ], [ "intussusception in adults is rare and is responsible for approximately 15% of all adult intestinal obstructions", ". these can be classified by location into enteroenteric , colocolonic , ileocolic and ileocaecal , respectively .", "the presenting symptoms are usually non - specific and chronic , with pain and symptoms of intermittent intestinal obstruction , such as vomiting , bloating and/or diarrhoea or constipation being the major complaints .", "the classic paediatric triad of cramping abdominal pain , bloody diarrhoea and a tender abdominal mass is rare in adults .", "as opposed to the paediatric population , where the aetiology is normally idiopathic , in adults , the cause is usually a structural pathology , with two - thirds of colonic cases being a malignant neoplasm , as opposed to one - third of those occurring in the small intestine [ 11 , 12 , 13 ] .", "abdominal ct is now regarded as the modality of choice for investigating such lesions . due to the preponderance for a structural aetiology ,", "the management of such cases is almost always via laparotomy , with no role for the use of air insufflation as utilised in the paediatric population .", "intraoperative reduction of the intussuscepted bowel is possible ; however , there are associated risks such as tumour seeding and anastomotic complications due to the pathological bowel tissue .", "it may , however , be carried out if the lesion has been confirmed as benign in the preoperative period , and can be performed by milking the lesion in a distal to proximal direction , followed by a limited resection [ 15 , 16 ] .", "a literature search revealed 13 cases of duodenal / ampullary adenomas causing duodeno - duodenal intussusception , and to the best of our knowledge , no case of an ampullary adenoma causing symptomatic intussusception with recurrent chest infections was previously reported .", "incidental duodenal polyps are reported in the literature at a rate of 0.34.6% and have a variety of pathological origins , including adenomas , submucosal tumours and hamartomas amongst others .", "ampullary adenomas are thought to be precursors to adenocarcinomas , as such tissue is present in 90% of the resected ampullary adenocarcinoma specimens , whilst based on autopsy studies , they may occur at a rate of 0.040.12% in the general population [ 1 , 2 , 3 ] . due to their location", ", ampullary adenomas may clinically manifest via obstruction of the main gastrointestinal tract or the biliary and/or pancreatic ducts .", "however , they are usually asymptomatic and found incidentally at endoscopy , whilst some patients may present with symptoms , most commonly gastric outlet obstruction or gastrointestinal bleeding [ 8 , 9 ] .", "rarer presenting complaints may include acute pancreatitis , biliary obstruction or , as in this case , intussusception .", "depending on the clinical urgency of definitive treatment , such lesions may be further investigated using a wide range of modalities , including ultrasonography , ct , endoscopic ultrasonography ( eus ) , magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography .", "eus has proven to be a vital technique in the local assessment of periampullary tumours and has a higher sensitivity to both us and ct when detecting such lesions and assessing muscular ( suggesting invasion , and therefore malignancy ) and vascular involvement ( to assess resectability ) . however , ercp is also extremely useful and has a similar sensitivity to eus in detecting ampullary tumours .", "there are three broad management strategies for ampullary adenomas : surveillance , endoscopic resection and surgery .", "endoscopic resection may be possible in adenomas with low - grade dysplasia , which are < 1 cm in size and demonstrate benign characteristics at endoscopy .", "however , chini and draganov suggest that they believe endoscopic resection to be an appropriate course of action even in the presence of high - grade dysplasia if the two above criteria are met . the presence of an ampullary adenocarcinoma warrants surgical resection if the candidate is fit .", "however , there have been sporadic case reports where such malignancies have been removed endoscopically [ 22 , 23 , 24 , 25 , 26 ] .", "however , debate surrounding their management exists , as some groups speculate that the progression from adenoma to carcinoma is a low percentage , whilst others argue that the quality of forceps biopsy via endoscopy may miss occult foci of adenocarcinoma within the adenoma .", "asymptomatic patients may be treated with endoscopic management with endoscopic surveillance as follow up ; however , there is no current consensus as to the ideal surveillance post endoscopic resection ." ], [ "clinicians must ensure that patients who are previously well and present recurrently with chest infections are fully investigated and that they consider the possibility of a lesion causing gastric outlet obstruction .", "clinicians should consider dedicated abdominal imaging , and if a mass is identified , undergo endoscopy to further characterise the lesion and develop an on - going management plan with appropriate specialist input .", "the index of suspicion should of course be higher if the patient goes on to further develop signs of an obstructing lesion , such as persistent vomiting ." ], [ "" ], [ "" ] ]

[ "this is a nonrandomized prospective comparative case study on 57 eyes seeking refractive surgery who were not indicated for laser corneal refractive surgery .", "patients with refraction error between 2.00 d and 14.50 d who could use both artiflex and artisan , were free to choose the type of the lens . other patients underwent artisan implantation .", "ophthalmic examinations before surgery included cyclo - refraction , uncorrected visual acuity ( ucva ) , best - corrected visual acuity ( bcva ) , topography , keratometry , endothelial cell count , pupil diameter , and anterior chamber ( ac ) depth measurement . according to patient history and examination , patients who had the following conditions underwent piol implantation surgery : insufficient corneal thickness for refractive corneal surgery , inappropriate corneal curvature and topography , high myopia more than 8 d which could not undergo corneal refractive surgery , negative history of any previous refractive surgery , sufficient internal ac depth ( 3 mm or greater ) , no history of intraocular pressure raise and endothelial cell density of 2500 cell / mm or more .", "lens dioptric power was calculated by van der heijde formula , using refractive error , refractive cylinder power , ac depth , and topographically derived keratometry values .", "emmetropia was the target power of the surgery , when the precise emmetropic lens was not available our choice was to a slight residual myopia .", "all surgeries were performed under general anesthesia , for artisan lens implantation , a superior 5 or 6 mm length corneal incision was created at the 12 o'clock position , followed by performing two stab incisions located at 2 and 10 o'clock .", "the lens was inserted into the ac under protection of intracameral ophthalmic viscoelastic material injection .", "artiflex lens implantation was performed similarly , although the lens was inserted through a 3.2 mm incision .", "other steps were carried out same as artisan implantation ; finally , the wound was closed by stromal hydration without the necessity of suturing .", "one year after surgery , cyclo - refraction , bcva , ucva , cs , hoa and patient satisfaction were assessed .", "cs was determined by csv-1000 chart , and refraction was obtained with topcon auto refractometer and then modified by subjective refraction .", "night vision , day vision , halo , photophobia , dryness , pain , and patient satisfaction were assessed by questionnaire with numerical scales ( 0 = weak , 1 = moderate , 2 = good , 3 = excellent for day and night vision 0 = none , 1 = mild , 2 = moderate , 3 = severe for halo , photophobia , dryness , and pain ) .", "the chart consists of four rows corresponding to spatial frequencies of 3 , 6 , 12 , and 18 cycles per degree ( cpd ) .", "the distance between the patient and the chart was 2.5 m. the test was performed with the patients monocular bcva to eliminate the effects of sia and residual post - operative refraction .", "the aberrations analyzed were classified in terms of hoa , vertical trefoil , vertical coma ; horizontal trefoil , horizontal coma , quatrefoil , second astigmatism , and fourth order spherical aberration .", "comparison between artisan and artiflex data were analyzed using parametric paired student 's t - test , p < 0.05 was considered statistically significant .", "this is a nonrandomized prospective comparative case study on 57 eyes seeking refractive surgery who were not indicated for laser corneal refractive surgery .", "patients with refraction error between 2.00 d and 14.50 d who could use both artiflex and artisan , were free to choose the type of the lens . other patients underwent artisan implantation .", "ophthalmic examinations before surgery included cyclo - refraction , uncorrected visual acuity ( ucva ) , best - corrected visual acuity ( bcva ) , topography , keratometry , endothelial cell count , pupil diameter , and anterior chamber ( ac ) depth measurement . according to patient history and examination , patients who had the following conditions underwent piol implantation surgery : insufficient corneal thickness for refractive corneal surgery , inappropriate corneal curvature and topography , high myopia more than 8 d which could not undergo corneal refractive surgery , negative history of any previous refractive surgery , sufficient internal ac depth ( 3 mm or greater ) , no history of intraocular pressure raise and endothelial cell density of 2500 cell / mm or more .", "lens dioptric power was calculated by van der heijde formula , using refractive error , refractive cylinder power , ac depth , and topographically derived keratometry values .", "emmetropia was the target power of the surgery , when the precise emmetropic lens was not available our choice was to a slight residual myopia .", "all surgeries were performed under general anesthesia , for artisan lens implantation , a superior 5 or 6 mm length corneal incision was created at the 12 o'clock position , followed by performing two stab incisions located at 2 and 10 o'clock .", "the lens was inserted into the ac under protection of intracameral ophthalmic viscoelastic material injection .", "artiflex lens implantation was performed similarly , although the lens was inserted through a 3.2 mm incision .", "other steps were carried out same as artisan implantation ; finally , the wound was closed by stromal hydration without the necessity of suturing .", "one year after surgery , cyclo - refraction , bcva , ucva , cs , hoa and patient satisfaction were assessed .", "cs was determined by csv-1000 chart , and refraction was obtained with topcon auto refractometer and then modified by subjective refraction .", "night vision , day vision , halo , photophobia , dryness , pain , and patient satisfaction were assessed by questionnaire with numerical scales ( 0 = weak , 1 = moderate , 2 = good , 3 = excellent for day and night vision 0 = none , 1 = mild , 2 = moderate , 3 = severe for halo , photophobia , dryness , and pain ) .", "the chart consists of four rows corresponding to spatial frequencies of 3 , 6 , 12 , and 18 cycles per degree ( cpd ) .", "the distance between the patient and the chart was 2.5 m. the test was performed with the patients monocular bcva to eliminate the effects of sia and residual post - operative refraction .", "the aberrations analyzed were classified in terms of hoa , vertical trefoil , vertical coma ; horizontal trefoil , horizontal coma , quatrefoil , second astigmatism , and fourth order spherical aberration .", "comparison between artisan and artiflex data were analyzed using parametric paired student 's t - test , p < 0.05 was considered statistically significant .", "a total of 57 eyes were included in the study ; of which , 24 eyes were in artisan group and 33 eyes were in artiflex group .", "the preoperative spherical equivalent ( se ) and bcva were obtained and compared , no statistically significant difference was noticed in the mean preoperative se ( 10.39 8.43 d in artisan group and 10.39 2.29 d in artiflex group ; p = 0.999 ) and bcva of two groups ( 0.25 0.21 logarithm of the minimum angle of resolution [ logmar ] for artisan group and 0.19 0.18 logmar for artiflex group ; p = 0.56 ) .", "the mean postoperative se was 0.93 1.28 d in the artisan group and 0.54 0.82 in the artiflex group , no significant difference was seen in in postoperative se between two groups ( p = 0.19 ) .", "one year after the operation , 54% of artisan - treated eyes ( 15/24 ) and 75.7% ( 25/33 ) of artiflex treated eyes were within 1.00 d of intended emmetropia [ figs . 1 and 2 ] . in the artisan group , 37.5% of the treated eyes ( 9/24 eyes ) had three or more snellen line improvement of postoperative bcva , 12.5% ( 3/24 eyes ) had two snellen line improvement and 25% ( 6/24 eyes ) had loss of two or more snellen lines . in the artiflex group ,", "51.5% of the treated eyes ( 17/33 eyes ) had three or more snellen line improvement of bcva , 15.1% ( 5/33 eyes ) had two line improvement , and no eyes had loss of two or more snellen lines of bcva [ fig . 3 ] .", "the bar graph shows the postoperative defocus equivalent 1 year after artisan ( a ) and artiflex ( b ) implantation bar graph of change in best - corrected visual acuity from the preoperative examination to postoperative examination in terms of the number of snellen line change .", "artisan ( a ) , artiflex ( b ) mesopic contrast sensitivity at spatial frequencies of 3 , 6 , 12 and 18 degree per cycles in this study , total hoa in artisan - treated eyes was significantly greater than artiflex - treated eyes ( p = 0.044 ) with a mean hoa of 0.44 0.15 root mean square ( rms ) for artisan and 0.35 0.15 rms for artiflex .", "there was no significant difference in the vertical trefoil , vertical coma , horizontal trefoil , horizontal coma , quatrefoil , second astigmatism , and fourth order spherical aberration .", "the evaluation of cs in mesopic conditions showed higher cs in artiflex - treated eyes at three spatial frequencies 6 , 12 , and 18 cpd ( p = 0.003 , p = 0.007 , and p = 0.00 , respectively ) , and no significant difference was seen between two lenses at 3 cpd spatial frequency [ table 1 and fig .", "contrast sensitivity data after phakic intra - ocular lens implantation subjective comparison of day and night vision in artisan versus artiflex treated eyes the mean of sia was 1.65 0.82 d in artisan group , and 1.31 1.05 in artiflex group , which showed no statistically significant difference between the groups ( p = 0.19 ) . in a subjective evaluation of day vision", ", all patients of the artiflex group reported good and excellent vision , in artisan group , 71% of patients reported good and excellent vision , whereas 7.1% complained of weak day vision . in the assessment of night vision ,", "50% of patients treated with artiflex reported good and excellent vision and no one complained of weak night vision . in artisan group ,", "71.4% reported good and excellent vision , whereas 21.4% reported weak night vision [ fig .", "subjective comparison of halo , photophobia , pain and dryness in artisan versus artiflex treated eyes among artiflex treated eyes , 50% reported moderate halos and in the artisan group 40% reported moderate to severe halos . about 12.5% of artiflex treated eyes experienced moderate photophobia , whereas 35.7% of artisan treated eyes experienced moderate to severe photophobia . in the artiflex group ,", "only 12.5% of treated eyes caused mild pain , whereas 36.7% of artisan treated eyes caused mild to moderate pain .", "moderate to severe dryness was reported in 25% of eyes treated with artiflex , whereas no artisan treated eye experienced moderate or severe dryness [ fig . 6 ] .", "the bar graph of patient 's satisfaction in artisan versus artiflex groups satisfaction more than 60% was reported in 100% of patients in artiflex group versus 60% of patients in artisan group [ fig .", "the bar graph presents the postoperative spherical equivalent 1 year after artisan ( a ) and artiflex ( b ) implantation .", "as it is well - confirmed , the recently food and drug administration approved pmma artisan piol is an effective mean for the correction of refractive errors and have been shown to have acceptable safety and efficacy .", "however , studies have revealed instances of significant induced astigmatism , attributed to the larger incisions performed in the implantation of the artisan lens .", "artiflex iris - fixated piol , a foldable version of artisan , is an improvement of the iris - supported piol concept , with a lower incidence of sia .", "artiflex piol haptics are made of pmma and the foldable optical zone is made of silicon .", "the main purpose of this investigation is to evaluate the differences in optic quality between rigid piol versus flexible piol by comparing wavefront aberration , cs and patient satisfaction in patients who underwent artisan and artiflex iris - fixated iol implantation . in this study ,", "mean postoperative residual se were 0.93 1.28 d and 0.54 0.82 d in the artisan and artiflex group , respectively , no significant difference was seen in postoperative se between two groups ( p = 0.19 ) .", "about 54% of artisan group and 75.7% of artiflex group were within 1.00 d of intended emmetropia . in a previous study , 58% and 83.9% of artisan and artiflex group had a residual postoperative se within 1.00 d , which was significantly better in artiflex group .", "another study reported that emmetropia ( 1.00 d ) was obtained in 60% and 91.7% of artisan and artiflex group , respectively , the difference was statistically significant [ table 2 ] .", "percentage of postoperative refraction within1.00 d of intended emmetropia in a retrospective comparative case series on 27 eyes , postoperative se was within 0.5 d in 76.2% and 85.7% of artisan and artiflex treated eyes , respectively . in a study of evaluating the outcome of iris - claw piol in fifty high myopic eyes , postoperative se was evaluated , which showed that 38% and 68% of the eyes were in the range of 0.5 d and 1.00 d , respectively .", "mean postoperative ucva was 0.34 0.25 logmar in artisan group and 0.12 0.15 logmar in artiflex group .", "mean postoperative bcva was 0.21 0.13 and 0.06 0.08 logmar in the artisan and artiflex groups , respectively [ table 3 ] .", "the differences of ucva and bcva in two groups were statistically significant ( p = 0.00 ) . in another study ,", "postoperative bcva between artisan and artiflex groups was not significantly different ( p = 0.9 ) .", "postoperative visual acuity ( logarithm of the minimum angle of resolution ) in artisan group versus artiflex group in this study , although vertical trefoil , vertical coma , horizontal trefoil , horizontal coma , secondary astigmatism , quatrefoil and fourth order spherical aberration were not significantly different between the artiflex and artisan groups , total hoa was higher in artisan group , that may be due to the slight differences in each component , which totally increases the hoa ( p = 0.044 ) . in a previous study , assessment of hoas after implantation of rigid versus foldable", "iris - fixated lenses , showed a significant decrease in postoperative spherical aberration among artiflex group and a significant increase in postoperative spherical aberration in artisan group . in both groups ,", "vertical trefoil and spherical aberration were higher in artisan group , p = 0.039 and p = 0.001 , respectively .", "assessment of cs in mesopic condition showed no significant difference between two groups at 3 cpd spatial frequency .", "cs was significantly higher in artiflex - treated eyes at three spatial frequencies 6 , 12 and 18 cpd ( p = 0.003 , p = 0.007 , and p = 0.00 , respectively ) .", "a previous investigation showed that mesopic cs was slightly better in artiflex group in comparison with artisan group , but the difference was not statistically significant , they also measured photopic cs function and found that both groups had poorer performance compared to the normal range .", "performance with the artisan lens was slightly better than performance with artiflex under photopic conditions , but worse under mesopic conditions ; neither differences were statistically significant . in this study , we have used artisan iol for patients not eligible for the available artiflex ( six myopic eye more than 14.5 d and one hyperopic eye ) , comparison of this two groups may have some biases because of other probable conditions such as retinal involvement or amblyopia , which can alter the results , this was one of our study limitations .", "in this study , 25% of patients with artisan lens lost two or more lines of bcva , we called the patients back and examined them again carefully , we recognized that in one patient both eyes had refractive error postoperatively in spite of implanting a lens with the same power of his preoperative se improvement in bcva was not seen with correction of this refractive error by trial - frame . in the other patients , we could not find any reason for the decrease in the bcva , unless considering pigment deposition on the iol as a reason for the visual loss ." ]

[ "<S> aim of study : the aim of this study is to assess wavefront aberration and contrast sensitivity ( cs ) after implantation of foldable iris claw artiflex- and rigid iris claw </S>", "<S> artisan- phakic intraocular lenses ( piols).materials and methods : a nonrandomized prospective comparative case study was performed on 57 eyes ; of which , 54 were myopia and 3 were hyperopia . </S>", "<S> twenty - four patients had artisan piol implantation and 33 had artiflex piol implantation . </S>", "<S> higher - order aberration ( hoa ) and cs were obtained 1 year after surgery.results:total hoa in artisan group was greater than artiflex group ( p = 0.044 ) with a mean hoa of 0.44 0.15 root mean square ( rms ) for artisan and 0.35 0.15 rms for artiflex . </S>", "<S> although , there were no significant differences in the vertical trefoil , vertical coma , horizontal trefoil , horizontal coma , secondary astigmatism , quatrefoil , and fourth order spherical aberration in two groups . </S>", "<S> cs in mesopic conditions was better in artiflex - treated eyes at three spatial frequencies of 6 , 12 , and 18 cycles per degree ( cpd ) ( p = 0.003 , p = 0.007 , and p = 0.00 , respectively ) , and no significant difference was seen between two lenses at 3 cpd.conclusion:although the components of hoa were not significantly different between two groups , total hoa was higher in artisan group , which may be due to the slight differences in each component , increasing the hoa as a total . </S>", "<S> cs was significantly better in artiflex group . </S>" ]

[ "Materials and Methods", "Patient selection and examination", "Surgical technique", "Outcome measures", "Statistical analysis", "Results", "Discussion", "Financial support and sponsorship", "Conflicts of interest" ]

[ [ "this is a nonrandomized prospective comparative case study on 57 eyes seeking refractive surgery who were not indicated for laser corneal refractive surgery .", "patients with refraction error between 2.00 d and 14.50 d who could use both artiflex and artisan , were free to choose the type of the lens . other patients underwent artisan implantation .", "ophthalmic examinations before surgery included cyclo - refraction , uncorrected visual acuity ( ucva ) , best - corrected visual acuity ( bcva ) , topography , keratometry , endothelial cell count , pupil diameter , and anterior chamber ( ac ) depth measurement . according to patient history and examination , patients who had the following conditions underwent piol implantation surgery : insufficient corneal thickness for refractive corneal surgery , inappropriate corneal curvature and topography , high myopia more than 8 d which could not undergo corneal refractive surgery , negative history of any previous refractive surgery , sufficient internal ac depth ( 3 mm or greater ) , no history of intraocular pressure raise and endothelial cell density of 2500 cell / mm or more .", "lens dioptric power was calculated by van der heijde formula , using refractive error , refractive cylinder power , ac depth , and topographically derived keratometry values .", "emmetropia was the target power of the surgery , when the precise emmetropic lens was not available our choice was to a slight residual myopia .", "all surgeries were performed under general anesthesia , for artisan lens implantation , a superior 5 or 6 mm length corneal incision was created at the 12 o'clock position , followed by performing two stab incisions located at 2 and 10 o'clock .", "the lens was inserted into the ac under protection of intracameral ophthalmic viscoelastic material injection .", "artiflex lens implantation was performed similarly , although the lens was inserted through a 3.2 mm incision .", "other steps were carried out same as artisan implantation ; finally , the wound was closed by stromal hydration without the necessity of suturing .", "one year after surgery , cyclo - refraction , bcva , ucva , cs , hoa and patient satisfaction were assessed .", "cs was determined by csv-1000 chart , and refraction was obtained with topcon auto refractometer and then modified by subjective refraction .", "night vision , day vision , halo , photophobia , dryness , pain , and patient satisfaction were assessed by questionnaire with numerical scales ( 0 = weak , 1 = moderate , 2 = good , 3 = excellent for day and night vision 0 = none , 1 = mild , 2 = moderate , 3 = severe for halo , photophobia , dryness , and pain ) .", "the chart consists of four rows corresponding to spatial frequencies of 3 , 6 , 12 , and 18 cycles per degree ( cpd ) .", "the distance between the patient and the chart was 2.5 m. the test was performed with the patients monocular bcva to eliminate the effects of sia and residual post - operative refraction .", "the aberrations analyzed were classified in terms of hoa , vertical trefoil , vertical coma ; horizontal trefoil , horizontal coma , quatrefoil , second astigmatism , and fourth order spherical aberration .", "comparison between artisan and artiflex data were analyzed using parametric paired student 's t - test , p < 0.05 was considered statistically significant ." ], [ "this is a nonrandomized prospective comparative case study on 57 eyes seeking refractive surgery who were not indicated for laser corneal refractive surgery .", "patients with refraction error between 2.00 d and 14.50 d who could use both artiflex and artisan , were free to choose the type of the lens . other patients underwent artisan implantation .", "ophthalmic examinations before surgery included cyclo - refraction , uncorrected visual acuity ( ucva ) , best - corrected visual acuity ( bcva ) , topography , keratometry , endothelial cell count , pupil diameter , and anterior chamber ( ac ) depth measurement . according to patient history and examination , patients who had the following conditions underwent piol implantation surgery : insufficient corneal thickness for refractive corneal surgery , inappropriate corneal curvature and topography , high myopia more than 8 d which could not undergo corneal refractive surgery , negative history of any previous refractive surgery , sufficient internal ac depth ( 3 mm or greater ) , no history of intraocular pressure raise and endothelial cell density of 2500 cell / mm or more ." ], [ "lens dioptric power was calculated by van der heijde formula , using refractive error , refractive cylinder power , ac depth , and topographically derived keratometry values .", "emmetropia was the target power of the surgery , when the precise emmetropic lens was not available our choice was to a slight residual myopia .", "all surgeries were performed under general anesthesia , for artisan lens implantation , a superior 5 or 6 mm length corneal incision was created at the 12 o'clock position , followed by performing two stab incisions located at 2 and 10 o'clock .", "the lens was inserted into the ac under protection of intracameral ophthalmic viscoelastic material injection .", "artiflex lens implantation was performed similarly , although the lens was inserted through a 3.2 mm incision .", "other steps were carried out same as artisan implantation ; finally , the wound was closed by stromal hydration without the necessity of suturing ." ], [ "one year after surgery , cyclo - refraction , bcva , ucva , cs , hoa and patient satisfaction were assessed .", "cs was determined by csv-1000 chart , and refraction was obtained with topcon auto refractometer and then modified by subjective refraction .", "night vision , day vision , halo , photophobia , dryness , pain , and patient satisfaction were assessed by questionnaire with numerical scales ( 0 = weak , 1 = moderate , 2 = good , 3 = excellent for day and night vision 0 = none , 1 = mild , 2 = moderate , 3 = severe for halo , photophobia , dryness , and pain ) .", "the chart consists of four rows corresponding to spatial frequencies of 3 , 6 , 12 , and 18 cycles per degree ( cpd ) .", "the distance between the patient and the chart was 2.5 m. the test was performed with the patients monocular bcva to eliminate the effects of sia and residual post - operative refraction .", "the aberrations analyzed were classified in terms of hoa , vertical trefoil , vertical coma ; horizontal trefoil , horizontal coma , quatrefoil , second astigmatism , and fourth order spherical aberration ." ], [ "comparison between artisan and artiflex data were analyzed using parametric paired student 's t - test , p < 0.05 was considered statistically significant ." ], [ "a total of 57 eyes were included in the study ; of which , 24 eyes were in artisan group and 33 eyes were in artiflex group .", "the preoperative spherical equivalent ( se ) and bcva were obtained and compared , no statistically significant difference was noticed in the mean preoperative se ( 10.39 8.43 d in artisan group and 10.39 2.29 d in artiflex group ; p = 0.999 ) and bcva of two groups ( 0.25 0.21 logarithm of the minimum angle of resolution [ logmar ] for artisan group and 0.19 0.18 logmar for artiflex group ; p = 0.56 ) .", "the mean postoperative se was 0.93 1.28 d in the artisan group and 0.54 0.82 in the artiflex group , no significant difference was seen in in postoperative se between two groups ( p = 0.19 ) .", "one year after the operation , 54% of artisan - treated eyes ( 15/24 ) and 75.7% ( 25/33 ) of artiflex treated eyes were within 1.00 d of intended emmetropia [ figs . 1 and 2 ] . in the artisan group , 37.5% of the treated eyes ( 9/24 eyes ) had three or more snellen line improvement of postoperative bcva , 12.5% ( 3/24 eyes ) had two snellen line improvement and 25% ( 6/24 eyes ) had loss of two or more snellen lines . in the artiflex group ,", "51.5% of the treated eyes ( 17/33 eyes ) had three or more snellen line improvement of bcva , 15.1% ( 5/33 eyes ) had two line improvement , and no eyes had loss of two or more snellen lines of bcva [ fig . 3 ] .", "the bar graph shows the postoperative defocus equivalent 1 year after artisan ( a ) and artiflex ( b ) implantation bar graph of change in best - corrected visual acuity from the preoperative examination to postoperative examination in terms of the number of snellen line change .", "artisan ( a ) , artiflex ( b ) mesopic contrast sensitivity at spatial frequencies of 3 , 6 , 12 and 18 degree per cycles in this study , total hoa in artisan - treated eyes was significantly greater than artiflex - treated eyes ( p = 0.044 ) with a mean hoa of 0.44 0.15 root mean square ( rms ) for artisan and 0.35 0.15 rms for artiflex .", "there was no significant difference in the vertical trefoil , vertical coma , horizontal trefoil , horizontal coma , quatrefoil , second astigmatism , and fourth order spherical aberration .", "the evaluation of cs in mesopic conditions showed higher cs in artiflex - treated eyes at three spatial frequencies 6 , 12 , and 18 cpd ( p = 0.003 , p = 0.007 , and p = 0.00 , respectively ) , and no significant difference was seen between two lenses at 3 cpd spatial frequency [ table 1 and fig .", "contrast sensitivity data after phakic intra - ocular lens implantation subjective comparison of day and night vision in artisan versus artiflex treated eyes the mean of sia was 1.65 0.82 d in artisan group , and 1.31 1.05 in artiflex group , which showed no statistically significant difference between the groups ( p = 0.19 ) . in a subjective evaluation of day vision", ", all patients of the artiflex group reported good and excellent vision , in artisan group , 71% of patients reported good and excellent vision , whereas 7.1% complained of weak day vision . in the assessment of night vision ,", "50% of patients treated with artiflex reported good and excellent vision and no one complained of weak night vision . in artisan group ,", "71.4% reported good and excellent vision , whereas 21.4% reported weak night vision [ fig .", "subjective comparison of halo , photophobia , pain and dryness in artisan versus artiflex treated eyes among artiflex treated eyes , 50% reported moderate halos and in the artisan group 40% reported moderate to severe halos . about 12.5% of artiflex treated eyes experienced moderate photophobia , whereas 35.7% of artisan treated eyes experienced moderate to severe photophobia . in the artiflex group ,", "only 12.5% of treated eyes caused mild pain , whereas 36.7% of artisan treated eyes caused mild to moderate pain .", "moderate to severe dryness was reported in 25% of eyes treated with artiflex , whereas no artisan treated eye experienced moderate or severe dryness [ fig . 6 ] .", "the bar graph of patient 's satisfaction in artisan versus artiflex groups satisfaction more than 60% was reported in 100% of patients in artiflex group versus 60% of patients in artisan group [ fig .", "the bar graph presents the postoperative spherical equivalent 1 year after artisan ( a ) and artiflex ( b ) implantation ." ], [ "as it is well - confirmed , the recently food and drug administration approved pmma artisan piol is an effective mean for the correction of refractive errors and have been shown to have acceptable safety and efficacy .", "however , studies have revealed instances of significant induced astigmatism , attributed to the larger incisions performed in the implantation of the artisan lens .", "artiflex iris - fixated piol , a foldable version of artisan , is an improvement of the iris - supported piol concept , with a lower incidence of sia .", "artiflex piol haptics are made of pmma and the foldable optical zone is made of silicon .", "the main purpose of this investigation is to evaluate the differences in optic quality between rigid piol versus flexible piol by comparing wavefront aberration , cs and patient satisfaction in patients who underwent artisan and artiflex iris - fixated iol implantation . in this study ,", "mean postoperative residual se were 0.93 1.28 d and 0.54 0.82 d in the artisan and artiflex group , respectively , no significant difference was seen in postoperative se between two groups ( p = 0.19 ) .", "about 54% of artisan group and 75.7% of artiflex group were within 1.00 d of intended emmetropia . in a previous study , 58% and 83.9% of artisan and artiflex group had a residual postoperative se within 1.00 d , which was significantly better in artiflex group .", "another study reported that emmetropia ( 1.00 d ) was obtained in 60% and 91.7% of artisan and artiflex group , respectively , the difference was statistically significant [ table 2 ] .", "percentage of postoperative refraction within1.00 d of intended emmetropia in a retrospective comparative case series on 27 eyes , postoperative se was within 0.5 d in 76.2% and 85.7% of artisan and artiflex treated eyes , respectively . in a study of evaluating the outcome of iris - claw piol in fifty high myopic eyes , postoperative se was evaluated , which showed that 38% and 68% of the eyes were in the range of 0.5 d and 1.00 d , respectively .", "mean postoperative ucva was 0.34 0.25 logmar in artisan group and 0.12 0.15 logmar in artiflex group .", "mean postoperative bcva was 0.21 0.13 and 0.06 0.08 logmar in the artisan and artiflex groups , respectively [ table 3 ] .", "the differences of ucva and bcva in two groups were statistically significant ( p = 0.00 ) . in another study ,", "postoperative bcva between artisan and artiflex groups was not significantly different ( p = 0.9 ) .", "postoperative visual acuity ( logarithm of the minimum angle of resolution ) in artisan group versus artiflex group in this study , although vertical trefoil , vertical coma , horizontal trefoil , horizontal coma , secondary astigmatism , quatrefoil and fourth order spherical aberration were not significantly different between the artiflex and artisan groups , total hoa was higher in artisan group , that may be due to the slight differences in each component , which totally increases the hoa ( p = 0.044 ) . in a previous study , assessment of hoas after implantation of rigid versus foldable", "iris - fixated lenses , showed a significant decrease in postoperative spherical aberration among artiflex group and a significant increase in postoperative spherical aberration in artisan group . in both groups ,", "vertical trefoil and spherical aberration were higher in artisan group , p = 0.039 and p = 0.001 , respectively .", "assessment of cs in mesopic condition showed no significant difference between two groups at 3 cpd spatial frequency .", "cs was significantly higher in artiflex - treated eyes at three spatial frequencies 6 , 12 and 18 cpd ( p = 0.003 , p = 0.007 , and p = 0.00 , respectively ) .", "a previous investigation showed that mesopic cs was slightly better in artiflex group in comparison with artisan group , but the difference was not statistically significant , they also measured photopic cs function and found that both groups had poorer performance compared to the normal range .", "performance with the artisan lens was slightly better than performance with artiflex under photopic conditions , but worse under mesopic conditions ; neither differences were statistically significant . in this study , we have used artisan iol for patients not eligible for the available artiflex ( six myopic eye more than 14.5 d and one hyperopic eye ) , comparison of this two groups may have some biases because of other probable conditions such as retinal involvement or amblyopia , which can alter the results , this was one of our study limitations .", "in this study , 25% of patients with artisan lens lost two or more lines of bcva , we called the patients back and examined them again carefully , we recognized that in one patient both eyes had refractive error postoperatively in spite of implanting a lens with the same power of his preoperative se improvement in bcva was not seen with correction of this refractive error by trial - frame . in the other patients , we could not find any reason for the decrease in the bcva , unless considering pigment deposition on the iol as a reason for the visual loss ." ], [ "" ], [ "" ] ]

[ "chronic lymphocytic leukemia ( cll ) is the most commonly found leukemia in the adult population of the western world and of clinical interest because of its prevalence .", "it is a neoplasm of monomorphic small round b lymphocytes which can be observed in peripheral blood , bone marrow , and/or lymph nodes .", "it is likely that cll has a multifactorial mode of inheritance with both genetic and environmental components .", "however , of all hematologic neoplasms , cll is reported to have the highest genetic predisposition and like many other hematologic malignancies , development of cll is found to be much higher in males than in females with a corresponding m / f sex ratio of at least 1.5 or higher .", "although the clinical ( phenotypic ) m / f sex ratio for cll has been well documented , the genetic ( genotypic ) m / f sex ratios associated with abnormal fluorescence in situ hybridization ( fish ) probes have not . for these reasons", ", cll represents an attractive cancer entity in which to evaluate what genetic impact , if any , gender may have on its development . genetic studies of sex ratio disparities in human neoplasms have been few , perhaps , because of the relative inaccessibility of investigational materials including appropriate databases which could be informative .", "it follows that the genetic basis for this phenomenon remains largely unknown and our understanding of it very limited .", "since our laboratory has collected clinical and laboratory data over many years on major categories of cancers including cll , review and analyses of these data presented an opportunity to examine certain aspects of this question . in order to better study the nature of the multifactorial components in cll , we determined the representation of genetic abnormalities detected by four defined fish probes , with respect to male and female cll patients . by this approach ,", "our study addressed not only what the m / f ratio is in cll patients having the clinical phenotype , but also the m / f ratio in patients who have a genotype which included specific fish abnormalities .", "these results provided a genetic basis for the notion that the fish abnormalities found underlie the phenotypic m / f sex ratio and also that they may be sex chromosomes ( x and/or y ) influenced .", "cll fish results in the database are derived from testing done on cytological preparations made from peripheral blood or bone marrow specimens submitted for cll evaluation .", "fish analyses were conducted using a fish panel of dna probes specific for the atm gene ( 11q22.3-q23.1 ) , chromosome 12 centromere ( d12z3 locus ) , the d13s319 locus ( 13q14.3 ) , and the tp53 gene ( 17p13.1 ) commercially available from vysis ( an abbott company ) , downers grove , il .", "the prognostic value and characterization of these dna probes have been reported in the literature .", "the technical protocols for fish testing ( hybridization of probes and detection of hybridization signals ) were those recommended by the manufacturer .", "microscopic studies were conducted by experienced technologists , then reviewed and interpreted by board - certified cytogeneticists .", "these fish test results were then archived in a computer - based database from which data were retrieved and statistically analyzed .", "data of fish results from all integrated oncology laboratories , a business unit of esoterix genetic laboratories , llc , using the same dna fish probes to evaluate cll patients were collected , reviewed , and statistically evaluated .", "the data contained entries of cll - fish panels performed during the period of 11/13/2005 through 10/20/2009 .", "these panels were specifically used for cll studies , and results were used only when the entire cll panel was applied in order to clearly identify only those patients having or suspected of having cll .", "also , cll panel probe results were those used only for the initial fish evaluation of newly diagnosed cll patient - and did not involve repeat studies on the same patient . this investigation was conducted in a fashion in which laboratory results had no patient - identity associations , in order to conform to privacy guidelines and patient confidentiality .", "this data was then imported into sas9.2 and formatted by sas data step and analyzed by sas procedures for one side binomial test and chi - square test .", "the one - side binomial tests were used to evaluate the sex ratio with respect to the abnormalities found for each of the probes used .", "the first category represented all abnormal panels having only a single abnormality per panel , for each probe .", "the second category included those having any abnormality with respect to a given probe whether it appeared as a single abnormality or in combination with other abnormalities .", "the binomial test took into account the frequency of an abnormality or abnormalities ( according to the categories describe above ) in a pool of data ( total number of normal and abnormal panels ) for each of the fish probes considered , and compared the results obtained for the male group with results obtained for the female group .", "essentially , the mean value for positive results ( all having the same abnormality + ) versus total results ( positive results plus normal having 0 abnormalities ) , was compared between males and females for each of the probe categories described above . the odds ratio ( or ) and 95% confidence interval ( ci ) values were derived from the one side binomial test while the p - values were determined by the chi - square tests .", "cll fish results in the database are derived from testing done on cytological preparations made from peripheral blood or bone marrow specimens submitted for cll evaluation .", "fish analyses were conducted using a fish panel of dna probes specific for the atm gene ( 11q22.3-q23.1 ) , chromosome 12 centromere ( d12z3 locus ) , the d13s319 locus ( 13q14.3 ) , and the tp53 gene ( 17p13.1 ) commercially available from vysis ( an abbott company ) , downers grove , il .", "the prognostic value and characterization of these dna probes have been reported in the literature .", "the technical protocols for fish testing ( hybridization of probes and detection of hybridization signals ) were those recommended by the manufacturer .", "microscopic studies were conducted by experienced technologists , then reviewed and interpreted by board - certified cytogeneticists .", "these fish test results were then archived in a computer - based database from which data were retrieved and statistically analyzed .", "data of fish results from all integrated oncology laboratories , a business unit of esoterix genetic laboratories , llc , using the same dna fish probes to evaluate cll patients were collected , reviewed , and statistically evaluated .", "the data contained entries of cll - fish panels performed during the period of 11/13/2005 through 10/20/2009 .", "these panels were specifically used for cll studies , and results were used only when the entire cll panel was applied in order to clearly identify only those patients having or suspected of having cll .", "also , cll panel probe results were those used only for the initial fish evaluation of newly diagnosed cll patient - and did not involve repeat studies on the same patient . this investigation was conducted in a fashion in which laboratory results had no patient - identity associations , in order to conform to privacy guidelines and patient confidentiality .", "this data was then imported into sas9.2 and formatted by sas data step and analyzed by sas procedures for one side binomial test and chi - square test .", "the one - side binomial tests were used to evaluate the sex ratio with respect to the abnormalities found for each of the probes used .", "the first category represented all abnormal panels having only a single abnormality per panel , for each probe .", "the second category included those having any abnormality with respect to a given probe whether it appeared as a single abnormality or in combination with other abnormalities .", "the binomial test took into account the frequency of an abnormality or abnormalities ( according to the categories describe above ) in a pool of data ( total number of normal and abnormal panels ) for each of the fish probes considered , and compared the results obtained for the male group with results obtained for the female group . essentially , the mean value for positive results ( all having the same abnormality + ) versus total results ( positive results plus normal having 0 abnormalities ) , was compared between males and females for each of the probe categories described above .", "the odds ratio ( or ) and 95% confidence interval ( ci ) values were derived from the one side binomial test while the p - values were determined by the chi - square tests .", "a characterization of the study population evaluated by the cll - fish panel is given in table 1 , and a summary of general observations are provided in table 2 .", "for example , table 2 shows that there were a total of 2773 ( 59.0% ) individual abnormal fish panels in the 4698 total fish panels applied to the cll patient study group . of the abnormal panels ,", "1711 were male and 1062 were female for an m / f sex ratio of 1.61 .", "other comparisons of the genders concerning data collected by this study are also reported in table 2 .", "table 3 shows the m / f ratios of fish results for the study population of cll patients in categories of abnormal results ( either as a single abnormality or as a single abnormality plus multiple abnormalities ) , normal results and total results ( normal and abnormal ) with respect to gender .", "there were 2035 results that appeared as only single abnormalities , and 3606 results that appeared as single plus other abnormalities combined ( artificially inflated since abnormalities were used multiple times ) . the most frequently found single abnormality was the deletion of 13q14.3 ( 1327 abnormal results : 65.2% ) , followed by trisomy 12 ( 472 abnormal : 23.2% ) , then deletion of 11q22.3 ( 138 abnormal : 6.8% ) , and least frequent was deletion of 17p13.1 ( 98 abnormal : 4.8% ) .", "the sex ratio values for the fish probes ranged from a low value of 1.39 to a high value of 2.54 . with the exception of the sex ratio found for the atp gene ( 2.54 ) , the rest of the sex ratios for fish abnormalities ranged from 1.39 to 1.54 , clustering around 1.5 . from table 3", ", it should be noted that the ab / nor ratio for each of the probes was not significantly different from each other except for the atm probe .", "this suggest that proportionality of abnormal results reflect the phenotypic representation of cll clinically in our study population except for abnormalities of the atm gene which is over - represented in male from that expected .", "figure 1 presents the distribution of patients ages in the cll study population in which the age range for males and females is very similar . at early ( less than 30 years ) and", "late age groups ( greater than 80 years ) the sex ratios between male and females do not appear to be significant .", "results from the chi - square tests and odds ratio analyses are presented in table 4 .", "of the four fish probes , only the deletion of the 11q22.3 ( atm gene ) demonstrated statistical significance in the chi - square test ( p - value of 0.0049 ) .", "the or value of 1.7045 is also significantly different from the or values found for the rest of the fish probes , indicating the male has 70.45% or 64.35% higher risk to have abnormal fish test results for the atm - fish probe .", "this was true for deletions of the atm gene when found as a sole abnormality ( or=1.7045 ) or in combination with other abnormalities which included the deletion of the atm gene ( or=1.6435 ) .", "both these two categories of single and multiple probe abnormalities strongly indicated that the deletion of the atm gene was disproportionately over - represented in male cll patients than in female cll patients , and that this difference could not be explained by chance alone .", "these data lead to the conclusion that this mutation was not only found in a higher percentage in males compared to females , but that it was distinct from the m / f sex ratio of ~1.5 found for the other fish probes [ trisomy 12 , del(13q14.3 ) , del(17p13.1 ) ]", ". the or values for the m / f sex ratios of trisomy 12 , deletion 13q , and deletion 17p ( tp53 gene ) fish probe abnormalities in either single or multiple combinations were not significantly different from 1.0 and could be explained by chance alone .", "therefore , the m / f sex ratio for each of the cll - fish probe abnormalities appeared to be about the same and not significantly different from an m / f sex ratio of 1.5 .", "although sex ratios in lymphoid neoplasms and other tumors show male predominance , there remains an incomplete understanding of these observations . our study makes this m / f ratio determination based on a study population of patients clinically diagnosed with , or suspected of having cll for whom fish studies were conducted .", "based on this clinical assignment , a ratio of 1.51 was found , which is congruent with previous studies , indicating similar values or higher .", "these observations suggest that cll may have a sex chromosomeinfluenced component determining its transmission . ", "we also examined the question of what the m / f ratio is in those cll patients who had fish abnormalities detected by specific genetic probes and found that 3 of the 4 probes had values close to 1.5 and that 1 probe ( atm gene ) had a significantly higher m / f ratio of 2.54 .", "the coincidence that , like the clinical m / f ratio , the genetic m / f ratio for 3 probes may simply suggest that in the cll study population which already has a disproportionately high number of male patients , the likelihood of detecting a genetic abnormality is equally high ( in males ) and that it may be sex chromosome influenced .", "however , for the atm - fish probe , the m / f ratio was significantly higher ( ~2.5 ) than the ~1.5 ratio found for the other probes and suggests that for this mutation , a special set of conditions necessary for the development of cll are more effectively enhanced in male than in female patients .", "m / f values vary depending on the type of cancer and the age of the patient", ". there are three possible categories of cancers ( hematologic and non - hematologic ) with respect to altered m / f ratios having values ranging from less than 0.50 to over 20.0 .", "generally , these categories include cancers in which : i ) males have a higher risk , ii ) females are more susceptible , and iii ) males and females are equally represented .", "however , these reported m / f values are phenotypic m / f values which do not take genetic aspects into account .", "our survey confirms that in our cll study population , as indicated in earlier reports the phenotypic m / f sex value is very close to 1.5 .", "m / f sex ratio corresponds to cancers in the first category in which common mechanisms of cancer development may exist .", "the purpose for using the fish data from the cll study population was to initiate a characterization of what the genotypic m / f sex ratio is for each of the fish probes used .", "surprisingly , very much like the phenotypic m / f ratio of ~1.5 found , our study demonstrated a genotypic m / f sex ratio higher than 1.0 ( 1.39 - 2.5 ) with respect to all cll fish probes .", "it is important to note that the genotypic m / f sex ratio of ~2.5 found for the deletion of the atm gene , is markedly skewed in males , and suggests a special mechanism which may involve atm gene functions which could include dna fidelity , homologous recombinational repair and chromosomal stability .", "losses and mutations of the atm gene have been demonstrated in a number of neoplasms including t - lymphocytic leukemia ( t - pll ) , b - cell chronic lymphocytic leukaemia ( b - cll ) and in mantle cell lymphoma ( mcl ) .", "these reports suggest that loss of a tumor suppressor gene [ or loss of heterozygosity ( loh ) ] may be one of many steps leading to a cancerous state in complex diseases with multifactorial contributions .", "gender dependent susceptibility to complex diseases could include polygenic mechanisms , epigenetic modulations , and sex - chromosome linked genes .", "two possible explanations can account for the skewed m / f ratios found in our study ( including the atm fish probe showing especially high level of deletions in male cll patients ) .", "explanation # 1 involves the non pseudoautosomal regions ( non - par ) of the x and y chromosome , and explanation # 2 which concerns the pseudoautosomal region ( par ) of the sex chromosomes .", "explanation 1 : the genotypic m / f sex ratio of ~1.5 was found for trisomy 12 , deletion of d13s319 at chromosome 13q14.3 , and deletion of the tp53 gene .", "these hypothetical x - chromosome linked genes appear as a single dose in male cells ( hemizygous ) and as two ( non - par , and not x - chromosome inactivated ) doses in female cells . if there is a predisposing mutation for any of the three fish autosomal markers in either males or females , the cll - x gene product will continue normal function with no cll development .", "however , if the cll - x gene is subsequently mutated in males ( hemizygous ) , gene function of the cll - gene markers would also be impaired resulting in cll . however , in females ( heterozygous ) the second non - mutated cll - x gene salvages the gene dosage requirement with no development of cll .", "mutations of both cll - x genes in females would be necessary for subsequent cll development .", "this explanation is consistent with the observation that cll typically has an adult age onset , that relatively fewer females succumb to cll given the salvaging effect of the double doses of the xlinked cll genes they possess , and offers a plausible biological / genetic basis for the ~1.5 sex ratio found for these three important cll genetic markers . since atm loss is considerably higher in males than females , with a genotypic sex ratio of ~2.5 , additional factors are implicated .", "other highly skewed phenotypic m / f sex ratios reported in hematological , nonhematological cancers , and in certain solid tumors , can be as high as 28.7 such as for kaposi sarcoma .", "for these entities it may be that mutations are not necessarily disabled by 100% loss of function . in males after the first atm mutation , a partially functioning cll - x gene might contribute to the loss of the 2 atm gene .", "however , in females the two partially functioning cll - x genes may be result in sufficient dna repair to prevent atm loss at a higher level than that found in males .", "this secondary salvage pathway would provide for a lower rate of atm gene loss and may represent a relative protective mechanism favoring females .", "there may also be other models that could explain gender ratio distortions involving multiple dna - repair related gene families . ", "our hypothesis of x - linked dna repair - related genes is supported by recent mapping of dna repair genes ( which include the ape1 , cetn2 , trex2 , ube2a , and rpa4 genes ) on the x chromosome .", "explanation 2 : the par is homologous in the human x and y chromosomes where they pair during meiosis , and where there are 2 doses for every gene found in this region .", "compelling evidence shows that the par plays a role in the m / f ratio found in mantle cell lymphoma ( mcl ) which may also apply to cll .", "the study demonstrated male predominance in mantle cell lymphoma ( mcl ) with loss of the y chromosome and homozygous deletions within the par .", "in all except 3 of the 21 mcl cases studied , the loss of the y chromosome was demonstrated .", "furthermore , 2 of the 3 cases ( which showed no y chromosome loss ) had biallelic losses of par1 in xp / yp , and of 16 cases in which there was y chromosome loss , an additional case showed biallelic loss of par1 .", "the genotypic 1.5 m / f ratio for the 3 probes used in our study could also be ex plained by mutations / losses of the par region of the x and y chromosomes .", "a similar linkage to the par has been proposed for hodgkin s lymphoma in which the risk for brothers of affected males or sisters of affected females was higher than the risk for siblings of the opposite gender . also supporting", "this explanation is that chemokines and chemokine receptors may play a role in b - cell malignancies , and that related cytokine - receptor genes map to the par of sex chromosomes .", "increasingly , it appears that gender plays a major role in defining not only the identity and nature of some neoplasms but also the mechanisms involved in their origin and progression ." ]

[ "<S> distorted sex ratios occur in hematologic disorders . </S>", "<S> for example , chronic lymphocytic leukemia ( cll ) displays disproportionate sex ratios with a large male excess . </S>", "<S> however , the underlying genetics for these disparities are poorly understood , and gender differences for specific cytogenetic abnormalities have not been carefully investigated . </S>", "<S> we sought to provide an initial characterization of gender representation in genetic abnormalities in cll by using fluorescence in situ hybridization ( fish ) . </S>", "<S> we confirm the well known skewed male - tofemale ( m / f sex ratio ) of ~1.5 in our cll study population , but also determine the genotypic m / f sex ratio values corresponding to specific fish dna probes . genetic changes in cll detectable by four fish probes were statistically compared with respect to gender . </S>", "<S> initial fish evaluations of 4698 cll patients were retrospectively examined and new findings of the genotypic m / f sex ratios for these probes are reported . </S>", "<S> this study represents the largest cll survey conducted in the united states using fish probes . </S>", "<S> the cll database demonstrated that fish abnormalities ( trisomy 12 , 13q14.3 deletion and 17p13.1 deletion ) probes had skewed m / f ratios of ~1.5 . </S>", "<S> also , by statistical analysis it was shown that atm gene loss ( 11q22.3q23.1 deletion ) solely or with other abnormalities was considerably higher in males with an m / f ratio of 2.5 and significantly different from m / f ratios of 1.0 or 1.5 . </S>", "<S> we hypothesize that interactions involving these autosomal abnormalities ( trisomy 12 , and deletions of 11q22.3 , 13q14.3 , and 17p13.1 ) , and the sex chromosomes may provide the genetic basis for the altered phenotypic m / f ratio in cll . </S>" ]

[ "Introduction", "Materials and Methods", "Chronic lymphocytic leukemia-fluorescence in situ hybridization panel", "Database", "Statistical analyses", "Results", "Discussion" ]

[ [ "chronic lymphocytic leukemia ( cll ) is the most commonly found leukemia in the adult population of the western world and of clinical interest because of its prevalence .", "it is a neoplasm of monomorphic small round b lymphocytes which can be observed in peripheral blood , bone marrow , and/or lymph nodes .", "it is likely that cll has a multifactorial mode of inheritance with both genetic and environmental components .", "however , of all hematologic neoplasms , cll is reported to have the highest genetic predisposition and like many other hematologic malignancies , development of cll is found to be much higher in males than in females with a corresponding m / f sex ratio of at least 1.5 or higher .", "although the clinical ( phenotypic ) m / f sex ratio for cll has been well documented , the genetic ( genotypic ) m / f sex ratios associated with abnormal fluorescence in situ hybridization ( fish ) probes have not . for these reasons", ", cll represents an attractive cancer entity in which to evaluate what genetic impact , if any , gender may have on its development . genetic studies of sex ratio disparities in human neoplasms have been few , perhaps , because of the relative inaccessibility of investigational materials including appropriate databases which could be informative .", "it follows that the genetic basis for this phenomenon remains largely unknown and our understanding of it very limited .", "since our laboratory has collected clinical and laboratory data over many years on major categories of cancers including cll , review and analyses of these data presented an opportunity to examine certain aspects of this question . in order to better study the nature of the multifactorial components in cll , we determined the representation of genetic abnormalities detected by four defined fish probes , with respect to male and female cll patients . by this approach ,", "our study addressed not only what the m / f ratio is in cll patients having the clinical phenotype , but also the m / f ratio in patients who have a genotype which included specific fish abnormalities .", "these results provided a genetic basis for the notion that the fish abnormalities found underlie the phenotypic m / f sex ratio and also that they may be sex chromosomes ( x and/or y ) influenced ." ], [ "cll fish results in the database are derived from testing done on cytological preparations made from peripheral blood or bone marrow specimens submitted for cll evaluation .", "fish analyses were conducted using a fish panel of dna probes specific for the atm gene ( 11q22.3-q23.1 ) , chromosome 12 centromere ( d12z3 locus ) , the d13s319 locus ( 13q14.3 ) , and the tp53 gene ( 17p13.1 ) commercially available from vysis ( an abbott company ) , downers grove , il .", "the prognostic value and characterization of these dna probes have been reported in the literature .", "the technical protocols for fish testing ( hybridization of probes and detection of hybridization signals ) were those recommended by the manufacturer .", "microscopic studies were conducted by experienced technologists , then reviewed and interpreted by board - certified cytogeneticists .", "these fish test results were then archived in a computer - based database from which data were retrieved and statistically analyzed .", "data of fish results from all integrated oncology laboratories , a business unit of esoterix genetic laboratories , llc , using the same dna fish probes to evaluate cll patients were collected , reviewed , and statistically evaluated .", "the data contained entries of cll - fish panels performed during the period of 11/13/2005 through 10/20/2009 .", "these panels were specifically used for cll studies , and results were used only when the entire cll panel was applied in order to clearly identify only those patients having or suspected of having cll .", "also , cll panel probe results were those used only for the initial fish evaluation of newly diagnosed cll patient - and did not involve repeat studies on the same patient . this investigation was conducted in a fashion in which laboratory results had no patient - identity associations , in order to conform to privacy guidelines and patient confidentiality .", "this data was then imported into sas9.2 and formatted by sas data step and analyzed by sas procedures for one side binomial test and chi - square test .", "the one - side binomial tests were used to evaluate the sex ratio with respect to the abnormalities found for each of the probes used .", "the first category represented all abnormal panels having only a single abnormality per panel , for each probe .", "the second category included those having any abnormality with respect to a given probe whether it appeared as a single abnormality or in combination with other abnormalities .", "the binomial test took into account the frequency of an abnormality or abnormalities ( according to the categories describe above ) in a pool of data ( total number of normal and abnormal panels ) for each of the fish probes considered , and compared the results obtained for the male group with results obtained for the female group .", "essentially , the mean value for positive results ( all having the same abnormality + ) versus total results ( positive results plus normal having 0 abnormalities ) , was compared between males and females for each of the probe categories described above . the odds ratio ( or ) and 95% confidence interval ( ci ) values were derived from the one side binomial test while the p - values were determined by the chi - square tests ." ], [ "cll fish results in the database are derived from testing done on cytological preparations made from peripheral blood or bone marrow specimens submitted for cll evaluation .", "fish analyses were conducted using a fish panel of dna probes specific for the atm gene ( 11q22.3-q23.1 ) , chromosome 12 centromere ( d12z3 locus ) , the d13s319 locus ( 13q14.3 ) , and the tp53 gene ( 17p13.1 ) commercially available from vysis ( an abbott company ) , downers grove , il .", "the prognostic value and characterization of these dna probes have been reported in the literature .", "the technical protocols for fish testing ( hybridization of probes and detection of hybridization signals ) were those recommended by the manufacturer .", "microscopic studies were conducted by experienced technologists , then reviewed and interpreted by board - certified cytogeneticists .", "these fish test results were then archived in a computer - based database from which data were retrieved and statistically analyzed ." ], [ "data of fish results from all integrated oncology laboratories , a business unit of esoterix genetic laboratories , llc , using the same dna fish probes to evaluate cll patients were collected , reviewed , and statistically evaluated .", "the data contained entries of cll - fish panels performed during the period of 11/13/2005 through 10/20/2009 .", "these panels were specifically used for cll studies , and results were used only when the entire cll panel was applied in order to clearly identify only those patients having or suspected of having cll .", "also , cll panel probe results were those used only for the initial fish evaluation of newly diagnosed cll patient - and did not involve repeat studies on the same patient . this investigation was conducted in a fashion in which laboratory results had no patient - identity associations , in order to conform to privacy guidelines and patient confidentiality ." ], [ "this data was then imported into sas9.2 and formatted by sas data step and analyzed by sas procedures for one side binomial test and chi - square test .", "the one - side binomial tests were used to evaluate the sex ratio with respect to the abnormalities found for each of the probes used .", "the first category represented all abnormal panels having only a single abnormality per panel , for each probe .", "the second category included those having any abnormality with respect to a given probe whether it appeared as a single abnormality or in combination with other abnormalities .", "the binomial test took into account the frequency of an abnormality or abnormalities ( according to the categories describe above ) in a pool of data ( total number of normal and abnormal panels ) for each of the fish probes considered , and compared the results obtained for the male group with results obtained for the female group . essentially , the mean value for positive results ( all having the same abnormality + ) versus total results ( positive results plus normal having 0 abnormalities ) , was compared between males and females for each of the probe categories described above .", "the odds ratio ( or ) and 95% confidence interval ( ci ) values were derived from the one side binomial test while the p - values were determined by the chi - square tests ." ], [ "a characterization of the study population evaluated by the cll - fish panel is given in table 1 , and a summary of general observations are provided in table 2 .", "for example , table 2 shows that there were a total of 2773 ( 59.0% ) individual abnormal fish panels in the 4698 total fish panels applied to the cll patient study group . of the abnormal panels ,", "1711 were male and 1062 were female for an m / f sex ratio of 1.61 .", "other comparisons of the genders concerning data collected by this study are also reported in table 2 .", "table 3 shows the m / f ratios of fish results for the study population of cll patients in categories of abnormal results ( either as a single abnormality or as a single abnormality plus multiple abnormalities ) , normal results and total results ( normal and abnormal ) with respect to gender .", "there were 2035 results that appeared as only single abnormalities , and 3606 results that appeared as single plus other abnormalities combined ( artificially inflated since abnormalities were used multiple times ) . the most frequently found single abnormality was the deletion of 13q14.3 ( 1327 abnormal results : 65.2% ) , followed by trisomy 12 ( 472 abnormal : 23.2% ) , then deletion of 11q22.3 ( 138 abnormal : 6.8% ) , and least frequent was deletion of 17p13.1 ( 98 abnormal : 4.8% ) .", "the sex ratio values for the fish probes ranged from a low value of 1.39 to a high value of 2.54 . with the exception of the sex ratio found for the atp gene ( 2.54 ) , the rest of the sex ratios for fish abnormalities ranged from 1.39 to 1.54 , clustering around 1.5 . from table 3", ", it should be noted that the ab / nor ratio for each of the probes was not significantly different from each other except for the atm probe .", "this suggest that proportionality of abnormal results reflect the phenotypic representation of cll clinically in our study population except for abnormalities of the atm gene which is over - represented in male from that expected .", "figure 1 presents the distribution of patients ages in the cll study population in which the age range for males and females is very similar . at early ( less than 30 years ) and", "late age groups ( greater than 80 years ) the sex ratios between male and females do not appear to be significant .", "results from the chi - square tests and odds ratio analyses are presented in table 4 .", "of the four fish probes , only the deletion of the 11q22.3 ( atm gene ) demonstrated statistical significance in the chi - square test ( p - value of 0.0049 ) .", "the or value of 1.7045 is also significantly different from the or values found for the rest of the fish probes , indicating the male has 70.45% or 64.35% higher risk to have abnormal fish test results for the atm - fish probe .", "this was true for deletions of the atm gene when found as a sole abnormality ( or=1.7045 ) or in combination with other abnormalities which included the deletion of the atm gene ( or=1.6435 ) .", "both these two categories of single and multiple probe abnormalities strongly indicated that the deletion of the atm gene was disproportionately over - represented in male cll patients than in female cll patients , and that this difference could not be explained by chance alone .", "these data lead to the conclusion that this mutation was not only found in a higher percentage in males compared to females , but that it was distinct from the m / f sex ratio of ~1.5 found for the other fish probes [ trisomy 12 , del(13q14.3 ) , del(17p13.1 ) ]", ". the or values for the m / f sex ratios of trisomy 12 , deletion 13q , and deletion 17p ( tp53 gene ) fish probe abnormalities in either single or multiple combinations were not significantly different from 1.0 and could be explained by chance alone .", "therefore , the m / f sex ratio for each of the cll - fish probe abnormalities appeared to be about the same and not significantly different from an m / f sex ratio of 1.5 ." ], [ "although sex ratios in lymphoid neoplasms and other tumors show male predominance , there remains an incomplete understanding of these observations . our study makes this m / f ratio determination based on a study population of patients clinically diagnosed with , or suspected of having cll for whom fish studies were conducted .", "based on this clinical assignment , a ratio of 1.51 was found , which is congruent with previous studies , indicating similar values or higher .", "these observations suggest that cll may have a sex chromosomeinfluenced component determining its transmission . ", "we also examined the question of what the m / f ratio is in those cll patients who had fish abnormalities detected by specific genetic probes and found that 3 of the 4 probes had values close to 1.5 and that 1 probe ( atm gene ) had a significantly higher m / f ratio of 2.54 .", "the coincidence that , like the clinical m / f ratio , the genetic m / f ratio for 3 probes may simply suggest that in the cll study population which already has a disproportionately high number of male patients , the likelihood of detecting a genetic abnormality is equally high ( in males ) and that it may be sex chromosome influenced .", "however , for the atm - fish probe , the m / f ratio was significantly higher ( ~2.5 ) than the ~1.5 ratio found for the other probes and suggests that for this mutation , a special set of conditions necessary for the development of cll are more effectively enhanced in male than in female patients .", "m / f values vary depending on the type of cancer and the age of the patient", ". there are three possible categories of cancers ( hematologic and non - hematologic ) with respect to altered m / f ratios having values ranging from less than 0.50 to over 20.0 .", "generally , these categories include cancers in which : i ) males have a higher risk , ii ) females are more susceptible , and iii ) males and females are equally represented .", "however , these reported m / f values are phenotypic m / f values which do not take genetic aspects into account .", "our survey confirms that in our cll study population , as indicated in earlier reports the phenotypic m / f sex value is very close to 1.5 .", "m / f sex ratio corresponds to cancers in the first category in which common mechanisms of cancer development may exist .", "the purpose for using the fish data from the cll study population was to initiate a characterization of what the genotypic m / f sex ratio is for each of the fish probes used .", "surprisingly , very much like the phenotypic m / f ratio of ~1.5 found , our study demonstrated a genotypic m / f sex ratio higher than 1.0 ( 1.39 - 2.5 ) with respect to all cll fish probes .", "it is important to note that the genotypic m / f sex ratio of ~2.5 found for the deletion of the atm gene , is markedly skewed in males , and suggests a special mechanism which may involve atm gene functions which could include dna fidelity , homologous recombinational repair and chromosomal stability .", "losses and mutations of the atm gene have been demonstrated in a number of neoplasms including t - lymphocytic leukemia ( t - pll ) , b - cell chronic lymphocytic leukaemia ( b - cll ) and in mantle cell lymphoma ( mcl ) .", "these reports suggest that loss of a tumor suppressor gene [ or loss of heterozygosity ( loh ) ] may be one of many steps leading to a cancerous state in complex diseases with multifactorial contributions .", "gender dependent susceptibility to complex diseases could include polygenic mechanisms , epigenetic modulations , and sex - chromosome linked genes .", "two possible explanations can account for the skewed m / f ratios found in our study ( including the atm fish probe showing especially high level of deletions in male cll patients ) .", "explanation # 1 involves the non pseudoautosomal regions ( non - par ) of the x and y chromosome , and explanation # 2 which concerns the pseudoautosomal region ( par ) of the sex chromosomes .", "explanation 1 : the genotypic m / f sex ratio of ~1.5 was found for trisomy 12 , deletion of d13s319 at chromosome 13q14.3 , and deletion of the tp53 gene .", "these hypothetical x - chromosome linked genes appear as a single dose in male cells ( hemizygous ) and as two ( non - par , and not x - chromosome inactivated ) doses in female cells . if there is a predisposing mutation for any of the three fish autosomal markers in either males or females , the cll - x gene product will continue normal function with no cll development .", "however , if the cll - x gene is subsequently mutated in males ( hemizygous ) , gene function of the cll - gene markers would also be impaired resulting in cll . however , in females ( heterozygous ) the second non - mutated cll - x gene salvages the gene dosage requirement with no development of cll .", "mutations of both cll - x genes in females would be necessary for subsequent cll development .", "this explanation is consistent with the observation that cll typically has an adult age onset , that relatively fewer females succumb to cll given the salvaging effect of the double doses of the xlinked cll genes they possess , and offers a plausible biological / genetic basis for the ~1.5 sex ratio found for these three important cll genetic markers . since atm loss is considerably higher in males than females , with a genotypic sex ratio of ~2.5 , additional factors are implicated .", "other highly skewed phenotypic m / f sex ratios reported in hematological , nonhematological cancers , and in certain solid tumors , can be as high as 28.7 such as for kaposi sarcoma .", "for these entities it may be that mutations are not necessarily disabled by 100% loss of function . in males after the first atm mutation , a partially functioning cll - x gene might contribute to the loss of the 2 atm gene .", "however , in females the two partially functioning cll - x genes may be result in sufficient dna repair to prevent atm loss at a higher level than that found in males .", "this secondary salvage pathway would provide for a lower rate of atm gene loss and may represent a relative protective mechanism favoring females .", "there may also be other models that could explain gender ratio distortions involving multiple dna - repair related gene families . ", "our hypothesis of x - linked dna repair - related genes is supported by recent mapping of dna repair genes ( which include the ape1 , cetn2 , trex2 , ube2a , and rpa4 genes ) on the x chromosome .", "explanation 2 : the par is homologous in the human x and y chromosomes where they pair during meiosis , and where there are 2 doses for every gene found in this region .", "compelling evidence shows that the par plays a role in the m / f ratio found in mantle cell lymphoma ( mcl ) which may also apply to cll .", "the study demonstrated male predominance in mantle cell lymphoma ( mcl ) with loss of the y chromosome and homozygous deletions within the par .", "in all except 3 of the 21 mcl cases studied , the loss of the y chromosome was demonstrated .", "furthermore , 2 of the 3 cases ( which showed no y chromosome loss ) had biallelic losses of par1 in xp / yp , and of 16 cases in which there was y chromosome loss , an additional case showed biallelic loss of par1 .", "the genotypic 1.5 m / f ratio for the 3 probes used in our study could also be ex plained by mutations / losses of the par region of the x and y chromosomes .", "a similar linkage to the par has been proposed for hodgkin s lymphoma in which the risk for brothers of affected males or sisters of affected females was higher than the risk for siblings of the opposite gender . also supporting", "this explanation is that chemokines and chemokine receptors may play a role in b - cell malignancies , and that related cytokine - receptor genes map to the par of sex chromosomes .", "increasingly , it appears that gender plays a major role in defining not only the identity and nature of some neoplasms but also the mechanisms involved in their origin and progression ." ] ]

[ "number of diseases comes under the category of silent killers as they gradually consume one without causing any serious symptoms in the early stages .", "it is defined as a localized resorptive process that commences on the surface of the root below the epithelial attachment and the coronal aspect of the supporting alveolar process , namely the zone of the connective tissue attachment .", "there is a homeostatic balance of pulp and periodontal ligament preventing clastic insults , which is due to an intact cementoblast and odontoblast cell layer .", "any breach in these layers may down - regulate osteoprotegrin and up - regulate receptor activator of nuclear factor ligand causing resorption .", "the severance of the protective layer may be commonly due to orthodontic treatment , dental trauma , or bleaching .", "a study has also revealed that an interleukin-1 polymorphism significantly increases the risk external resorption .", "heithersay classified icr into four classes according to the extent and severity of the lesion within the tooth .", "class 1 - 3 has a better prognosis , while class 4 is not amenable for treatment and might require extraction and replacement with implant retained crown restoration .", "the lesion classically presents as an asymmetrical radiolucency with ragged or irregular margins in the cervical region of the tooth .", "early lesions might be radiolucent ; however , more advanced lesions might have a mottled appearance caused by the osseous nature of the advanced lesions .", "unless proper treatment is initiated , the resorption continues unabated leading to irreversible destruction of tooth structure .", "early diagnosis , elimination of the resorption and restorative management are the keys to a successful outcome .", "this article discusses an icr managed by endodontic treatment , surgical management of resorptive defect by reverse sandwich technique and periodontal regeneration using platelet rich fibrin ( prf ) , prf membrane and bone graft .", "a 35-year - old non - smoking male patient presented for consultation at the endodontic department with a chief complaint of swelling and pus discharge in the upper left canine [ figures 1a and 2a ] .", "after performing sensitivity tests , tooth # 11 was diagnosed as having pulpal necrosis . clinical examination revealed localized gingival recession and gingival enlargement in relation to tooth # 11 .", "probing pocket depth on the mesial surface of the tooth # 11 was 10 mm with a high bleeding score .", "based on the clinical and radiological finding , we came to the diagnosis of localized chronic periodontitis and pulpal necrosis with external inflammatory root resorption .", "the treatment plan was informed to the patient , which included patient motivation , oral hygiene reinforcement , scaling and root planning , endodontic procedure and lastly surgical intervention .", "patient and his parents were informed of possible discomforts and potential risks concerning the planned treatment procedure and prognosis .", "( a ) pre - operative clinical photograph showing swelling and pus discharge in the upper left canine .", "( b ) surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication .", "( c ) the resorptive defects were restored with microfilled composite to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement .", "( d ) the bony defect was filled with freeze - dried bone allograft and platelet rich fibrin .", "( f ) follow - up after 2 years shows normal probing depth , no gingival recession , and no loss of clinical attachment loss ( a ) pre - operative intraoral periapical showing bone loss in the mesial aspect of upper left canine with two resorptive regions .", "( d ) at 2 years follow - up showing significant bone fill in phase one therapy oral prophylaxis was performed .", "endodontic access cavity was prepared after placing a rubber dam and disinfecting the area with 2% of chlorhexidine digluconate ( calypso , septodont , india ) .", "working length was determined by using the electronic apex locator root zx ( j. morita mfg .", "corporation , kyoto , japan ) and file in - radiograph with 15 number k - files .", "the root canal was cleaned and shaped by rotary ni - ti protaper system along with glyde ( dentsply maillefer company , usa ) using the crown down technique .", "the root canal was copiously irrigated with 2.5% sodium hypochlorite ( novo dental product , india ) .", "access cavity was temporized with calcium hydroxide and temporary endodontic restorative material ( term ) .", "root canal was irrigated again with normal saline and dried using paper points . before obturation , master points were seated to test their suitability to canals and radiograph was taken .", "the canals were obturated with selected master gutta - percha cone ( variable taper ) and ah - plus sealer ( dentsply maillefer company , usa ) .", "the coronal gutta - percha cones were sheared off using heated instrument and vertical compaction was done using the heated pluggers at the canal orifices .", "the access cavity was restored with a composite ( clearfil majesty ; kuraray , osaka , japan ) .", "the surgical procedure was performed under local infiltration anesthesia ( 2% of lidocaine with 1:100,000 adrenaline ) on vestibular mucosa .", "an intrasulcular incision was made from the distal surface of the maxillary left central incisor to the distal surface of the maxillary left canine and full - thickness mucoperiosteal flap was elevated .", "surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication [ figure 1b ] .", "granulomatous tissue within the defect area was removed and 90% of trichloroacetic acid was applied to the resorptive defect for coagulation necrosis .", "the resorptive defects were restored with microfilled composite ( durafill vs ; heraeus kulzer gmbh , dormagen , germany ) to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement ( rmgic ) ( fuji ii lc ; gc corporation , tokyo , japan ) to a smooth finish with a cervical matrix and light cured for 20 s [ figure 1c ] .", "a test dose of 12 ml sample of whole blood was drawn intravenously from the patient 's right antecubital vein and centrifuged ( remi model r-8c with 12 ml 15 ml swing out head ) under 3000 rpm for 10 min to obtain the prf , which was jelly like in consistency .", "prf clot started to release its serum ( prf - clot exudates ) and was ready for compression into the membrane .", "the bony defect was filled with freeze - dried bone allograft ( lifenet , virginia beach , va ) and prf [ figure 1d ] . without delay autologous prf membrane", "was placed [ figure 1e ] and the flaps were secured with 4 - 0 polyglactin 910 sutures ( vicryl , ethicon , inc . , piscataway , nj ) .", "after the surgery , the patient was prescribed amoxicillin 1 g twice a day for 1 week and 0.2% of chlorhexidine mouthwash 15 ml twice a day for 2 weeks .", "the desired gingival contour was achieved ; the patient was asymptomatic 1 week after the surgery and the sutures were removed .", "patient was followed - up for 6 months , [ figure 2b ] 1 year [ figure 2c ] and 2 year . at 2-year follow - up period ,", "periodontal status of related tooth demonstrated mild mobility with normal probing depth , no gingival recession and no loss of clinical attachment [ figures 1f and 2d ] .", "endodontic access cavity was prepared after placing a rubber dam and disinfecting the area with 2% of chlorhexidine digluconate ( calypso , septodont , india ) .", "working length was determined by using the electronic apex locator root zx ( j. morita mfg .", "corporation , kyoto , japan ) and file in - radiograph with 15 number k - files .", "the root canal was cleaned and shaped by rotary ni - ti protaper system along with glyde ( dentsply maillefer company , usa ) using the crown down technique .", "the root canal was copiously irrigated with 2.5% sodium hypochlorite ( novo dental product , india ) .", "access cavity was temporized with calcium hydroxide and temporary endodontic restorative material ( term ) .", "root canal was irrigated again with normal saline and dried using paper points . before obturation , master points were seated to test their suitability to canals and radiograph was taken .", "the canals were obturated with selected master gutta - percha cone ( variable taper ) and ah - plus sealer ( dentsply maillefer company , usa ) .", "the coronal gutta - percha cones were sheared off using heated instrument and vertical compaction was done using the heated pluggers at the canal orifices .", "the access cavity was restored with a composite ( clearfil majesty ; kuraray , osaka , japan ) .", "the surgical procedure was performed under local infiltration anesthesia ( 2% of lidocaine with 1:100,000 adrenaline ) on vestibular mucosa .", "an intrasulcular incision was made from the distal surface of the maxillary left central incisor to the distal surface of the maxillary left canine and full - thickness mucoperiosteal flap was elevated .", "surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication [ figure 1b ] .", "granulomatous tissue within the defect area was removed and 90% of trichloroacetic acid was applied to the resorptive defect for coagulation necrosis .", "the resorptive defects were restored with microfilled composite ( durafill vs ; heraeus kulzer gmbh , dormagen , germany ) to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement ( rmgic ) ( fuji ii lc ; gc corporation , tokyo , japan ) to a smooth finish with a cervical matrix and light cured for 20 s [ figure 1c ] .", "a test dose of 12 ml sample of whole blood was drawn intravenously from the patient 's right antecubital vein and centrifuged ( remi model r-8c with 12 ml 15 ml swing out head ) under 3000 rpm for 10 min to obtain the prf , which was jelly like in consistency .", "prf clot started to release its serum ( prf - clot exudates ) and was ready for compression into the membrane .", "the bony defect was filled with freeze - dried bone allograft ( lifenet , virginia beach , va ) and prf [ figure 1d ] . without delay autologous prf membrane", "was placed [ figure 1e ] and the flaps were secured with 4 - 0 polyglactin 910 sutures ( vicryl , ethicon , inc . ,", "after the surgery , the patient was prescribed amoxicillin 1 g twice a day for 1 week and 0.2% of chlorhexidine mouthwash 15 ml twice a day for 2 weeks .", "the desired gingival contour was achieved ; the patient was asymptomatic 1 week after the surgery and the sutures were removed .", "patient was followed - up for 6 months , [ figure 2b ] 1 year [ figure 2c ] and 2 year . at 2-year follow - up period ,", "periodontal status of related tooth demonstrated mild mobility with normal probing depth , no gingival recession and no loss of clinical attachment [ figures 1f and 2d ] .", "treatment depends on the severity , location , whether the defect has perforated the root canal system and the restorability of the tooth . as smaller lesions offer the most favorable long - term outcome , endodontic treatment followed by surgical treatment", "the true nature of the defect could be assessed only after surgical exploration . to treat the icr lesions ,", "it is usually necessary , to curette away the granulomatous tissue from the adjacent periodontium to sever the blood supply to the resorbing cells and fills the defect with an inert filling material .", "periodontal regeneration is the next aim , which includes reconstruction of lost or injured tissue so that the form and function of the lost structures are restored .", "the removal of granulomatous tissue the surgical defect was filled by the reverse sandwich technique microfilled composite as a liner followed by lamination with rmgic .", "microfilled resin was used because they tend to flex with the tooth rather than debond and rmgic was biocompatible to periodontal tissues .", "the development of subgingival plaque over rmgic is prevented by achieving a smooth finish using the cervical matrix .", "the regenerative process is a complex biologic process in itself , requiring intricately regulated interaction between cells , signaling molecules ( local and systemic ) and extra cellular matrices in which these entities interact .", "prf is a scaffold that not only acts as carriers for growth factors and proteins , but also allow cellular infiltration and subsequent integration of the newly formed tissue within the native one .", "it is biocompatible , non - cytotoxic and non - immunogenic to prevent adverse effects on recruited cells and neighboring tissue .", "this scaffold material along with growth factors and cells pave the way to accelerate bone and periodontal regeneration .", "prf can up - regulate phosphorylated extracellular signal - regulated protein kinase expression and suppress osteoclastogenesis by promoting the secretion of osteoprotegerin in osteoblasts cultures .", "prf membrane has a very significant slow sustained release of key growth factors for at least 1 week and up to 28 days , which means that the membrane stimulates its environment for a significant time during wound healing .", "hydroxyapetite ( ha ) was added to prf in our study , this was because it has been demonstrated that the treatment of intrabony defects with prf results in significant improvements of pocket depth , clinical attachment level and bone fill compared with baseline and secondly , that ha increases the clinical effects observed with prf in the treatment periapical defects .", "significant bone fill was obtained in our case after a 2-year follow - up period ; thanks to the synergistic combination of prf and ha ." ]

[ "<S> invasive cervical resorption ( icr ) is a localized resorptive process that commences on the surface of the root below the epithelial attachment and the coronal aspect of the supporting alveolar process , namely the zone of the connective tissue attachment early diagnosis , elimination of the resorption and restorative management are the keys to a successful outcome . </S>", "<S> treatment done was a combined non - surgical root canal therapy , surgical treatment to expose the resorptive defect and the resorptive defect was filled up with reverse sandwich technique and finally the bony defect filled with platelet rich fibrin ( prf ) , hydroxylapatite and prf membrane . </S>", "<S> significant bone fill was obtained in our case after a 2 year follow - up period . </S>", "<S> this case report presents a treatment strategy that might improve the healing outcomes for patients with icr . </S>" ]

[ "Introduction", "Case Report", "Treatment procedure", "Preparation of PRF membrane", "Discussion" ]

[ [ "number of diseases comes under the category of silent killers as they gradually consume one without causing any serious symptoms in the early stages .", "it is defined as a localized resorptive process that commences on the surface of the root below the epithelial attachment and the coronal aspect of the supporting alveolar process , namely the zone of the connective tissue attachment .", "there is a homeostatic balance of pulp and periodontal ligament preventing clastic insults , which is due to an intact cementoblast and odontoblast cell layer .", "any breach in these layers may down - regulate osteoprotegrin and up - regulate receptor activator of nuclear factor ligand causing resorption .", "the severance of the protective layer may be commonly due to orthodontic treatment , dental trauma , or bleaching .", "a study has also revealed that an interleukin-1 polymorphism significantly increases the risk external resorption .", "heithersay classified icr into four classes according to the extent and severity of the lesion within the tooth .", "class 1 - 3 has a better prognosis , while class 4 is not amenable for treatment and might require extraction and replacement with implant retained crown restoration .", "the lesion classically presents as an asymmetrical radiolucency with ragged or irregular margins in the cervical region of the tooth .", "early lesions might be radiolucent ; however , more advanced lesions might have a mottled appearance caused by the osseous nature of the advanced lesions .", "unless proper treatment is initiated , the resorption continues unabated leading to irreversible destruction of tooth structure .", "early diagnosis , elimination of the resorption and restorative management are the keys to a successful outcome .", "this article discusses an icr managed by endodontic treatment , surgical management of resorptive defect by reverse sandwich technique and periodontal regeneration using platelet rich fibrin ( prf ) , prf membrane and bone graft ." ], [ "a 35-year - old non - smoking male patient presented for consultation at the endodontic department with a chief complaint of swelling and pus discharge in the upper left canine [ figures 1a and 2a ] .", "after performing sensitivity tests , tooth # 11 was diagnosed as having pulpal necrosis . clinical examination revealed localized gingival recession and gingival enlargement in relation to tooth # 11 .", "probing pocket depth on the mesial surface of the tooth # 11 was 10 mm with a high bleeding score .", "based on the clinical and radiological finding , we came to the diagnosis of localized chronic periodontitis and pulpal necrosis with external inflammatory root resorption .", "the treatment plan was informed to the patient , which included patient motivation , oral hygiene reinforcement , scaling and root planning , endodontic procedure and lastly surgical intervention .", "patient and his parents were informed of possible discomforts and potential risks concerning the planned treatment procedure and prognosis .", "( a ) pre - operative clinical photograph showing swelling and pus discharge in the upper left canine .", "( b ) surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication .", "( c ) the resorptive defects were restored with microfilled composite to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement .", "( d ) the bony defect was filled with freeze - dried bone allograft and platelet rich fibrin .", "( f ) follow - up after 2 years shows normal probing depth , no gingival recession , and no loss of clinical attachment loss ( a ) pre - operative intraoral periapical showing bone loss in the mesial aspect of upper left canine with two resorptive regions .", "( d ) at 2 years follow - up showing significant bone fill in phase one therapy oral prophylaxis was performed .", "endodontic access cavity was prepared after placing a rubber dam and disinfecting the area with 2% of chlorhexidine digluconate ( calypso , septodont , india ) .", "working length was determined by using the electronic apex locator root zx ( j. morita mfg .", "corporation , kyoto , japan ) and file in - radiograph with 15 number k - files .", "the root canal was cleaned and shaped by rotary ni - ti protaper system along with glyde ( dentsply maillefer company , usa ) using the crown down technique .", "the root canal was copiously irrigated with 2.5% sodium hypochlorite ( novo dental product , india ) .", "access cavity was temporized with calcium hydroxide and temporary endodontic restorative material ( term ) .", "root canal was irrigated again with normal saline and dried using paper points . before obturation , master points were seated to test their suitability to canals and radiograph was taken .", "the canals were obturated with selected master gutta - percha cone ( variable taper ) and ah - plus sealer ( dentsply maillefer company , usa ) .", "the coronal gutta - percha cones were sheared off using heated instrument and vertical compaction was done using the heated pluggers at the canal orifices .", "the access cavity was restored with a composite ( clearfil majesty ; kuraray , osaka , japan ) .", "the surgical procedure was performed under local infiltration anesthesia ( 2% of lidocaine with 1:100,000 adrenaline ) on vestibular mucosa .", "an intrasulcular incision was made from the distal surface of the maxillary left central incisor to the distal surface of the maxillary left canine and full - thickness mucoperiosteal flap was elevated .", "surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication [ figure 1b ] .", "granulomatous tissue within the defect area was removed and 90% of trichloroacetic acid was applied to the resorptive defect for coagulation necrosis .", "the resorptive defects were restored with microfilled composite ( durafill vs ; heraeus kulzer gmbh , dormagen , germany ) to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement ( rmgic ) ( fuji ii lc ; gc corporation , tokyo , japan ) to a smooth finish with a cervical matrix and light cured for 20 s [ figure 1c ] .", "a test dose of 12 ml sample of whole blood was drawn intravenously from the patient 's right antecubital vein and centrifuged ( remi model r-8c with 12 ml 15 ml swing out head ) under 3000 rpm for 10 min to obtain the prf , which was jelly like in consistency .", "prf clot started to release its serum ( prf - clot exudates ) and was ready for compression into the membrane .", "the bony defect was filled with freeze - dried bone allograft ( lifenet , virginia beach , va ) and prf [ figure 1d ] . without delay autologous prf membrane", "was placed [ figure 1e ] and the flaps were secured with 4 - 0 polyglactin 910 sutures ( vicryl , ethicon , inc . , piscataway , nj ) .", "after the surgery , the patient was prescribed amoxicillin 1 g twice a day for 1 week and 0.2% of chlorhexidine mouthwash 15 ml twice a day for 2 weeks .", "the desired gingival contour was achieved ; the patient was asymptomatic 1 week after the surgery and the sutures were removed .", "patient was followed - up for 6 months , [ figure 2b ] 1 year [ figure 2c ] and 2 year . at 2-year follow - up period ,", "periodontal status of related tooth demonstrated mild mobility with normal probing depth , no gingival recession and no loss of clinical attachment [ figures 1f and 2d ] ." ], [ "endodontic access cavity was prepared after placing a rubber dam and disinfecting the area with 2% of chlorhexidine digluconate ( calypso , septodont , india ) .", "working length was determined by using the electronic apex locator root zx ( j. morita mfg .", "corporation , kyoto , japan ) and file in - radiograph with 15 number k - files .", "the root canal was cleaned and shaped by rotary ni - ti protaper system along with glyde ( dentsply maillefer company , usa ) using the crown down technique .", "the root canal was copiously irrigated with 2.5% sodium hypochlorite ( novo dental product , india ) .", "access cavity was temporized with calcium hydroxide and temporary endodontic restorative material ( term ) .", "root canal was irrigated again with normal saline and dried using paper points . before obturation , master points were seated to test their suitability to canals and radiograph was taken .", "the canals were obturated with selected master gutta - percha cone ( variable taper ) and ah - plus sealer ( dentsply maillefer company , usa ) .", "the coronal gutta - percha cones were sheared off using heated instrument and vertical compaction was done using the heated pluggers at the canal orifices .", "the access cavity was restored with a composite ( clearfil majesty ; kuraray , osaka , japan ) .", "the surgical procedure was performed under local infiltration anesthesia ( 2% of lidocaine with 1:100,000 adrenaline ) on vestibular mucosa .", "an intrasulcular incision was made from the distal surface of the maxillary left central incisor to the distal surface of the maxillary left canine and full - thickness mucoperiosteal flap was elevated .", "surgical exploration revealed that two lesions were present on the mesial surface of upper left canine and there was no palatal intercommunication [ figure 1b ] .", "granulomatous tissue within the defect area was removed and 90% of trichloroacetic acid was applied to the resorptive defect for coagulation necrosis .", "the resorptive defects were restored with microfilled composite ( durafill vs ; heraeus kulzer gmbh , dormagen , germany ) to a thickness of about 1 mm and light cured for 20 s. the rest of the defects were restored with resin modified glass ionomer cement ( rmgic ) ( fuji ii lc ; gc corporation , tokyo , japan ) to a smooth finish with a cervical matrix and light cured for 20 s [ figure 1c ] ." ], [ "a test dose of 12 ml sample of whole blood was drawn intravenously from the patient 's right antecubital vein and centrifuged ( remi model r-8c with 12 ml 15 ml swing out head ) under 3000 rpm for 10 min to obtain the prf , which was jelly like in consistency .", "prf clot started to release its serum ( prf - clot exudates ) and was ready for compression into the membrane .", "the bony defect was filled with freeze - dried bone allograft ( lifenet , virginia beach , va ) and prf [ figure 1d ] . without delay autologous prf membrane", "was placed [ figure 1e ] and the flaps were secured with 4 - 0 polyglactin 910 sutures ( vicryl , ethicon , inc . ,", "after the surgery , the patient was prescribed amoxicillin 1 g twice a day for 1 week and 0.2% of chlorhexidine mouthwash 15 ml twice a day for 2 weeks .", "the desired gingival contour was achieved ; the patient was asymptomatic 1 week after the surgery and the sutures were removed .", "patient was followed - up for 6 months , [ figure 2b ] 1 year [ figure 2c ] and 2 year . at 2-year follow - up period ,", "periodontal status of related tooth demonstrated mild mobility with normal probing depth , no gingival recession and no loss of clinical attachment [ figures 1f and 2d ] ." ], [ "treatment depends on the severity , location , whether the defect has perforated the root canal system and the restorability of the tooth . as smaller lesions offer the most favorable long - term outcome , endodontic treatment followed by surgical treatment", "the true nature of the defect could be assessed only after surgical exploration . to treat the icr lesions ,", "it is usually necessary , to curette away the granulomatous tissue from the adjacent periodontium to sever the blood supply to the resorbing cells and fills the defect with an inert filling material .", "periodontal regeneration is the next aim , which includes reconstruction of lost or injured tissue so that the form and function of the lost structures are restored .", "the removal of granulomatous tissue the surgical defect was filled by the reverse sandwich technique microfilled composite as a liner followed by lamination with rmgic .", "microfilled resin was used because they tend to flex with the tooth rather than debond and rmgic was biocompatible to periodontal tissues .", "the development of subgingival plaque over rmgic is prevented by achieving a smooth finish using the cervical matrix .", "the regenerative process is a complex biologic process in itself , requiring intricately regulated interaction between cells , signaling molecules ( local and systemic ) and extra cellular matrices in which these entities interact .", "prf is a scaffold that not only acts as carriers for growth factors and proteins , but also allow cellular infiltration and subsequent integration of the newly formed tissue within the native one .", "it is biocompatible , non - cytotoxic and non - immunogenic to prevent adverse effects on recruited cells and neighboring tissue .", "this scaffold material along with growth factors and cells pave the way to accelerate bone and periodontal regeneration .", "prf can up - regulate phosphorylated extracellular signal - regulated protein kinase expression and suppress osteoclastogenesis by promoting the secretion of osteoprotegerin in osteoblasts cultures .", "prf membrane has a very significant slow sustained release of key growth factors for at least 1 week and up to 28 days , which means that the membrane stimulates its environment for a significant time during wound healing .", "hydroxyapetite ( ha ) was added to prf in our study , this was because it has been demonstrated that the treatment of intrabony defects with prf results in significant improvements of pocket depth , clinical attachment level and bone fill compared with baseline and secondly , that ha increases the clinical effects observed with prf in the treatment periapical defects .", "significant bone fill was obtained in our case after a 2-year follow - up period ; thanks to the synergistic combination of prf and ha ." ] ]

[ "due to industrial development and the consequent increase in daily comfort , physical \n activities have gradually decreased , leading to increased body weight with reduced physical \n strength , as well as more sedentary lifestyles and significantly decreased amounts of \n exercise . due to incorrect exercise habits , even among those who exercise regularly , bad \n posture over extended periods , and inappropriate daily life and work habits ,", "the resulting continuous back tension has increased the \n number of patients with low back pain1 .", "mennell2 presented the term joint \n dysfunction for arthrokinematic dysfunction in the absence of pathological changes in the \n joints , including capsules and ligaments .", "he also attributed muscular pain and muscle spasm \n to difficulties with normal arthrokinematic mobilization in joint capsules , which limit \n joint movement when patients attempted to move joints suffering from the symptoms of joint \n dysfunction .", "joint mobilization can be performed to achieve a neurophysiological effect to \n reduce muscle pain and guarding , and a mechanical effect such as stretch or burst of \n contracted tissues .", "one study reported increased active exercise by patients as a result of \n joint mobilization3 .", "the physiological effects of joint mobilization , which is aimed at increasing the range of \n joint motion and pain reduction , can be explained by the gate control theory proposed by \n melzack and wall4 .", "the vicious cycle of \n muscle pain and spasm can be broken by closing the gate where the pain stimulus is largely \n transmitted through thin filaments , which have slow stimulus conduction velocity , while \n proprioceptive neurons of thick filaments are stimulated .", "the present study aimed to determine the effect of an 8-week program of joint mobilization \n on changes in pelvic obliquity and pain level in seventeen female university students aged \n in their 20 s with sacroiliac joint dysfunction by dividing them into two groups : a joint \n mobilization group , and a control group .", "seventeen subjects were selected from female university students aged in their 20 s \n attending n university in cheon - an city , korea .", "the subjects had sacroiliac joint syndrome , \n but experienced no problems with daily living and had no previous experience of joint \n mobilization exercise .", "the subjects were randomly assigned to a joint mobilization group of \n eight , and a control group of nine who performed joint mobilization exercise . the mobilization with movement ( mwm ) group was 21.131.46 years old , 158.596.91 cm tall , \n and 59.5912.93 kg in weight . the control group was 23.202.15 years old ,", "inbody 720 ( biospace , korea ) was used to measure the subjects body composition while \n 4d - formetric ( germany ) was used for pelvic analysis .", "body fat and lean body mass were measured using inbody 7.0 ( biospace , korea ) . the direct \n segmental multi - frequency bioelectrical impedance analysis method ( dsm - bia ) was used for \n body composition measurement .", "a pressure footstool ( pedoscan , diers , germany ) and a trunk measurement system ( formetric \n 4d , diers , germany ) , a 3d image processing apparatus with high resolution for the vertebrae \n used in the studies of lippold and colleagues5 and schroder6 , \n were used to measure 3d trunk images of vertebrae and pelvis obliquity , as well as static \n balance ability .", "the posterior innominate and anterior innominate methods were used for joint mobilization \n ( mobilization with movement ) .", "these two methods have been well described in the literature \n by mulligan7 , both procedures were \n repeated 10 times for one set , and for three sets ( figs \n 1 and 2fig .", "posterior innominate of mwm anterior innominate of mwm spss pc for windows ( version 18.0 ) was used for data processing . in order to analyze the \n inter - group effect , two - way anova with repeated measures", "if a main effect was \n found , the paired t - test was conducted to test the difference between the groups , and \n one - way anova was conducted to determine the difference of each parameter between the \n groups .", "all the subjects understood the purpose of this study and provided their written informed \n consent prior to their participation in the study in accordance with the ethical principles \n of the declaration of helsinki .", "the pelvic obliquity dl - dr decreased by 3.25 from 4.88 before the intervention to 1.63 \n after the mobilization exercise in the control mwm group , but increased by 0.22 from 1.89 \n to 2.11 in the control group .", "the pelvic torsion l - r decreased by 0.5 from 2.50 to 2.00 in the mwm group , but \n increased by 0.66 from 1.56 to 2.22 in the control group .", "the trunk length increased by 8.38 mm from 440.25 mm to 448.63 mm in the mwm group , but \n decreased by 1.89 mm from 451.67 mm to 449.78 mm in the control group ( table 1)table 1 .", "change of pelvis malpositiongrouppre - testpost - testpelvic obliquity dl - dr ( )mwm4.882.481.631.92**control1.891.692.111.76pelvic l - r torsion ( )mwm2.500.762.000.53*control1.561.012.221.20**trunk length ( mm)mwm440.2517.62448.6315.62*control451.6716.82449.7815.93***p<0.05 , * * p < 0.01 : paired t - test . # # p<0.01 : independent t - test . * p<0.05 , * * p < 0.01 : paired t - test .", "# # p<0.01 : independent t - test the mean velocity decreased by 0.28 mm from 1.26 mm to 0.98 mm in the mwm group , but \n increased by 0.1 mm from 1.13 mm to 1.23 mm in the control group .", "the sway area decreased by 0.14 cm from 0.21 cm to \n 0.07 cm in the mwm group , but increased by 0.07 cm from \n 009 cm to 0.16 cm in the control group . the mean frequency decreased by 0.42 hz from 0.97hzto 0.55 hz in the mwm group , but was \n unchanged at 0.66 hz in the control group ( table \n 2table 2 .", "change of static stabilitygrouppre - testpost - testmean velocity ( cm / s)mwm1.260.270.980.12*control1.130.131.230.14sway area ( cm)mwm0.210.220.070.03control0.090.040.160.11mean frequency ( hz)mwm0.970.520.550.28*control0.660.260.660.18*p<0.05 , * * p < 0.01 : paired t - test . # # p<0.05 : independent t - test ) . * p<0.05 , * * p < 0.01 : paired t - test . # # p<0.05 : independent t - test", "the sacroiliac joint refers to the posterior joint of the bony pelvis between the sacrum \n and the ilium of the pelvis . with its extremely limited mobility and small joint \n mobilization", ", this joint rarely causes any pathological problem except for body imbalance \n due to sacroiliac joint obliquity and changes in iliac , ischium , and length of the \n ilium8 .", "the analysis of the pelvis \n obliquity showed the intervention of this study provided the greatest statistically \n significant interaction effect on left and right pelvis obliquity ( p<0.001 ) .", "furthermore , \n a group performing combined joint mobilization and functional exercise showed statistically \n significant differences between before and after the exercise whereas the control group \n performing simple joint mobilization did not ( p<0.001 ) . in a previous study , yang9 reported the measurement results of changes \n around the sacroiliac joint after 12 weeks of rolling massage for patients with chronic low \n back pain .", "he found that ilium deviation was reduced by about 2.37 mm , sacroiliac joint \n deviation by about 2.25 mm , and ischium deviation by about 2.5 mm , resulting in a \n significant difference overall in the sacroiliac joint - related areas ( p<0.001 ) .", "however , \n lee10 reported that changes in low back \n pain found in experimental and control groups after manipulative therapy administered to \n patients with sacroiliac joint dysfunction with muscle imbalance differed significantly \n between the two groups before and after the experiment ( p<0.001 ) , whereas changes in \n pelvis rotation between the experimental and control groups showed significant difference \n between before and after the experiment ( p>0.05 ) , in contrast to our study s results .", "we \n attribute difference in these study results to the difference between our joint mobilization \n and lee s manipulative therapy , and to the lack of any functional exercise . for pain control , it is advantageous to discover and treat specific lesions causing the \n pain ( trigger point , over - loaded muscle , weakened or abnormal movement types , or joint \n dysfunction ) , not only to reduce the symptoms ( pain ) but also to induce functional \n recovery11 .", "the results for the joint mobilization intervention used in this study show that two groups \n of showed statistically significant decreases of pain compared to the control group \n ( p<0.001 ) . in a previous study , lee12 reported \n significant reductions of low back pain , functional disorder level , and low back instability \n in patients with chronic low back pain after lower extremity strengthening exercise along \n with low back stabilization exercise ( p<0.05 ) .", "he also claimed that a program of combined \n exercise performing low back stabilization exercise and lower extremity strengthening \n exercise was more effective at decreasing low back pain , functional disorder level , and low \n back instability than a stabilization exercise alone .", "han13 reported that a combination of \n functional exercises resulted in significant pain relief from 4.61 to 1.94 on a subjective \n pain index ( vas ) , while simple exercise resulted in non - significant pain relief from 3.93 to \n 1.57 .", "furthermore , im14 reported that vas \n showed a significant difference between before and after chuna therapy and spinal \n stabilization exercises for 16 weeks .", "significant differences were also found between two \n groups : a group of single treatment with chuna therapy and a group of combined treatment of \n chuna therapy and spinal stabilization exercise ." ]

[ "<S> [ purpose ] the present study aimed to determine the effect of an 8-week program of joint \n mobilization on changes in pelvic obliquity and pain level in seventeen female university \n students aged in their 20 s with sacroiliac joint dysfunction by dividing them into two \n groups : a joint mobilization group ( mwm ) and a control group . </S>", "<S> [ subjects ] seventeen \n subjects were selected from female university students aged in their 20 s attending n \n university in cheon - an city , korea , the subjects had sacroiliac joint syndrome , but \n experienced no problems with daily living and had no previous experience of joint \n mobilization exercise . </S>", "<S> the subjects were randomly assigned to a joint mobilization group \n of eight and a control group of nine who performed joint mobilization exercise . </S>", "<S> [ methods ] \n body fat and lean body mass were measured using inbody 7.0 ( biospace , korea ) . the direct \n segmental multi - frequency bioelectrical impedance analysis method ( dsm - bia ) was used for \n body composition measurement . </S>", "<S> a pressure footstool ( pedoscan , diers , germany ) and a trunk \n measurement system ( formetric 4d , diers , germany ) , a 3d image processing apparatus with \n high resolution for vertebrae , were used to measure 3d trunk images of the vertebrae and \n pelvis obliquity , as well as static balance ability . </S>", "<S> [ result ] the mwm group showed a \n significantly better balance than the control group . </S>", "<S> in addition , the results of the \n left / right and the front / rear balance abilities were significantly better than those of \n the control group . [ conclusion ] this study proved that a combination of mobilization with \n movement and functional training was effective in reducing pelvis malposition and pain , \n and improving static stability control . </S>" ]

[ "INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION" ]

[ [ "due to industrial development and the consequent increase in daily comfort , physical \n activities have gradually decreased , leading to increased body weight with reduced physical \n strength , as well as more sedentary lifestyles and significantly decreased amounts of \n exercise . due to incorrect exercise habits , even among those who exercise regularly , bad \n posture over extended periods , and inappropriate daily life and work habits ,", "the resulting continuous back tension has increased the \n number of patients with low back pain1 .", "mennell2 presented the term joint \n dysfunction for arthrokinematic dysfunction in the absence of pathological changes in the \n joints , including capsules and ligaments .", "he also attributed muscular pain and muscle spasm \n to difficulties with normal arthrokinematic mobilization in joint capsules , which limit \n joint movement when patients attempted to move joints suffering from the symptoms of joint \n dysfunction .", "joint mobilization can be performed to achieve a neurophysiological effect to \n reduce muscle pain and guarding , and a mechanical effect such as stretch or burst of \n contracted tissues .", "one study reported increased active exercise by patients as a result of \n joint mobilization3 .", "the physiological effects of joint mobilization , which is aimed at increasing the range of \n joint motion and pain reduction , can be explained by the gate control theory proposed by \n melzack and wall4 .", "the vicious cycle of \n muscle pain and spasm can be broken by closing the gate where the pain stimulus is largely \n transmitted through thin filaments , which have slow stimulus conduction velocity , while \n proprioceptive neurons of thick filaments are stimulated .", "the present study aimed to determine the effect of an 8-week program of joint mobilization \n on changes in pelvic obliquity and pain level in seventeen female university students aged \n in their 20 s with sacroiliac joint dysfunction by dividing them into two groups : a joint \n mobilization group , and a control group ." ], [ "seventeen subjects were selected from female university students aged in their 20 s \n attending n university in cheon - an city , korea .", "the subjects had sacroiliac joint syndrome , \n but experienced no problems with daily living and had no previous experience of joint \n mobilization exercise .", "the subjects were randomly assigned to a joint mobilization group of \n eight , and a control group of nine who performed joint mobilization exercise . the mobilization with movement ( mwm ) group was 21.131.46 years old , 158.596.91 cm tall , \n and 59.5912.93 kg in weight . the control group was 23.202.15 years old ,", "inbody 720 ( biospace , korea ) was used to measure the subjects body composition while \n 4d - formetric ( germany ) was used for pelvic analysis .", "body fat and lean body mass were measured using inbody 7.0 ( biospace , korea ) . the direct \n segmental multi - frequency bioelectrical impedance analysis method ( dsm - bia ) was used for \n body composition measurement .", "a pressure footstool ( pedoscan , diers , germany ) and a trunk measurement system ( formetric \n 4d , diers , germany ) , a 3d image processing apparatus with high resolution for the vertebrae \n used in the studies of lippold and colleagues5 and schroder6 , \n were used to measure 3d trunk images of vertebrae and pelvis obliquity , as well as static \n balance ability .", "the posterior innominate and anterior innominate methods were used for joint mobilization \n ( mobilization with movement ) .", "these two methods have been well described in the literature \n by mulligan7 , both procedures were \n repeated 10 times for one set , and for three sets ( figs \n 1 and 2fig .", "posterior innominate of mwm anterior innominate of mwm spss pc for windows ( version 18.0 ) was used for data processing . in order to analyze the \n inter - group effect , two - way anova with repeated measures", "if a main effect was \n found , the paired t - test was conducted to test the difference between the groups , and \n one - way anova was conducted to determine the difference of each parameter between the \n groups .", "all the subjects understood the purpose of this study and provided their written informed \n consent prior to their participation in the study in accordance with the ethical principles \n of the declaration of helsinki ." ], [ "the pelvic obliquity dl - dr decreased by 3.25 from 4.88 before the intervention to 1.63 \n after the mobilization exercise in the control mwm group , but increased by 0.22 from 1.89 \n to 2.11 in the control group .", "the pelvic torsion l - r decreased by 0.5 from 2.50 to 2.00 in the mwm group , but \n increased by 0.66 from 1.56 to 2.22 in the control group .", "the trunk length increased by 8.38 mm from 440.25 mm to 448.63 mm in the mwm group , but \n decreased by 1.89 mm from 451.67 mm to 449.78 mm in the control group ( table 1)table 1 .", "change of pelvis malpositiongrouppre - testpost - testpelvic obliquity dl - dr ( )mwm4.882.481.631.92**control1.891.692.111.76pelvic l - r torsion ( )mwm2.500.762.000.53*control1.561.012.221.20**trunk length ( mm)mwm440.2517.62448.6315.62*control451.6716.82449.7815.93***p<0.05 , * * p < 0.01 : paired t - test . # # p<0.01 : independent t - test . * p<0.05 , * * p < 0.01 : paired t - test .", "# # p<0.01 : independent t - test the mean velocity decreased by 0.28 mm from 1.26 mm to 0.98 mm in the mwm group , but \n increased by 0.1 mm from 1.13 mm to 1.23 mm in the control group .", "the sway area decreased by 0.14 cm from 0.21 cm to \n 0.07 cm in the mwm group , but increased by 0.07 cm from \n 009 cm to 0.16 cm in the control group . the mean frequency decreased by 0.42 hz from 0.97hzto 0.55 hz in the mwm group , but was \n unchanged at 0.66 hz in the control group ( table \n 2table 2 .", "change of static stabilitygrouppre - testpost - testmean velocity ( cm / s)mwm1.260.270.980.12*control1.130.131.230.14sway area ( cm)mwm0.210.220.070.03control0.090.040.160.11mean frequency ( hz)mwm0.970.520.550.28*control0.660.260.660.18*p<0.05 , * * p < 0.01 : paired t - test . # # p<0.05 : independent t - test ) . * p<0.05 , * * p < 0.01 : paired t - test . # # p<0.05 : independent t - test" ], [ "the sacroiliac joint refers to the posterior joint of the bony pelvis between the sacrum \n and the ilium of the pelvis . with its extremely limited mobility and small joint \n mobilization", ", this joint rarely causes any pathological problem except for body imbalance \n due to sacroiliac joint obliquity and changes in iliac , ischium , and length of the \n ilium8 .", "the analysis of the pelvis \n obliquity showed the intervention of this study provided the greatest statistically \n significant interaction effect on left and right pelvis obliquity ( p<0.001 ) .", "furthermore , \n a group performing combined joint mobilization and functional exercise showed statistically \n significant differences between before and after the exercise whereas the control group \n performing simple joint mobilization did not ( p<0.001 ) . in a previous study , yang9 reported the measurement results of changes \n around the sacroiliac joint after 12 weeks of rolling massage for patients with chronic low \n back pain .", "he found that ilium deviation was reduced by about 2.37 mm , sacroiliac joint \n deviation by about 2.25 mm , and ischium deviation by about 2.5 mm , resulting in a \n significant difference overall in the sacroiliac joint - related areas ( p<0.001 ) .", "however , \n lee10 reported that changes in low back \n pain found in experimental and control groups after manipulative therapy administered to \n patients with sacroiliac joint dysfunction with muscle imbalance differed significantly \n between the two groups before and after the experiment ( p<0.001 ) , whereas changes in \n pelvis rotation between the experimental and control groups showed significant difference \n between before and after the experiment ( p>0.05 ) , in contrast to our study s results .", "we \n attribute difference in these study results to the difference between our joint mobilization \n and lee s manipulative therapy , and to the lack of any functional exercise . for pain control , it is advantageous to discover and treat specific lesions causing the \n pain ( trigger point , over - loaded muscle , weakened or abnormal movement types , or joint \n dysfunction ) , not only to reduce the symptoms ( pain ) but also to induce functional \n recovery11 .", "the results for the joint mobilization intervention used in this study show that two groups \n of showed statistically significant decreases of pain compared to the control group \n ( p<0.001 ) . in a previous study , lee12 reported \n significant reductions of low back pain , functional disorder level , and low back instability \n in patients with chronic low back pain after lower extremity strengthening exercise along \n with low back stabilization exercise ( p<0.05 ) .", "he also claimed that a program of combined \n exercise performing low back stabilization exercise and lower extremity strengthening \n exercise was more effective at decreasing low back pain , functional disorder level , and low \n back instability than a stabilization exercise alone .", "han13 reported that a combination of \n functional exercises resulted in significant pain relief from 4.61 to 1.94 on a subjective \n pain index ( vas ) , while simple exercise resulted in non - significant pain relief from 3.93 to \n 1.57 .", "furthermore , im14 reported that vas \n showed a significant difference between before and after chuna therapy and spinal \n stabilization exercises for 16 weeks .", "significant differences were also found between two \n groups : a group of single treatment with chuna therapy and a group of combined treatment of \n chuna therapy and spinal stabilization exercise ." ] ]